Pub Microbial Strategies For Crop Improvement

Microbial Strategies for Crop Improvement
Mohammad Saghir Khan l Almas Zaidi l
Javed Musarrat
Editors
Microbial Strategies
for Crop Improvement
Editors
Dr. Mohammad Saghir Khan Dr. Almas Zaidi
Aligarh Muslim University Aligarh Muslim University
Faculty Agricultural Sciences Faculty Agricultural Sciences
Dept. Agricultural Microbiology Dept. Agricultural Microbiology
Aligarh-202002 Aligarh-202002
India India
khanms17@rediffmail.com
Prof. Javed Musarrat

Aligarh Muslim University
Faculty Agricultural Sciences
Dept. Agricultural Microbiology
Aligarh-202002
India
ISBN 978-3-642-01978-4 e-ISBN 978-3-642-01979-1

DOI: 10.1007/978-3-642-01979-1
Springer Dordrecht Heidelberg London New York
Library of Congress Control Number: 2009928629
# Springer-Verlag Berlin Heidelberg 2009

This work is subject to copyright. All rights are reserved, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,
reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication
or parts thereof is permitted only under the provisions of the German Copyright Law of September 9,
1965, in its current version, and permission for use must always be obtained from Springer. Violations
are liable to prosecution under the German Copyright Law.
The use of general descriptive names, registered names, trademarks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant
protective laws and regulations and therefore free for general use.
Cover design: WMXDesign GmbH, Heidelberg, Germany
Printed on acid-free paper
Springer is part of Springer Science+Business Media (www.springer.com)

Foreword
With an ever-increasing human population, the demand placed upon the agriculture
sector to supply more food is one of the greatest challenges for the agrarian
community. In order to meet this challenge, environmentally unfriendly agrochem-
icals have played a key role in the green revolution and are even today commonly
recommended to circumvent nutrient deficiencies of the soils. The use of agro-
chemicals is, though, a major factor for improvement of plant production; it causes
a profound deteriorating effect on soil health (soil fertility) and in turn negatively
affects the productivity and sustainability of crops. Concern over disturbance to the
microbial diversity and consequently soil fertility (as these microbes are involved in
biogeochemical processes), as well as economic constraints, have prompted funda-
mental and applied research to look for new agro-biotechnologies that can ensure
competitive yields by providing sufficiently not only essential nutrients to the plants
but also help to protect the health of soils by mitigating the toxic effects of certain
pollutants. In this regard, the role of naturally abundant yet functionally fully
unexplored microorganisms such as biofertilizers assume a special significance in
the context of supplementing plant nutrients, cost and environmental impact under
both conventional practices and derelict environments. Therefore, current develop-
ments in sustainability involve a rational exploitation of soil microbial communities
and the use of inexpensive, though less bio-available, sources of plant nutrients,
which may be made available to plants by microbially-mediated processes. These
organisms affect plant growth directly by fixation of atmospheric nitrogen, solubi-
lization of minerals such as phosphorus, or by production of plant growth regulators
(phytohormones), or indirectly by improving growth-restricting conditions either
via production of antagonistic substances, by inducing resistance against pathogens,
and by improving soil properties by leaving organic residues. In addition, the
rhizobacterial strains can improve plant stress tolerance to drought, salinity, and
metal toxicity leading thereby to increased plant growth. Plant growth-promoting
rhizobacteria also increase the growth of plants through the synthesis of specific
enzymes, which induce physiological changes in plants. For example, ethylene
plays a critical role in various developmental processes and regulates nod factor
signaling and nodule formation. At higher concentrations, ethylene inhibits growth
v
vi Foreword
and development of plants. However, bacterial enzyme 1-aminocyclopropane-1-

carboxylate (ACC) deaminase alleviates the stress induced by an ethylene-
mediated impact on plants. Other organism of great practical significance to plant
system is arbuscular mycorrhizal (AM) fungi, which play a major role in plant
resource capture and nutrient cycling. Thus, microbial populations could participate
in many key ecosystem processes such as those involved in the biogeochemical
cycling of essential nutrients and biological control of plant pathogens, and,
consequently, may affect plant development under different agro-ecosystems. The
alternative approach to traditional biofertilizers could be the development of bacte-
rial biofilms that may be used effectively as biofertilizers for various crops. Soil
microbial communities play an important role in crop improvement in different
agro-ecosystems by decomposing various organic matters, but due in part to the
scarcity of convenient methods for exploration, our understanding of the different
degrees and dynamics of microbial community including structure and functional
diversity is so far limited. In addition, soil microbial communities are constantly
under environmental pressure. Therefore, we need a better understanding of how
different factors including plant genotypes, soil management practices, and agro-
chemicals influence the microbial variations in soil environments. Furthermore,
there is also a need to examine the rate of these responses to environmental changes
and the factors that influence the rate of change and how these responses are related
to community composition. Pathogenic microorganisms affecting plant health are a
major and chronic threat to food production and ecosystem stability worldwide. To
address such problems, pesticides are considered as a relatively reliable method of
crop protection, but may instead cause several negative effects including develop-
ment of pathogen resistance to the applied agents and their nontarget environmental
impacts. Furthermore, there are also a number of fastidious diseases for which
chemical solutions are few, ineffective, or nonexistent. Control of soil-borne
pathogens by applying microbes or microbial products (biopesticides) provides a
viable alternative way of reducing the use of chemicals in crop improvement.
Worldwide, salinity is one of the most important abiotic stresses that limit crop
growth and productivity. Ion imbalance and hyper-osmotic stress in plants caused
by high concentrations of salt often lead to oxidative stress conditions for plants.
Soil salinization may be due to natural causes, and is common in the hot and dry
regions of the world, or it may be a consequence of inadequate irrigation manage-
ment practices. In this context, the use of microorganisms offers an attractive
alternative to facilitate the growth of plants in otherwise inhibitory levels of salt.
An understanding of these mechanisms is likely to lead to the development of
simple and practical approaches to dealing with this problem in the field. Further-
more, in the Mediterranean basin, a millenarian history of overexploitation has
led to the loss of most primeval forests and an increase of the surface area covered
by shrublands that represent stages of degradation of mature forests. In this
situation, environmental characteristics act as barriers to succession and, hence,
human intervention by applying reforestation practice is usually necessary to
improve recovery of woodlands. To address such problems, some native plant
species could act as ‘‘nurse plants’’ through their positive impacts on soil abiotic
Foreword vii
characteristics and also through positive influence on soil microbiota, especially on

symbiotic microorganisms. The beneficial effects of these plant nurses on the
growth of Mediterranean tree species, the soil bio-functioning, and the underlining
of the benefits of using native plant species to rehabilitate degraded areas especially
in stressful conditions are suggested. Release of heavy metals without proper
treatment poses a serious threat to soil health because of their nonbiodegradability
and persistence. The remediation of heavily contaminated soils involving microbes
offers a promising and inexpensive approach to design strategies for crop improve-
ment in soils contaminated with heavy metals. In some instances, indeed, the
availability of metal-tolerant microbes with very specific mechanisms of action
has contributed substantially to our understanding of plant improvement under
stressed environments.
Despite the significant scientific developments, the role of microbial commu-
nities in productivity of crops and sustainability of environment is not fully
exploited for the benefit of crop improvement under different agro-ecosystems
around the world. The beneficial microbial systems involved in plant growth
promotion are very often crop- and region-specific besides soil-specific in natural
ecosystems. The advent of molecular and biotechnological tools has, however,
made it possible to tailor the genetic traits of such microbes to make them suitable
for a specific environment.
In recent years, the volume of research published on the contribution of microbes
in crop improvement has increased dramatically. This can be attributed to several
factors: in general, there has been an increasing interest in the application of
inexpensive natural bio-resource, microbes, within the scientific and agrarian com-
munities; there has been the advent of molecular engineering techniques which help
to identify and design microbes with specific activities; and finally this has
prompted industries to become much more involved in the exploitation of beneficial
microbes at a commercial scale. Progress in this exciting and applied field of
microbiology has been so pronounced that there has been a strong need for a
book on this subject. Although the research in this area has been published in
various scientific media, they are unlikely to provide sufficient information on the
themes covered in this book. Given the large and growing body of information
available on microbial effects in crop improvement, it is becoming difficult to
maintain familiarity with the subject. University students and teachers, agronomists
in particular and scientists in the private sectors in general, require access to this
information. In this book ‘‘Microbial Strategies for Crop Improvement’’, we have
attempted to meet such needs, and to integrate the relevant information on micro-
bial strategies employed to improve crop productivity in different agro-ecological
niches into a single volume. Broadly, this book addresses the multidisciplinary
nature and many fascinating aspects of microbiological approaches for crop im-
provement in both conventional and stressed soils where quality and safety are the
key concerns. The major goal of microbial strategies for crop improvement is
intended to provide a cross-section of latest accomplishments and envisaged future
directions in these areas. Furthermore, the information provided here gives a
holistic view of the basic concepts and practical utility of microbes. We hope that
viii Foreword
the reader will find this book a useful source of information on many aspects of crop
improvement through microbial applications as well as a starting point for more
advanced reading and research in this area. This compilation is expected to help
students, teachers, industry regulators, and nonspecialist readers to get balanced and
up-to-date information on potential application of microbes in crop improvement.
This book is thus likely to benefit the people working in the area of agronomy,
biotechnology, environmental biology, microbiology, plant physiology, plant pro-
tection, and soil science.
The contributions by eminent academicians and professionals for this volume
have ensured a good equilibrium between theory and practice without compromis-
ing the basic conceptual framework of the concerned subject. Qualified editors and
authors from different countries have tried to bring in quality and the most attractive
presentation. This book will thus present an all-inclusive contemporary treatise on
strategic aspects of the diverse microbial communities providing solutions to many
of common agronomic problems and indeed some bizarre ones facing the agrarian
communities. The book also provides an opportunity to the readers to understand
the complexity of microbes and exploit these natural resources for the benefit of
crops. This book will serve as an important and comprehensive source material
because it includes recent data focusing mainly on the microbial strategies for
improvement and sustainability of crops leading thereby to achieve global food
security.
Preface
The dramatic increase in the use of chemical fertilizers for achieving optimum
yields has become an integral component of present day agricultural practices. The
frequent and imbalanced application of such fertilizers is not only expensive but
also pollutes the environment at a faster rate and makes soils unsuitable for
cultivation. In addition, soil deterioration (soil salinization), disturbances in com-
position and functional properties of soil microbial communities and, consequently,
loss of soil fertility following various soil management practices has further
compounded the agronomic problems. To overcome such environmentally unde-
sirable activities, we need to develop a viable substitute that could address those
problems more effectively in a sustained manner. In this context, the microbiolo-
gical strategies involving the use of functionally diverse groups of microbes as vital
components of soil ecosystems provides an inexpensive alternative to chemical
fertilizers. In recent times, much interest has been generated in exploiting microbial
strategies to facilitate plant growth and development, and in some cases they have
been commercialized for different crops. The majority of organic growers adopt
these microbial technologies without proper knowledge and understanding of it,
and use them to inoculate seeds/soils to provide nutrients like phosphate, nitrogen,
and other phyto-compounds. In addition, microbes have also attracted worldwide
attention due to their role in disease management and remediation of salinized/
polluted soils. Thus, the microbial communities in general are the potential tools for
sustainable production of crops and the trend for the future. Scientific researchers,
however, involve multidisciplinary approaches to understand the complexity and
practical utility of the wide spectrum of microbes for the benefits of crops. The
success of crop improvement, however, depends largely on the performance of
microbes and the willingness and acceptance by the growers. Substantial amounts
of research work has been done to highlight the role of microbes in the improve-
ment of crops, but very little attempt is made to organize such findings in a way that
could substantially help students/teachers/scientists and to progressive farmers.
‘‘Microbial Strategies for Crop Improvement’’ written by experts in the field
provides a comprehensive source of information on strategies and concepts of
microbial technology for the improvement of crops in different agro-ecosystems.
ix
x Preface
The book presents strategies for the management of salinized soils and crop dis-
eases, and explores means of integrating various approaches to achieve desired
levels of crop yield under both conventional and derelict soils. Traditional applica-
tions for molecular plant–microbe interactions in crop improvement (e.g., nitrogen
fixation in legume–rhizobia symbioses and the improvement of plant P nutrition by
arbuscular mycorrhizae) are broadly covered in the book. This book also presents a
broad and updated view of the phosphate-solubilising microbes and role of metal-
tolerant microbes in providing protection to plants grown in metal-contaminated
soils. Thus, it provides a reference point that will be an invaluable resource for
crop improvement. The preparation and application of microbial biofilm inoculants,
the role of bacterial ACC deaminase in the development of functional symbiosis
between rhizobia and legumes, and functional diversity among plant growth-
promoting rhizobacteria are also discussed. The book further describes how the
plant growth-promoting rhizobacteria facilitate plant growth and how advanced
information strategies can be used to manipulate and modify the soil environment.
Plant growth-promoting diazotrophs and productivity of wheat on the Canadian
Prairies is discussed separately. The common mechanisms regulating symbiosis and
development in biopesticides to control pathogenic microbes are discussed, making
it possible to further examine their role in the crop improvement. The other factors
affecting the crop productivity include the health of soil and how soil management
practices affects the fertility of soils. Special attention is paid to highlight the role of
a fertile soil in crop improvement and to understand the impact of various manage-
ment practices on the variability of microbes both in terms of community structure
and their function. This book is useful for a wide audience of students/researchers/
practitioners specializing in different areas of microbiology and plant science, agro-
chemistry, soil biology, molecular biology, and other related disciplines.
We very sincerely wish to acknowledge our colleague authors who participated in
this endeavor from different countries and who have assisted in the development of
‘‘Microbial Strategies for Crop Improvement’’ by providing the recent information
and comprehensive scripts without which it would have been extremely difficult to
complete this herculean task. Also, we want to thank our scientific colleagues who
generously spared their valuable time to respond to our queries regarding the pre-
paration of this book and who have informally made suggestions to us to improve the
overall presentation of the book. We also wish to thank research scholars working
with us, who have helped immensely in making this dream a reality. We also grate-
fully thank the publisher of this book.
We are grateful to our families for their support during the compilation of this book.
Finally, we believe this book may have some conceptual or printing mistakes
arising unintentionally during preparation for which we apologise in anticipation
and, if pointed out to us, will certainly correct in subsequent printings/editions. We
also invite suggestions and healthy criticism from the readers of this book so that the
information on the subject it contains can be improved in future.
India, March 2009 Mohammad Saghir Khan

Almas Zaidi
Javed Musarrat
Contents
1 The Use of Microorganisms to Facilitate the Growth of Plants

in Saline Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Elisa Gamalero, Graziella Berta, and Bernard R. Glick
2 Recent Advances in Plant Growth Promotion by

Phosphate-Solubilizing Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Almas Zaidi, Mohammad Saghir Khan, Munees Ahemad, Mohd Oves,
and P.A. Wani
3 Developing Beneficial Microbial Biofilms on Roots of Non legumes:

A Novel Biofertilizing Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Gamini Seneviratne, RMMS Thilakaratne, APDA Jayasekara, KACN
Seneviratne, KRE Padmathilake, and MSDL De Silva
4 Role of 1-Aminocyclopropane-1-carboxylate deaminase

in Rhizobium–Legume Symbiosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Javed Musarrat, Abdulaziz A AlKhedhairy, Saud Al-Arifi,
and Mohammad Saghir Khan
5 Strategies for Crop Improvement in Contaminated Soils

Using Metal-Tolerant Bioinoculants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Anju Rani and Reeta Goele
6 Functional Diversity Among Plant Growth-Promoting Rhizobacteria:

Current Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Mohammad Saghir Khan, Almas Zaidi, P. A. Wani, Munees
Ahemad, and Mohammad Oves
7 Plant Growth Promoting Rhizobacteria and Sustainable

Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Azeem Khalid, Muhammad Arshad, Baby Shaharoona,
and Tariq Mahmood
xi
xii Contents
8 Soil Health – A Precondition for Crop Production . . . . . . . . . . . . . . . . . . . 161

Niharendu Saha and Biswapati Mandal
9 Recent Advances in Biopesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

Parvez Qamar Rizvi, Rummana A. Choudhury, and Arshad Ali
10 Benefits of Arbuscular Mycorrhizal Fungi to Sustainable

Crop Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
M. Vosátka and J. Albrechtová
11 Enhancement of Rhizobia–Legumes Symbioses and Nitrogen

Fixation for Crops Productivity Improvement . . . . . . . . . . . . . . . . . . . . . . . . 227
Hamdi Hussein Zahran
12 Monitoring the Development of Nurse Plant Species

to Improve the Performances of Reforestation Programs
in Mediterranean Areas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
R. Duponn gois, M. Hafidi, J. Thioulouse, A. Galiana, L. Ouahmane,
B. Dreyfus, and Y. Prin
13 Pea Cultivation in Saline Soils: Influence of Nitrogen Nutrition . . . . 267

Etelvina Figueira
14 Plant Growth-Promoting Diazotrophs and Productivity of Wheat

on the Canadian Prairies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
Anthony O. Anyia, Daniel J. Archambault, Carols J. Bécquer,
and Jan J. Slaski
15 Factors Affecting the Variation of Microbial Communities

in Different Agro-Ecosystems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Munees Ahemad, Almas Zaidi, Md Saghir Khan, and Mohammad Oves
16 Strategies for Utilizing Arbuscular Mycorrhizal Fungi and

Phosphate-Solubilizing Microorganisms for Enhanced Phosphate
Uptake and Growth of Plants in the Soils of the Tropics . . . . . . . . . . . . 325
Nelson Walter Osorio and Mitiku Habte
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353
The Editors
Mohammad Saghir Khan, Ph.D. is an Associate Professor at the Department of

Agricultural Microbiology, Aligarh Muslim University, Aligarh, India. Dr. Khan
received his M.Sc. from Aligarh Muslim University, Aligarh, India and his Ph.D.
(Microbiology) from Govind Ballabh Pant University of Agriculture and Technol-
ogy, Pantnagar, India. He has been teaching microbiology to postgraduate students
for the last 13 years and has research experience of 17 years. In addition to teaching,
Dr. Khan is engaged in guiding students for their doctoral degree in microbiology.
He has published over 50 scientific papers including, original research articles,
review articles, and book chapters in various national and international publication
media. Dr. Khan has also edited a book published by one of the leading publishers.
His interest in the various aspects and applications of microbiology coupled with
his classroom and laboratory experience makes him uniquely qualified to author
this book. He is deeply involved in research activities focusing mainly on rhizo-
biology, microbiology, environmental microbiology, especially, heavy metals–
microbes–legume interaction, bioremediation, pesticide–PGPR–plant interaction,
biofertilizers and rhizo-immunology.
Almas Zaidi, received her M.Sc. and Ph.D. (Agricultural Microbiology) from
Aligarh Muslim University, Aligarh, India, and currently serving as Guest faculty/
Assistant Professor at the Department of Agricultural Microbiology, Aligarh Mus-
lim University, Aligarh. She has been teaching microbiology at postgraduate level
for the last 5 years and has research experience of 13 years. She has published about
40 research papers and reviews in journals of national and international repute. She
has also contributed chapters to different books. Dr. Zaidi has also edited a book
published by one of the leading publishers. Her main focus of research is to address
problems related with rhizo-microbiology, microbiology, environmental microbi-
ology, and biofertilizer technology.
Javed Musarrat, M.Sc., Ph.D. (Biochemistry), former Chairman of the Depart-
ment of Agricultural Microbiology and Ex-Dean, Faculty of Agricultural Sciences,
Aligarh Muslim University, Aligarh, India is presently working as a Professor,
DNA Research Chair at the King Saud University, Riyadh. He has been teaching
xiii
xiv The Editors
biochemistry, microbiology, and molecular biology to postgraduate students for the

last 21 years and has research experience of about 25 years. He has contributed
more than 50 national and international scientific publications. Recently,
Dr. Musarrat has edited a book published by one of the leading publishers. He is
associated with several scientific bodies such as DBT, CSIR, UGC, ICAR, and
UPCST, in various capacities. His major area of interest includes the molecular
microbiology, microbial ecology, and genetic toxicology.
Contributors
Munees Ahemad, Department of Agricultural Microbiology, Faculty of

Agricultural Sciences, Aligarh Muslim University, Aligarh, U.P., India
Pervaze Ahmad Wani, Department of Agricultural Microbiology, Faculty of

Abdulaziz A Al Khedhairy, DNA Research Chair, College of Science, King Saud

University, Riyadh, SA
Saud Al-Arifi, DNA Research Chair, College of Science, King Saud University,
Riyadh, SA
J. Albrechtová, Charles University in Prague, Faculty of Science, Department of

Plant Physiology, Vinicna 5, 128 44 Prague, Czech Republic
Arshad Ali, Department of Plant Protection, Faculty of Agricultural Sciences,

Aligarh Muslim University, Aligarh; Uttar Pradesh, India
Anthony O. Anyia, Alberta Research Council, Bioresource Technologies,

P.O. Bag 4000, Vegreville, Alberta, Canada, T9C 1T4
Daniel J. Archambault, Laurentian University, 935 Ramsey Lake Road, Sudbury,

Ontario, Canada, P3E 2C6
Muhammad Arshad, Institute of Soil and Environmental Sciences, University of

Agriculture, Faisalabad 38040, Pakistan
Carols J. Bécquer, Ministerio de la Agricultura, Instituto de Ganaderı́a Tropical,

Estación Experimental Sancti-Spiritus, Cuba
xv
xvi Contributors
Graziella Berta, Dipartimento di Scienze dell’Ambiente e della Vita, Università

del Piemonte Orientale, ‘‘A. Avogadro’’, Alessandria, Italy
Rummana A. Choudhury, Department of Plant Protection, Faculty of

Agricultural Sciences, Aligarh Muslim University, Aligarh; Uttar Pradesh, India
M.S.D.L. De Silva, Tea Research Institute of Sri Lanka, Talawakele, Sri Lanka
B. Dreyfus, IRD. UMR 113. CIRAD/INRA/IRD/SUPAGRO/UM2. Laboratoire

des Symbioses Tropicales et Méditerranéennes (LSTM). Montpellier, France
R. Duponnois, IRD. UMR 113. CIRAD/INRA/IRD/SUPAGRO/UM2. Laboratoire

des Symbioses Tropicales et Méditerranéennes (LSTM). Montpellier. France
Etelvina Figueira, Centre for Cell Biology, Departamento de Biologia,

Universidade e Aveiro, Campus, de Santiago, 3810-193 Aveiro, Portugal
A. Galiana, CIRAD. UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2. Laboratoire

des Symbioses Tropicales et Méditerranéennes (LSTM). Montpellier. France
Elisa Gamalero, Dipartimento di Scienze dell’Ambiente e della Vita, Università

del Piemonte Orientale, ‘‘A. Avogadro’’, Alessandria, Italy
Bernard R. Glick, Department of Biology, University of Waterloo, Waterloo,

Ontario, Canada
Mitiku Habte, University of Hawai’i at Manoa, College of Tropical Agriculture

and Human Resources, St. John Room 102, 3190 Maile Way, Honolulu, HI,
USA, 96822
M. Hafidi, Equipe Ecologie et Environnement, Faculté des Sciences Semlalia,

Université Cadi Ayyad. Marrakech. Maroc
Hamdi Hussein Zahran, Botany Department, Faculty of Science, Beni-Suef

University, Beni-Suef 62511, Egypt
A.P.D.A. Jayasekara, Tea Research Institute of Sri Lanka, Talawakele, Sri Lanka
Azeem Khalid, Department of Environmental Sciences, PMAS Arid Agriculture

University, Rawalpindi 46300, Pakistan
Tariq Mahmood, Department of Environmental Sciences, PMAS Arid

Agriculture University, Rawalpindi 46300, Pakistan
Contributors xvii
Biswapati Mandal, AICRP on Soil Test Crop Response Correlation, Directorate

of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani- 741235 Nadia,
West Bengal, India
Javed Musarrat, Department of Agricultural Microbiology, Faculty of

Agricultural Sciences, Aligarh Muslim University, Aligarh, U.P., India; DNA
Research Chair, College of Science, King Saud University, Riyadh, SA
Nelson-Walter Osorio, Universidad Nacional de Colombia at Medellin, College

of Sciences, Soil Microbiology Research Group, Calle 59A No. 63-20, Medellin,
Colombia
L. Ouahmane, Equipe Ecologie et Environnement, Faculté des Sciences

Semlalia, Université Cadi Ayyad. Marrakech. Maroc
Mohammad Oves, Department of Agricultural Microbiology, Faculty of

K.R.E. Padmathilake, Biological Nitrogen Fixation Project, Institute of

Fundamental Studies, Hantana Road, Kandy, Sri Lanka
Y. Prin, CIRAD. UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2. Laboratoire des

Symbioses Tropicales et Méditerranéennes (LSTM). Montpellier. France
Parvez Qamar Rizvi, Department of Plant Protection, Faculty of Agricultural

Sciences, Aligarh Muslim University, Aligarh; Uttar Pradesh, India
Anju Rani, Department of Microbiology, Govind Ballabh Pant University of

Agriculture & Technology, Pantnagar, Uttaranchal – 263145, India
Goel Reeta, Department of Microbiology, Govind Ballabh Pant University of

Agriculture & Technology, Pantnagar, Uttaranchal – 263145, India
Mohammad Saghir Khan, Department of Agricultural Microbiology, Faculty of

Niharendu Saha, AICRP on Soil Test Crop Response Correlation, Directorate

of Research, Bidhan Chandra Krishi Viswavidyalaya, Kalyani- 741235 Nadia,
West Bengal, India
Gamini Seneviratne, Biological Nitrogen Fixation Project, Institute of

K.A.C.N. Seneviratne, Royal Botanic Gardens, Peradeniya, Sri Lanka

xviii Contributors
Baby Shaharoona, Institute of Soil and Environmental Sciences, University of

Agriculture, Faisalabad 38040, Pakistan
Jan J. Slaski, Alberta Research Council, Bioresource Technologies, P.O. Bag

4000, Vegreville, Alberta, Canada, T9C 1T4
R.M.M.S. Thilakaratne, Biological Nitrogen Fixation Project, Institute of

J. Thioulouse, Université Lyon 1; CNRS; UMR 5558, Laboratoire de Biométrie

et Biologie Evolutive, 43 boulevard du 11 novembre 1918, Villeurbanne F-69622,
France
M. Vosátka, Institute of Botany, Academy of Sciences of the Czech Republic, 252

43 Pruhonice, Czech Republic
Almas Zaidi, Department of Agricultural Microbiology, Faculty of Agricultural

Sciences, Aligarh Muslim University, Aligarh, U.P., India
Chapter 1
The Use of Microorganisms to Facilitate
the Growth of Plants in Saline Soils
Elisa Gamalero, Graziella Berta, and Bernard R. Glick
Abstract Worldwide, salinity is one of the most important abiotic stresses that
limits crop growth and productivity. Ion imbalance and hyperosmotic stress in
plants caused by high concentrations of salt often lead to oxidative stress conditions
for plants. Soil salinization may be due to natural causes, and is common in the hot
and dry regions of the world, or it may be a consequence of inadequate irrigation
management practices. It has been estimated that around 20% of the world’s
cultivated lands and up to half of all irrigated lands are affected by high salinity.
Moreover, at the present time, there is more arable land being lost through salinity
than is gained through the clearing of forests. In this chapter, the ability of plant
beneficial microorganisms, notably plant growth-promoting (PGP) bacteria and
mycorrhizae, to facilitate plant growth in the presence of salt is reviewed and
discussed. Particular attention is paid to the development of a fundamental under-
standing of precisely how these microorganisms enable plants to proliferate in the
presence of otherwise inhibitory levels of salt. A better understanding of these
mechanisms is likely to lead to the development of simple and practical approaches
for dealing with this problem in the field.
1.1 Introduction
Salinity is an enormous worldwide problem for agriculture, especially for crops that
are grown under irrigation. This is because salt is inhibitory to the growth of a large
number of different plants (Cuartero and Fernandez-Munoz 1999). Moreover, the
E. Gamalero (*) and G. Berta

Dipartimento di Scienze dell’Ambiente e della Vita, Università del Piemonte Orientale “A. Avogadro”,
Alessandria, Italy
e‐mail: elisa.gamalero@unipmn.it
B.R. Glick
Department of Biology, University of Waterloo, Waterloo, ON Canada
M.S. Khan et al. (eds.), Microbial Strategies for Crop Improvement, 1

DOI: 10.1007/978-3-642-01979-1_1, # Springer‐Verlag Berlin Heidelberg 2009
2 E. Gamalero et al.
amount of salt-affected land worldwide is estimated to be >900 million hectares

which is approximately 6% of the total global land mass, or about 20% of the
world’s cultivated area (Flowers 2004). Importantly, around half of the land
devoted to the growth of irrigated crops is adversely affected by salt.
Soil salinity inhibits plant growth and development with adverse effects such as
osmotic stress, Na+ and Cl toxicity, ethylene production, plasmolysis, nutrient
imbalance, production of reactive oxygen species (ROS), and interference with
photosynthesis (Sairam and Tyagi 2004). The consequence of these physiological
changes is an inhibition of seed germination, seedling growth and vigor, flowering
and fruit set. Early responses of plants to drought and salinity are very similar; both
are attributed to water stress. Water stress-induced metabolic processes include a
decrease in photosynthesis, the production of ROS and generation of the plant
hormone abscisic acid (Bartels and Sunkar 2005). When plants are exposed to high
salinity, a decrease in the growth rate followed by a gradual recovery to a new
reduced growth rate is the plant’s first response to the decrease in water potential
caused by salt, rather than to any salt-specific toxicity (Verslues et al. 2006). Once
taken up, Na+ may be translocated to shoots in the rapidly moving transpiration
stream in the xylem (Smith et al. 1980). Although Na+ can return to roots via the
phloem, this flow is minimal so that leaves or shoots typically accumulate higher
concentrations of Na+ than roots (Tester and Davenport 2003). Moreover, most
crops translocate little Na+ to reproductive or storage structures such as seeds as
these organs are fed mainly through the phloem. On the other hand, vegetative
tissues are supplied mainly by the xylem flow and therefore tend to be higher in Na+
levels. The metabolic toxicity of Na+ is mainly attributed to the Na+ competition
with K+ for binding sites essential for cellular functioning (Blaha et al. 2000; Tester
and Davenport 2003).
Chlorine ion, an essential micronutrient for higher plants, is involved in the
oxygen-evolving reactions of photosynthesis, maintaining electrical neutrality
across membranes, and adjusting the vacuolative osmotic condition. Root cells
take up Cl from soil through anion channels; the Cl then traverses the root by a
symplastic pathway to reach the xylem. Like Na+, floral tissues generally have
lower Cl levels than other shoot parts, and tissues that are fed predominantly
through the phloem (e.g., fruits and seeds) tend to have the lowest Cl concentra-
tions (White and Broadley 2001). Salinity-induced stress in plants is partly the result
of ethylene production (O’Donnell et al. 1996; Cuartero and Fernandez-Munoz
1999; Blumwald 2000; Mayak et al. 2004; Shibli et al. 2007). For instance,
ethylene production was stimulated from two- to about tenfold in tomato
(Lycopersicon esculentum) and Arabidopsis plants that were exposed to salinity stress
(Richard and El-Abd 1989; Hall and Smith 1995). In this regard, in chickpea
(Cicer arietinum), Kukreja et al. (2005) not only observed a salinity-induced increase
in ethylene evolution, but also increases both in 1-aminocyclopropane-1-carboxylate
(ACC) content, the immediate precursor of ethylene, and in the activity of the
enzyme ACC oxidase. Furthermore, the relationship between salinity stress and ethylene
production was consistent with the observation that aminoethoxyvinylglycine
(AVG), a chemical inhibitor of ethylene biosynthesis, alleviated salinity-induced
1 The Use of Microorganisms to Facilitate the Growth of Plants in Saline Soils 3
plant responses such as increased hook closure and thickness of seedlings

(El Beltagy et al. 1979).
Salt-tolerant plants may be grouped into two categories: salt-excluders and
salt-includers. The former group of plants adapt to a saline environment by avoiding
salt, whereas the includers take up salt and sequester it. Biochemical strategies to
cope with salt stress include (1) selective accumulation or exclusion of ions,
(2) control of ion uptake by roots and transport into leaves, (3) compartmentalization
of ions at the cellular and whole-plant levels, (4) synthesis of compatible solutes,
(5) alteration of membrane structure, (6) induction of antioxidative enzymes, and
(7) induction of plant hormones (Khan and Rizvi 1994; Parida and Das 2005).
However, it is important to bear in mind that the salt tolerance of any particular
species is likely to vary with the growth phase of the plant, the ionic composition of
the soil, and the overall health of the plant.
1.2 Mechanisms Used by Plant Growth-Promoting Bacteria
Plant beneficial bacteria are of two general types: those that form a symbiotic
relationship, which involves formation of specialized structures or nodules on
host plant roots, and those that are free-living in the soil; the latter are often
found near, on, or even within the roots of plants (Kloepper et al. 1989; van Peer
and Schippers 1989 Frommel et al. 1991). Beneficial free-living soil bacteria are
often referred to as plant growth-promoting rhizobacteria (PGPR) and are found in
association with the root surfaces of many different plants. However, to be inclusive
of the many different types of bacteria that facilitate plant growth, the term plant
growth-promoting bacteria (PGPB), is preferred (Bashan and Holguin 1998).
Moreover, while numerous free-living soil bacteria are considered to be PGPB,
not all bacterial strains of a particular genus and species have identical metabolic
capabilities. Thus, for example, some Pseudomonas putida strains may actively
promote plant growth while others have no measurable effect on plants.
PGPB can function either indirectly or directly (Glick 1995). Indirect promotion
of plant growth occurs when bacteria decrease or prevent some of the deleterious
effects of a phytopathogenic organism (a fungus or a bacterium) by any one or more
of several different mechanisms. On the other hand, direct promotion of plant growth
by PGPB generally entails providing the plant with a compound that is synthesized
by the bacterium or facilitating the uptake of nutrients from the environment.
Direct promotion of plant growth can occur in several different ways. PGPB may
fix atmospheric nitrogen and supply it to plants; synthesize and secrete siderophores
which can solubilize and sequester iron from the soil and provide it to plant cells;
synthesize different phytohormones, including auxins, cytokinins and gibberelins;
solubilize minerals such as phosphorus which then become more readily available
for plant growth; and they may synthesize an enzyme that can modulate plant
ethylene levels (Brown 1974; Kloepper et al. 1986, 1989; Davison 1988; Lambert
and Joos 1989; Glick 1995; Patten and Glick 1996, 2002). In addition, a particular
bacterium may affect plant growth and development using any one, or more, of these
mechanisms. Moreover, since many PGPB possess several traits that enable them to
facilitate plant growth, a bacterium may utilize different traits at various times
during the life cycle of the plant, and may vary considerably in its effectiveness
depending upon the plant host and the soil composition. Interestingly, PGPB generally
have little or no measurable effect on plant growth when the plants are cultivated in
nutrient-rich soil and grown under optimal conditions in the absence of stress.
One of the major mechanisms that many PGPB use to facilitate plant growth is
the lowering of plant ethylene levels through the action of the enzyme ACC
deaminase (Glick 1995, 2004; Glick et al. 1998, 2007a, b). A model was previously
developed to explain the role of ACC deaminase in the promotion of plant growth
(Glick et al. 1998). In this model, the ACC deaminase-expressing bacteria bind to
the surface of either plant seeds or roots and, in response to exuded tryptophan
(or other small molecules), the bacteria synthesize and secrete indole acetic acid (IAA)
(Fallik et al. 1994; Patten and Glick 2002), some of which is taken up by the plant.
Together with the endogenous pool of plant IAA, the IAA taken up by the plant can
stimulate plant cell proliferation and elongation and/or it can induce synthesis of the
plant enzyme ACC synthase which converts S-adenosylmethionine to ACC (Kende
1993). Some of the newly synthesized ACC is exuded by the plant, along with other
small molecules, and taken up by the bacteria where it is cleaved by ACC deaminase
to form a-ketobutyrate and ammonia, both of which are readily metabolized by
the bacteria (Penrose and Glick 2001; Whipps 1990). Some of the ACC that
remains within the plant root or seed may be converted to ethylene when the
enzyme ACC oxidase is present (Kende 1993). In essence, bacteria that express
ACC deaminase, and are bound to the surface of roots or seeds, act as a sink for
ACC, thereby decreasing the amount of ethylene that can form when the plant is
stressed (Glick et al. 1998; Glick 2004). In this way, ACC deaminase-expressing
PGPR protect plants against the growth inhibition that might otherwise result
following flooding, extremes of temperature, the presence of organic or inorganic
toxicants, phytopathogens, drought, or high salt (Glick 2004).
Given that the overwhelming majority of rhizobacteria are able to synthesize and
secrete IAA (Patten and Glick 1996), the question arises why the IAA produced by
these bacteria does not eventually result in inhibitory levels of ethylene in response to
the IAA (activating ACC synthase transcription). In fact, as the level of ethylene rises,
it progressively blocks IAA signal transduction (Dharmasiri and Estell 2004; Glick
et al. 2007a). Thus, ACC deaminase works synergistically with IAA so that when a
bacterium expresses ACC deaminase, the enzyme lowers the ethylene repression of
auxin response factor synthesis, and allows IAA to “do its job” and increase plant
growth without producing excessive amounts of ethylene (Glick et al. 2007a).
1.3 Mechanisms Used by Plant Growth-Promoting Fungi
There is an enormous amount of literature regarding the use of fungi to protect

agricultural plants from salt-induced damage. However, microorganisms promoting
salt tolerance in trees have received relatively little attention. Thus, while numerous
reports have highlighted the beneficial role of arbuscular mycorrhizal (AM) fungi,
only a few of them have focused on the positive effects induced by ectomycorrhizal
(ECM) fungi which are typically found in association with many trees in nature.
Surprisingly, to our knowledge, only one report (Waller et al. 2005) deals with the
alleviation of salt stress by the employment of a nonmycorrhizal soil fungus. The
mechanisms by which PGP fungi (both mycorrhizal and nonmycorrhizal ones)
enhance plant tolerance to salinity are described in the following section.
The main mechanisms thought to be involved in salt stress alleviation by AM
fungi may be summarized as follows: (1) improvement of mineral nutrition leading
to plant growth promotion; (2) variation in the plant accumulation of Na and K as
well as soluble sugars and electrolytes; (3) modification of some physiological
processes and enzymatic activities involved in plant antioxidative reactions; and
(4) alteration of the root architecture facilitating water uptake by the plant. Obviously,
differential gene expression/protein synthesis induced by the establishment of
the symbiosis between the fungi and plant under salt stress is at the base of
these strategies. Early studies demonstrated that improved P nutrition increases
crop yields under saline conditions (Hirrel and Gerdemann 1978; Champagnol 1979).
In addition, several papers (Poss et al. 1985; Awad et al. 1990; Azcón and Atrash
1997; Ruiz-Lozano et al. 1996; Cantrell and Linderman 2001; Feng et al. 2002;
Colla et al. 2008) have compared the effect of AM fungi with P applications on
plant growth in saline conditions. It is now evident that the increased salinity
tolerance in mycorrhizal plants is based on AM fungi-catalyzed effects, such as
the improvement of the plant’s water status, and the enhancement of nutrient uptake
from the soil (Smith and Read 1997). Moreover, the improvement of plant nutrition
and water uptake may be attributed to mycorrhizal-induced modifications of the
root architecture, which result in more numerous, thicker and/or more branched
roots in herbaceous plants, and in a strong increase in the number of laterals of
highest order in woody plants (Berta et al. 1993, 2002, 2005; Gamalero et al. 2002,
2004). Furthermore, studies on the effect of salinity on mycorrhizal plants have
shown that AM roots maintain a high K+/Na+ ratio (Allen and Cunningham 1983;
Pfeiffer and Bloss 1988; Sannazzaro et al. 2006; Giri et al. 2004, 2007).
Some physiological processes, such as increased carbon dioxide exchange rate,
stomatal conductance and water use efficiency, all involved in osmoregulation, are
facilitated by AM fungi (Ruiz-Lozano et al. 1996). Depending on the particular
fungal species involved, AM plants under water deficit conditions are more efficient
in taking up water than non-AM plants under the same conditions (Marulanda et al.
2003; Khalvati et al. 2005). An improved osmoregulation capacity in AM maize
(Zea mays L. cv. Yedan 13) plants is indicated by the higher soluble sugar and
electrolyte concentrations recorded (Feng et al. 2002). Furthermore, salt is known
to reduce the activity or abundance of Hg-sensitive water channels, termed aqua-
porins, in plants (Carvajal et al. 1999, 2000). In salt-stressed tomato plants
preinoculated with a mixture of Glomus geosporum and G. intraradices, the content
of aquaporins in roots is reduced, while the content of these proteins in leaves is
significantly increased, suggesting a major impact of AM fungi on the distribution
of water throughout the plant. In addition, it is likely that AM fungi facilitate the
mobilization of water in the presence of salt by mediating its transfer from the root
to the shoot (Ouziad et al. 2006).
A higher relative water content, leading to the prevention of leaf dehydration

caused by drought or salinity, has been measured in the leaves of common beans
(Phaseolus vulgaris) inoculated with the AM fungus G. intraradices (Aroca et al. 2007).
In agreement with the previously mentioned beneficial effect of AM fungi on
the host plant water status, the variation in the relative water content of AM
plants is associated with a reduction of the transpiration stream and an increase
of free exuded sap flow (Aroca et al. 2007).
Damage to plants that are induced by salt stress may also be a consequence
of the production of ROS (Hernandez et al. 1995). In this regard, plants
with high concentrations of antioxidants or antioxidative enzymes are typically
more resistant to damage by ROS (Spychalla and Desbough 1990; Dionisio-Sese
and Tobita 1998; Jiang and Zhang 2002). It has been well established that AM
fungi affect the expression of various antioxidative enzymes. For example, in
soybean (Glycine max) plants that were grown in the presence of salt and were
inoculated with a salt-adapted isolate of the AM fungus G. etunicatum, the
amount of peroxidase and polyphenoloxidase activity was found to increase
(Ghorbanli et al. 2004).
Clear positive effects of AM fungi on the formation of nodules and the expression
of antioxidative activity by leguminous plants have recently been described
(Garg and Manchanda 2008). Thus, although pigeonpea (Cajanus cajan) plants
exposed to salt had a higher number of nodules compared to untreated plants,
these nodules were characterized by reduced biomass, relative permeability, lipid
peroxidation, acetylene-reducing activity (ARA), and leghemoglobin content.
Inoculation with G. mosseae increased nodulation, leghemoglobin content,
nitrogenase activity, and the activity of several antioxidant enzymes (superoxide
dismutase, catalase, ascorbate peroxidase, peroxidise, and glutathione reductase),
suggesting that these factors may be responsible for the positive effects of
mycorrhiza to stress-induced premature nodule senescence. The synthesis of the
plant polyamines putrescine, spermidine and spermine is often affected by
stressful environmental conditions such high salt concentrations (Krishnamurthy
and Bhagwat 1989; Aziz et al. 1998; Simon-Sarkadi et al. 2002). Recently,
Sannazzaro et al. (2007) found higher amounts of total free polyamines in AM
plants (Lotus glaber) compared to non-AM ones suggesting that the modulation of
polyamine content could be one of the mechanisms involved in the adaptation of
plant by AM fungi to saline soils.
AM fungi may influence the level of the plant hormone, abscisic acid
(Danneberg et al. 1992). This hormone plays a key role in mediating plant responses
to several stresses (Zhang et al. 2006) For instance, it promotes stomatal closure to
reduce transpirational water loss and activates a number of genes involved in
stress responses. As observed by Jahromi et al. (2008), the abscisic acid content
in lettuce (Lactuca sativa) roots colonized by the AM fungus G. intraradices was
significantly higher than in non-AM lettuce roots. Furthermore, in the presence of
salt, non-AM roots accumulated more abscissic acid than roots colonized by the
AM fungus. Thus, AM fungi may alter the expression of abscisic acid and
thereby modulate gene expression that results in protection of the plant against
salt stress.
In addition to the other mechanisms used by AM fungi, it has recently been
demonstrated that modifications of plant morphogenetic parameters and photosynthetic
efficiency induced by these organisms may be involved in supporting the plant’s
development in salt conditions (Gamalero et al. 2009). Thus, leaves of cucumber
plants inoculated with the AM fungus Gigaspora rosea and grown under salt stress
were more abundant and had a higher average and total leaf projected area than
uninoculated plants. Roots of AM plants under salt stress were more developed than
those of uninoculated plants grown in presence or absence of salt. It is likely that the
higher total root length, surface area and tip number found in the roots of AM plants
may be more efficient at supporting plant growth in saline soils. In addition, while
the presence of salt caused a fivefold reduction in leaf photosynthetic efficiency
(PI), AM plants exposed to salt showed PI values comparable to plants grown in
absence of salt (Gamalero et al. 2009). It is likely that the observed improvement
of photosynthetic activity in the presence of AM fungi is related to the increase of
chlorophyll content in the leaves of AM plants (Ruiz-Lozano et al. 1996; Giri and
Mukerji 2004; Sannazzaro et al. 2006; Zuccarini 2007).
Although several in vitro studies (Hutchison 1990; Chen et al. 2001; Kernaghan
et al. 2002; Bois et al. 2006; Langenfeld-Heyser et al. 2007) have demonstrated the
potential of salt-tolerant ECM fungi to enhance the tolerance of trees to salinity,
little is known about the mechanisms involved. However, variations in the plant
water status and in the Na+ and K+ content induced by the interaction of the plant
with ECM have been reported. For example, Muhsin and Zwiazek (2002a, b) found
that the ECM fungus Hebeloma crustuliniforme enhanced the root hydraulic
conductance of white spruce (Picea glauca) trees in the presence of salt. Reduction
of Na+ and Cl–, with a concomitant increase of PO4–2 and K+, in the tissues of
seagrape (Coccoloba uvifera L.) seedlings colonized by the ECM Scleroderma
bermudense indicate a higher osmoregulating capacity of ECM plants in saline soils
(Bandou et al. 2006). Similar results were obtained by Nguyen et al. (2006) in ECM
spruce and pine. Nevertheless, this salt exclusion mechanism seems to be host
plant-specific (Yi et al. 2008). In fact, while salt-treated aspen (Populus tremuloides
Michx.) colonized by the ECM fungus H. crustuliniforme had higher Na+ and Cl–
than non-ECM plants, Na+ and Cl– concentrations in ECM and non-ECM birch
(Betula papyrifera Marsh.) did not differ. Nevertheless, no information exists about
the possibility of salt tolerance in these trees when colonized by AM fungi.
Finally, it has been found that the PGP-endophytic fungus Piriformospora indica
protects barley (Hordeum vulgare) seedlings from the stress induced by moderate
concentration of salt as well as biotic stress, such as infection with the necrotrophic
fungus Fusarium culmorum. This beneficial activity appears to be a consequence of
an increase of antioxidant activity in the plant and the activation of systemic
resistance induced by the colonization of the plant by P. indica (Waller et al. 2005).
From this brief overview, it is apparent that the mechanisms employed by PGP
fungi are quite different from those used by PGPB. Moreover, these two types
of microorganisms colonize plant roots (and possibly other tissues) very differ-
ently. Thus, it should be possible, by better understanding of how plants,
bacteria, and mycorrhizae interact with one another, to eventually design/engi-
neer this tripartite relationship so that it is most beneficial for the promotion of
plant growth in the presence of salt and other environmental stresses.
1.4 Amelioration of Salt Stress by Plant Growth-Promoting

Bacteria
Numerous reports on the role of soil bacteria in mitigating the inhibitory effects of
salt on growth and development of plants are available. For the most part, the
mechanistic basis of this protection is unknown. However, several studies have
suggested a variety of possible explanations for this behavior.
It is well known that a variety of environmental stresses, including the presence
of salt, induce the production of ethylene in a number of different plants (Jones and
El-Abd 1989; Abeles et al. 1992; Hall and Smith 1995; Kukreja et al. 2005; Parida
and Das 2005). While the physiology of salt-stressed plants is often dramatically
altered compared to plants grown in the absence of salt (Munns 2002; Sairam and
Tyagi 2004; Parker et al. 2006), many of the changes observed (including increased
production of osmolytes and antioxidants) may be secondary effects of increased
ethylene levels within the plant. In this regard, it has been demonstrated that the
NTHK1 gene from tobacco, which encodes an ethylene receptor, is specifically
induced by salt (Zhang et al. 2001; Cao et al. 2006). If ethylene does indeed act as a
stress signal activating the synthesis of a range of salt-stress response genes in
plants then it should be possible, by attenuating plant ethylene levels, to significantly
modify the stress response of the plant to salt. Based on the idea that bacterial
strains that contain the enzyme ACC deaminase can lower ethylene levels throughout
the plants where these bacteria are bound to the roots, Mayak et al. (2004)
reasoned that these bacteria should not only lower the level of stress ethylene
following exposure of plants to salt, they should also prevent much of the growth
inhibition that might otherwise ensue. Mayak et al. (2004) collected soil samples
from dried river beds in the arid and salty Arava region in the southern part of the
Negev desert in Israel. PGPB isolated from these soil samples were selected to
assess their ability to produce ACC deaminase and, on the basis of preliminary
testing, one bacterium, Achrobacter piechaudii ARV8, was selected for further
characterization. This strain not only significantly lowered the ethylene produced
by tomato plants following the addition of salt, but also dramatically increased
both root and shoot growth in the presence of salt concentrations up to 200 mM.
This pioneering work served as the basis for a spate of experimental work on several
different plants by scientists from all over the world. Since then, laboratories
from India, Pakistan, China and Canada have reported successfully employing
ACC deaminase-expressing bacteria to promote plant growth in the presence
of high levels of salt (Saravanakumar and Samiyappan 2006; Cheng et al. 2007;
Nadeem et al. 2007; Yue et al. 2007). These studies have included groundnut
(Saravanakumar and Samiyappan 2006), maize (Nadeem et al. 2007), cotton (Yue
et al. 2007), and canola (Cheng et al. 2007). Importantly, Saravanakumar and
Samiyappan (2006) demonstrated that the protection against growth inhibition that
ACC deaminase-expressing bacteria provide to plants grown in salt-impacted soil is
evident in the field as well as in a laboratory or greenhouse setting. In the experiments
reported by Cheng et al. (2007), only wild-type P. putida UW4 and not an ACC
deaminase minus mutant of this bacterium protected canola plants against growth
inhibition by salt. This result provides a clear demonstration of the role of ethylene in
growth inhibition by high salt. In addition to the work mentioned above, we are aware
of the unpublished results of researchers in Uzbekistan and Iran which are also
consistent with the successful use of ACC deaminase-expressing bacteria to promote
plant growth in the presence of high levels of salt, both in the laboratory and in the field.
It was found, in a recent study of tomatoes treated with salt, that while the
chemical ethylene inhibitors CoCl2 and NiCl2 significantly reduced ethylene
accumulation in the headspace of the reaction vessel, thereby reducing epinasty,
the negative effects of the salt on growth and other physiological parameters were
essentially unchanged (Shibli et al. 2007). These results were interpreted as indicat-
ing that ethylene does not make a major contribution to the inhibition of plant
growth under saline conditions (Shibli et al. 2007). The differences between the
results with chemical inhibitors of ethylene production and those observed with
bacteria that express ACC deaminase may reflect artifacts that are part of the closed
system including tomato microshoots that were utilized to study the effects of salt
plus chemical inhibitors. On the other hand, several studies that employed PGPB
that contained ACC deaminase were conducted with whole plants in various types
of soil (including in the field) reflecting a more natural situation. Moreover, in
addition to the studies reported using ACC deaminase-expressing bacteria, transgenic
canola plants that express a bacterial ACC deaminase gene under the control of
a root-specific promoter were significantly protected against the growth inhibitory
effects of 50, 100 and 200 mM salt compared to nontransformed plants
(Sergeeva et al. 2006), consistent with the fact that ethylene is a major contributor
to growth inhibition in the presence of salt.
A number of researchers have reported that treatment of plants with either
Azospirillum lipoferum or A. brasilense can mitigate some of the inhibitory effects
of salt stress on wheat, maize, beans or lettuce (Bacilio et al. 2004; Hamdia et al.
2004; Rabie and Almadini 2005; Barassi et al. 2006). Since at least some of these
strains do not possess ACC deaminase activity (Holguin and Glick 2001), those
bacteria must utilize mechanisms other than lowering ethylene with ACC
deaminase to protect plants. In this regard, it is possible (but not proven) that
bacterial IAA, synthesized by these Azospirillum spp. Strains, is responsible for
the promotion of plant growth in the presence of salt.
A number of other bacterial mechanisms have been implicated, but not proven,
to be involved in ameliorating some of the inhibitory effects of high salt concentrations
on plant growth and development. In one study, several different commercial
seed treatments, primarily consisting of various Bacillus spp. strains, were tested
for the ability to protect squash plants against salt stress (Yildrim et al. 2006).
These workers concluded that these seed treatments worked by altering the
uptake of minerals into the plant thereby increasing the K+/Na+ ratio which is
positively correlated with plant growth. Other workers have suggested that
N-acyl-homoserine lactone quorum-sensing signaling molecules can mediate
the ability of two different strains of the PGPB Burkholderia graminis to promote
tomato plant growth and to induce protection against salt stress (Barriuso et al.
2008). While the data presented clearly indicate the involvement of quorum
sensing as a component of the overall functioning of these bacteria, quorum
sensing per se is unlikely to fully explain the mechanistic basis behind the ability of
these strains to protect plants against salt stress.
In a novel approach to developing an understanding of some of the mechanisms
used by PGPB to facilitate plant growth in the presence of salt, protein changes that
occur in the bacterium Pseudomonas fluorescens MSP-393 as a consequence of salt
stress were elaborated using a proteomics approach (Paul et al. 2006). Thus, when
P. fluorescens MSP-393 was incubated in the presence of high salt, the synthesis of
a number of unique proteins was induced. Not surprisingly, the majority of the
proteins that could be identified were homologous to stress proteins from other
prokaryotes. It was therefore suggested that, by maintaining its normal metabolic
capabilities in the presence of salt, a salt-stressed PGPB is still able to facilitate
plant growth (by whatever, unspecified, mechanism(s) it normally employs).
Finally, two separate laboratories have reported the isolation and characterization
of different bacterial strains, all of which confer some measure of salt tolerance
on treated plants, specifically maize and wheat (Prı́ncipe et al. 2007; Egamberdieva
et al. 2008). These studies evaluated various biological activities of these bacteria
such as IAA production, biocontrol activity, siderophore production, phosphate
solubilization, plant growth promotion with and without salt, and bacterial genus
and species. Unfortunately, from the data presented, no correlation between salt
stress amelioration activity and any one or two biological traits measured was
found. Thus, while it may be straightforward to select soil bacteria that can
ameliorate some of the inhibitory effects of salt stress, these studies do not provide
any clear indication as to the mechanism (s) that are employed by these bacteria.
1.5 Amelioration of Salt Stress by Plant Growth-Promoting

Fungi
Despite the fact that some AM fungi naturally occur in saline environments
(Pond et al. 1984; Rozema et al. 1986; Ho 1987; van Duin et al. 1989; Cooke and
Lefor 1990; Sengupta and Chaudhuri 1990; Hoefnagels et al. 1993; Johnson-Green
et al. 1995), the reports on the effects of salts on AM fungi are contradictory. For
instance, some researchers observed inhibition of AM colonization by salt
(Ojala et al. 1983; Duke et al. 1986; Rozema et al. 1986; Dixon et al. 1993; Johnson-
Green et al. 1995; Juniper 1996; McMillen et al. 1998), while others noted that the
development of the symbiosis remained unaffected by salt application (Levy et al.
1983; Hartmond et al. 1987). It is likely that AM fungi isolated from saline soils are
better adapted to this environmental condition and, therefore, are more efficient at
plant growth promotion under salt stress. Surprisingly, in one instance, it was found
that AM fungi from nonsaline soil performed better in the amelioration of plant
growth under salt stress than AM fungi from saline soils (Cantrell and Linderman
2001). On the other hand, in these experiments, AM fungi from saline soils
colonized roots more intensively than AM fungi from nonsaline soils, indicating
that, in this case, the plant beneficial effects of AM fungi were independent of the
root colonization efficiency and the hypothetical adaptation of the microorganism
to salt. Similar results were obtained for two strains of Glomus mosseae, one
isolated from a saline soil and the other from a nonsaline soil (Tian et al. 2004).
The fungus from the saline soil induced a high accumulation of sodium and chloride
and had very little effect on the alleviation of salt stress in cotton (Gossypium
hirsutum) plants suggesting that the main mechanism involved in the protection of
these plants from the detrimental effects of salinity by AM fungi is related to the
capability of the fungi to influence the uptake of sodium and chloride. This
notwithstanding, performing salt-acclimatization of AM fungi, by exposing the
fungal isolate to gradual increases of NaCl concentration, has been reported to
increase their efficiency in supporting plant growth in saline conditions
(Sharifi et al. 2007). In addition, it is important to stress that a preferential associa-
tion exists between plants and AM fungi (Bever et al. 2002; Gollotte et al. 2004;
Copetta et al. 2006; Pivato et al. 2007) that could help to select the appropriate/
specific fungus to better tolerate salt stress.
Since AM fungi are obligate symbionts, differentiation between the AM fungal
response to environmental stress and the AM fungal response to plant-mediated
factors is not straightforward. In an effort to sort it out, several workers have
attempted to understand how AM fungi react to environmental stimuli by focusing
on the precolonization phase and spore germination (Daniels and Trappe 1980;
Elias and Safir 1987; Gianinazzi-Pearson et al. 1989). Following this approach,
Juniper and Abbott (2006) observed that the main effect of soil salinity is a delay
of spore germination and inhibition of hyphal growth from propagules of AM fungal
isolates. In addition, in the presence of salt, the number of branched absorbing
structures and the rate of sporulation both significantly declined (Jahromi et al. 2008).
Despite numerous contradictory results, especially in the earlier literature, the
more recent literature that is our main focus document a significant extent of plant
protection by AM fungi. As pointed out by Cantrell and Linderman (2001), it may
be important to preinoculate plants with AM fungi in order to firmly establish their
symbiotic relationship which might otherwise be inhibited/prevented by salt in the
soil. In fact, AM lettuce and onion (Allium cepa L.) transplants grown in saline soil
had higher shoot biomass than noninoculated plants indicating that preinoculated
AM plants grow better than non-AM plants under saline conditions (Cantrell and
Linderman 2001). The composite application of AM fungi has been reported to be
more efficient than single inoculation in alleviating plant salt stress. As an example,
the growth of Acacia auriculiformis under salinity stress varied with the fungal
species used, but the mixed inoculum (Glomus fasciculatus and macrocarpum)
resulted in the highest root colonization, biomass production, K+ concentration,
PO42 level, and chlorophyll content at all salinity levels (Giri et al. 2003). The
contribution of this mixed inoculum to plant growth is not limited to improved
nutrition, but also to variation in root development so that plants treated with
the two fungi had very branched roots. This AM-mediated modification of root
architecture is probably caused by the decreased meristematic activity of root
apices as shown in leek (Berta et al. 2002). AM fungi also increase the number of
adventitious roots, thereby improving nutrient uptake and water balance in the host
plant under salinity stress. The root modifications previously described occur in
herbaceous plants, where AM fungi also delay root senescence (Lingua et al. 1999).
Different modifications are described in AM woody plants where root turnover may
be facilitated. These two strategies (delayed senescence and root turnover) enable
AM fungi to accomplish the same result, namely a more vital and efficient root
system, that could allow plants to better tolerate abiotic stresses, including salt
stress. The positive effects of AM fungi on plants under salt conditions may also
lead to increases in fruit yield. As an example, in tomato, fruit fresh yield was
enhanced by 29% under nonsaline and by 60% under saline water conditions
following preinoculation of transplants with G. mosseae (Al-Karaki 2006). It
would be interesting to ascertain whether the same occurs in other fruit plants,
whose root systems are strongly influenced by AM fungi (Schellenbaum et al. 1991;
Berta et al. 1995; Manschke et al. 1995).
AM fungi can also stimulate the rhizobial symbiosis efficiency of leguminous
plants exposed to salt. As observed by Giri et al. (2004), in saline soil, plants of
Sesbania aegyptiaca and S. grandiflora inoculated with Glomus macrocarpum had
higher root and shoot dry biomass production, chlorophyll content and PO4 2,
N and Mg+2 concentrations, than non-AM seedlings. Moreover, the number of
nodules was significantly higher in AM than non-AM plants. Interestingly, the
mycorrhizal dependency of both Sesbania species in salt conditions increased with
plant age suggesting that, under salinity, plants need AM fungi not only for
acclimatization but also for continued nutrient uptake for their development
(Giri et al. 2004). In addition, the mycorrhizal dependency is higher for salt-sensitive
than for salt-tolerant plant varieties (Sannazzaro et al. 2006).
Although Basiodiomycetes are generally considered to be relatively salt-sensitive
(Tresner and Hayes 1971), several isolates of Laccaria, Hebeloma, and Pisolithus
have been characterized as relatively salt tolerant (Chen et al. 2001; Kernaghan
et al. 2002). In addition, several in vitro studies highlight the salt tolerance of
ECM fungi, generally ascribed to salt exclusion and osmoregulation, and their
potential exploitation under field conditions (Chen et al. 2001; Kernaghan et al.
2002; Bois et al. 2006). Experiments performed under greenhouse conditions
confirmed the beneficial effects of ECM fungi on plant growth in the presence of
salt. Thus, seagrape plants colonized by S. bermudense were exposed to a wide
range of NaCl levels. The beneficial effects of the fungus on plant growth and
mineral nutrition occurred both under salt stress and nonstress conditions,
suggesting that the ECM effect in improving plant growth is not a specific
process induced by salinity. In addition, as already reported for AM fungi, the
mycorrhizal dependency of seagrape increased with increasing NaCl levels
(Bandou et al. 2006).
It has recently been shown that the fungus Paxillus involutus can tolerate high
NaCl concentrations (Langenfeld-Heyser et al. 2007). The establishment of symbiosis
between this fungus and the salt-sensitive poplar hybrid Populus canescens
positively affected plant biomass and reduced Na+ uptake. The plant’s symptoms
of salt stress (e.g., leaf degradation) were delayed but not fully prevented by the
symbiosis (Langenfeld-Heyser et al. 2007). It has been suggested that the fungus
might provide a physical barrier against salt uptake into the root (Muhsin and
Zwiazek 2002b). However, microscopical analysis performed by energy-dispersive
X-ray microanalysis showed that tissues of both mycorrhizal and nonmycorrhizal
roots contained comparable amounts of Na+ and Cl–. In addition, outer cell walls
adjacent to mycorrhizal hyphae contained higher sodium concentrations than
those of nonmycorrhizal plants indicating that mycorrhizal roots have access to
more NaCl than nonmycorrhizal roots and that the hyphal mantle does not
provide a physical barrier against NaCl influx.
Under saline conditions, the effect of ECM fungi may vary according to the plant.
In their work, Yi et al. (2008) compared the impact of the ECM H. crustuliniforme
on trembling aspen (P. tremuloides Michx.) and paper birch (B. papyrifera) in
the presence of salt. Despite the fact that both tree species are relatively tolerant
to NaCl, ECM inoculation in NaCl-treated plants increased the biomass.
However, roots of NaCl-treated aspen inoculated with H. crustuliniforme had over
twofold higher concentrations of sodium compared with nonmycorrhizal ones. On
the contrary, Na+ and Cl– concentrations in mycorrhizal and nonmycorrhizal birch
did not differ. What is clear from the bulk of the data examining the interaction of
mycorrhizae and plants in the presence of salt is that these fungi typically protect
plants from growth inhibition by salt. On the other hand, notwithstanding many
years of experimentation with different plants and mycorrhizal fungi in different
soils, the detailed mechanisms used by these PGP fungi remain elusive. However, it
is likely that through the use of microarray technology, proteomics and metabolomics
to study the changes in both plants and mycorrhizae that occur when these two
organisms interact will provide greater insight into these fundamental mechanisms.
1.6 Examples of Generating Salt Tolerance with Bacteria

and Mycorrhizae
Many of the manuscripts mentioned earlier in this chapter summarize the host of
beneficial effects of either PGPB or fungi on salt-stressed plants. In addition,
several pieces of evidence suggest the possibility of additive or synergistic effects
between these beneficial microorganisms in supporting plant growth in saline

conditions. However, the interactions among PGPB, PGP fungi, plants and stressful
conditions have not been studied in depth. Moreover, despite the fact that
mycorrhizal helper bacteria (MHB) (Garbaye and Bowen 1989), as well as fungal
endophytic bacteria (Bonfante-Fasolo and Scannerini 1977), were discovered some
time ago for ECM fungi, to our knowledge no studies have been performed on the
possible enhancement of plant tolerance to salt stress by the combination of ECM
fungi and these bacteria. Studies dealing with the coinoculation of bacteria and
fungi on plants exposed to salt stress have mainly been focused on the rhizobia–AM
fungi–legume tripartite symbioses (Hatimi 1999; Diouf et al. 2005; Rabie and
Almadini 2005).
In Northern Africa, the salt-tolerant tree Acacia cyanophylla is used to stabilize
the coastal dunes, increase saline soil fertility and produce large amounts of wood
and forage. Soil salinization and the low availability of water induce severe
reductions of plant height and dry biomass production (Hatimi 1999). Nevertheless,
the inhibitory physiological alterations of A. cyanophylla that were induced by salt
stress were moderated in the presence of Bradyrhizobium sp. Moreover, the toler-
ance of the plant to salinity increased significantly when this symbiosis between A.
cyanophylla and Bradyrhizobium sp. was associated with a mixture of endomycor-
rhizal fungi isolated from coastal dune soils. In plants grown in the presence of salt
and treated with both microsymbionts, the observed reduction of dry matter was
lower than when plants were inoculated with any one of these organisms
(Hatimi 1999). Similar results were obtained by combining Glomus intraradices
and Bradyrhizobia sp. with salt-stressed Acacia auriculiformis (A. Cunn. ex Benth.)
or Acacia mangium (Willd.) seedlings (Diouf et al. 2005).
An increase of plant tolerance to salt stress was observed in fava bean
(Vicia faba) plants inoculated with Glomus clarum. And the presence of nitrogen
fixing rhizobia in mycorrhizal fava bean plants resulted in an additional increase of
plant tolerance to salt. Moreover, the inhibitory effects of salinity on nitrogen
fixation were significantly reduced by preinoculating the fava bean plants with
the AM fungus (Rabie and Almadini 2005).
A clear example of the impact that salt stress can have on the interactions
between mycorrhiza and bacteria has recently been described by Gamalero et al.
(2008a, 2009). Under nonstressful conditions, the ACC deaminase-expressing
strain P. putida UW4 stimulated the development of the AM fungus G. rosea,
and this positive cooperation between microorganisms induced synergistic effects
on plant growth. Unfortunately, with this system, this synergism disappeared when
the plants were exposed to high levels of salt (Gamalero et al. 2009). The lack of
these positive interactions in the presence of salt may be explained in several
different ways. For example, plants exposed to salt stress can release different
root exudates that may decrease or abolish the synergism between the microorgan-
isms, even to the point where they are in competition with one another. Alternatively,
since salt stress can induce an increase in the level of ethylene in the stressed
plant, under these conditions, the amount of ACC deaminase that is synthesized by
the bacterial strain may become insufficient to reduce the ethylene due to both the
salt stress and to the localized plant response that occurs as a direct consequence of
AM colonization. Given that both PGPB and fungi can separately ameliorate some
of the effects of salt stress, there is a paucity of studies where these two types of
microorganisms are utilized together. Nevertheless, there is every reason to
believe that, with the right combinations of bacteria and fungi, the salt stress
of many important agricultural plants may be decreased significantly. In turn,
this would increase crop productivity on saline soils and facilitate agricultural
practice on some marginal lands.
1.7 Conclusion
1. Given the current reluctance on the part of many consumers worldwide to

embrace the use as foods of genetically modified plants, it may be advantageous
to consider the use of either PGPB or fungi (or a combination of the two) as a
means of promoting plant growth in the presence of otherwise inhibitory levels
of salt.
2. Using PGPB and/or fungi instead of genetically manipulated plants is also
advantageous when one considers the large number of different plants, the
many cultivars of those plants, and the multiplicity of genes that would need
to be engineered into plants to ensure that they are salt tolerant.
3. PGPB or fungi may be readily selected and/or manipulated to maximally protect
a wide range of different salt-stressed plants. Thus, it is easier to select and/or
modify a few dozen soil bacteria than hundreds or even thousands of different
plant cultivars.
4. The wide-scale use of PGPB and/or fungi may decrease the worldwide
dependence on agricultural chemicals. Moreover, it is a technology that is readily
accessible to farmers in both developed and developing countries.
5. The successful implementation of this approach has already been demonstrated
in the field to a limited extent. However, the large-scale use of this technology
would benefit from additional studies, particularly those directed toward under-
standing how the synergism between PGPB and fungi might be facilitated.
Acknowledgments The work from our laboratories that is cited in this manuscript has been
financially supported by grants from the Italian MIUR and by the Natural Sciences and Engineering
Research Council of Canada.
References
Abeles FB, Morgan PW, Saltveit ME Jr (1992) Ethylene in plant biology. Academic, New York
Al-Karaki GN (2006) Nursery inoculation of tomato with arbuscular mycorrhizal fungi and
subsequent performance under irrigation with saline water. Sci Hort 109:1–7
Allen EB, Cunningham GL (1983) Effects of vesicular arbuscular mycorrhizae on Distichlis

spicata under three salinity levels. New Phytologist 93: 227–236
Aroca R, Porcel R, Ruiz-Lozano JM (2007) How does arbuscular mycorrhizal symbiosis regulate
root hydraulic properties and plasma membrane aquaporins in Phaseolus vulgaris under
drought, cold or salinity stresses? New Phytol 173:808–816
Awad AS, Edwards DG, Campbell LC (1990) Phosphorus enhancement of salt tolerance of
tomato. Crop Sci 30:123–128
Azcón R, Atrash EEI (1997) Influence of arbuscular mycorrhizae and phosphorus fertilization on
growth, nodulation and N2 fixation in Medicago sativa at four salinity levels. Biol Fertil Soil
24:81–86
Aziz A, Martin-Tanguy J, Larher F (1998) Stress-induced changes in polyamine and tyramine
levels can regulate proline accumulation in tomato leaf discs treated with sodium chloride.
Physiol Plant 104:195–202
Bacilio M, Rodriguez H, Moreno M, Hernandez J-P, Bashan Y (2004) Mitigation of salt stress in
wheat seedlings by a gfp-tagged Azospirillum lipoferum. Biol Fertil Soil 40:188–193
Bandou E, Lebailly F, Muller F, Dulormne M, Toribio A, Chabrol J, Courtecuisse R, Plenchette C,
Prin Y, Duponnois R, Thiao M, Sylla S, Dreyfus B, Bâ AM (2006) The ectomycorrhizal fungus
Scleroderma bermudense alleviates salt stress in seagrape (Coccoloba uvifera L.) seedlings.
Mycorrhiza 16:559–565
Barassi CA, Ayrault G, Creus CM, Sueldo RJ, Sobrero MT (2006) Seed inoculation with
Azospirillum mitigates NaCl effects on lettuce. Sci Hort 109:8–14
Barriuso J, Solano BR, Fray RG, Cámara M, Hartmann A, Mañero FJG (2008) Transgenic tomato
plants alter quorum sensing in plant growth-promoting rhizobacteria. Plant Biotechnol J
6:442–452
Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58
Bashan Y, Holguin G (1998) Proposal for the division of plant growth-promoting rhizobacteria
into two classifications: biocontrol-PGPB (plant growth-promoting bacteria) and PGPB. Soil
Biol Biochem 30:1225–1228
Berta G, Fusconi A, Trotta A (1993) VA mycorrhizal infection and the morphology and function
of root systems. Environ Exp Bot 33:159–173
Berta G, Trotta A, Fusconi A, Hooker J, Munro M, Atkinson D, Giovannetti M, Marini S, Fortuna
P, Tisserant B, Gianinazzi-Pearson V, Gianinazzi S (1995) Arbuscular mycorrhizal induced
changes to plant growth and root system morphology in Prunus cerasifera L. Tree Physiol
15:281–293
Berta G, Fusconi A, Hooker JE (2002) Arbuscular mycorrhizal modifications to plant root
systems: scale, mechanisms and consequences. In: Gianinazzi S, Schüepp H, Barea JM,
Haselwandter K (eds) Mycorrhizal technology in agriculture. Birkäuser, Basel, pp 71–85
Berta G, Sampo S, Gamalero E, Massa N, Lemanceau P (2005) Suppression of Rhizoctonia
root-rot of tomato by Glomus mossae BEG12 and Pseudomonas fluorescens A6RI is associated
with their effect on the pathogen growth and on the root morphogenesis. Eur J Plant Pathol
111:279–288
Bever JD, Pringle A, Schultz PA (2002) Dynamics within the plant-arbuscular mycorrhizal fungal
mutualism: testing the nature of community feedback. In: van der Heijden MGA, Sanders IE (eds)
Mycorrhizal ecology. Springer, Heidelberg, Germany, pp 267–292
Blaha G, Stelzl U, Spahn CM, Agrawal RK, Frank J, Nierhaus KH (2000) Preparation of
functional ribosomal complexes and effect of buffer conditions on tRNA positions observed
by cryoelectron microscopy. Method Enzymol 317:292–309
Blumwald E (2000) Sodium transport and salt tolerance in plants. Curr Opin Cell Biol 12:431–434
Bois G, Bertrand A, Piché Y, Fung M, Khasa DP (2006) Growth, compatible solute and salt
accumulation of five mycorrhizal fungal species grown over a range of NaCl concentrations.
Bonfante-Fasolo P, Scannerini S (1977) Cytological observations on the mycorrhiza Endogone
flammicorona-Pinus strobus. Allionia 22:23–34
Brown ME (1974) Seed and root bacterization. Annu Rev Phytopathol 12:181–197
Cantrell IC, Linderman RG (2001) Preinoculation of lettuce and onion with VA mycorrhizal fungi
reduces deleterious effects of soil salinity. Plant Soil 233:269–281
Cao W-H, Liu J, Zhou Q-Y, Cao Y-R, Zheng SF, Du B-X, Zhang J-S, Chen SY (2006) Expression
of tobacco ethylene receptor NTHK1 alters plant responses to salt stress. Plant Cell Environ
29:1210–1219
Carvajal M, Martinez V, Alcarez CF (1999) Physiological function of water channels as affected
by salinity in roots of paprika pepper. Physiol Plant 105:95–101
Carvajal M, Cerda A, Martinez V (2000) Does calcium ameliorate the negative effect of NaCl on
melon root water transport by regulating aquaporin activity? New Phytol 145:439–447
Champagnol F (1979) Relationships between phosphate nutrition of plants and salt toxicity.
Phosphorus Agric 76:34–43
Chen DM, Ellul S, Herdman K, Cairnay JWG (2001) Influence of salinity on biomass production
by Australian Pisolithus spp. isolates. Mycorrhiza 11:231–236
Cheng Z, Park E, Glick BR (2007) 1-Aminocyclopropane-1-carboxylate deaminase from Pseudo-
monas putida UW4 facilitates the growth of canola in the presence of salt. Can J Microbiol
53:912–918
Colla G, Rouphael Y, Cardarelli M, Tullio M, Rivera CM, Rea E (2008) Alleviation of salt stress
by arbuscular mycorrhizal in zucchini plants grown at low and high phosphorus concentration.
Biol Fertil Soil 44:501–509
Cooke JC, Lefor MW (1990) Comparison of vesicular–arbuscular mycorrhizae in plants from
disturbed and adjacent undisturbed regions of a coastal salt marsh in Clinton, Connecticut,
USA. Environ Manage 14:131–137
Copetta A, Lingua G, Berta G (2006) Effects of three AM fungi on growth, distribution of
glandular hairs, and essential oil production in Ocimum basilicum L. var. Genovese. Mycor-
rhiza 16:485–494
Cuartero J, Fernandez-Munoz R (1999) Tomato and salinity. Sci Hortic 78:83–125
Daniels BA, Trappe JM (1980) Factors affecting spore germination of the vesicular–arbuscular
mycorrhizal fungus Glomus epigaeus. Mycologia 72:457–471
Danneberg G, Latus C, Zimmer W, Hundeshagen B, Schneider-Poetsch HJ, Bothe H (1992)
Influence of vesicular-arbuscular mycorrhiza on phytohormone balances in maize (Zea mays L.).
J Plant Physiol 141:33–39
Davison J (1988) Plant beneficial bacteria. Biotechnology 6:282–286
Dharmasiri N, Estell M (2004) Auxin signaling and regulated protein degradation. Trends Plant
Sci 9:302–308
Dionisio-Sese ML, Tobita S (1998) Antioxidant responses of rice seedling to salinity stress. Plant
Sci 135:1–9
Diouf D, Duponnois R, Ba AT, Neyra M, Lesueur D (2005) Symbiosis of Acacia auriculiformis
and Acacia mangium with mycorrhizal fungi and Bradyrhizobium spp. improves salt tolerance
in greenhouse conditions. Funct Plant Biol 32:1143–1152
Dixon RK, Garg VK, Rao MV (1993) Inoculation of Leucaena and Prosopis seedlings with
Glomus and Rhizobium species in saline soil: rhizosphere relations and seedling growth.
Arid Soil Res Rehabil 7:133–144
Duke ER, Johnson CR, Koch KE (1986) Accumulation of phosphorus, dry matter and betaine
during NaCl stress of split-root citrus seedlings colonised with vesicular–arbuscular mycorrhizal
fungi on zero, one or two halves. New Phytol 104:583–590
Egamberdieva D, Kamilova F, Validov S, Gafurova L, Kucharova Z, Lugtenberg BJJ (2008) High
incidence of plant growth-stimulating bacteria associated with the rhizosphere of wheat grown
on salinated soil in Uzbekistan. Environ Microbiol 10:1–9
El Beltagy A, Khalifa M, Hall M (1979) Salinity in relation to ethylene. Egypt J Hortic
6:269–271
Elias KS, Safir GR (1987) Hyphal elongation of Glomus fasciculatus in response to root exudates.
Appl Environ Microbiol 53:1928–1933
Fallik E, Sarig S, Okon Y (1994) Morphology and physiology of plant roots associated with
Azospirillum. In: Okon Y (ed) Azospirillum/Plant associations. CRC, Boca Raton, FL, pp 77–85
Feng G, Zhang FS, Li XL, Tian CY, Tang C, Rengel Z (2002) Improved tolerance of maize plants
to salt stress by arbuscular mycorrhiza is related to higher accumulation of soluble sugars in
roots. Mycorrhiza 12:185–190
Flowers T (2004) Improving crop salt tolerance. J Exp Bot 55:307–319
Frommel MI, Nowak J, Lazarovits G (1991) Growth enhancement and development modifications
of in vitro grown potato (Solanum tuberosum ssp. tuberosum) as affected by a nonfluorescent
Pseudomonas sp. Plant Physiol 96:928–936
Gamalero E, Martinotti MG, Trotta A, Lemanceau P, Berta G (2002) Morphogenetic modifica-
tions induced by Pseudomonas fluorescens A6RI and Glomus mosseae BEG12 in the root
system of tomato differ according to plant growth conditions. New Phytol 155:293–300
Gamalero E, Trotta A, Massa N, Copetta A, Martinotti MG, Berta G (2004) Impact of two
fluorescent pseudomonads and an arbuscular mycorrhizal fungus on tomato plant growth,
root architecture and P acquisition. Mycorrhiza 14:185–192
Gamalero E, Berta G, Massa N, Glick BR, Lingua G (2008) Synergistic interactions between
the ACC deaminase-producing bacterium Pseudomonas putida UW4 and the AM fungus
Gigaspora rosea positively affect cucumber plant growth. FEMS Microbiol Ecol 64:459–467
Gamalero E, Berta G, Massa N, Glick BR, Lingua G (2009) Interactions between Pseudomonas
putida UW4 and Gigaspora rosea BEG9 and their consequences on the growth of cucumber
under salt stress conditions. This paper has been accepted for publication in Journal of Applied
Microbiology 10.1111/j.1356-2672.2009.04414.x
Garbaye J, Bowen GD (1989) Stimulation of ectomycorrhizal infection of Pinus radiata by some
microorganisms associated with the mantle of ectomycorrhizas. New Phytol 112:383–388
Garg N, Manchanda G (2008) Effect of arbuscular mycorrhizal inoculation on salt-induced nodule
senescence in Cajanus cajan (Pigeonpea). J Plant Growth Regul 27:115–124
Ghorbanli M, Ebrahimzadeh H, Sharifi M (2004) Effects of NaCl and mycorrhizal fungi on
antioxidative enzymes in soybean. Biol Plant 48:575–581
Gianinazzi-Pearson V, Branzanti B, Gianinazzi S (1989) In vitro enhancement of spore germination
and early hyphal growth of a vesicular–arbuscular mycorrhizal fungus by host root
exudates and plant flavonoids. Symbiosis 7:243–255
Giri B, Mukerji KG (2004) Mycorrhizal inoculant alleviates salt stress in Sesbania aegyptiaca and
Sesbania grandiflora under field conditions: evidence for reduced sodium and improved
magnesium uptake. Mycorrhiza 14:307–312
Giri B, Kapoor R, Agarwal L, Mukerji KG (2004) Pre-inoculation with arbuscular mycorrhizae
helps Acacia auriculiformis grow in degraded Indian wasteland soil. Communications in soil
science and plant analysis 35: 193–204
Giri B, Kapoor R, Mukerji KG (2003) Influence of arbuscular mycorrhizal fungi and salinity on
growth, biomass, and mineral nutrition of Acacia auriculiformis. Biol Fertil Soil 38:170–175
Giri B, Kapoor R, Mukerji KG (2007) Improved tolerance of Acacia nilotica to salt stress by
arbuscular mycorrhiza, Glomus fasciculatum may be partly related to elevated K/Na ratios in
root and shoot tissues. Microb Ecol 54:753–760
Glick BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol
41:109–117
Glick BR (2004) Bacterial ACC deaminase and the alleviation of plant stress. Adv Appl Microbiol
56:291–312
Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentrations by
plant growth promoting bacteria. J Theor Biol 190:63–68
Glick BR, Cheng Z, Czarny J, Duan J (2007a) Promotion of plant growth by ACC deaminase-
containing soil bacteria. Eur J Plant Pathol 119:329–339
Glick BR, Todorovic B, Czarny J, Cheng Z, Duan J, McConkey B (2007b) Promotion of plant
growth by bacterial ACC deaminase. Crit Rev Plant Sci 26:227–242
Gollotte A, van Tuinen D, Atkinson D (2004) Diversity of arbuscular mycorrhizal fungi colonising
roots of the grass species Agrostis capillaris and Lolium perenne in a field experiment.
Hall MA, Smith AR (1995) Ethylene and the responses of plants to stress. Bulg J Plant Physiol
21:71–79
Hamdia MA, Shaddad MAK, Doaa MM (2004) Mechanisms of salt tolerance and interactive
effects of Azospirillum brasilense inoculation on maize cultivars grown under salt stress
conditions. Plant Growth Regul 44:165–174
Hartmond U, Schaesberg NV, Graham JH, Syvertsen JP (1987) Salinity and flooding stress effects
on mycorrhizal and nonmycorrhizal citrus rootstock seedlings. Plant Soil 104:37–43
Hatimi A (1999) Effect of salinity on the association between root symbionts and Acacia
cyanophylla Lind.: growth and nutrition. Plant Soil 216:93–101
Hernandez JA, Olmos E, Corpas FJ, Sevilla F, del Rio LA (1995) Salt induced oxidative stress in
chloroplasts of pea plants. Plant Sci 105:151–167
Hirrel MC, Gerdemann JW (1980) Improved growth of onion and bell pepper in saline soils by two
vesicular–arbuscular mycorrhizal fungi. Soil Sci Soc Am J 44:654–655
Ho I (1987) Vesicular–arbuscular mycorrhizae of halophytic grasses in the Alvard desert of
Oregon. Northwest Sci 61:148–151
Hoefnagels MH, Broome SW, Shafer SR (1993) Vesicular–arbuscular mycorrhizae in salt marshes
in North Carolina. Estuaries 16:851–858
Holguin G, Glick BR (2001) Expression of the ACC deaminase gene from Enterobacter cloacae
UW4 in Azospirillum brasilense. Microb Ecol 41:281–288
Hutchinson LJ (1990) Studies on the systematic of ectomycorrhizal fungi in axenic culture. IV.
The effects of some selected fungi toxic compounds upon linear growth. Canadian Journal of
Botany 68:2172–2178
Jahromi F, Aroca R, Porcel R, Ruiz-Lozano JM (2008) Influence of salinity on the in vitro
development of Glomus intraradices and on the in vivo physiological and molecular responses
of mycorrhizal lettuce plants. Microb Ecol 55:45–53
Jiang M, Zhang J (2002) Water stress-induced abscisic acid accumulation triggers the increased
generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in
maize leaves. J Exp Bot 53:2401–2410
Johnson-Green PC, Kenkel NC, Booth T (1995) The distribution and phenology of arbuscular
mycorrhizae along an inland salinity gradient. Can J Bot 73:1318–1327
Jones RA, El-Abd SO (1989) Prevention of salt-induced epinasty by a-aminooxyacetic acid and
cobalt. Plant Growth Regul 8:315–323
Juniper S (1996) The effect of sodium chloride on some vesicular–arbuscular mycorrhizal fungi.
PhD Thesis, The University of Western Australia
Juniper S, Abbott LK (2006) Soil salinity delays germination and limits growth of hyphae from
propagules of arbuscular mycorrhizal fungi. Mycorrhiza 16:371–379
Kende H (1993) Ethylene biosynthesis. Annu Rev Plant Physiol Plant Mol Biol 44:283–307
Kernaghan G, Hambling B, Fung M, Khasa D (2002) In vitro selection of Boreal ectomycorrhizal
fungi for use in reclamation of saline–alkaline habitats. Restor Ecol 10:1–9
Khalvati MA, Hu Y, Mozafar A, Schmidhalter U (2005) Quantification of water uptake by
arbuscular mycorrhizal hyphae and its significance for leaf growth, water relations, and gas
exchange of barley subjected to drought stress. Plant Biol 7:706–712
Khan MA, Rizvi Y (1994) Effect of salinity, temperature, and growth-regulators on the germination
and early seedling growth of Atriplex Griffithii var stocksii. Can J Bot 72:475–479
Kloepper JW, Scher FM, Laliberte M, Tipping B (1986) Emergence-promoting rhizobacteria:
description and implications for agriculture. In: Swinburne TR (ed) Iron, siderophores, and
plant disease. Plenum, New York, pp 155–164
Kloepper JW, Lifshitz R, Zablotowicz RM (1989) Free-living bacterial inocula for enhancing crop
productivity. Trends Biotechnol 7:39–43
Krishnamurthy R, Bhagwat KA (1989) Polyamines as modulators of salt tolerance in rice

cultivars. Plant Physiol 91:500–504
Kukreja S, Nandwal AS, Kumar N, Sharma SK, Sharma SK, Unvi V, Sharma PK (2005) Plant
water status, H2O2 scavaging enzymes, ethylene evolution and membrane integrity of Cicer
arietinum as affected by salinity. Biol Plant 49:305–308
Lambert B, Joos H (1989) Fundamental aspects of rhizobacterial plant growth promotion research.
Trends Biotechnol 7:215–219
Langenfeld-Heyser R, Gao J, Ducic T, Ph T, Lu CF, Fritz E, Gafur A, Polle A (2007) Paxillus
involutus mycorrhiza attenuate NaCl-stress responses in the salt-sensitive hybrid poplar
Populuscanescens. Mycorrhiza 17:121–131
Levy Y, Dodd J, Krikun J (1983) Effect of irrigation water salinity and rootstock on the vertical
distribution of vesicular–arbuscular mycorrhiza in citrus roots. New Phytol 95:397–403
Lingua G, Sgorbati S, Citterio A, Fusconi A, Trotta A, Gnavi E, Berta G (1999) Arbuscular
mycorrhizal colonization delays nucleus senescence in leek root cortical cells. New Phytol
141:161–169
Manschke M, Berta G, Gianinazzi S, Moncousin C (1995) Effect of endomycorrhizal infection on
root system development in the apple rootstock (Malus domestica Borkh.) M26, morphogenesis.
In: Azcon C, Barea JM, Ocampo J (eds) Mycorrhizas in integrated systems: from genes to
plant development. . Office for Official Publications of the European Communities, Brussels,
Luxemburg, pp 349–352
Marulanda A, Azcon R, Ruiz-Lozano JM (2003) Contribution of six arbuscular mycorrhizal
fungal isolates to water uptake by Lactuca sativa plants under drought stress. Physiol Plant
119:526–533
Mayak S, Tirosh T, Glick BR (2004) Plant growth-promoting bacteria that confer resistance in
tomato to salt stress. Plant Physiol Biochem 42:565–572
McMillen B, Juniper S, Abbott LK (1998) Inhibition of hyphal growth of a vesicular–arbuscular
mycorrhizal fungus in soil containing sodium chloride limits the spread of infection from
spores. Soil Biol Biochem 30:1639–1646
Muhsin TM, Zwiazek JJ (2002a) Colonization with Hebeloma crustuliniforme increases water
conductance and limits shoot sodium uptake in white spruce (Picea glauca) seedlings. Plant
Soil 238:217–225
Muhsin TM, Zwiazek JJ (2002b) Ectomycorrhizas increase apoplastic water transport and root
hydraulic conductivity in Ulmus americana seedlings. New Phytol 153:153–158
Munns R (2002) Comparative physiology of salt and water stress. Plant Cell Environ 25:239–250
Nadeem SM, Zahair ZA, Naveed M, Arshad M (2007) Preliminary investigations on inducing salt
tolerance in maize through inoculation with rhizobacteria containing ACC deaminase activity.
Can J Microbiol 53:1141–1149
Nguyen H, Calvo Polanco M, Zwiazek JJ (2006) Gas exchange and growth responses of ectomy-
corrhizal Picea mariana, Picea glauca, and Pinus banksiana seedlings to NaCl and Na2SO4.
Plant Biol 8:646–652
O’Donnell PJ, Calvert C, Atzorn R, Wasternack C, Leyser HMO, Bowles DJ (1996) Ethylene as a
signal mediating the wound response of tomato plants. Science 274:1914–1917
Ojala JC, Jarrell WM, Menge JA, Johnson ELV (1983) Influence of mycorrhizal fungi on the
mineral nutrition and yield of onion in saline soil. Agron J 75:255–259
Ouziad F, Wilde P, Schmelzer E, Hildebrandt U, Bothe H (2006) Analysis of expression of
aquaporins and Na+/H+ transporters in tomato colonized by arbuscular mycorrhizal fungi and
affected by salt stress. Environ Exp Bot 57:177–186
Parida AK, Das AB (2005) Salt tolerance and salinity effects on plants: a review. Ecotoxicol
Environ Saf 60:324–349
Parker R, Flowers TJ, Moore AL, Harpham NVJ (2006) An accurate and reproducible method for
proteome profiling of the effects of salt stress in the rice leaf lamina. J Exp Bot 57:1109–1118
Patten CL, Glick BR (1996) Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol
42:207–220
Patten CL, Glick BR (2002) The role of bacterial indoleacetic acid in the development of the host
plant root system. Appl Environ Microbiol 68:3795–3801
Paul D, Dineshkumar N, Nair S (2006) Proteomics of a plant growth-promoting rhizobacterium,
Pseudomonas fluorescens MSP-393, subjected to salt shock. World J Microbiol Biotechnol
22:369–374
Penrose DM, Glick BR (2001) Levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in
exudates and extracts of canola seeds treated with plant growth-promoting bacteria. Can J
Microbiol 47:368–372
Pfeiffer CM, Bloss HE (1988) Growth and nutrition of guayule (Parthenium argentatum) in a
saline soil as influenced by vesicular arbuscular mycorrhiza and phosphorus fertilization. New
Phytol 108:315–321
Pivato B, Mazurier S, Lemanceau P, Siblot S, Berta G, Mougel C, van Tuinen D (2007) Medicago
species affect the community composition of arbuscular mycorrhizal fungi associated with
roots. New Phytol 176:197–210
Pond EC, Menge JA, Jarrell WM (1984) Improved growth of tomato in salinized soil by vesicular–
arbuscular mycorrhizal fungi collected from saline soils. Mycologia 76:74–84
Poss JA, Pond EC, Menge JA, Jarrell WM (1985) Effect of salinity on mycorrhizal onion and
tomato in soil with and without additional phosphate. Plant Soil 88:307–319
Prı́ncipe A, Alvarez F, Castro MG, Zachi L, Fischer SE, Mori GB, Jofré E (2007) Biocontrol
and PGPR features in native strains isolated from saline soils of Argentina. Curr Microbiol
55:314–322
Rabie GH, Almadini AM (2005) Role of bioinoculants in development of salt-tolerance of Vicia
faba under salinity stress. Afr J Biotechnol 4:210–222
Richard AJ, El-Abd SO (1989) Prevention of salt-induced epinasty by a-aminooxyacetic acid and
cobalt. Plant Growth Regul 8:315–323
Rozema J, Arp W, Van Diggelen J, Van Esbroek M, Broekman R, Punte H (1986) Occurrence and
ecological significance of vesicular–arbuscular mycorrhiza in the salt marsh environment. Acta
Bot Neerl 35:457–467
Ruiz-Lozano JM, Azcon R, Gomez M (1996) Alleviation of salt stress by arbuscular-mycorrhizal
Glomus species in Lactuca sativa plants. Physiol Plant 98:767–772
Sairam RK, Tyagi A (2004) Physiology and molecular biology of salinity stress tolerance in
plants. Curr Sci 86:407–421
Sannazzaro AI, Ruiz OA, Alberto EO, Menendez AB (2006) Alleviation of salt stress in
Lotus glaber by Glomus intraradices. Plant Soil 285:279–287
Sannazzaro AI, Echeverria M, Alberto EO, Ruiz OA, Menendez AB (2007) Modulation of
polyamine balance in Lotus glaber by salinity and arbuscular mycorrhiza. Plant Physiol
Biochem 45:39–46
Saravanakumar D, Samiyappan R (2006) ACC deaminase from Pseudomonas fluorescens
mediated saline resistance in groundnut (Arachis hypogea) plants. J Appl Microbiol
102:1283–1292
Schellenbaum L, Berta G, Ravolanirina F, Tisserant B, Giainazzi S, Fitter AH (1991) Influence of
endomycorrhizal infection on root morphology in a micropropagated woody plant species
(Vitis vinifera L.). Ann Bot 67:135–141
Sengupta A, Chaudhuri S (1990) Vesicular arbuscular mycorrhiza (VAM) in pioneer salt marsh
plants of the Ganges River Delta in West Bengal (India). Plant Soil 122:111–113
Sergeeva E, Shah S, Glick BR (2006) Growth of transgenic canola (Brassica napus cv. Westar)
expressing a bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene on high
concentrations of salt. World J Microbiol Biotechnol 22:277–282
Sharifi M, Ghorbanli M, Ebrahimzadeh A (2007) Improved growth of salinity-stressed soybean
after inoculation with salt pre-treated mycorrhizal fungi. J Plant Physiol 164:1144–1151
Shibli RA, Kushad M, Yousef GG, Lila MA (2007) Physiological and biochemical responses of
tomato microshoots to induced salinity stress with associated ethylene accumulation. Plant
Growth Regul 51:159–169
Simon-Sarkadi L, Kocsy G, Sebestyen Z (2002) Effect of salt stress on free amino acid and
polyamine content in cereals. Acta Biol Szegediensis 46:73–75
Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic, London, p 605
Smith GS, Middleton KR, Edmonds AS (1980) Sodium nutrition of pasture plants.1. Translocation
of sodium and potassium in relation to transpiration rates. New Phytol 84:603–612
Spychalla JP, Desbough SL (1990) Superoxide dismutase, catalase, and alpha-tocopherol content
of stored potato tubers. Plant Physiol 94:1214–1218
Tester M, Davenport R (2003) Na+ tolerance and Na+ transport in higher plants. Ann Bot 91:
503–527
Tian CY, Feng G, Li XL, Zhang FS (2004) Different effects of arbuscular mycorrhizal fungal
isolates from saline or non-saline soil on salinity tolerance of plants. Appl Soil Ecol
26:143–148
Tresner HD, Hayes JA (1971) Sodium chloride tolerance of terrestrial fungi. Appl Environ
van Duin WE, Rozema J, Ernst WHO (1989) Seasonal and spatial variation in the occurrence of
vesicular–arbuscular mycorrhiza in salt marsh plants. Agric Ecosyst Environ 29:107–110
van Peer R, Schippers B (1989) Plant growth responses to bacterization with selected Pseudomonas
spp. strains and rhizosphere microbial development in hydroponic cultures. Can J Micro-
biol 35:456–463
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK (2006) Methods and concepts in
quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water
status. Plant J 45:523–539
Waller F, Achatz B, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Huckelhoven R,
Neumann C, von Wettstein D, Franken P, Kogel KH (2005) The endophytic fungus
Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher
yield. Proc Natl Acad Sci USA 102:13386–13391
Whipps JM (1990) Carbon utilization. In: Lynch JM (ed) The rhizosphere. Wiley, Chichester, UK,
pp 59–97
White PJ, Broadley MR (2001) Chloride in soils and its uptake and movement within the plant:
A review. Ann Bot 88:967–988
Yi H, Calvo Polanco M, MacKinnonc MD, Zwiazek JJ (2008) Responses of ectomycorrhizal
Populus tremuloides and Betula papyrifera seedlings to salinity. Environ Exp Bot 62:357–363
Yildrim E, Taylor AG, Spittler TD (2006) Ameliorative effects of biological treatments on growth
of squash plants under salt stress. Sci Hortic 111:1–6
Yue HT, Mo WP, Li C, Zheng YY, Li H (2007) The salt stress relief and growth promotion effect
of Rs-5 on cotton. Plant Soil 297:139–145
Zhang J-S, Xi C, Shen Y-G, Shen S-Y (2001) A two component gene (NTHK1) encoding a
putative ethylene-receptor homolog is both developmentally and stress regulated in tobacco.
Theor Appl Genet 102:815–824
Zhang J, Jia W, Yang J, Ismail AM (2006) Role of ABA in integrating plant responses to drought
and salt stresses. Field Crop Res 97:111–119
Zuccarini P (2007) Mycorrhizal infection ameliorates chlorophyll content and nutrient uptake of
lettuce exposed to saline irrigation. Plant Soil Environ 53:283–289
Chapter 2
Recent Advances in Plant Growth Promotion
by Phosphate-Solubilizing Microbes
Almas Zaidi, Mohammad Saghir Khan, Munees Ahemad, Mohd Oves,

and P.A. Wani
Abstract Most soils contain large reserves of total phosphorus (P), but its fixation
and precipitation with soil constituents cause a major P-deficiency and severely
restrict the growth and yield of plants. The use of chemical P-fertilizers is obviously
the best means to circumvent P-deficiency, but their use is always limited due to its
spiraling cost. In order to increase the availability of P and to reduce the use of
chemical fertilizers, solubilization of insoluble P by phosphate-solubilizing micro-
organisms has provided an alternative to chemical phosphatic fertilizer. Besides
P, these organisms promote the growth of plants by N2 fixation, enhancement
of other plant nutrients, synthesizing phytohormones, suppressing plant diseases
(bio-control) and reducing the toxicity of ethylene through 1-aminocyclopropane-
1carboxylate (ACC) deaminase. In this chapter, attention is paid to understanding
the fundamental and molecular basis as to how precisely these microbes, notably
bacteria and fungi, help plants to grow better in P-deficient soils. Effective use of
such microbes is likely to result in an ideal cropping system with a lesser impact on
the environment through decreased application of chemical fertilizers.
2.1 Introduction
Phosphorus is one of the major nutrients limiting plant growth. In contrast, P is

required for growth and development of plants and promotes N2 fixation. It is also
involved in photosynthesis, energy transfer, signal transduction, macromolecular
biosynthesis, and respiration (Saber et al. 2005; Fernández et al. 2007). However,
most of the soils throughout the world are P-deficient (Batjes 1997) and, therefore,
require P to replenish the P-demand by crop plants. Worldwide, 5.7 billion hectares
M.S. Khan(*), A. Zaidi, M. Ahemad, M. Oves and P.A. Wani

Faculty of Agricultural Sciences, Department of Agricultural Microbiology, Aligarh Muslim
University, Aligarh, U.P., India
e-mail: khanms17@rediffmail.com

24 A. Zaidi et al.
contain too little available P for sustaining optimal crop production (Hinsinger
2001), and P-ion concentration in most soils varies from 0.1 to 10mM while
P required for optimal growth ranges from 1 to 5mM for grasses and from 5 to
60mM for high P-demanding crops, such as, tomato (Lycopersicon esculentum) and
pea (Pisum sativum) (Raghothama 1999). Suboptimal levels of P can, however,
lead to a 5–15% loss in the yield of plants (Hinsinger 2001). To circumvent the
P-deficiency, synthetic P-fertilizers are applied. The repeated and injudicious
applications of these fertilizers, however, lead to (1) the loss of soil fertility, (2)
disturbance to microbial diversity and their associated metabolic activities, and (3)
reduced yield of agronomic crops. Moreover, after application, a considerable
amount of P is rapidly transformed into less available forms by forming a complex
with Al or Fe in acid soils or with Ca in calcareous soils (Goldstein 1986; Toro
2007) before plant roots have had a chance to absorb it. This has led to the search
for environment-friendly and economically feasible alternative strategies for
improving crop production in low or P-deficient soils. Phosphorus is present in
soils both in organic and inorganic forms. Of these, organic forms, as found in
humus and other organic materials including decayed plant, animal and microbial
tissues, is an important reservoir of immobilized P accounting for about 20–80% of
total soil P (Richardson 1994). Organic P compounds can be slowly mineralized as
available inorganic P or they can be immobilized as part of the soil organic matter
(Mckenzie and Roberts 1990). The process of mineralization/solubilization or
immobilization affected by rhizosphere microbes, especially phosphate-solubiliz-
ing microorganisms (PSM), serves as an alternative to chemical phosphatic fertili-
zers and provides the available forms of P to plants (Bojinova et al. 2008; Oliveira
et al. 2008).
Besides providing P to the plants, the PSM(s) also facilitate the growth of plants
by stimulating the efficiency of N2 fixation, accelerating the accessibility of other
trace elements and by synthesizing important growth promoting substances (Wani
et al. 2007a; Mittal et al. 2008), including siderophores (Wani et al. 2007b) and
antibiotics (Lipping et al. 2008), and providing protection to plants against soil-
borne pathogens (Hamdali et al. 2008). Accordingly, these microbial communities
when used singly (Poonguzhali et al. 2008; Chen et al. 2008) or in combination with
other rhizosphere microbes (Zaidi and Khan 2006; Wani et al. 2007c; Vikram and
Hamzehzarghani 2008) have shown substantial measurable effects on plants in
conventional agronomic soils. Furthermore, bacterial strains capable of solubilizing
P have also been isolated from various niches of saline–alkali soils. For example,
strain RMLU-26, identified as Xanthomonas campestris with the ability to efficiently
solubilize P, was subjected to N-methyl-N0 -nitro-N-nitrosoguanidine (NTG) for
development of mutants. The wild-type and mutant strains of X. campestris
revealed a differential response to various stress factors (high pH, temperature,
and salt concentration). Both the wild and mutant strains revealed substantial
P-solubilization, but percent P2O5 solubilization by both strains revealed a steep
decline in tricalcium phosphate (TCP) solubilization with an increase in NaCl
concentration from 0.5 to 10% along with a concomitant drop in pH of the medium
2 Recent Advances in Plant Growth Promotion by Phosphate-Solubilizing Microbes 25
from 8 to 4.5 in wild-type and 4 in mutant strain. However, the mutant strain
displayed a 1.5- to 2-fold increase in P-solubilization compared to the wild-type
strain when grown in the presence of NaCl. The overall improved tolerance of the
strains to alkalinity and salinity could be due to accumulation and/or secretion
of specific solute (xanthan), suggesting that these strains could also be used as a bio-
inoculant under stressed soil environments (Sharan et al. 2008). In the following
section, particular attention is paid to identifying such microbes and the mechan-
isms by which they solubilize insoluble P and promote the growth of plants. Special
attention is further paid to the exploitation of microbial communities endowed with
P-solubilizing activity for their use in agricultural practices in different agro-
ecological niches.
2.2 Strategies for Isolation and Inoculant Development
Phosphate-solubilizing microbes form an integral bio-component of soils and affect

the fertility of soils through biogeochemical cycles. Numerous rhizosphere micro-
organisms capable of dissolving insoluble P have been reported (Henri et al. 2008;
Hameeda et al. 2008). Of these, the important genera of P-solubilizing bacteria
include Bacillus and Pseudomonas (Illmer and Schinner 1992; Wani et al. 2007a),
while Aspergillus and Penicillium form the important fungal genera (Souchie et al.
2006; Pandey et al. 2008). Other bacteria reported as P-solubilizers include Rho-
dococcus, Arthrobacter, Serratia, Chryseobacterium, Gordonia, Phyllobacterium,
Arthrobacter, Delftia sp. (Wani et al. 2005; Chen et al. 2006), Azotobacter (Kumar
et al. 2001), Xanthomonas (de Freitas et al. 1997), and Enterobacter, Pantoea, and
Klebsiella (Chung et al. 2005). Furthermore, symbiotic nitrogenous rhizobia, which
fix atmospheric nitrogen into ammonia and export the fixed nitrogen to the host
plants, have also shown PS activity. For instance, Rhizobium leguminosarum bv.
trifolii (Abril et al. 2007), R. leguminosarum bv. viciae (Alikhani et al. 2007) and
Rhizobium species nodulating Crotalaria species (Sridevi et al. 2007) improved
plant P-nutrition by mobilizing inorganic and organic P. These organisms are
ubiquitous but vary in density and mineral PS (mps) ability from soil to soil
or from one production system to another. They are generally isolated from
rhizosphere and nonrhizosphere soils, rhizoplane, phyllosphere, and RP deposit
area soil and even from stressed soils using serial plate dilution method or by
enrichment culture technique. The viable microbial preparations possessing
P-solubilizing activity are generally termed microphos. The microphos production
involves three critical stages: (1) screening and selection and in vitro evaluation of
P-solubilizing potentials of the microbial strains, (2) selection of carriers, mixing of
inocula with selected carriers and proper development of microbial inoculants, and
(3) testing of the quality of inoculants in terms of persistence of P-solubilizing
activity, viable microbial load per gram of carrier, and proper distribution to the
farmers. Since 1948, when Gerretsen suggested that microbes could dissolve not
26 A. Zaidi et al.
(Plate A) (Plate B)
(Plate C) (Plate D)
Fig. 2.1 Solubilization of tricalcium phosphate on Pikovskaya by species of (Plate A) Serratia
(Plate B) Bacillus (Plate C) Aspergillus and (Plate D) Penicillium
easily available forms of soil P and play an important role in providing P to plants,
numerous methods and media, such as Pikovskaya (Pikovskaya 1948), bromophe-
nol blue dye method (Gupta et al. 1994), and National Botanical Research Institute
P (NBRIP) medium (Nautiyal 1999), have been proposed.
Both bacterial and fungal strains exhibiting PS activity are detected by the
formation of clear halo (a sign of solubilization) around their colonies (Fig. 2.1).
Due to inconsistency and variations in PS activity, these cultures are repeatedly
subcultured. Once the efficient PS organisms have been selected, the release of P by
PS organisms is quantitatively assayed. The phosphate-solubilizing microbes
showing greater solubilization (both qualitatively and quantitatively) of insoluble
P under in vitro conditions are selected for bulk production for ultimate transmis-
sion to the farmers. For instance, use of fungi as inoculants for increasing plant
P-nutrition has been demonstrated by the successful commercial release of Penicil-
lium bilaiae (JumpStart; Philom Bios, Saskatoon, Canada) and Penicillium radicum
(PR-70 RELEASE; Bio-Care Technology, Somersby, Australia).
2.3 Mechanisms of P-Solubilization: A General Account
The solubilization of P-compounds by naturally abundant P-solubilizing microbes

is common under in vitro conditions (Souchie et al. 2007; Song et al. 2008). Indeed,
soil microorganisms are effective in releasing P from inorganic P through solubili-
zation (Toro 2007; Wani et al. 2007b, c) and from organic pools of total soil P by
mineralization (Bishop et al. 1994; Ponmurugan and Gopi 2006). The microbial
biomass in soil also contains a significant quantity of immobilized P that is
accessible for uptake by plants (Brookes et al. 1984; Oberson et al. 2001). The
mechanisms used by PS microbes to solubilize mineral phosphate have been
investigated by several workers (Cunningham and Kuiack 1992; Illmer and Schin-
ner 1995; Song et al. 2008). Generally, the mps trait is correlated with the produc-
tion of organic acids (OA) via the direct oxidation pathway that occurs on the outer
face of the cytoplasmic membrane, with concomitant drop in pH value. The inverse
correlation between pH of the culture and the release of P consolidates the hypoth-
esis of OA involvement in the solubilization of insoluble P. The OA(s) released by
PS microbes, notably bacteria (Table 2.1) and fungi (Table 2.2), chelate mineral
ions or drop the pH to bring P into solution (Maliha et al. 2004; Pradhan and Sukla
2005). The OA produced both by bacteria and fungi in turn leads to acidification of
Table 2.1 Organic acids involved in P-solubilization and produced by PS bacteria

Bacterial communities Organic acids produced References
Burkholderia cepacia DA23 Gluconic acid Song et al. (2008)
Pseudomonas corrugata Gluconic, 2-ketogluconic acid Trivedi and Sa
(NRRL B-30409) (2008)
Citrobacter sp. DHRSS Acetic and gluconic acid Patel et al. (2008)
Burkholderia, Serratia, Ralstonia and Gluconic acid Elizabeth et al.
Pantoea (2007)
Bacillus, Rhodococcus, Arthrobacter, Citric acid, gluconic acid, Chen et al.
Serratia and one Chryseobacterium, lactic acid, succinic acid, (2006)
Delftia, Gordonia, Phyllobacterium, propionic acid
Arthrobacter ureafaciens,
Phyllobacterium myrsinacearum,
Rhodococcus erythropolis and Delftia sp.
Enterobacter intermedium 2-ketogluconic Hwangbo et al.
(2003)
Bacillus amyloliquefaciens, B. licheniformis, Lactic, itaconic, isovaleric, Vazquez et al.
B. atrophaeus, Penibacillus macerans, isobutyric, acetic (2000)
Vibrio proteolyticus, xanthobacter agilis,
Enterobacter aerogenes, E. taylorae,
E. asburiae, Kluyvera cryocrescens,
Pseudomonas aerogenes, Chryseomonas
luteola
Pseudomonas cepacia Gluconic, 2-ketgluconic Bar-Yosef et al.
(1999)
Bacillus polymyxa, B. licheniformis, Bacillus Oxalic, citric Gupta et al.
spp. (1994)
28 A. Zaidi et al.
Table 2.2 Organic acids produced by phosphate solubilizing fungi

Organism Predominant acids References
Aspergillus niger Gluconic acid, oxalic acid Chuang et al. (2007)
Penicillium oxalicum Malic acid, gluconic acid, Shin et al. (2006)
oxalic acid
Aspergillus flavus, A. niger, Oxalic, citric, gluconic succinic Maliha et al. (2004)
Penicillium canescens
Penicillium rugulosum Citric, gluconic acid Reyes et al. (2001)
A. niger Succinic acid Vazquez et al. (2000)
Penicillium variabile Gluconic acid Fenice et al. (2000)
Penicillium rugulosum Gluconic Reyes et al. (1999)
Penicillium radicum Gluconic Whitelaw et al.
(1999)
P. variabile Gluconic Vassilev et al. (1996)
A. niger Citric, oxalic,gluconic Illmer et al. (1995)
A. awamori, A. foetidus, A. terricola, Oxalic, citric Gupta et al. (1994)
A. amstelodemi, A. tamari
A. japonicus, A. foetidus Oxalic, citric gluconic succinic, Singal et al. (1994)
tartaric acid
microbial cells and their surroundings and, consequently, the release of P-ions from
the P-mineral by H+ substitution for Ca2+ (Goldstein 1994). However, a lot of fixed
P in acidic soil (such as red soil) accumulates Fe or Al ions, and no correlation was
found between pH and the amount of P-solubilized (Asea et al. 1988). It is still
unexplained why no substantial amounts of OA production could be detected from
some PS microorganisms (Illmer and Schinner 1992; Chen et al. 2006). For this
reason, alternative possibilities other than OA for insoluble inorganic P-solubiliza-
tion have been proposed. Such mechanisms include the release of H+, production
of chelating substances and inorganic acids (Khan et al. 2007b). In this context,
Illmer and Schinner (1995) suggested that the OA released by the bacterial cells are
not the only means of P-solubilization and, hence, acidification does not seem to be
the only mechanism of solubilization, as the ability to reduce the pH in some cases
did not correlate with the ability to solubilize mineral P. The chelating ability of the
OA is also important, as it has been shown that the addition of 0.05 M EDTA to the
medium has the same solubilizing effect as inoculation with Penicillium bilaii
(Kucey 1988). In another study, Altomare et al. (1999) investigated the capability
of the plant growth-promoting and bio-control fungus Trichoderma harzianum
T-22 to solubilize in vitro insoluble minerals including rock phosphate. OA were
not detected in culture filtrates and, hence, the authors concluded that the insoluble
P could be solubilized by mechanisms other than acidification process. In this study,
the fungal-solubilizing activity was attributed both to chelation and to reduction
processes, which could also play a role in the bio-control of plant pathogens.
Among nodule bacteria, e.g., Rhizobium/Bradyrhizobium, the PS activity of Rhizo-
bium was associated with the production of 2-ketogluconic acid which was abol-
ished by the addition of NaOH, indicating that PS activity of this organism
was entirely due to its ability to reduce the pH of the medium (Halder and
Passimilation from liquid

(indirect dissolution)
Organic acids
Respiratory H2CO3 production/chelation of
production cations bound to P
hosphate
of p so
ms l
ub
is
an
iliz
Mech
ation
Acid phosphatases,
phytases inorganic acids production
H2S production release of H+/H+ release

accompanying NH4+
NH4+-N
Exopolysaccharides
production
Fig. 2.2 Mechanisms of P-solubilization by phosphate solubilizing bacteria
Chakrabarty 1993). However, the detailed biochemical and molecular mechanism

of P-solubilization by symbiotic nodule bacteria are not conclusive. In addition to
OA, the solubilization of insoluble P by inorganic acid, e.g., HCl (Kim et al. 1997),
and involvement of the H+ pump, as reported in Penicillium rugulosum, is sug-
gested (Reyes et al. 1999).
Recently, four bacterial strains of Enterobacter sp. (EnHy-401), Arthrobacter sp.
(ArHy-505), Azotobacter sp. (AzHy-510) and Enterobacter sp. (EnHy-402), pos-
sessing the ability to solubilize TCP, were used to assess the role of exopolysac-
charide (EPS) in the solubilization of P (Yi et al. 2008). These PS bacteria produced
a significant amount of EPS and demonstrated a strong ability for P-solubilization.
Of these, the strain EnHy-401 with the highest EPS and OA production had a
stronger capacity for P-solubilization than the others. Further studies demonstrated
that addition of EPS into the medium could increase the amount of P-solubilized by
OA, but it failed to release P from TCP alone. The synergistic effects of EPS and
OA on TCP-solubilization varied with the origin and the concentration of EPS in
medium. EPS produced by EnHy-401 was most effective in promoting P release
at an optimal concentration in the medium. The increase in P-solubilization by
EPS was attributed mainly to the participation of EPS which led to a change in
the homeostasis of P-solubilization, pushing it towards P-dissolving by holding
free P in the medium, consequently resulting in greater P released from insoluble P.
It was, therefore, suggested that EPS with the ability of P-holding may be a novel
important factor in the microbial dissolution of TCP except for OA. A general
mechanism how PS organisms bring out solubilization of insoluble P is presented in
Fig. 2.2.
30 A. Zaidi et al.
2.3.1 Enzymatic Dissolution of Phosphates by Phosphate-

Solubilizing Microbes
Organic compounds containing P is mineralized in soil by phosphatases, phytases

and phosphonatases and C–P lyases. The efficiency of microbial phosphatases,
a widely distributed exoenzyme, in dissolution and mineralization of organic
P-compounds in the rhizosphere and P-uptake by plants has been reported (Rodriguez
and Fraga 1999; Rodriguez et al. 2006). Among phosphatases, acid phosphatase
(To-O et al. 2000) is commonly found in fungi (To-O et al. 1997; Omar and
Abd-Alla 2000) and activates the release of inorganic P from P-esters (Nozawa
et al. 1998). Acid phosphatase has been detected in vacuoles and vesicles (Ruch
and Motta 1987; Saito 1995) and other intracellular and extracellular (Ruch and
Motta 1987) organs of fungi like phenotypic mutants of Aspergillus tubingensis
(Varenyam et al. 2007). For instance, the enzyme was found in the vacuoles of
ungerminated conidia of Colletotrichum graminicola, and also during germination
(Schadeck et al. 1998a, b). Under in vitro conditions, phosphatase activity is
identified by a distinct zone of clearing (transparent yellow halos) around
fungal colonies on Pikovskaya medium or agar plates supplemented with 1-naphthyl
P as a chromogenic substrate (Goud et al. 2008). Aspergillus, Emmericella and
Penicillium, isolated from arid and semiarid regions of India, have shown phytin and
glycerophosphate hydrolyzing activity (Yadav and Tarafdar 2003). In this report,
the extracellular (E) phosphatases released by fungi were less than their intracellular
(I) counterpart, and the E:I ratio of fungi ranged from 0.39 to 0.86 for acid
phosphatase and 0.29 to 0.41 for alkaline phosphatases. The efficiency of hydrolysis
of organic P-compounds of fungi varied from 2.12 to 4.85mg min1 g1 for glycer-
ophosphate to 0.92–2.10mg min1 g1 for phytin. The trend of efficiency was:
Aspergillus sp. > Emmericella sp. > Penicillium sp. Similar phosphatase activity
is also reported for A. caespitosus and Mucor rouxii (Guimarães et al. 2006).
Another attractive use of P-dissolving enzymes is the solubilization of soil organic
P through phytate degradation mediated by enzyme phytase. In its basic form,
phytate is the primary source of inositol and the major stored form of P in plant
seeds and pollen, and is a major component of organic P in soil (Richardson 1994).
Although the ability of plants to obtain P directly from phytate is very limited, yet the
growth and P-nutrition of Arabidopsis plants supplied with phytate was significantly
improved when they were genetically transformed with the phytase gene (phyA)
derived from Aspergillus niger (Richardson et al. 2001a). This led to an increase in
P-nutrition to such an extent that the growth and P-content of the plant was
equivalent to control plants supplied with inorganic P. A similar increase in utiliza-
tion of inositol P by plants in the presence of PS fungus (A. niger) capable of
producing phytase is reported (Richardson et al. 2001b; Vassilev et al. 2007).
Therefore, developing inoculants with high phosphatase and phytase activity
would be of great practical interest for augmenting plant nutrition and reducing
P-pollution in soil.
Fig. 2.3 Mechanism of growth promotion by phosphate solubilizing bacteria
2.4 Mechanism of Plant Growth Promotion

by P-Solubilizing Microbes
PS microbes are well known for making soluble P accessible for uptake by plants.
They can also facilitate growth and development of plants by producing essential
nutrients (Thomas et al. 2005) or by changing the concentration of plant growth-
promoting substances including phytohormones such as indoleacetic acid (Wani
et al. 2007a, b), through asymbiotic or symbiotic N2 fixation (Zaidi 1999; Zaidi
and Khan 2007), soil conditioning, exhibiting bio-control activity (Pandey et al.
2006), by synthesizing siderophores (Vassilev et al. 2006), antibiotics, and
cyanide (Lipping et al. 2008), by synthesizing an ACC deaminase that can
modulate plant ethylene levels ( Anandham et al. 2008; Poonguzhali et al.
2008), and by solubilizing or reducing the toxicity of metals (bioremediation)
(Khan et al. 2009). These mechanisms can probably be active simultaneously or
sequentially at different stages of plant growth. However, the intrinsic ability of
PS microbes for synthesizing the growth-promoting substances varies consider-
ably under different ecological niches. The mechanisms involved in plant growth
promotion (Fig. 2.3) and growth regulators produced by PS organisms are pre-
sented in Table 2.3.
32 A. Zaidi et al.
Table 2.3 Growth-promoting substances released by phosphate solubilizing bacteria

Phosphate solubilizing bacteria Plant growth-promoting traits References
Dyella ginsengisoli, Burkholderia Siderophore, IAA, salicylic acid, ACC Anandham et al.
kururiensis, Pandoraea sp. deaminase (2008)
strain ATSB30
Pseudomonas sp. ACC deaminase, IAA, siderophore Poonguzhali et al.
(2008)
Bacillus subtilis IAA, siderophore, antifungal activity Singh et al. (2008)
Serratia marcescens IAA, siderophore, HCN Selvakumar et al.
(2008)
Pseudomonas fluorescens ACC deaminase Shaharoona et al.
(2008)
Acinetobacter sp., ACC deaminase, IAA, antifungal Indiragandhi et al.
Pseudomonas sp. activity, N2-fixation (2008)
Enterobacter sp. ACC deaminase, IAA, siderophore Kumar et al. (2008)
Burkholderia ACC deaminase, IAA, siderophore, Jiang et al. (2008)
heavy metal solubilization
Pseudomonas jessenii ACC deaminase, IAA, siderophore, Rajkumar and
heavy metal solubilization Freitas (2008)
Pseudomonas aeruginosa ACC deaminase, IAA, siderophore Ganesan (2008)
Azotobacter sp., Mesorhizobium IAA, siderophore, antifungal activity, Ahmad et al. (2008)
sp., Pseudomonas sp., ammonia production, HCN
Bacillus sp.
P. aeruginosa, P. plecoglossicida Siderophore, IAA, protease, cellulase Jha et al. (2008)
and P. mosselii and HCN
Bacillus spp. IAA, siderophores, ammonia Wani et al. (2007a,
production, HCN, chromium 2007b, 2007c)
reduction, metal solubilization
Mesorhizobium loti MP6 HCN, IAA Chandra et al. (2007)
Pseudomonas sp., Bacillus sp. IAA, siderophore Rajkumar et al.
(2006)
2.4.1 Phosphate-Solubilizing Microbes as Bio-Control Agent
Phosphate-solubilizing microorganisms as a group form an integral component of

soils. In addition to providing P to plants, PS microorganisms also act as a bio-
control agent and promote the growth of plants by suppressing the soilborne
phytopathogens. Several in vitro studies show the potential of PS microorganisms
for the simultaneous synthesis and release of pathogen-suppressing metabolites,
mainly siderophores, and lytic enzymes (Pandey et al. 2006; Rane et al. 2008).
Potential application of PS microbes as bio-control agents is reviewed and
discussed.
Bio-control ability of PS bacterium Pseudomonas aeruginosa ID 4365, a bio-
control agent of groundnut (Arachis hypogaea) phytopathogens of marine origin,
was previously attributed to the production of pyoverdin type of siderophores.
However, pyoverdin-rich supernatants of this organism have shown better antifun-
gal activity compared to equivalent amounts of purified pyoverdin indicating the
presence of undetected metabolite(s) in pyoverdin-rich supernatants. In addition
to pyoverdin, the strain produced additional siderophores, i.e., pyochelin and

salicylic acid, and two broad spectrum antifungal compounds, i.e., pyocyanin and
phenazine-1-carboxylic acid, and exhibited antifungal activity (Rane et al. 2008).
Similarly, P-solubilizer Pseudomonas putida exhibited antifungal activity against
phytopathogenic fungi in Petri dish assays, and produced chitinase, b-1,3-gluca-
nase, salicylic acid, siderophore, and hydrogen cyanide. The plant growth-promotion
and antifungal properties were demonstrated through a maize (Zea mays)-based
bioassay under greenhouse conditions. Although the bacterial inoculation demon-
strated a substantial increase in plant biomass, it stimulated bacterial and sup-
pressed fungal counts in the rhizosphere. This study leads to a better
understanding of the microbial diversity of the colder regions as well as to under-
standing the potential biotechnological applications of native microbes (Pandey
et al. 2006). Among the fungal P-solubilizers, Trichoderma species are the most
commonly studied bio-control microorganisms which also exhibit plant growth-
promoting activity (Harman and Bjorkman 1998). While the mechanisms of
bio-control have been well investigated, those responsible for the growth promo-
tion by T. harzianum have not been extensively studied. In one study, Altomare
et al. (1999) investigated the capability of the plant growth-promotion and bio-
control activity of T. harzianum T-22 to solubilize in vitro insoluble minerals
including RP. OA were not detected in the culture filtrates and, hence, the authors
concluded that acidification was probably not the major mechanism of solubiliza-
tion as the pH never fell below 5. The fungal-solubilizing activity was attributed
both to chelation and to reduction processes, which also play a role in the bio-
control of plant pathogens. A similar bio-control effect of PS filamentous fungi
against Fusarium wilt in tomato (Fusarium oxysporum f. sp. lycopersici; Fol)
was reported by Khan and Khan (2001). Root-dip applications of B. subtilis,
P. fluorescens, Aspergillus awamori, A. niger, and Penicillium digitatum declined
the rhizosphere population of Fol. Tomato yield was enhanced, being greatest
with A. awamori and P. digitatum. Direct soil–plant inoculation with A. niger,
A. awamori, and P. digitatum decreased the rhizosphere Fol population by
23–49% while the tomato yield increased by 28–53% in field experiments.
The authors propose that OA produced by these microorganisms may inhibit
fungal infection but that other metabolites such as bulbiformin and phenazin
could also be involved, particularly in the treatments with B. subtilis and
P. fluorescens. Research using P. variabile P16 demonstrated increased glu-
cose oxidase (GOD) production in the presence of polysaccharides, which were
found to serve as activators of defensive systems in this filamentous fungus
(Petruccioli et al. 1999). In fact, GOD activity can play a significant role in
antibiosis in the soil environment, and H2O2 enzymatically produced is cytotoxic
for microorganisms. In another report, PS fungus P. oxalicum showed strong
antibiotic activity against pathogenic fungi, including Sclerotinia sclerotiorum,
a widespread pathogenic fungus that severely attacks rapeseed (Brassica
napus) (Lipping et al. 2008). The combination of P-solubilization and bio-
control activity of PSM(s) could prove very effective in promoting the growth
of plants both in conventional and stressed soils of different agro-ecosystems.
34 A. Zaidi et al.
Table 2.4 Phosphate solubilizing microbes with bio-control activity

Strain species Bio-control Metabolite with bio- References
activity control activity
Pseudomonas aeruginosa + Siderophores and Rane et al. (2008)
ID 4365 phenazines
P. aeruginosa, + Protease, cellulase Jha et al. (2008)
P. plecoglossicida and
P. mosselii
P. putida + Chitinase, b-l,3- Pandey et al. (2006)
glucanase,
salicylic acid,
siderophore,
and hydrogen
cyanide
Aspergillus niger + Siderophores Vassilev et al. (2006)
A. niger, A. awamori + Organic acids Khan and Khan (2001,
Penicillium digitatum 2002)
P. variabile P16 Glucose oxidase Petruccioli et al. (1999)
Trichoderma harzianum T22 + Siderophores Altomare et al. (1999)
+ Proved, not proved: modified from Nikolay et al. (2006)
Some of the metabolites released both by PS bacteria and fungi are presented in
Table 2.4.
2.5 Molecular Engineering of P-Solubilizing Bacteria
Studies at the molecular level in order to understand how precisely the PS microbes
brings out the solubilization of insoluble P were inconclusive (Rodriguez et al.
2006). However, several phosphatase-encoding genes have been cloned and char-
acterized, and a few genes involved in mps have been isolated (Table 2.5) and
characterized (Fraga-Vidal et al. 2003; Krishnaraj and Goldstein 2001). Genetic
manipulation of PS bacteria to improve their ability to improve plant growth may
include cloning genes involved in both mps and organic P-solubilization (ops),
followed by their expression in selected rhizobacterial strains. For example, Gold-
stein and Liu (1987) have shown that mps activity is genetically coded in a gene
cluster on plasmids of microbes endowed with PS activity. They further transferred
this gene cluster to an E. coli strain that did not previously possess PS activity but
could demonstrate that the transferred gene was expressed in the transgenic E. coli
strain. They have also found that the gene expression and mps activity of bacteria
is affected by the presence of soluble P in the medium (feed-back regulation).
Chromosomal insertion of these genes under appropriate promoters is another
interesting approach (Rodriguez and Fraga 1999). Rodrı́guez et al. (2000) carried
out a genetic construction using the broad host range vector pKT230 and plasmid
pMCG898, which encode the Erwinia herbicola pyrroloquinoline quinone (PQQ)
synthase, a gene involved in mps. The final construct was transformed and
Table 2.5 Cloning of genes involved in mineral phosphate solubilization

Microorganisms Gene Features Reference
or plasmid
Serratia pKG3791 Produces gluconic acid and Krishnaraj and Goldstein
marcescens solubilizes P (2001)
Rahnella aquatilis pKIM10 Solubilizes P and produces gluconic Kim et al. (1998)
acid in E. coli DH5a
Enterobacter pKKY Solubilizes P in E. coli 109; Does Kim et al. (1997)
agglomerans not lower pH
Pseudomonas Gab Y Produces gluconic acid and Babu-Khan et al. (1995)
cepacia solubilizes
mineral P in E. coli JM109; No
homology with PQQ genes
Erwinia herbicola Mps Produces gluconic acid and Goldstein and Liu (1987)
solubilizes mineral P in
E. coli HB101; Probably
involved in PQQ synthesis
expressed in E. coli MC1061, and the recombinant plasmids were transferred to

Burkholderia cepacia IS-16 and Pseudomonas sp. PSS recipient cells by conjuga-
tion. Clones containing recombinant plasmids produced higher clearing halos in
plates with insoluble P as the unique (P) source, in comparison to those strains
without plasmids, demonstrating the heterologous expression of the E. herbicola
gene in the recipient strains. This genetic manipulation allowed the increase in mps
ability of both strains, enhancing their potential as growth promoters of agricultural
crops. In addition, the subcloning of the gene encoding the PhoC acid phosphatase
from Morganella morganii (phoC gene), in a vector that permits stable chromo-
somal integration of this gene in plant growth-promoting bacteria, has been
reported (Fraga-Vidal et al. 2003). Another important rhizosphere-competent bac-
teria (Pseudomonas spp.) can form gluconic acid through the oxidative metabolism
and overexpression of PQQ synthase and glucose dehydrogenase (GDH) genes
make them functionally a better PS organism. Interestingly, in addition to its role in
P-solubilization, PQQ also plays an important role in beneficial traits, such as
antifungal activity and induced systemic resistance (ISR) of Enterobacter inter-
medium, possibly by acting as a cofactor for several enzymes including GDH (Han
et al. 2008). In yet another approach, the mps genes can directly be transferred into
the target bacteria by over-/underexpression of genes followed by the selection of
transformants with mps ability. Such an approach has been used to obtain mps
genes from Synechosystis PCC 6803 in E. coli (Gyaneshwar et al. 1998). However,
it remains to be seen if this will also be effective in other bacteria. Genetic
engineering could also help in increasing the survival of the inoculant strains by
incorporating the abilities to utilize certain nutrients better than the rest of the
microbial populations (Glick and Bashan 1997). In addition, genes for utilization of
salicylate were transferred to growth-promoting bacteria and the recombinant
bacterium was able to survive and enhance plant growth better than the wild-type
(Colbert et al. 1993).
36 A. Zaidi et al.
2.6 Crop Improvement by P-Solubilizing Microbes
2.6.1 Inoculation Effects of Phosphate-Solubilizing Bacteria
Phosphate-solubilizing bacteria in general enhance the growth of plant by providing

soluble P to plants. However, they also affect the growth and development of plants
by other mechanisms. For example, PS strains of Pseudomonas capable of synthe-
sizing IAA, ACC deaminase, and siderophores in vitro, increased the root elonga-
tion and biomass of Chinese cabbage (Brassica rapa) but had no effect on P-uptake
of plants, suggesting that the growth promotion by PS strain could be due to the
production of phytohormones or mechanisms other than P-solubilization (Poon-
guzhali et al. 2008). Similarly, a significant increase in biomass and total P of winter
wheat (Triticum aestivum) following a single inoculation of Phosphobacterium
(strain 9320-SD) under both pot and field conditions has been reported; although
no obvious difference was found in plant height (Chen et al. 2006). Cold-tolerant PS
Serratia marcescens with inherent plant growth-promoting traits (IAA, HCN and
siderophore production) has also been found to significantly enhanced plant bio-
mass and nutrient uptake of wheat seedlings grown in cold temperatures (Selvakumar
et al. 2008). Two plant growth-promoting PSB, Pseudomonas fluorescens and
P. fluorescens biotype F, having ACC deaminase showed a profound effect on
growth, yield, and nutrient use efficiency of wheat under simultaneously varying
levels of all the three major nutrients, N, P, and K (at 0, 25, 50, 75, and 100% of
recommended doses). Results of pot and field trials revealed that the growth-
promoting efficacy of these strains decreased with increasing rates of NPK added
to the soil. In most of the cases, significant negative linear correlations were
recorded between percentage increases in growth and yield parameters of wheat
caused by inoculation and increasing levels of applied NPK fertilizers. They
speculated that, under low fertilizer application, the ACC deaminase activity of
PS strains might have caused reduction in the synthesis of stress (nutrient)-induced
inhibitory levels of ethylene in the roots through ACC hydrolysis into NH3 and a-
ketobutyrate. This study suggested that Pseudomonads could be used in combina-
tion with appropriate doses of fertilizers for better plant growth and savings of
fertilizers (Shaharoona et al. 2008). A similar increase in biomass of maize plants
following inoculation of two efficient strains of Serratia marcescens (EB 67) and
Pseudomonas sp. (CDB 35) has been reported (Hameeda et al. 2008). Of these PS
bacteria, strain EB 67 increased the biomass by 99% while strain CDB 35 demon-
strated an increase of 94%. Increase in plant biomass at 48 and 96 days after sowing
was 66 and 50%, respectively, with EB 67 and 51 and 18%, respectively, with CDB
35 under field conditions. Seed bacterization with strain EB 67 and CDB 35
increased the grain yield of field-grown maize by 85 and 64%, respectively.
Similarly, B. subtilis having antifungal activity against Macrophomina phaseolina
and other phytopathogens, including Fusarium oxysporum and Rhizoctonia solani
and plant growth-promoting attributes (IAA and siderophore production) isolated
from chirpine (Pinus roxburghii) rhizosphere, resulted in 44 and 94% increases in
root and shoot dry weights of chirpine, respectively (Singh et al. 2008). Phosphate-
solubilizing Acinetobacter sp. (PSGB04) and Pseudomonas sp. (PRGB06) with
plant growth-promoting traits (N2 fixation, IAA, salicylic acid production) isolated
from the larval guts of diamondback moths (Plutella xylostella) have also been
tested for their effects on growth of canola (Brassica napus) and tomato (Indir-
agandhi et al. 2008). Acinetobacter sp. (PSGB04) significantly increased root
length (41%), seedling vigor, and dry biomass (30%) of the canola test plants,
whereas a substantial increase, greater than that of the control, was also recorded for
the tomato plants when seeds were treated with Pseudomonas sp. (PRGB06)
possessing antifungal activity.
Phosphate-solubilizing microbes cohabit in the rhizosphere with other agrono-
mically beneficial microbes and could play an additive or synergistic role in the
growth promotion of plants. Subsequently, dramatic increases in yield of various
crops following seed or soil inoculation with PS organisms and other plant growth-
promoting rhizobacteria (Afzal and Bano 2008) or AM fungus (Khan and Zaidi
2006; Ehteshami et al. 2007) under different agro-ecosystems have been reported.
The inoculation of PS bacteria Pseudomonas striata and N2-fixing bacterium
Rhizobium sp. (Vigna) substantially increased the yield of greengram (Vigna
radiata L.) (Khan et al. 1997, 1998) and nodulation, available P of soil as well as
dry matter of the plants, grain yield, and P- and N-uptake by chickpea (Cicer
aeritinum) (Wani et al. 2007b, c) compared to single inoculation of either PS
bacteria or N2 fixers. Similarly, the seed inoculation of chickpea with single, dual
and triple inoculations of Rhizobium, Bacillus subtilis (OSU-142) and PS Bacillus
megaterium (M-3) increased biological and chemical properties of chickpea (Elk-
oca et al. 2008). Substantial increases in the seed yield under different inoculation
treatments ranged between 18% (Rhizobium) and 31% (Rhizobium + OSU-142 +
M-3) over control. Generally, the increases in seed and total biomass yields were
more pronounced in dual and triple inoculations. In another study, Wani et al.
(2007c) demonstrated synergistic effects of N2-fixing and PS rhizobacteria on
chickpea plants. Legume grain yield and concentration and uptake of N and P
were significantly increased following coinoculation with Mesorhizobium and PS
Pseudomonas and Bacillus spp. The inoculation of M. ciceri with Azotobacter
chroococcum and Bacillus tripled the seed yield and resulted in the highest grain
protein (295 mg g1). An 8% increase in P concentration above the uninoculated
control was observed in the case of a single inoculation with Pseudomonas, while
the P-uptake was highest (2.14-fold above the uninoculated control) when M. ciceri
was applied with A. chroococcum and Bacillus. They showed that the multiple
inoculations with rhizospheric microorganisms can synergistically facilitate growth
and yields and increase concentrations and uptake of N and P by field-grown
chickpea, as also reported by Valverde et al. (2006). In another study, Canbolat
et al. (2006) observed a significant increase in seedling growth, total dry matter
accumulation and available P in barley (Hordeum vulgare) bioprimed with
N2-fixing and P-dissolving Bacilli suggesting that the N2-fixing and PS bacterial
strains could be used to save fertilizer application. It is also reported that PS
P. putida, possessed with antifungal activity, antibiotic resistance and ability to
38 A. Zaidi et al.
produce chitinase, b-1,3-glucanase, salicylic acid, siderophore, and HCN signifi-

cantly increased the plant biomass in a maize-based bioassay under greenhouse
conditions (Pandey et al. 2006). The effect of a combined inoculation of Rhizobium,
a P-solubilizing B. megaterium subsp. phosphaticum strain-PB and a bio-control
fungus Trichoderma spp. on the performance of chickpea under glasshouse and
field conditions is also reported (Rudresh et al. 2005b). Tripartite inoculations
exhibited increased germination, nutrient uptake, plant height, number of branches,
nodulation, yield, and total biomass of chickpea compared to either individual
inoculations or an uninoculated control. Increased growth and yield parameters
were more pronounced when T. harzianum was inoculated along with the PS
bacterium and Rhizobium.
Furthermore, the PS bacteria forms a strong association with AM fungi in
P-deficient soils or RP-enriched soils and can release some P-ions from an other-
wise sparingly soluble P source, which is tapped and translocated by the AM fungal
hyphae to the plants. In this context, the composite inoculation of PS bacteria and
AM fungi has been found to improve plant growth in a more sustainable manner in
soil deficient in P (Zaidi et al. 2004; Zaidi and Khan 2007). During this intergeneric
interaction, the AM fungi increases plant growth via improved uptake of nutrients,
especially P, due to the exploration by the external hyphae of the soil beyond the
root-hair zone where P is depleted. The AM fungi also produce plant hormones and
increase the activity of N2-fixing organisms in the root zone. In contrast, the PS
bacteria could alter the composition of root exudates and plasticity which in turn
may affect the colonization and development of AM fungi. These factors together
facilitate the colonization and establishment of AM fungus onto the root system of
plants and also alter microbial composition of rhizospheres, which in turn affect the
competition between inoculated and native soil organisms. However, a thorough
understanding of interactions between organisms of agronomic importance is
required so that a bio-inoculant with multifaceted activity could be developed.
Phosphate-solubilizing microbes are reported to reduce the toxicity of metals
and protect the plants against the toxic effects of these metals and consequently
enhance the growth and yield of plants in contaminated soils (Wani et al. 2007a).
For instance, Rajkumar et al. (2006) assessed the plant growth-promoting activity
of Cr (vi) resistant plant growth-promoting bacteria having PS potential, Pseudo-
monas sp. and Bacillus sp., recovered from heavy metal contaminated soils, on the
Indian mustard (Brassica juncea) with different concentrations of Cr (vi) added to
soil. Inoculation of both strains promoted the growth of plants at 95.3 and 198.3g of
Cr (vi)/g soil by protecting the plants against the inhibitory effects of chromium,
and probably due to the solubilization of P and production of IAA and siderophores.
In another report, a heavy metal- and antibiotic-resistant bacterial strain, Burkhol-
deria sp., isolated from metal-contaminated soils capable of solubilizing inorganic
P, producing IAA, siderophore and ACC deaminase, was found to appreciably
increase the biomass of maize and tomato plants (Jiang et al. 2008). Rajkumar and
Freitas (2008) reported that the inoculation of metal-resistant plant growth-promoting
Pseudomonas sp. (PsM6) and P. jessenii (PjM15) strains possessing intrinsic ability
of P-solubilization ability for the utilization of ACC as the sole N source and
production of IAA. increased the growth and the uptake of Ni, Cu and Zn by Ricinus
communis, grown in noncontaminated and contaminated soil. Application of cad-
mium-resistant plant growth-promoting P. aeruginosa exhibiting P-solubilization,
ACC deaminase activity, siderophore production, and auxin synthesis, when used
as inoculant for blackgram (Vigna mungo L.) plants grown in soil treated with a
gradient of CdCl2 concentration, reduced the toxicity of metal to plants (Ganesan
2008) . These and other associated data suggest that such PS microbes could be used
as biofertilizer not only to provide P to plants but could also play an important role
in reducing/detoxifying the effects of heavy metals in derelict soils.
2.6.2 Inoculation Effects of Phosphate-Solubilizing Fungi
The inoculation of P-solubilizing fungi is a promising technique because it can

increase P-availability in soils fertilized with RP. Accordingly, several authors have
reported a profound increase in dry weight yield, and in N- and P-contents of
Brassica chinensis Linn. (Chuang et al. 2007) and soybean and faba bean (Vicia
faba) (Abd-Alla et al. 2001) through inoculation of PS fungi. Recently, Mittal et al.
(2008) observed the effect of six PS fungi, including two strains of A. awamori and
four strains of P. citrinum, on growth and seed production of chickpea cv. GPF2
plants. The PS fungi was biocompatible and produced growth-promoting hormone,
indole acetic acid (IAA), varying in concentration from 2.5 to 9.8g ml1. Inocula-
tion of four strains of P. citrinum exhibited a smaller stimulatory effect and
increased the shoot height by 7%, seed number by twofold, and seed weight by
87% above uninoculated plants. However, a consortium of all the six fungal isolates
showed no stimulatory effect on chickpea plants. Similarly, the inoculation of
P. oxalicum (CBPS-3F-Tsa) used either alone or along with fused phosphates
(FP) and RP, increased the growth and N and P accumulation in maize plants
compared to control (Shin et al. 2006), while the mutant strain of P. regulosum
(Mps+) also stimulated the growth of maize plants as indicated by a 3.6–28.6%
increase in dry matter yield. In the presence of RP, P-uptake by maize plants
inoculated with the two mutants, Mps++ and Mps, was not always in agreement
with their P-solubilizing phenotypes (Reyes et al. 2002). Also, a substantial increase
in plant height (1.4 times), plant weight (5.2–8.1 times) and root length (1.1–1.2
times) of maize following inoculation of PS fungus Penicillium sp. PS-113 is
reported (Kang and Choi 1999). Babana and Antoun (2006) isolated two TPR
(Tilemsi phosphate rock)-solubilizing fungi (TSM) A. awamori Nakazawa C1 and
P. chrysogenum Thom C13 with high P-solubilizing trait from the rhizosphere of
three wheat cultivars (Alkama Beri, Hindi Tossom and Tetra) and tested against
wheat cv. Tetra fertilized with 30 kg ha1 P (added as TPR or DAP). A. awamori
Nakazawa C1 increased the root dry matter yields by 60% while P. chrysogenum
Thom C13 enhanced the root dry matter yields by 40%. A 3-year field trial on a
Gangetic alluvial soil of India was conducted to examine how P demands of rice (Oryza
sativa)–wheat cropping systems might be met with heavy initial dressings of RP.
40 A. Zaidi et al.
Preplant inoculation of rice seedling roots or wheat seeds with P-solubilizing

fungus (A. Awamori) led to a yield increase over noninoculated treatments of 0.09–
0.22 tha1 in rice and 0.15–0.45 tha1 in wheat. The agronomic efficiency and
recovery efficiency of fertilizer P in the rice–wheat system were highest (57.2 kg
grain/kg P and 40.4%, respectively) under DAP-fertilized treatments. The agro-
nomic efficiency ranged from 14.3 to 44.4 kg grain/kg P and the recovery efficiency
ranged from 7.7 to 26.4% for phosphate rock treatments. The agronomic efficiency
and recovery efficiency increased with increasing initial phosphate rock application
rate and with P-solubilizing fungus inoculation (Dwivedi et al. 2004). In another
study, PS fungi, A. niger, A. fumigatus and P. pinophilum, significantly increased the
yield components of wheat and faba bean plants, P. pinophilum being the most
efficient, which increased the yield of wheat grains by 28.9 and 32.8% in the soil
treated with RP and superphosphate, respectively. Similarly, it increased the pro-
duction of faba bean seeds by 14.7 and 29.4% with the same treatments, and the
uptake of P by both plants significantly increased due to inoculation of the soil with
the tested fungi (Wahid and Mehana 2000). Inoculation of Trichoderma spp. has
also shown a substantial increase in growth and yield parameters of chickpea under
both glasshouse and field trials (Rudresh et al. 2005).
Volcanic soils in the south of Chile have an elevated quantity of total P, which
is hardly available due to its high P-fixation capacity. One strategy for increasing
the availability of P for the vegetables that grow there would be to use PS fungi. In
one assay conducted in a greenhouse on a volcanic soil, the effect of inoculation
with Penicillium albidum on the growth of red clover (Trifolium pratense) includ-
ing active inoculum [In (+)], inactive inoculum [In ()] and without inoculum
[In (0)] was reported. The In (+) significantly increased the root growth of the plants
and the P mobilized by the shoot with In (+) was twofold higher than in the In (0) or
In () treatments. In soil, available P was not different among the treatments but
phosphatase activity in In (+) was higher in comparison to In (0). The author
suggested that P. albidum could be developed as fungal inoculant to increase the
productivity of crops in volcanic soils of Chile (Morales et al. 2007). Wakelin et al.
(2007) tested the PS fungi P. radicum, P. bilaiae (strain RS7B-SD1), and an
unidentified Penicillium sp. designated strain KC6-W2 for their ability to increase
the growth and P nutrition of wheat and lentil (Lens culinaris) in three soils of
neutral to alkaline pH reaction. The strongest plant growth-promoting strain was
found Penicillium sp. (KC6-W2), which increased shoot growth and dry mass.
Levels of increase by Penicillium sp. KC6-W2 ranged from 6.6 to 19% and were
associated with increased uptake of P to the shoot. Inoculation of seed with
P. radicum increased lentil growth by 5.5% in soil from Tarlee but did not affect
plant growth in the eight other experiments. However, when significant, stimulation
of plant growth promotion by P. bilaiae RS7B-SD1 was strong and showed a 15%
increase in lentil shoot dry matter.
Nitrogen and P are the two major plant nutrients whose composite inoculation
has shown a greater impact on the performance of various crops than those observed
for single inoculation treatments. For example, when PS bacterium (Bacillus ) and
asymbiotic N2 fixer (A. chroococcum) were used in combination with PS fungus,
P. variabile, significantly enhanced the seed yield, grain protein and N- and P-
accumulation in wheat (Khan and Zaidi 2007). The increase in overall performance
of wheat was attributed to the ability of PS fungi to solubilize inorganic P. During
interactions, the phytostimulator, A. chroococcum, besides providing N, produced
considerable amounts of growth-promoting substances in the rhizosphere (Kucey
et al. 1989). Combining an improved nutrient supply with N (A. chroococcum) and
P (PS fungus) with plant growth promotion appears to have additive and possibly
even multiplicative effects. Furthermore, a plant growth-promoting rhizobacterium,
Azospirillum brasilense SP7, when applied with a bio-control fungus possessing PS
activity (T. harzianum Rifai 1295-22) and RP at 1 mg ha1, significantly increased
seed yield, total N and total P of field-grown beans (Mehmet et al. 2005). However,
variations in the effectiveness of microbial combinations under field conditions are
reported which may possibly be due to (1) variations in the survivability and
colonization efficiency of the inoculated microbial cultures in the soils, (2) strong
competition from the natural microbiota of the field soils, leading possibly to the
exclusion of inoculated cultures from the rhizosphere, (3) differential rhizosphere
effect of plants in harboring a target microbial strain (Pal 1998), (4) the modulation
of the PS activity by specific root exudates (Goldstein et al. 1999; Dakora and
Phillips 2002), (5) availability of inadequate nutrients in the rhizosphere to produce
enough OA, (6) variation in the persistence of PS activity, and (7) genetic instability
among inoculated strains. Furthermore, there are reports that also suggest the
inhibitory effect of fungi on crop improvement (Zaidi et al. 2003). This inhibition
has been due to high OA-secreting ability of fungi which in turn limits the growth of
even associative partners that require neutral or alkaline growth conditions for their
active metabolism. Moreover, the inhibitory effect of PS fungi on the associative
partners could be due to the release of toxins in the growing environment which
might affect the functional symbioses between rhizobia and their specific host
plants. These data suggests that, before carrying out in situ experiments, the
compatibility between the two associate members must be checked in vitro.
The use of AM fungi has been shown to possess the ability to increase nutrient
uptake of plants by developing associations with roots of more than 80% of higher
plant species (Marschner and Dell 1994; Schreiner et al. 1997). The combined
inoculation of N2 fixers, P-solubilizers and AM fungi has been found to stimulate
plant growth more than inoculation of either organism used alone in certain situa-
tions when the soil is P-deficient (Khan et al. 2007). During this intergeneric
pairing, the P-solubilizers interact well with the AM fungi in P-deficient soils or
soils having RP (Poi et al. 1989). The PS fungi release some P-ions from otherwise
sparingly soluble P sources which is tapped and translocated by the AM fungal
hyphae to the plant (Azcon-Aguilar et al. 1986). Moreover, the P-solubilizers
survives longer around mycorrhizal roots compared to nonmycorrhizal roots, and
acts synergistically with AM fungi leading to increased plant growth. In this
context, the coinoculation of AM fungus (G. fasciculatum) and PS fungus
(P. variabile or Penicillium sp.) has been shown to facilitate chickpea (Zaidi and
Khan 2007) and greengram (Khan and Zaidi 2006) growth more than single
inoculation. However, when Mesorhizobium or Bradyrhizobium sp. (Vigna) was
42 A. Zaidi et al.
simultaneously applied with dual inoculation of AM fungus and P-solubilizing

fungi, the seed yield, grain protein and nutrient (N and P) accumulation was further
enhanced in these legumes. The simplest interpretation of this fact is that mycor-
rhizal endophyte could be stimulated in quantity, efficiency, and longevity. The
main effect of this mycorrhiza in improving plant growth is through improved
uptake of nutrients, especially P, due to the exploration by the external hyphae of
the soil beyond the root-hair zone where P is depleted. In addition, root exudation
and plasticity might change by the activity of PS fungi, which could also affect
mycorrhizal development. Moreover, the triple inoculation of nitrogen fixer
(Mesorhizobium/Bradyrhizobium), PS fungus (P. variabile) and AM fungus
(G. fasciculatum) synergistically enhanced the dry matter accumulation, seed
yield, grain protein and N and P of chickpea (Zaidi et al. 2003; Zaidi and Khan
2007) and greengram (Khan and Zaidi 2006). These results strongly suggested that
a relationship existed between root colonization, P-uptake and growth promotion,
which in turn profoundly enhanced the yield of crops.
2.7 Conclusion
In agricultural practices, to circumvent the P-deficiency, chemical P fertilizers are

applied. However, the excessive and injudicious applications of these fertilizers
leads to a severe threat to microbial diversity, soil microbial community structure,
soil fertility and consequently the productivity of crops in different agro-ecosystems.
Microbiologists and soil scientists are thus searching for an alternative to these P
problems. Since the majority of soils the world over are deficient in plant-available
P and since phosphatic fertilizers are expensive, focus is placed on the use of soil
microorganisms endowed with PS ability, which could be used as inoculants to
mobilize P from poorly available sources in soil. The use of such PSM(s) opens up a
new horizon for better plant productivity besides reducing the reliance on chemical
P and protecting the agro-ecosystems from the hazards of agrochemicals. The
protection of the soil environment by applying PSM(s) could become a major
breakthrough for plants grown in derelict soils. Moreover, the molecular engineer-
ing of these microbes has also provided a new insight into the promotion of crops in
P-deficient soils. In this context, novel, genetically engineered and soil- and region-
specific PSM(s) and technologies have to be developed, pilot tested and transferred
to farmers in a relatively short time in order to improve plant P-nutrition and agro-
ecosystem sustainability.
References
Abril A, Zurdo-Piñeiro JL, Peix A, Rivas R, Velázquez E (2007) Solubilization of phosphate by a

strain of Rhizobium leguminosarum bv. trifolii isolated from Phaseolus vulgaris in El Chaco
Arido soil (Argentina). In: Velazquez E, Rodriguez-Berrueco C (eds) Book Series: Develop-
ments in Plant and Soil Sciences. Springer, The Netherlands, pp 135–138
Abd-Alla MH, Omar SA, Omar SA (2001) Survival of rhizobia/bradyrhizobia and a rock phos-
phate solubilising fungus on various carriers from some agro-industrial wastes and their effects
on nodulation and growth of fababean and soybean. J Plant Nutr 24: 261–272
Afzal A, Bano A (2008) Rhizobium and phosphate solubilizing bacteria improve the yield and
phosphorus uptake in wheat (Triticum aestivum). Int J Agric Biol 10:85–88
Ahmad F, Ahmad I, Khan MS (2008) Screening of free-living rhizospheric bacteria for their
multiple plant growth promoting activities. Microbiol Res 163:173–181
Alikhani HA, Saleh-Rastin N, Antoun H (2007) Phosphate solubilization activity of rhizobia
native to Iranian soils. In: Velazquez E, Rodriguez-Berrueco C (eds) Book Series: Develop-
ments in plant and soil sciences. Springer, The Netherlands, pp 135–138
Altomare C, Norvell WA, Bjorkman T, Harman GE (1999) Solubilization of phosphates and
micronutrients by the plant growth promoting and biocontrol fungus Trichoderma harzianum
rifai 1295–22. Appl Environ Microbiol 65:2926–2933
Anandham R, Gandhi PI, Madhaiyan M, Sa T (2008) Potential plant growth promoting traits and
bioacidulation of rock phosphate by thiosulfate oxidizing bacteria isolated from crop plants.
J Basic Microbiol . doi:10.1002/jobm.200700380
Asea PEA, Kucey RMN, Stewart JWB (1988) Inorganic phosphate solubilization by two Penicil-
lium species in solution culture and soil. Soil Biol Biochem 20:459–464
Azcon-Aguilar C, Diaz-Rodriguez R, Barea JM (1986) Effect of soil microorganisms on spore
germination and growth on the vesicular arbuscular mycorrhizal fungus Glomus moseae. Trans
Br Mycol Soc 86:337–340
Babana AH, Antoun H (2006) Biological system for improving the availability of Tilemsi phosphate
rock for wheat (Triticum aestivum L.) cultivated in Mali. Nutr Cycl Agroecosys 76:285–295
Babu-Khan S, Yeo C, Martin WL, Duron MR, Rogers R, Goldstein A (1995) Cloning of a mineral
phosphate-solubilizing gene from Pseudomonas cepacia. Appl Environ Microbiol 61:972–978
Bar-Yosef B, Rogers RD, Wolfram JH, Richman E (1999) Pseudomonas cepacia mediated rock
phosphate solubilization in kaolinite and montmorillonite suspensions. Soil Sci Soc Am
J 63:1703–1708
Batjes NH (1997) A world data set of derived soil properties by FAO–UNESCO soil unit for global
modelling. Soil Use Manage 13:9–16
Bishop ML, Chang AC, Lee RWK (1994) Enzymatic mineralization of organic phosphorus in a
volcanic soil in Chile. Soil Sci 157:238–243
Bojinova D, Velkova R, Ivanova R (2008) Solubilization of Morocco phosphorite by Aspergillus
niger. Biores Technol 99:7348–7353
Brookes PC, Powlson DS, Jenkinson DS (1984) Phosphorus in the soil microbial biomass. Soil
Canbolat MY, Bilen S, Cakmakci R, Sahin F, Aydin A (2006) Effect of plant growth-promoting
bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and
rhizosphere microflora. Biol Fertil Soils 42:350–357
Chandra S, Choure K, Dubey RC, Maheshwari DK (2007) Rhizosphere competent Mesorhizobium
loti mp6 induces root hair curling, inhibits Sclerotinia sclerotiorum and enhances growth of
Indian mustard (Brassica campestris). Brazil J Microbiol 38:124–130
Chen YP, Rekha PD, Arun AB, Shen FT, Lai WA, Young CC (2006) Phosphate solubilizing
bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl Soil
Ecol 34:33–41
Chen Z, Ma S, Liu LL (2008) Studies on phosphorus solubilizing activity of a strain of phospho-
bacteria isolated from chestnut type soil in China. Biores Technol 99:6702–6707
Chuang CC, Kuo YL, Chao CC, Chao WL (2007) Solubilization of inorganic phosphates and plant
growth promotion by Aspergillus niger. Biol Fertil Soils 43:575–584
Chung H, Park M, Madhaiyan M, Seshadri S, Song J, Cho H, Sa T (2005) Isolation and
characterization of phosphate solubilizing bacteria from the rhizosphere of crop plants of
Korea. Soil Biol Biochem 37:1970–1974
44 A. Zaidi et al.
Colbert SF, Hendson M, Ferri M, Schroth MN (1993) Enhanced growth and activity of a biocontrol
bacterium genetically engineered to utilize salicylate. Appl Microbiol 59:2071–2076
Cunningham J, Kuiack C (1992) Production of citric and oxalic acids and solubilization of calcium
phosphate by Penicillium bilaii. Appl Environ Microbiol 58:1451–1458
Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient
environments. Plant Soil 245:35–47
De Freitas JR, Banerjee MR, Germida JJ (1997) Phosphate-solubilizing rhizobacteria enhance the
growth and yield but not phosphorus uptake of canola (Brassica napus L.). Biol Fertil Soils
24:358–364
Dwivedi BS, Singh VK, Dwivedi V (2004) Application of phosphate rock, with or without
Aspergillus awamori inoculation, to meet phosphorus demands of rice–wheat systems in the
Indo–Gangetic plains of India. Aust J Exp Agric 44:1041–1050
Ehteshami SMR, Aghaalikhani M, Khavazi K, Chaichi MR (2007) Effect of phosphate solubiliz-
ing microorganisms on quantitative and qualitative characteristics of maize (Zea mays L.)
under water deficit stress. Pak J Biol Sci 10:3585–3591
Elizabeth P, Miguel S, Ball Maria M, Andrés YL (2007) Isolation and characterization of mineral
phosphate-solubilizing bacteria naturally colonizing a limonitic crust in the south-eastern
Venezuelan region. Soil Biol Biochem 39:2905–2914
Elkoca E, Kantar F, Sahin F (2008) Influence of nitrogen fixing and phosphorus solubilizing
bacteria on the nodulation, plant growth, and yield of chickpea. J Plant Nutr 31:157–171
Fenice M, Selbman L, Federici F, Vassilev N (2000) Application of encapsulated Penicillium
variabile P16 in solubilization of rock phosphate. Biores Technol 73:157–162
Fernández LA, Zalba P, Gómez MA, Sagardoy MA (2007) Phosphate-solubilization activity of
bacterial strains in soil and their effect on soybean growth under greenhouse conditions. Biol
Fertil Soils 43:805–809
Fraga-Vidal R, Rodriguez HM, De Villegas TG (2003) Vector for chromosomal integration of
the phoC gene in plant growth-promoting bacteria. In: Velazquez E, Rodrguez-Barrueco C
(eds) First international meeting on microbial phosphate solubilization. Springer, Berlin,
pp 239–244
Ganesan V (2008) Rhizoremediation of cadmium soil using a cadmium-resistant plant growth-
promoting rhizopseudomonad. Curr Microbiol 56:403–407
Glick BR, Bashan Y (1997) Genetic manipulation of plant growth promoting bacteria to enhance
biocontrol of phytopathogens. Biotechnol Adv 15:353–378
Goldstein AH (1986) Bacterial solubilization of mineral phosphates: historical perspectives and
future prospects. Am J Altern Agric 1:57–65
Goldstein AH (1994) Involvement of the quinoprotein glucose dehydrohenase in the solubilization
of exogenous phosphates by gran-negative bacteria. In: Torriani-Gorini A, Yagil E, Silver S
(eds) Phosphate in microorganisms: cellular and molecular biology. ASM, Washington DC, pp
197–203
Goldstein AH, Liu ST (1987) Molecular cloning and regulation of a mineral phosphate solubiliz-
ing gene from Erwinia herbicola. Biotechnology 5:72–74
Goldstein AH, Braverman K, Osorino N (1999) Evidence for mutualism between a plant growing
in a phosphate limited desert environment and a mineral phosphates solubilizing (MPS)
rhizobacterium. FEMS Microbiol Ecol 30:295–300
Goud MJP, Goud JVS, Charya MAS (2008) Replica plate screening method for detecting
phosphatase activity in basidiomycetes using 1-napthyl phosphate as a chromogenic substrate.
Sci World J 3:13–15
Guimarães LHS, Peixoto-Nogueira SC, Michelin M, Rizzatti ACS, Sandrim VC, Zanoelo F,
Aquino ACMM, Junior AB, De Lourdes M, Polizeli TM (2006) Screening of filamentous
fungi for production of enzymes of biotechnological interest. Brazil J Microbiol 37:474–480
Gupta RR, Singal R, Shanker A, Kuhad RC, Saxena RK (1994) A modified plate assay for
secreening phosphate solubilizing microorganisms. Gen Appl Microbiol 40:255–260
Gyaneshwar P, Naresh K, Parekh LJ (1998) Cloning of mineral phosphate solubilizing genes from
Synechocystis PCC 6803. Curr Sci 74:1097–1099
Halder AK, Chakrabarty PK (1993) Solubilization of inorganic phosphate by Rhizobium. Folia
Hamdali H, Hafidi M, Virolle MJ, Ouhdouch Y (2008) Rock phosphate-solubilizing Actinomy-
cetes: screening for plant growth-promoting activities. World J Microbiol Biotechnol 24:2565–
2575
Hameeda B, Harini G, Rupela OP, Wani SP, Reddy G (2008) Growth promotion of maize by
phosphate-solubilizing bacteria isolated from composts and macrofauna. Microbiol Res
163:234–242
Han SH, Kim CH, Lee JH, Park JY, Cho SM, Park SK, Kim KY, Krishnan HB, Kim YC (2008)
Inactivation of pqq genes of Enterobacter intermedium 60–2G reduces antifungal activity and
induction of systemic resistance. FEMS Microbiol Lett 282:140–146
Harman GE, Bjorkman T (1998) Potential and existing uses of Trichoderma and Gliocladium for
plant disease control and plant growth enhancement. In: Harman GE, Kubicek CP (eds)
Trichoderma and Gliocladium, vol 2. Taylor and Francis, London, UK, pp 229–265
Henri F, Laurette NN, Annette D, John Q, Wolfgang M, François-Xavier E, Dieudonne N (2008)
Solubilization of inorganic phosphates and plant growth promotion by strains of Pseudomonas
fluorescens isolated from acidic soils of Cameroon. Afr J Microbiol Res 2:171–178
Hinsinger P (2001) Bio-availability of soil inorganic P in the rhizosphere as affected by root
induced chemical changes: a review. Plant Soil 237:173–195
Hwangbo H, Park RD, Kim YW, Rim YS, Park KH, Kim TH, Such JS, Kim KY (2003)
2-ketogluconic acid production and phosphate solubilization by Enterobacter intermedium.
Curr Microbiol 47:87–92
Illmer P, Schinner F (1992) Solubilization of inorganic phosphates by microorganisms isolated
from forest soil. Soil Biol Biochem 24:389–395
Illmer P, Schinner F (1995) Solubilization of inorganic calcium phosphates-solubilization
mechanisms soil. Soil Biol Biochem 27:257–263
Illmer P, Barbato A, Schinner F (1995) Solubilization of hardly soluble AlPO4 with P-solubilizing
microorganisms. Soil Biol Biochem 27:265–270
Indiragandhi P, Anandham R, Madhaiyan M, Sa TM (2008) Characterization of plant growth-
promoting traits of bacteria isolated from larval guts of diamondback moth Plutella xylostella
(Lepidoptera: Plutellidae). Curr Microbiol 56:327–333
Jha BK, Pragash MG, Cletus J, Raman G, Sakthivel N (2008) Simultaneous phosphate solubiliza-
tion potential and antifungal activity of new fluorescent pseudomonad strains, Pseudomonas
aeruginosa, P. plecoglossicida and P. mosselii. World J Microbiol Biotechnol. doi:10.1007/
s11274-008-9925-x
Jiang C, Sheng X, Qian M, Wang Q (2008) Isolation and characterization of a heavy metal-
resistant Burkholderia sp. from heavy metal-contaminated paddy field soil and its potential in
promoting plant growth and heavy metal accumulation in metal-polluted soil. Chemosphere
72:157–164
Kang SC, Choi MC (1999) Solid culture of phosphate solubilising fungus. Sanoeb Misaegmul
Haghoeji 27:1–7
Khan MR, Khan SM (2001) Biomanagement of Fusarium wilt of tomato by the soil application of
certain phosphate-solubilizing microorganisms. Int J Pest Manag 47:227–231
Khan MR, Khan SM (2002) Effect of root-dip treatment with certain phosphate-solubilizing
microorganisms on the Fusarium wilt of tomato. Biores Technol 85:213–215
Khan MS, Zaidi A (2006) Influence of composite inoculations of phosphate solubilizing organisms
and an arbuscular mycorrhizal fungus on yield, grain protein and phosphorus and nitrogen
uptake by greengram. Arch Agron Soil Sci 52:579–590
Khan MS, Zaidi A (2007) Synergistic effects of the inoculation with plant growth promoting
rhizobacteria and arbuscular mycorrhizal fungus on the performance of wheat. Turk J Agric
For 31:355–362
46 A. Zaidi et al.
Khan MS, Aamil M, Zaidi A (1997) Associative effect of Bradyrhizobium sp. (vigna) and
phosphate solubilizing bacteria on moongbean [Vigna radiata(L.) wilczek]. Biojournal
10:101–106
Khan MS, Aamil M, Zaidi A (1998) Moongbean response to inoculation with nitrogen fixing and
phosphate solubilizing bacteria. In: Deshmukh AM (ed) Biofertilizers and biopesticides.
Technoscience, Jaipur, pp 40–48
Khan MR, Khan SM, Mohiddin FA (2007a) Effect of certain fungal and bacterial phosphate
solubilizing microorganisms on the fusarial wilt of tomato. In: Velázquez E, Rodrı́guez-
Barrueco C (eds) First International Meeting on Microbial Phosphate Solubilization. Springer,
The Netherlands, pp 357–361
Khan MS, Zaidi A, Wani PA (2007b) Role of phosphate solubilizing microorganisms in sustain-
able agriculture: A review. Agron Sustain Dev 27:29–43
Khan MS, Zaidi A, Wani PA, Oves M (2009) Role of plant growth promoting rhizobacteria in the
remediation of metal contaminated soils. Environ Chem Lett 7: 1–19
Kim KY, Mcdonald GA, Jordan D (1997) Solubilization of hydroxypatite by Enterobacter
agglomerans and cloned Escherichia coli in culture medium. Biol Fertil Soils 24:347–352
Kim KY, Jordan D, Krishnan HB (1998) Expression of genes from Rahnella aquatilis that are
necessary for mineral phosphate solubilization in Escherichia coli. FEMS Microbiol Lett
159:121–127
Krishnaraj PU, Goldstein AH (2001) Cloning of a Serratia marcescens DNA fragment that
induces quinoprotein glucose dehydrogenase-mediated gluconic acid production in Escher-
ichia coli in the presence of stationary phase Serratia marcescens. FEMS Microbiol Lett
205:215–220
Kucey RMN (1988) Effect of Penicillium bilaji on the solubility and uptake of P and micronu-
trients from soil by wheat. Can J Soil Sci 68:261–270
Kucey RMN, Janzen HH, Legget ME (1989) Microbial mediated increases in plant available
phosphorus. Adv Agron 42:199–228
Kumar V, Behl RK, Narula N (2001) Establishment of phosphate-solubilizing strains of Azoto-
bacter chroococcum in the rhizosphere and their effect on wheat cultivars under greenhouse
conditions. Microbiol Res 156:87–93
Kumar KV, Singh N, Behl HM, Srivastava S (2008) Influence of plant growth promoting bacteria
and its mutant on heavy metal toxicity in Brassica juncea grown in fly ash amended soil.
Chemosphere 72:678–683
Lipping Y, Jiatao X, Daohong J, Yanping F, Guoqing L, Fangcan L (2008) Antifungal substances
produced by Penicillium oxalicum strain PY-1–potential antibiotics against plant pathogenic
fungi. World J Microbiol Biotechnol 24:909–915
Maliha R, Samina K, Najma A, Sadia A, Farooq L (2004) Organic acids production and phosphate
solubilization by phosphate solubilizing microorganisms under in vitro conditions. Pak J Biol
Sci 7:187–196
Marschner H, Dell B (1994) Nutrient uptake in mycorrhizal symbiosis. Plant Soil 159:89–102
Mckenzie RH, Roberts TL (1990) Soil and fertilizers phosphorus update. Alberta Soil Science
Workshop Proceedings, Edmonton, Alberta, pp 84–104
Mehmet O, Cevdet A, Oral D, Ali SM (2005) Single and double inoculation with Azospirillum/
Trichoderma : the effects on dry bean and wheat. Biol Fertil Soils 41:262–272
Mittal V, Singh O, Nayyar H, Kaur J, Tewari R (2008) Stimulatory effect of phosphate-solubilizing
fungal strains (Aspergillus awamori and Penicillium citrinum) on the yield of chickpea (Cicer
arietinum L. cv. GPF2). Soil Biol Biochem 40:718–727
Morales A, Alvear M, Valenzuela E, Rubio R, Borie F (2007) Effect of inoculation with Penicilli-
um albidum, a phosphate-solubilizing fungus, on the growth of Trifolium pratense cropped in a
volcanic soil. J Basic Microbiol 47:275–280
Nautiyal CS (1999) An efficient microbiological growth medium for screening of phosphate
solubilizing microorganisms. FEMS Microbiol Lett 170:265–270
Nikolay V, Maria V, Iana N (2006) Simultaneous P-solubilizing and biocontrol activity of

microorganisms: potentials and future trends. Appl Microbiol Biotechnol 71:137–144
Nozawa M, Hu HY, Fujie K, Tanaka H, Urano K (1998) Quantitative detection of Enterobacter
cloacae strain HO-I In bioreactor for chromate wastewater treatment using polymerase chain
reaction (PCR). Water Res 32:3472–3476
Oberson A, Friesen DK, Rao IM, Bühler S, Frossard E (2001) Phosphorus transformations in an
oxisol under contrasting land-use systems: The role of the microbial biomass. Plant Soil
237:197–210
Oliveira CA, Alves VMC, Marriel IE, Gomes EA, Scotti MR, Carneiro NP, Guimarães CT,
Schaffert RE, Sá NMH (2008) Phosphate solubilizing microorganisms isolated from rhizo-
sphere of maize cultivated in an oxisol of the Brazilian Cerrado Biome. Soil Biol Biochem .
doi:10.1016/j.soilbio.2008.01.012
Omar SA, Abd-Alla MH (2000) Physiological aspects of fungi isolated from root nodules of faba
bean (Vicia faba L.). Microbiol Res 154:339–347
Pal SS (1998) Interaction of an acid tolerant strain of phosphate solubilizing bacteria with a few
acid tolerant crops. Plant Soil 198:169–177
Pandey A, Trivedi P, Kumar B, Palni LMS (2006) Characterization of a phosphate solubilizing and
antagonistic strain of Pseudomonas putida (B0) isolated from a sub-alpine location in the
Indian Central Himalaya. Curr Microbiol 53:102–107
Pandey A, Das N, Kumar B, Rinu K, Trivedi P (2008) Phosphate solubilization by Penicillium spp.
isolated from soil samples of Indian Himalayan region. World J Microbiol Biotechnol 24:
97–102
Patel DK, Archana G, Kumar GN (2008) Variation in the nature of organic acid secretion and
mineral phosphate solubilization by Citrobacter sp. DHRSS in the presence of different sugars.
Curr Microbiol 56:168–174
Petruccioli M, Federici F, Bucke C, Keshavarz T (1999) Enhancement of glucose oxidase
production by Penicillium variabile P16. Enzyme Microb Technol 24:397–401
Pikovskaya RI (1948) Mobilization of phosphorus in soil in connection with vital activity of some
microbial species. Microbiology 17:362–370
Poi SC, Ghosh G, Kabi MC (1989) Response of chickpea (Cicer aeritinum L.) to combined
inoculation with Rhizobium, phosphobacteria and mycorrhizal organisms. Zentral fur Micro-
biol 114:249–253
Ponmurugan P, Gopi C (2006) In vitro production of growth regulators and phosphatase activity
by phosphate solubilizing bacteria. Afr J Biotechnol 5:348–350
Poonguzhali S, Madhaiyan M, Sa T (2008) Isolation and identification of phosphate solubilizing
bacteria from chinese cabbage and their effect on growth and phosphorus utilization of plants.
J Microbiol Biotechnol 18:773–777
Pradhan N, Sukla LB (2005) Solubilization of inorganic phosphates by fungi isolated from
agriculture soil. Afr J Biotechnol 5:850–854
Raghothama KG (1999) Phosphate acquisition. Annu Rev Plant Physiol Mol Biol 50:665–693
Rajkumar M, Freitas H (2008) Influence of metal resistant-plant growth-promoting bacteria on the
growth of Ricinus communis in soil contaminated with heavy metals. Chemosphere 71:
834–842
Rajkumar M, Nagendran R, Lee KJ, Lee WH, Kim SZ (2006) Influence of plant growth promoting
bacteria and Cr6+ on the growth of Indian mustard. Chemosphere 62:741–748
Rane MR, Sarode PD, Chaudhari BL, Chincholkar SB (2008) Exploring antagonistic metabolites
of established biocontrol agent of marine origin. Appl Biochem Biotechnol 151:665–675
Reyes I, Bernier L, Simard RR, Antoun H (1999) Effect of nitrogen source on the solubilization of
different inorganic phosphates by an isolate of Penicillium rugulosum and two UV induced
mutants. FEMS Micobiol Ecol 28:281–290
Reyes I, Bernier L, Antoun H (2002) Rock phosphate solubilization and colonization of maize
rhizosphere by wild and genetically modified strains of Penicillium regulosum. Microb Ecol
44:39–45
48 A. Zaidi et al.
Reyes I, Baziramakenga R, Bernier L, Antoun H (2001) Solubilization of phosphate rocks and

minerals by a wild type strain and two UV induced mutants of Penicillium regulosum. Soil Biol
Biochem 33: 1741–1747
Richardson AE (1994) Soil microorganisms and phosphorous availability. In: Pankhurst CE,
Doube BM, Gupta VVSR (eds) Soil biota: management in sustainable farming systems.
CSIRO, Victoria, Australia, pp 50–62
Richardson AE, Hadobas PA, Hayes JE (2001a) Extracellular secretion of Aspergillus phytase from
Arabidopsis roots enables plants to obtain phosphorous from phytate. Plant J 25:641–649
Richardson AE, Hadobas PA, Hayes JE, O’Hara CP, Simpson RJ (2001b) Utilization of phospho-
rus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of
soil micro-organisms. Plant Soil 229:47–56
Rodriguez H, Fraga R (1999) Phosphate solubilizing bacteria and their role in plant growth
promotion. Biotechnol Adv 17:319–339
Rodriguez H, Fraga R, Gonzalez T, Bashan Y (2006) Genetics of phosphate solubilization and its
potential applications for improving plant growth-promoting bacteria. Plant Soil 287:15–21
Rodrı́guez H, Gonzalez T, Selman G (2000) Expression of a mineral phosphate solubilizing gene
from Erwinia herbicola in two rhizobacterial strains. J Biotechnol 84:155–161
Ruch DG, Motta JJ (1987) Ultrastructure and cytochemistry of dormant basidiospores of Psilocybe
cubensis. Mycologia 79:387–398
Rudresh DL, Shivaprakash MK, Prasad RD (2005a) Tricalcium phosphate solubilizing abilities of
Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer
arietinum L.). Can J Microbiol 51:217–222
Rudresh DL, Shivaprakash MK, Prasad RD (2005b) Effect of combined application of Rhizobium,
phosphate solubilizing bacterium and Trichoderma spp. on growth, nutrient uptake and yield of
chickpea (Cicer aritenium L.). Appl Soil Ecol 28:139–146
Saber K, Nahla L, Ahmed D, Chedly A (2005) Effect of P on nodule formation and N fixation in
bean. Agron Sustain Dev 25:389–393
Saito M (1995) Enzyme activities of the internal hypha and germinated spores of an arbuscular
mycorrhizal fungus, Gigaspora margarita. New Phytol 129:425–431
Schadeck RJG, Buchi DF, Leite B (1998a) Ultrastructural aspects of Colletotrichum graminicola
conidium germination, appressorium formation and penetration on cellophane membranes:
focus on lipid reserves. J Submicrosc Cytol Pathol 30:555–561
Schadeck RJG, Leite B, Buchi DF (1998b) Lipid mobilization and acid phosphatase activity in
lytic compartments during conidium dormancy and appressorium formation of Colletotrichum
graminicola. Cell Struct Funct 23:333–340
Schreiner RP, Mihara KL, Mcdaniel H, Bethlenfalvay GJ (1997) Mycorrhizal fungi influence plant
and soil functions and interactions. Plant Soil 188:199–209
Selvakumar G, Mohan M, Kundu S, Gupta AD, Joshi P, Nazim S, Gupta HS (2008) Cold
tolerance and plant growth promotion potential of Serratia marcescens strain SRM (MTCC
8708) isolated from flowers of summer squash (Cucurbita pepo). Lett Appl Microbiol
46:171–175
Shaharoona B, Naveed M, Arshad M, Zahir ZA (2008) Fertilizer-dependent efficiency of Pseu-
domonads for improving growth, yield, and nutrient use efficiency of wheat (Triticum aestivum
L.). Appl Microbiol Biotechnol 79:147–155
Sharan A, Shikha DNS, Gaur R (2008) Xanthomonas campestris, a novel stress tolerant, phos-
phate-solubilizing bacterial strain from saline–alkali soils. World J Microbiol Biotechnol
24:753–759
Shin W, Ryu J, kim Y, Yang J, Madhaiyan M, Sa T (2006) Phosphate solubilization and growth
promotion of maize (Zea mays L.) by the rhizosphere soil fungus Penicillium oxalicum.18th
World Congress of Soil Science. July 9–15, Philadelphia, Pennsylvania, USA
Singal R, Gupta R, Saxena RK (1994) Rock phosphate solubilization under alkaline conditions by
Aspergillus japonicus and A. foetidus. Folia Microbiol 39:33–36
Singh N, Pandey P, Dubey R, Maheshwari DK (2008) Biological control of root rot fungus
Macrophomina phaseolina and growth enhancement of Pinus roxburghii (Sarg.) by rhizo-
sphere competent Bacillus subtilis BN1. World J Microbiol Biotechnol 24:1669–1679
Song OR, Lee SJ, Lee YS, Lee SC, Kim KK, Choi YL (2008) Solubilization of insoluble inorganic
phosphate by Burkholderia cepacia DA23 isolated from cultivated soil. Brazil J Microbiol
39:151–156
Souchie EL, Azcón R, Barea JM, Saggin-Júnior OJ, Silva EMR (2006) Phosphate solubilization
and synergism between P-solubilizing and arbuscular mycorrhizal fungi. Pesquisa Agrope-
cuária Brasileira 41:1405–1411
Souchie EL, Abboud ACS, Caproni AL (2007) In vitro phosphate solubilization by rhizospheric
microorganisms from pigeonpea. Biosci J 23:53–60
Sridevi M, Mallaiah KV, Yadav NCS (2007) Phosphate solubilization by Rhizobium isolates from
Crotalaria species. J Plant Sci 2:635–639
Thomas GV, Shantaram MV, Saraswathy N (2005) Occurrence and activity of phosphate solubi-
lizing fungi from coconut plantation soils. J Plant Sci 87:357–364
To-o K, Kamasaka H, Kusaka K, Kuriki T, Kometani K, Okada S (1997) A novel acid phosphatase
from Aspergillus niger KU-8 that specifically hydrolyzes C-6 phosphate groups of phosphoryl
oligosaccharides. Biosci Biotechnol Biochem 61:1512–1517
To-o K, Kamasaka H, Kuriki T, Okada S (2000) Substrate selectivity in Aspergillus niger
KU-8 acid phosphatase II using phosphoryl oligosaccharides. Biosci Biotechnol Biochem
64:1534–1537
Toro M (2007) Phosphate solubilizing microorganisms in the rhizosphere of native plants from
tropical savannas: An adaptive strategy to acid soils? In: Velazquez C, Rodriguez-Barrueco E
(eds) Developments in Plant and Soil Sciences. Springer, The Netherlands, pp 249-–252
Trivedi P, Sa TM (2008) Pseudomonas corrugata (NRRL B-30409) mutants increased phosphate
solubilization, organic acid production, and plant growth at lower temperatures. Curr Micro-
biol 56:140–144
Valverde A, Burgos A, Fiscella T, Rivas R, Velazquez E, Rodriguez-Barrueco C, Cervantes E,
Chamber M, Igual JM (2006) Differential effects of coinoculations with Pseudomonas jessenii
PS06 (a phosphate-solubilizing bacterium) and Mesorhizobium ciceri C-2/2 strains on the
growth and seed yield of chickpea under greenhouse and field conditions. Plant Soil 287:43–50
Varenyam A, Savant VV, Reddy MS (2007) Phosphate solubilization by a wild type strain and
UV-induced mutants of Aspergillus tubingensis. Soil Biol Biochem 39:695–699
Vassilev N, Fenice M, Federici F (1996) Rock phosphate solubilisation with gluconic acid
produced by immobilized Penicillium variable P16. Biotecnol Tech 20: 585–588
Vassilev N, Vassileva M, Nikolaeva I (2006) Simultaneous P-solubilizing and biocontrol activity
of microorganisms: potentials and future trends. Appl Microbiol Biotechnol 71:137–144
Vassilev N, Vassileva M, Bravo V, Fernández-Serrano M, Nikolaeva I (2007) Simultaneous
phytase production and rock phosphate solubilization by Aspergillus niger grown on dry
olive wastes. Ind Crop Prod 26:332–336
Vazquez P, Holguin G, Puente M, Elopez CA, Bashan Y (2000) Phosphate solubilizing micro-
organisms associated with the rhizosphere of mangroves in a semi arid coastal lagoon. Biol
Fertil Soil 30:460–468
Vikram A, Hamzehzarghani H (2008) Effect of phosphate solubilizing bacteria on nodulation and
growth parameters of greengram (Vigna radiate L. Wilczec). Res J Microbiol 3:62–72
Wahid OA, Mehana TA (2000) Impact of phosphate solubilizing fungi on the yield and phospho-
rus uptake by wheat and faba bean plants. Microbiol Res 155:221–227
Wakelin SA, Gupta VVSR, Harvey PR, Ryder MH (2007) The effect of Penicillium fungi on plant
growth and phosphorus mobilization in neutral to alkaline soils from southern Australia. Can J
Wani PA, Zaidi A, Khan AA, Khan MS (2005) Effect of phorate on phosphate solubilization and
indole acetic acid (IAA) releasing potentials of rhizospheric microorganisms. Ann Plant Prot.
Sci 13:139–144
50 A. Zaidi et al.
Wani PA, Khan MS, Zaidi A (2007a) Chromium reduction, plant growth promoting potentials and
metal solubilization by Bacillus sp. isolated from alluvial soil. Curr Microbiol 54:237–243
Wani PA, Khan MS, Zaidi A (2007b) Co-inoculation of nitrogen fixing and phosphate solubilizing
bacteria to promote growth, yield and nutrient uptake in chickpea. Acta Agron Hung 55:
315–323
Wani PA, Khan MS, Zaidi A (2007c) Synergistic effects of the inoculation with nitrogen fixing
and phosphate solubilizing rhizobacteria on the performance of field grown chickpea. J Plant
Nutr Soil Sci 170:283–287
Whitelaw MA, Harden TJ, Helyar KR (1999) Phosphate solubilization in solution culture by the
soil fungus Penicillium radicum. Soil Biol Biochem 32:655–665
Yadav RS, Tarafdar JC (2003) Phytase and phosphatase producing fungi in arid and semi-arid soils
and their efficiency in hydrolyzing different organic P compounds. Soil Biol Biochem 35:
745–751
Yi Y, Huang W, Ge Y (2008) Exopolysaccharide: a novel important factor in the microbial
dissolution of tricalcium phosphate. World J Microbiol Biotechnol 24:1059–1065
Zaidi A (1999) Synergistic interactions of nitrogen fixing microorganisms with phosphate mobi-
lizing microorganisms. PhD Thesis, Aligarh Muslim University, Aligarh
Zaidi A, Khan MS (2006) Co-inoculation effects of phosphate solubilizing microorganisms and
Glomus fasciculatum on green gram Bradyrhizobium symbiosis. Turk J Agric For 30:223–230
Zaidi A, Khan MS (2007) Stimulatory effects of dual inoculation with phosphate solubilizing
microorganisms and arbuscular mycorrhizal fungus on chickpea. Aust J Exp Agric 47:1016–
1022
Zaidi A, Khan MS, Amil M (2003) Interactive effect of rhizotrophic microorganisms on yield and
nutrient uptake of chickpea (Cicer arietinum L.). Eur J Agron 19:15–21
Zaidi A, Khan MS, Aamil M (2004) Bio-associative effect of rhizospheric microorganisms on
growth, yield and nutrient uptake of greengram. J Plant Nutr 27:599–610
Chapter 3
Developing Beneficial Microbial Biofilms
on Roots of Non legumes: A Novel
Biofertilizing Technique
Gamini Seneviratne, RMMS Thilakaratne, APDA Jayasekara, KACN

Seneviratne, KRE Padmathilake, and MSDL De Silva
Abstract Biofilms are often complex communities of multiple microbial species

and remain attached to surfaces or with interfaces. Such beneficial biofilms can be
developed in vitro and be used as biofertilizers (biofilmed biofertilizers, BBs) and
biocontroling agents for nonlegumes, when applied at high cell densities. This
chapter describes research studies conducted so far in this field with special
attention into development of biofilms of N2-fixing bacteria and P-solubilizing
fungi. When these two distinct microbes were cocultured in vitro, the bacteria
colonized fungal mycelia to form the biofilms. The biofilms showed higher rates
of biological nitrogen fixation and organic acid production, which was directly
proportional to the synthesis of indoleacetic acid-like substances, than microbes
when used alone. The plant growth-promoting effects of such BBs were evaluated
using rice (Oryza sativa), tea (Camellia sinensis), wheat (Triticum aestivum), and
anthurium (Anthurium andraeanum). The biofilms formed nodule-like structures or
“pseudonodules” on roots of such plants. For rice and tea, the results showed that
recommended chemical fertilizers may be reduced by about 50% while applying
BBs. Since this field of research is in its infancy, both laboratory and field experi-
ments are required to fully explore the potential of this emerging biotechnological
approach in the future.
G. Seneviratne (*), RMMS Thilakaratne and KRE Padmathilake

Biological Nitrogen Fixation Project, Institute of Fundamental Studies, Hantana Road, Kandy,
Sri Lanka
e‐mail: gaminis@ifs.ac.lk
APDA Jayasekara and MSDL De Silva
Tea Researh Institute of Sri Lanka, Talawakele, Sri Lanka
KACN Seneviratne
Royal Botanic Gardens, Peradeniya, Sri Lanka

52 G. Seneviratne et al.
3.1 Introduction
Although microorganisms have historically been studied as planktonic (freely

swimming) cells in many ecosystems, it is common to find assembling of micro-
organisms adherent to each other and/or surfaces and embedded in a matrix of
polymers, which are then known as biofilms (Harrison et al. 2005; Rudrappa et al.
2008). The assemblage leads to metabolic cooperation among the microbes (Davey
and O’toole 2000). Newly developed microbiological and molecular biological
methods clearly show that most bacteria live as biofilms formed on various biotic
and abiotic surfaces (Romanova et al. 2006).
A biofilm consists of microbial cells (algal, fungal, bacterial and/or other
microbial) and sticky extracellular polymeric substances (EPS), which provide
structure and protection to the community (Vandevivere and Kirchman 1993;
Seneviratne 2003). The EPS is composed of polysaccharides, proteins, nucleic
acids and other substances which help protect the biofilm organisms from various
environmental stress factors, such as UV radiation, extreme pH conditions, osmotic
shock, dehydration, antimicrobial substances, predators, etc. (Costerton et al. 1987;
Stewart and Costerton 2001; Romanova et al. 2006). As the biofilm microbes
behave like a group within the EPS coating, external environmental stresses and
attacks of competitors are tolerable (Seneviratne and Jayasinghearachchi 2005).
This is why microorganisms prefer to exist in the biofilm mode rather than the
planktonic stage. As an example, when Pseudomonas putida cells were incubated in
the presence of Saccharomyces cerevisiae in grape (Vitis vinifera) juice or in a
synthetic medium containing various concentrations of glucose, their stationary
phase survival improved dramatically (Romano and Kolter 2005), possibly through
biofilm formation. Similarly, when introduced into soils after coupling with a
common soil fungus to form biofilms, rhizobial cells were observed to perform
better than when they were used alone (Seneviratne and Jayasinghearachchi 2005).
Biofilms occur naturally in animals, plants and the environment. These bio-
filmed communities could be harmful/pathogenic or beneficial (Morikawa 2006).
Beneficial biofilms attached to the plant roots of some crops may help cycle
nutrients as well as biocontrol of pests and diseases and, consequently, improve
the productivity of crops. However, the density in the soil of such naturally
occurring beneficial biofilms is too low to have a significant effect. This was
reflected by the success of the microbial inoculation into a soil planted with rice
(G. Seneviratne, unpublished). Critical cell density-dependant quorum sensing is a
prerequisite for biofilm formation (Kong et al. 2006), which is frequently not
attainable in the soil solution under natural conditions. Further, the naturally
occurring biofilms are N-deficient for optimal action, which may be overcome by
incorporating N2 fixers to them (Seneviratne 2008). Therefore, the development of
such biofilms in vitro and their application as biofertilizers are essential for aug-
menting agricultural productivity (Bandara et al. 2006). This chapter highlights
the recent advances in research, focusing on the potential effect of the BBs on non
leguminous crops.
3 A Novel Biofertilizing Technique 53
3.2 Effects of Beneficial Microbial Biofilms
With the first in vitro development and observation of interactions between com-
mon nonmycorrhizal soil fungi (e.g., Penicillium spp.) and rhizobia forming the
biofilms (Seneviratne and Jayasinghearachchi 2003), a series of studies were con-
ducted to assess their potentials as microbial agents in plant growth. Microbes used
in this study were mainly N2-fixing bacteria and P-solubilizing fungi. When they
were cocultured in vitro, the bacteria attached and colonized on fungal mycelia to
form the biofilms, known as fungal–bacterial biofilm (FBB). When the bacterium is
a Rhizobium species, they are called fungal–rhizobial biofilm (FRB). It was
observed that the interaction in the FRB fixed N2 biologically, as revealed by
nitrogenase activity and N-accumulation, which was not observed when a rhizobial
strain was used alone as a monoculture (Jayasinghearachchi and Seneviratne
2004a). The rhizobial strain used here was Bradyrhizobium elkanii SEMIA 5019,
a soybean (Glycine max)-nodulating strain with a high N2-fixing capacity. A recent
study showed the enhanced release of organic acids and plant growth-promoting
substances by developed FBB/FRB, leading to an increase of 25% in dry matter
accumulation in early grown rice over the monocultured conventional inocula
(Seneviratne et al. 2008a). It was also observed that there was a significant negative
relationship between pH and indoleacetic acid-like substances (IAAS) production
in liquid culture media of the biofilms, but not in mixed cultures without biofilm
formation of a large collection of microbes (Seneviratne et al. 2008b). Thus, when
biofilms were formed, high acidity reflected high production of IAAS. The high
acidity is generally important for pathogen suppression. The biofilmed inocula can
also be used effectively to enhance biosolubilization of rock phosphate, due to high
acid production (Jayasinghearachchi and Seneviratne 2006a; Seneviratne and
Indrasena 2006). Moreover, the biofilmed inocula can be used for successful
establishment of introduced beneficial microorganisms in plants for biocontrol of
diseases. For instance, a Pleurotus ostreatus–Pseudomonas fluorescens biofilm
(FBB) increased endophytic colonization of tomato (Lycopersicon lycopersicum)
by P. fluorescens, a biocontroling agent, by over 1,000%, compared to inoculation
with P. fluorescens alone under in vitro conditions (Jayasinghearachchi and
Seneviratne 2006b).
3.3 Developing Beneficial Biofilms on Roots of Non legumes
It has now been clearly shown under axenic conditions that the monocultures of
Rhizobium sp. inoculated into nonleguminous plant roots develop biofilms on the
root surface (Santaella et al. 2008). Recent studies conducted under axenic condi-
tions demonstrated that the inoculation of the FRB helped maintain a higher cell
density of rhizobia on the root system of wheat than the inoculation of rhizobial
monocultures (Fig. 3.1). The monocultures developed clusters of rhizobial cells on
Fig. 3.1 Rhizobial biofilms (BF) developed on root hairs of wheat, when rhizobial monocultures
(a) or fungal–rhizobial biofilms (FRB) (b) was inoculated under axenic conditions. The inocula-
tion of the FRB helped maintain a higher cell density of rhizobia on the root hairs than the
inoculation of the monoculture
the root hairs whereas fungal mycelium of the FRB linked root hairs, which
provided support to maintain the higher cell density (Seneviratne et al. 2008b).
Thus, the FRB may act as nodule-like structures or “pseudonodules” capable of
fixing N2 biologically on roots of such non legumes, as reported earlier by Jayasin-
ghearachchi and Seneviratne (2004b). The FBB/FRB can also develop biofilms
Fig. 3.2 Root hairs of rice (a), tea (b) and Anthurium (c) colonized by microbial biofilms (BF),
when fungal–bacterial biofilms (FBB) or fungal–rhizobial biofilms (FRB) were inoculated
under axenic conditions. Darkness is due to cotton blue stain absorbed by the EPS produced
by the BF
on root hairs of other non legumes, when inoculated under axenic conditions. Root
hairs of rice, tea and Anthurium have been found heavily colonized with biofilm
formation, as evidenced by the EPS produced by the biofilms (Fig. 3.2). It was
reported recently that the adsorption of rhizobia to biotic surfaces like plant roots is
governed by the rhizobial adhesion protein RapA1 (Mongiardini et al. 2008). It can
therefore be speculated that this adhesion protein, which may also have contributed
to attachment and formation of the FBB/FRB, is common in many bacteria.
3.4 Effects of Biofilmed Biofertilizers on Plant

Growth and Yield
When the FBB/FRB formulated as the BBs were applied to the growing medium of
plants, they formed biofilms on the root system, as discussed earlier. These biofilms
have shown their positive effects on growth and yield of crops. For instance, in a
soil pot experiment, when rice was grown in the absence of chemical fertilizers, it
was found that the shoot growth of this crop improved when the number of
beneficial microbial species of the BBs was increased (Fig. 3.3). Conventional
biofertilizers with single microbial species or monocultures increased the shoot
biomass by only 7% compared to control. In contrast, the inoculation of BBs
containing three microbes FBB/FRB (two bacteria + one fungus) increased the
dry matter accumulation in shoots substantially by 25%. However, when a tripartite
culture of FBB/FRB was used with 50% of the recommended rates of chemical
fertilizers (urea 100 kg N ha 1, triple super phosphate 13 kg P ha 1 and muriate of
potash 28 kg K ha 1) for rice, plant biomass increased by ca. 55% compared to
those observed for 100% application of the recommended fertilizers alone
(Table 3.1). This was mainly attributed to increased root dry weight. Increasing
the recommended fertilizer level up to 100% with the BBs, however, did not
increase the plant growth. Both panicle formation and seed yield per hill were
comparable between the BBs + 50% of the recommended fertilizers and the 100%
of the recommended fertilizers alone (G. Seneviratne, unpublished).
In a nursery trial of tea with BBs of diazotrophic bacteria and/or recommended
chemical fertilizers (T65; sulfate of ammonia, mono-ammonium phosphate, sulfate
of potash and epsom salt; 15:20:15:15 (weight ratio) 80 kg per 10,000 plants during
nursery period), there was a positive correlation between net photosynthetic rate
and relative growth rate of leaf area (Fig. 3.4a). When two-bacterial BBs were
applied, there was a propensity of increasing photosynthesis compared to one-
bacterial BBs. Moderate application of 50% of the recommended fertilizers with
the two-bacterial BBs helped increase leaf growth compared to other microbial
fertilizer treatments and even 100% of the recommended fertilizer application. Soil
C after harvest of the tea plants of the nursery was positively related to shoot/root
ratio of the plants (Fig. 3.4b). The two-bacterial BBs with the moderate fertilizer
level showed the highest shoot/root ratio and the soil C, which could possibly be
due to increased growth of fine roots (Zavahir et al. 2008) and subsequent rapid
turnover. The application of recommended fertilizers alone resulted in a low shoot/
root ratio and soil C. The high shoot/root ratio with the two-bacterial BBs and the
moderate N application maintained a low transpiration rate (Fig. 3.4c). However,
the plants treated with the recommended fertilizers alone showed a high transpira-
tion rate, as reflected by early wilting when exposed to sunlight (Jayasekara et al.
2008). The application of BBs with the N fertilizer helped increase leaf N of tea due
to increased root growth (Fig. 3.4d). In a field experiment, young tea applied with
a
Shoot dry weight (g/plant) 0.4
0.35
0.3
0.25
Monocultures 2-Microbe 3-Microbe Soil alone
FBB/FRB FBB/FRB control
b 3
Plant dry weight (g/plant)
2.5
1.5
0.5
0
3-Microbe FBB/FRB 3-Microbe FBB/FRB Rec. fertilizer only
+ 50% Rec. fertilizer + Rec. fertilizer
c 18
16
14
12
10
8
6
4
2
0
Biofertilizer + 50% Rec. 100% Rec. chemical
chemical fertilizers fertilizers
Number of panicles per hill Seed weight (g/hill)
Fig. 3.3 Shoot growth of rice (a) in a soil pot experiment, when the number of beneficial microbial
species of the fungal–bacterial biofilms (FBB) and fungal–rhizobial biofilms (FRB), formulated and
applied as biofilmed biofertilizers (BBs) was increased, in comparison to microbial monocultures.
Total plant growth (b), when a three-microbe FBB/FRB was coupled with 50% or 100% of the
recommended chemical fertilizers, or 100% of the recommended fertilizers alone. Panicle forma-
tion and seed yield of rice (c), when the BBs were coupled with 50% of the recommended chemical
fertilizers or 100% of the recommended fertilizers alone. Vertical bars show standard error
Table 3.1 Dry matter accumulation in shoot, root and whole plant of rice grown in pot soil treated
with a tripartite FBB/FRB with 50% or 100% of recommended chemical fertilizer, or 100% of the
recommended fertilizer alone
Treatment Shoot dry Root dry Total dry
weight weight weight
(g per plant) (g per plant) (g per plant)
Tripartite FBB/FRB + 50% recommended 1.95a0.03 0.40a0.001 2.35a0.03
fertilizer
Tripartite FBB/FRB + 100% recommended 1.99a0.10 0.29b0.02 2.29ab0.12
fertilizer
100% recommended fertilizer alone 1.42a0.19 0.10c0.01 1.53b0.19
CV (%) 14.5 6.20 13.4
Mean SE. Values in each column followed by a same letter are not significantly different at 5%
probability level
four-microbe FRB (three bacteria + one fungus) alone produced a leaf weight of
506 kg ha 1 compared to 431 kg ha 1 with 100% of the recommended chemical
fertilizers alone, at the first tipping.
Anthurium plantlets treated with four-microbe FRB and 50% of recommended
chemical fertilizers in an inert particle medium showed a higher relative growth rate
of plant dry weight than that of the 100% of the recommended fertilizers alone,
in early growth (Fig. 3.5). The four-microbe FRB alone marginally supported the
plant growth, possibly due to low microbial biomass of the biofilm, in the absence
of the fertilizer nutrients. These findings suggested that the input of chemical
fertilizers can be reduced by 50% during the vegetative growth phase and possibly
for the entire crop of such non legumes, which could be a huge economic gain
in terms of fertilizer saving.
The BBs once applied to a root establish an association between the root and the
biofilm, as shown in the conceptual model presented in Fig. 3.6. The moderate
application of the chemical fertilizer nutrients helps increase the microbial biomass
of the biofilm, which in turn tends to increase the microbial efficiency or the
functionality, as the concentrations of the fertilizer nutrients, particularly N
depletes. The biofilm acts as a nodule-like structure or a pseudonodule-fixing N2.
This fixed N may be transferred to the root, and in return the root may supply carbon
sources to the biofilm, the processes of which need future investigations. The
release of organic acids by the biofilm helps suppress microbial pathogens (Browning
et al. 2006) as well as increase mineralization of soil nutrients in the rhizosphere
(Seneviratne and Jayasinghearachchi 2005). Moreover, plant growth hormones,
such as IAA produced by the biofilms (Bandara et al. 2006), should increase the
growth of roots and mycorrhizal fungi. In this manner, this association constitutes
an excellent metabolic cooperation that helps the healthy growth of the plant.
In addition, the BBs are also important in replenishing beneficial microbial com-
munities in deteriorated soils due to heavy use of chemical inputs and intensive
cropping (Seneviratne 2009).
a
Leaf area (RGR/wk) 0.011
0.01 r = 0.62 (p = 0.10) 2B+hN
0.009
0.008 +N
0.007 B+hN B+N
0.006 B–N 2B–N
2B+N
0.005
0.004 –B–N
0.003
0.002
2 3 4 5 6 7 8 9
Net photosynthetic rate (µmol/m2/s)
b 3.5
Shoot/root ratio
3 2B+hN
r = 0.97** 2B–N
2.5 B+hN
2B+N B+N
2 +N
1.5 -B–N
B–N
1
0.5 0.6 0.7 0.8 0.9 1
Soil C (%)
c 3.5
Shoot/root ratio
r = 0.69 (p = 0.06)
3 2B+hN
2.5 B+hN 2B–N

2B+N B+N
2 +N
1.5
–B–N
B–N
1
11 11.5 12 12.5 13 13.5 14 14.5
Transpiration rate (mM/cm2/s)
d 5.5
5 2B+N
4.5 r = 0.85*
Leaf N (%)
4 B+hN 2B+hN
3.5
3 +N
2.5 2B–N
–B–N B–N
2
1.5
14 16 18 20 22
Root lenght (cm/plant)
Fig. 3.4 Relationships among plant and soil parameters in a nursery trial of tea, when biofilmed
biofertilizers (BBs) of diazotrophic bacteria and/or recommended chemical fertilizers were
applied. Correlations between (a) net photosynthetic rate and relative growth rate of leaf area
(b) soil C after harvest of the plants and shoot/root ratio (c) shoot/root ratio and transpiration rate
and (d) leaf N and root growth. Treatments included no bacteria ( B), one (B) or two (2B)
bacteria, and half (hN) or full (N) recommendation of chemical fertilizers, or no fertilizers ( N)
0.07
Plant dry weight (RGR/wk)
0.06
0.05
0.04
0.03
0.02
0.01
0
50% Rec. 100% Rec. 4-Microbe FRB Control
fertilizer + 4- fertilizer alone
Microbe FRB
Fig. 3.5 Relative growth rate (RGR) of plant dry weight of Anthurium plantlets treated with a
four-microbe fungal–rhizobial biofilm (FRB) alone, four-microbe FRB and 50% of recommended
chemical fertilizers or 100% of the recommended fertilizers alone, in an inert particle medium
during early growth. Vertical bars show standard error
Conceptual Model: Root-Biofilm Association

N2
Excellent metabolic
Nodule-like structure
cooperation
Pathogens
IAA/H+
IAA/P....
Fig. 3.6 Conceptual model showing the association established between the root and the biofilm,
when the biofilmed biofertilizers (BBs) were applied to a root of a non legume
3.5 Conclusion
It is clear from various studies that the FBB/FRB when formulated as BBs and
applied to non legumes enhance the plant growth in the presence of even moderate
levels of chemical fertilizers. The biofilms with a higher number of beneficial
microbial species (higher order biofilms) increased the plant growth possibly due
to improved microbial activities. However, selection of combinations of microbes
possessing the highest efficiency, simultaneous biofertilizing and biocontroling
activities is a key factor in the preparation and formulation of BBs. Diverse forms
of the BBs can serve as a source to increase N2 fixation, promote nutrient uptake
and to manage plant diseases in different agro-ecosystems. Therefore, both labora-

tory and field trials are required to realize the full impact of this biotechnological
approach for sustainable crop production.
References
Bandara WMMS, Seneviratne G, Kulasooriya SA (2006) Interactions among endophytic bacteria

and fungi: effects and potentials. J Biosci 31:645–650
Browning M, Wallace DB, Dawson C, Alm SR, Amador JA (2006) Potential of butyric acid for
control of soil-borne fungal pathogens and nematodes affecting strawberries. Soil Biol Bio-
chem 38:401–404
Costerton JW, Geesy GG, Cheng KJ (1987) Bacterial biofilms in nature and disease. Annu Rev
Davey ME, O’toole GA (2000) Microbial biofilms: from ecology to molecular genetics. Microbiol
Mol Biol Rev 64:847–867
Harrison JJ, Turner RJ, Marques LLR, Ceri H (2005) Biofilms. Am Sci 93:508–515
Jayasekara APDA, Seneviratne G, De Silva MSDL, Jayasinghe LASP, Prematunga P (2008)
Preliminary investigations on the potential applications of biofilmed biofertilizers for tea
nurseries. In: Nainanayake NPAD, Everard JMDT (eds) Proceedings of the second symposium
on plantation crop research-export competitiveness through quality improvements. Coconut
Research Institute, Lunuwila, Sri Lanka, pp 170–175
Jayasinghearachchi HS, Seneviratne G (2004a) Can mushrooms fix atmosphericnitrogen? J Biosci
23:293–296
Jayasinghearachchi HS, Seneviratne G (2004b) A bradyrhizobial-Penicillium spp biofilm with
nitrogenase activity improves N2 fixing symbiosis of soybean. Biol Fertil Soil 40:432–434
Jayasinghearachchi HS, Seneviratne G (2006a) Fungal solubilization of rockphosphate is
enhanced by forming fungal-rhizobia biofilms. Soil Biol Biochem 38:405–408
Jayasinghearachchi HS, Seneviratne G (2006b) A mushroom-fungus helps improve endophytic
colonization of tomato by Pseudomonas fluorescence through biofilm formation. Res J Micro-
biol 1:83–89
Kong KF, Vuong C, Otto M (2006) Staphylococcus quorum sensing in biofilm formation and
infection. Int J Med Microbiol 296:133–139
Mongiardini EJ, Ausmees N, Pérez-Giménez J, Althabegoiti MJ, Quelas JI, López-Garcı́a SL,
Lodeiro AR (2008) The rhizobial adhesion protein RapA1 is involved in adsorption of rhizobia
to plant roots but not in nodulation. FEMS Microbiol Ecol 65:279–288
Morikawa M (2006) Beneficial biofilm formation by industrial bacteria Bacillus subtilis and
related species. J Biosci Bioeng 101:1–8
Romano JD, Kolter R (2005) Pseudomonas-Saccharomyces interactions: influence of fungal
metabolism on bacterial physiology and survival. J Bacteriol 187:940–948
Romanova YM, Smirnova TA, Andreev AL, Il’ina TS, Didenko LV, Gintsburg AL (2006)
Formation of biofilms as an example of the social behaviour of bacteria. Microbiology
75:481–485
Rudrappa T, Biedrzycki ML, Bais HP (2008) Causes and consequences of plant-associated
biofilms. FEMS Microbiol Ecol 64:153–166
Santaella C, Schue M, Berge O, Heulin T, Achouak W (2008) The exopolysaccharide of Rhizobium
sp. YAS34 is not necessary for biofilm formation on Arabidopsis thaliana and Brassica napus
roots but contributes to root colonization. Environ Microbiol 10:2150–2163
Seneviratne G (2003) Development of eco-friendly, beneficial microbial biofilms. Curr Sci
85:1395–1396
Seneviratne G (2008) Biological nitrogen fixation: potential biotechnological applications beyond

biofertilizers. Curr Sci 95:7
Seneviratne G (2009) Collapse of beneficial microbial communities and deterioration of soil
health: a cause for reduced crop productivity. Curr Sci 96:633
Seneviratne G, Indrasena IK (2006) Nitrogen fixation in lichens is important for improved rock
weathering. J Biosci 31:639–643
Seneviratne G, Jayasinghearachchi HS (2003) Mycelial colonization by Bradyrhizobia and
Azorhizobia. J Biosci 28:243–247
Seneviratne G, Jayasinghearachchi HS (2005) A rhizobial biofilm with nitrogenase activity alters
nutrient availability in a soil. Soil Biol Biochem 37:1975–1978
Seneviratne G, Zavahir JS, Bandara WMMS, Weerasekara MLMAW (2008a) Fungal-bacterial
biofilms: their development for novel biotechnological applications. World J Microbiol Bio-
technol 24:739–743
Seneviratne G, Kecskés ML, Kennedy IR (2008b) Biofilmed biofertilisers: novel inoculants for
efficient nutrient use in plants. In: Kennedy IR, Choudhury ATMA, Kecskés ML, Rose MT
(eds) Efficient nutrient use in rice production in Vietnam achieved using inoculant biofertili-
sers. Proceedings of a project (SMCN/2002/073) workshop held in Hanoi, Vietnam, 12–13
October 2007. ACIAR Proceedings No. 130, ACIAR, Canberra, pp 126–130
Stewart P, Costerton JW (2001) Antibiotic resistance of bacteria in biofilms. Lancet 358:135–138
Vandevivere P, Kirchman DL (1993) Attachment stimulates exopolysaccharide synthesis by a
bacterium. Appl Environ Microbiol 59:3280–3286
Zavahir JS, Jayasekara APDA, Seneviratne G, De Silva MSDL (2008) Potential application of
biofilms: a new approach for tea gardens. Tea Bull 20:1–6
Chapter 4
Role of 1-Aminocyclopropane-1-carboxylate
deaminase in Rhizobium–Legume Symbiosis
Javed Musarrat, Abdulaziz A Al Khedhairy, Saud Al-Arifi, and

Mohammad Saghir Khan
Abstract Rhizobia form symbiotic relationships with leguminous plants and con-
vert atmospheric nitrogen into ammonia which can be utilized by host legume
plants. Symbiotic nitrogen fixation is important not only for the production of
protein-rich legumes but also to improve the fertility of soils. Nodulation in the
roots of legumes is regarded as an initiating event in the onset of N2 fixation.
Inhibition of nodulation restricts the N2-fixing ability of legumes, which may occur
due to variety of reasons. For instance, ethylene, as a stress hormone, inhibits
nodulation in legumes. However, its action is naturally offset by an enzyme
1-aminocyclopropane-1-carboxylate (ACC) deaminase, produced by rhizobia.
This enzyme cleaves plant-produced ACC into ammonia and a-ketobutyrate there-
by lowering the ethylene level. As a consequence of decreased ethylene levels,
nodulation is increased. Moreover, the ACC deaminase-containing plant growth-
promoting rhizobacteria including symbiotic N2 fixers reduce the physiological
damage to plants caused by other environmental factors. In this chapter, the focus is
to understand how the rhizobial ACC deaminase lowers the ethylene level in
legumes and overcomes the inhibitory effects of ethylene on nodulation. The
strategy adopted by rhizobial species to promote nodulation by adjusting ethylene
levels could be exploited as an effective tool for legume improvement in different
agro-ecological niches.
J. Musarrat (*), A.A.Al Khedhairy and S. Al-Arifi

College of Science, King Saud University, Riyadh, SA
e-mail: musarratj1@yahoo.com
M.S. Khan
Department of Agricultural Microbiology, Faculty of Agricultural Sciences, Aligarh Muslim
University, Aligarh-202002, U.P., India

64 J. Musarrat et al.
4.1 Introduction
Microbial communities, in general, predominate in or around the plant root surfaces

compared to bulk soil, and play a crucial role in maintaining soil health and plant
development (Kloepper et al. 1980). This “rhizosphere effect” occurs primarily
owing to the release of as much as 40% of photosynthates (e.g., sugars, organic
acids and amino acids, etc.) from the plant roots (Lynch and Whipps 1991; Nelson
2004). The microorganisms use these exudates as nutrients and rapidly colonize the
roots of plants (VanLoon and Glick 2004). Root-colonizing bacteria commonly
referred to as “rhizobacteria” may remain confined to the root surface (rhizoplane),
or enter the root interior and behave as endophytes (Sturz et al. 2000). Such
rhizobacterial strains, including both free-living and symbiotic bacteria, capable
of facilitating plant growth after inoculation on to seeds or when already present in
soils, are called plant growth-promoting rhizobacteria (PGPR) (Kloepper et al.
1980; Zahir et al. 2004). Of these PGPR, soil bacteria belonging to various genera
of the order rhizobiales (collectively called rhizobia) invade legume roots in
nitrogen-limiting environments, leading to the formation of a highly specialized
organ, the root nodule, on compatible legume roots or stems, and converts atmo-
spheric N2 into NH3 and exports the fixed N to the host plants (Long 1989). Besides
promoting the growth of plants by providing N to plants, rhizobia also facilitate the
growth of plants by preventing the deleterious effects of one or more phytopatho-
genic organisms (e.g., Botrytis cinerea and Fusarium oxysporum ) by synthesizing
an array of antibiotics, such as phenazines (Krishnan et al. 2007) and phytohor-
mones like IAA (Mandal et al. 2007; Sridevi and Mallaiah 2007; Wani et al. 2007)
and siderophores (Storey et al. 2006; Wani et al. 2008) which can solubilize and
sequester iron and make it available to plants, and solubilize not easily available
forms of phosphorus (Abd-Alla 1994; Alikhani et al. 2006).
The gaseous hormone ethylene produced endogenously by plants has many
strong effects on plant development and has been shown to act as a secondary
signal in the induction of plant defenses (Ecker 1995). Ethylene has been involved
in many physiological processes, such as seed germination, tissue differentiation,
formation of root and shoot primordial, root elongation, lateral bud development,
flowering initiation, anthocyanin synthesis, flower opening and organ senescence,
fruit aroma and ripening, storage product hydrolysis, and leaf and fruit abscission
(Frankenberger and Arshad 1995; Spaink 1997). In addition, ethylene acts as a
messenger of biotic and abiotic stresses (Herder et al. 2006). It also plays a major
role in the beneficial rhizobial and arbuscular mycorrhizal symbioses (Guinel and
Geil 2002). Ethylene is produced and sensed in response to a wide variety of
environmental and developmental factors, including salinity, drought, water log-
ging, heavy metals, pathogenicity and nodulation (Abeles et al. 1992; Spaink 1997).
Despite acting as a plant growth regulator, ethylene at higher concentrations
inversely affects many physiological stages of plants including nodulation by
rhizobia in various legumes (Hirsch and Fang 1994; Oldroyd et al. 2001; VanLoon
et al. 2006). For instance, ethylene has been shown to inhibit nodule development in
4 Role of 1-Aminocyclopropane-1-carboxylate 65
alfalfa (Medicago sativa) (Ligero et al. 1991; Glick et al. 2007a) and pea (Pisum
sativum) (Guinel and Geil 2002; Cheng et al. 2008). However, Medicago truncatula,
a hypernodulating mutant sickle, is ethylene insensitive (Penmetsa and Cook
1997) and has shown altered auxin transport regulation during nodulation (Prayitno
et al. 2006).
Similar to the model proposed for free-living bacteria, the strains of N2-fixing
bacteria also synthesize a multimeric (homodimeric or homotrimeric) enzyme,
1-aminocyclopropane-1-carboxylate (ACC) deaminase (Glick 1995; Glick et al.
1999), which facilitates symbiosis by metabolizing the immediate biosynthetic
precursor (ACC) of ethylene (Glick et al. 1994; Mayak et al. 1999) into ammonia
and a-ketobutyrate (Glick et al. 1998). Hence, ACC deaminase decreases ethylene
levels in host plants (Ma et al. 2003a, 2004; Glick 2005) and consequently improves
the overall growth of plants (Glick et al. 2007b; Contesto et al. 2008). This enzyme
was first detected in Pseudomonas sp. strain ACP and the yeast, Hansenula
saturnus (Honma and Shimomura 1978; Honma 1993; Minami et al. 1998) and
since then, it has been reported in numerous other PGPR strains (Shaharoona et al.
2007; Naveed et al. 2008; Duan et al. 2008) including large number of symbiotic
N2-fixing bacteria, such as Rhizobium leguminosarum bv. viciae, R. hedysari,
Rhizobium spp., Mesorhizobium and Bradyrhizobium (Belimov et al. 2005; Hontzeas
et al. 2005; Blaha et al. 2006; Bajgiran et al. 2008). When the bacteria posses-
sing ACC deaminase are bound to the seed coat of a developing seedling, they
act as a sink for ACC ensuring that the ethylene level does not increase to the
point where root growth is impaired. By facilitating the formation of longer
roots, these bacteria may enhance the survival of some seedlings, especially
during the first few days after the seeds are planted. Plants treated with ACC
deaminase-containing bacteria have longer roots (Hall et al. 1996; Shah et al. 1998)
and help plants to better resist the inhibitory effects of stress ethylene on growth
imposed by heavy metals (Reed and Glick 2005; Safronova et al. 2006; Zhang et al.
2008), pathogens (Wang et al. 2000; Belimov et al. 2007), drought (Zahir et al. 2008;
Arshad et al. 2008), flooding (Grichko and Glick 2001; Farwell et al. 2007), and high
salt (Cheng et al. 2007; Yang et al. 2008; Jalili et al. 2008). Thus, understanding the
role of rhizobial ACC deaminase in reducing the effect of ethylene would help to
develop the rhizobial inoculant containing ACC deaminase activity that could
become useful in sustaining growth and development of legumes under stress
conditions by reducing stress-induced ethylene production.
4.2 Rhizobia–Legume Symbiosis: An Overview
The interaction between rhizobia and their corresponding specific legume host plants
leading to nodule formation is a complex process that requires a continuous and
adequate signal exchange between the plant and the bacteria (Perret et al. 2000;
Bartsev et al. 2004). The symbiotic associates show a high degree of mutual specificity
which is often mediated by the exchange of signal compounds (Long and Staskawicz
1993; Spaink 1997). During this interaction, rhizobia are attracted by root exudates
and colonize plant root surfaces. Flavonoids, the plant signal compounds present in
the exudates, trigger the transcription of bacterial nodulation (nod) genes leading
thereby to the synthesis of lipochito-oligosaccharide signals called Nod factors
(Perret et al. 2000). These signal compounds in turn cause the legume root hairs
to curl. Nod factors together with additional microbial signals, such as polysac-
charides and secreted proteins, allow bacteria attached to root hairs to penetrate
the root through a tubular structure called the infection thread, through which the
rhizobia enter, move into root hair, and subsequently reach to the dividing cortical
cells. When the thread reaches the primordium, the bacteria are released into the
plant cytoplasm, where they differentiate into endosymbiotic form, the N2-fixing
bacteroids. Inside the central nodule cells, rhizobia are housed as symbiosome
that are horizontally acquired organelles and are involved in the enzymatic
reduction of atmospheric nitrogen to ammonia and make this N accessible to
their hosts. In return, the bacteria are supplied with carbohydrates in a protected
environment. The host plant, however, regulates the number of nodules formed,
the maturation of nodules, and the N2 fixation of the nodules dependent upon
available nitrogen.
Despite substantial progress in biological sciences, the mechanisms regulating
legume root nodule development are still poorly understood, and very few regu-
latory genes have been cloned and characterized. For instance, EFD (ethylene
response factor, ERF, required for nodule differentiation), a gene that is upregulated
during nodulation in M. truncatula, has recently been characterized (Vernié et al.
2008). The EFD transcription factor belongs to the ERF group V, which contains
ERN1, 2, and 3, three ERFs involved in Nod factor signaling. The role of EFD in
the regulation of nodulation has been examined through the characterization of a
null deletion mutant (efd-1), RNA interference, and overexpression studies (Vernié
et al. 2008). EFD is a negative regulator of root nodulation and infection by
Rhizobium. It is required for the formation of functional nitrogen-fixing nodules
and affects the plant and bacteroid differentiation processes occurring beneath the
nodule meristem. Furthermore, EFD also activate a cytokinin primary response
gene Mt RR4 that encodes a type-A response regulator. Induction of Mt RR4 leads
to the inhibition of cytokinin signaling with the suppression of new nodule initiation
and activation of differentiation as cells leave the nodule meristem. Thus, a key
regulator linking early and late stages of nodulation and the regulation of the
cytokinin pathway are important both for nodule initiation and development.
Rhizobia, in general, produce both indeterminate and determinate types of
nodules. Indeterminate nodules are characterized by different zones: (1) the distal
meristem, where bacteria are internalized, (2) an interzone with amyloplast accu-
mulation and differentiation of bacteroids, and (3) a fixation zone that includes
plant cells and a senescent zone (Pawlowski and Bisseling 1996; Timmers et al.
1999; Jeroen et al. 2006). In comparison, determinate nodules are typically round
shaped and are derived from the cessation of meristem activity after nodule initia-
tion and growth of the nodule mainly by cell expansion (Jeroen et al. 2006).
4.3 Effect of Ethylene on Symbiosis
Legumes form a mutualistic symbiosis with rhizobia. Although the interaction is

beneficial to the plant, the symbiosis is tightly regulated. The gaseous plant
hormone ethylene has been found to be involved in many physiological events
including regulation of symbiotic process in legumes. For instance, many plants
require a burst of ethylene to break seed dormancy (Esashi and Esashi 1991) but,
following germination, a sustained high level of ethylene may inhibit root elonga-
tion (Jackson 1991). It is reported that root growth is stimulated at low concentra-
tions of ethylene but is markedly decreased at higher concentrations. Another
interesting response of plant species to ethylene is the massive production of root
hairs. Ethylene may also modify shoot growth (Bajgiran et al. 2008) and nodule
formation in legumes by rhizobia (Table 4.1). However, the mechanism by which
ethylene inhibits nodulation is unclear, and the position at which it acts during the
Table 4.1 Effect of ethylene on Rhizobium–legume symbiosis

Legume Host specific rhizobia Effect on nodulation References
Medicago Sinorhizobium Initiation of infection Oldroyd et al.
truncatula meliloti threads, Inhibits the (2001)
Nod factor signal
transduction
pathway, Inhibits
the maintenance of
calcium spiking
Glycine max cv. Bradyrhizobium Decrease in nodule Xie et al. (1996)
Gong jiao japonicum number
6301-1
Pisum sativum cv. Rhizobium Decrease in nodule Lee and LaRue
Rondo leguminosarum number (1992)
bv. viciae
Melilotus alba S. meliloti Decrease in nodule Lee and LaRue
U389 number (1992)
Pisum sativum cv. R. leguminosarum Decrease in nodule Lee and LaRue
Sparkle bv. viciae number; blockage (1992)
of infection thread
elongation in inner
cortex
Pisum sativum cv. R. leguminosarum Inhibition of root Goodlass and
Feltham First bv. viciae extension, decrease Smith (1979)
in nodule numbers
and nitrogen fixation
Trifolium repense R. trifoli Decrease in nodule Goodlass and
cv.Huia number and nitrogen Smith (1979)
fixation
Phaseolus vulgaris Rhizobium sp. Decrease in nodule Grobbelaar et al.
var. Pencil number and nitrogen (1971)
Podded Black fixation
Wax
Modified from Okazaki et al. (2004)
complex symbiotic process is inconclusive. For example, inoculation with sinor-

hizobia and bradyrhizobia enhanced ethylene production in the roots of alfalfa
and soybean (Glycine max) plants (Ligero et al. 1986, 1991; Suganuma et al.
1995). In contrast, there are several reports that suggest that exogenously applied
ethylene inhibits nodulation in legumes by blocking cortical cell division and
reducing the number of cortical infections (Lee and LaRue 1992; Spaink 1997).
The inhibition of nodule formation could be a result of developmental effects that
ethylene exerts on the plant. For example, ethylene is required for the elongation
of root hairs (Tanimoto et al. 1995; Pitts et al. 1998); a function clearly involved
in the invasion of rhizobia, but simultaneously inhibits cell division (Goodlass
and Smith 1979), a function that would be detrimental to nodule formation.
However, a report suggests that the induction of root hair growth by nod factors
occurs independently of ethylene (Heidstra et al. 1997). Moreover, the synthesis
of endogenous ethylene in legume (e.g., pea) controls the position of nodule
primodia formation (Heidstra et al. 1997). Additionally, the exogenously applied
ethylene inhibits infection thread elongation in inner cortex and root growth (Xie
et al. 1996), and also reduces nodule number (Peters and Crist-Estes 1989; Lee
and LaRue 1992) and the morphology of nodules (Fernandez-Lopez et al. 1998)
formed on the root systems and, consequently, nitrogen fixation. The production
of ethylene in legume roots has been found to increase after application of
rhizobial cells (Ligero et al. 1986), nitrate (Ligero et al. 1986) and nod factor
(van Spronsen et al. 1995), suggesting that these factors control nodulation
through their effects on the levels of ethylene. For example, the fate of rhizobial
infection in the root hairs of legumes has been proposed to be regulated by the
levels of ethylene in the underlying plant cortex (Guinel and Geil 2002); a low
level of ethylene, allowing proper disposition of the cytoskeleton, is probably
required for successful entry of the infection thread in the outermost layer of
cortical cells, whereas higher levels of the hormone induce abortion of the
infection thread by inducing crosslinking of its matrix glycoproteins. This hypo-
thesis is substantiated by much evidence, for instance, sickle, an ethylene-
insensitive mutant of barrel medic, has a very high persistence of infection threads
(Penmetsa and Cook 1997), while brz, a potential ethylene-oversensitive mutant
of pea, had much higher numbers of aborted infection threads than those observed
for wild-type plants. Ethylene effect has commonly been reported in legumes that
form indeterminate nodules. It has been shown to inhibit nodule development
in pea (Guinel and Sloetjes 2000; Guinel and Geil 2002), Trifolium repens
(Goodlass and Smith 1979), and M. sativa (Peters and Crist-Estes 1989). On the
other hand, the nodulation response of determinate nodules to ethylene is variable
and species-dependent. For example, ethylene inhibited nodulation in Lotus
japonicus and Macroptilium atropurpureum (Nukui et al. 2000) but failed to
alter nodulation in soybean (Schmidt et al. 1999; Suganuma et al. 1995), although
the number of nodules on soybean cv. Bragg was reduced by ethylene. Also,
Sesbania rostrata is less sensitive to ethylene because exogenous ACC or CEPA
(2-chloroethylphosphonic acid) did not negatively influence root nodulation
(Xie et al. 1996). In S. rostrata roots, differential ethylene concentrations may
determine the fate of nodule development by influencing the persistence of the

nodule meristem (Fernandez-Lopez et al. 1998). Differential ethylene concentra-
tions could be caused by mechanisms interfering with ethylene production,
diffusion, or perception. Ethylene, thus, (1) regulates the formation of the infec-
tion thread, (2) regulates the maintenance of the nodule meristems, as reported in
S. rostrata, and (3) inhibits the maintenance of calcium spiking after induction by
nod factor (Oldroyd et al. 2001).
However, when ethylene pressure is removed from plants, a substantial increase
in nodule formation occurred in M. trunculata (Oldroyd et al. 2001), M. sativa
(Nukui et al. 2000), L. japonicus (Bras et al. 2000), pea (Lorteau et al. 2001), and
M. atropurpureum (Nukui et al. 2000). Since legume root nodulation is dependent
on the density of Rhizobium in the root environment (Kucey and Hynes 1989), it is,
therefore, possible that ethylene controls nodulation by also limiting rhizobial
multiplication. To validate this hypothesis, Tamimi and Timko (2003) suggested
that ethylene could modulate an early step in the Nod factor signal transduction
pathway indicating that the effect of ethylene on nodulation is not limited to the
host plant but might also affect the bacterial symbiont.
4.4 Rhizobial Strategies for Decreasing Ethylene Levels
Legume production depends heavily on biologically fixed nitrogen (BNF) derived

from the symbiotic interactions between rhizobia and its specific host plants.
Despite a phytohormone, ethylene acts as a negative factor in the nodulation
process. Conversely, nodulation can be promoted when plants are treated with
ethylene inhibitors or antagonists (Peters and Crist-Estes 1989; Yuhashi et al.
2000). Interestingly, recent discoveries suggest that rhizobia employ several stra-
tegies to reduce the amount of ethylene and consequently its inhibitory effect on
nodulation. In this context, two potential strategies have been reported for lowering
the ethylene effect. One strategy involves an ethylene biosynthesis inhibitor, called
rhizobiotoxine, an enol–ether amino acid [2-amino-4-(2-amino-3-hydroxypro-
poxy)- trans-3-butenoic acid] produced by rhizobia (Okazaki et al. 2007) that
inhibits ACC synthase while the other involves an enzyme, ACC deaminase,
produced by the symbiotic nitrogen-fixing bacteria (Duan et al. 2008; Bajgiran
et al. 2008). Of these, rhizobiotoxine are secreted outside rhizobial cells probably
by a rhizobiotoxine transporter and delivered to plants. Although rhizobiotoxin is
best known for its chlorosis-inducing activity on leguminous plants (Owens and
Wright 1965; Owens 1973), the recent studies have shown that rhizobiotoxine plays
a positive role in nodule development through its inhibition of ethylene biosynthe-
sis. The positive and necessary role of rhizobiotoxine in mutualistic symbiosis was
demonstrated by Duodu et al. (1999) who suggested that rhizobiotoxine is required
for Bradyrhizobium elkanii for efficient nodulation in greengram (Vigna radiata L.
Wilczek). Since then, the role of rhizobiotoxine in symbiosis improvement has
been reported for other legumes (Yuhashi et al 2000; Sugawara et al. 2006).
The cumulative evidence suggests that rhizobitoxine-producing bacteria modu-

late plant–microbe interactions via ethylene in the rhizosphere and phyllosphere
environments. In addition, rhizobitoxine-producing capability might be utilized
as tools in agriculture and biotechnology (Sugawara et al. 2006). The reduction in
ethylene levels by synthesizing rhizobiotoxin is, however, limited to slow grow-
ing rhizobial species. Therefore, how fast growing rhizobia alleviate the toxicity
of ethylene needs to be addressed since they could enhance nodulation as well.
Hence, an alternative strategy for reduction in the effects of ethylene on plants
involves ACC deaminase. The extent of ACC deaminase in rhizobial strains
varies greatly. For example, out of the total 33 strains of rhizobia collected
from 30 different sites across Saskatchewan, Canada, only 11% had ACC deami-
nase activity (Duan et al. 2008), while in another study, around 38% Rhizobium
spp. displayed ACC activity (Ma et al. 2003a).
Rhizobia capable of producing ACC deaminase are able to reduce ethylene
concentration in the plant cells in the immediate vicinity of the infection threads.
Therefore, the infection threads containing ACC deaminase-producing rhizobial
cells can better suppress the defense signals in the plant cell and increase the
persistence of the infection threads. This leads to higher nodule numbers as
reported in plants inoculated with S. meliloti Rm11466 as well as a higher portion
of nodules formed by Rm11466 when the plants were coinoculated with both
S. meliloti Rm5356 and Rm11466. In addition, since a high percentage of
infection threads abort before a nodule is formed, infection threads containing
Rm11466 could suppress the development of those containing Rm5356 by reach-
ing nodule primordia and forming nodules faster. This also accounts for the
higher competitiveness of Rm11466 (Ma et al. 2004). Gage et al. (1996) observed
that S. meliloti proliferates inside the infection threads of alfalfa. The population
of bacteria inside the threads originates from the clonal expansion of a small
number of founder cells which enter the threads early, and only the bacterial cells
of the threads will eventually be released into the cytoplasm of the plant cell as
reported by Ma et al. (2004). It is also suggested that S. meliloti cells travel down
the infection threads by proliferation instead of swimming, since bacteria do
not have flagella when inside the threads. However, what nutrient source(s)
S. meliloti cells utilize to proliferate in the infection threads is not known.
Although the ACC concentrations in the infection threads are expected to be
low, the possible ability of the ACC deaminase-producing cells to utilize ACC
as an extra nutrient source makes the bacterium proliferate better in the infec-
tion threads than those that do not have this enzyme. Therefore, infecting cells
that produce ACC deaminase are more likely to reach nodule primordia and
form mature nodules, which results in the higher competitiveness of S. meliloti
Rm11466 than of Rm5356 .
In order to explain how ACC deaminase-containing PGPR can lower plant
ethylene levels and in turn stimulate plant growth, a model (Fig. 4.1) similar to
those originally proposed by Glick et al. (1998) has also been reported for reducing
the levels of ethylene in legumes by rhizobia (Ma et al. 2003a, 2004). According to
this model, the reduction in the concentration of ethylene by ACC deaminase
Fig. 4.1 Schematic representation of mechanisms through which the PGPR attached to either a
seed or plant root lowers ethylene concentration and prevents ethylene inhibition of root elon-
gation. The (⊥) indicates inhibition. IAA indicates indoleacetic acid, ACC 1-aminocyclopropane-
1- carboxylic acid and SAM, S-adenosyl-methionine (adapted from Glick et al. 1998)
involves: (1) binding or colonization of organisms onto the surface of either the
seed or root of a developing plant; (2) synthesis and release of IAA by bacterial
strains in response to tryptophan and other small molecules present in the seed or
root exudates; some of IAA secreted could be used by the plants; the IAA so
released, together with endogenous plant IAA, stimulate plant cell proliferation and
elongation, or it can also induce the activity of ACC synthase to produce ACC; (3)
exudation of the plant’s ACC along with other small molecules such as sugars,
organic acids and amino acids (Penrose and Glick 2001); (4) uptake of exudates by
the bacteria which is used as a source of nutrients; and (5) cleavage of ACC by ACC
deaminase forming ammonia and a-ketobutyrate, which are further metabolized by
the bacteria. Thus, the uptake and cleavage of ACC by ACC deaminase containing
N2 fixers decreases the amount of ACC and thereby acts as a sink for it. In turn, the
decreased level of ACC lowers the levels of ethylene and alleviates its potential
inhibitory effect and consequently help plants to grow better under ethylene
stressed environment.
4.5 Genes Involved in the Synthesis of ACC Deaminase

and Nodulation Enhancement
1-amino cyclopropane-1-carboxylic acid deaminase (AcdS) is an enzyme that

degrades the precursor of plant hormone ethylene. The AcdS activity has been
identified in many soil bacteria (Hameeda et al. 2006; Cheng et al. 2007) including
rhizobia, which is reported to play an important role in plant growth promotion.
Genes encoding ACC deaminase have been reported in PGPR (Venkadasamy et al.
2008) including B. japonicum (Kaneko et al. 2002), Mesorhizobium loti (Kaneko
et al. 2000; Sullivan et al. 2002; Uchiumi et al. 2004), R. leguminosarum and
R. gallicum (Ma et al. 2003a; Duan et al. 2008) and R. radiobacter. The ACC
deaminase genes from R. leguminosarum bv. viciae 128C53K and 99A1 showed
64% similarities with the gene reported in plant growth-promoting bacterium,
Pseudomonas putida UW4 (Shah et al. 1998). However, all these rhizobial strains
had relatively low ACC deaminase activities compared to that expressed by P.
putida UW4. Furthermore, the regulation study of ACC deaminase in strain
128C53K revealed its very low basal level of expression in the absence of ACC,
which could be induced by ACC concentrations as low as 1mM (Ma et al. 2003a),
suggesting that ACC deaminase in strain 128C53K could lower the ethylene
concentration in pea roots as a consequence of nodulation (Ma et al. 2003a). In
fact, an ACC deaminase knockdown strain 128C53K was approximately 30% less
efficient at nodulating pea plant roots than the wild-type. In contrast, introduction of
the ACC deaminase gene and its upstream regulatory gene, a leucine responsive
regulatory protein (LRP)-like gene (acdR) from strain 128C53K, into a strain of
S. meliloti, which does not produce this enzyme, made it 35–40% more effective
and competitive than the wild-type at nodulating M. sativa (Ma et al. 2004). ACC
deaminase genes in M. loti strains MAFF303099 and R7A are preceded by a nifA-
dependent (Owens et al. 1972) promoter and thus appear to be expressed only in the
symbiotic state (Sullivan et al. 2002; Uchiumi et al. 2004). Indeed, the ACC
deaminase genes of M. loti strains MAFF303099 were drastically upregulated
in bacteroids compared with the level of its transcription in free-living cells
(Uchiumi et al. 2004). In contrast, ACC deaminase genes in B. japonicum and
R. leguminosarum possess a s promoter (Peters and Crist-Estes 1989) with an LRP
(leucine responsible regulatory protein) box (Kaneko et al. 2002). Since the pro-
moter structure of s/LRP was involved in induction of ACC deaminase gene in
R. leguminosarum and E. cloacae (Ma et al. 2003a, b), ACC is probably an inducer
of the gene in B. japonicum chromosome. The proteome analysis of B. japonicum
bacteroids has shown that ACC deaminase is one of the major proteins in bacteroids
rather than the free-living cell, suggesting that ACC in host plants might induce
the ACC deaminase in rhizobia (Hoa et al. 2004).
An important question is how a decreased level of ethylene enhances nodulation.
To address this issue, several models have been proposed depicting the relationship
between signal transduction, ethylene sensing and the development of nodula-
tion (Stearns and Glick 2003). Thus, two strategies can be adopted: (1) construct
transgenic legumes with altered ethylene sensitivities; the expression of ethylene
receptors that cannot bind ethylene confers reduced ethylene sensitivity to heterol-
ogous plants in a genetically dominant manner (Bleecker 1999); and (2) engineer
rhizobia to enhance nodulation competitiveness on host legumes rather than by
single inoculation (Okazaki et al. 2003; Uchiumi et al. 2004). Nodulation competi-
tiveness, the ability of certain rhizobial strain to form nodules in a multistrain
environment, has practical importance in agriculture due to the fact that super-
nodulating strains often perform poorly under field conditions due to better
competing populations of inferior rhizobia in soils. It is, therefore, possible that
engineering rhizobia to exhibit high ACC deaminase activity is likely to delay the
nodule senescence by reducing ethylene production, and thereby promoting nodule
development and nitrogen fixation. Accordingly, nodules with high bacteroid ACC
deaminase activity are expected to grow larger and contain more bacteroids that
remain active for a longer period of time. For example, the ACC deaminase of
R. leguminosarum bv. viciae 128C53K has been previously reported to be able to
enhance nodulation of peas. The ACC deaminase structural gene (acdS) and its
upstream regulatory gene, a leucine-responsive regulatory protein (LRP)-like gene
(lrpL) from R. leguminosarum bv. viciae 128C53K, were introduced into
S. meliloti, which does not produce this enzyme, in two different ways: through a
plasmid vector and by in situ transposon replacement. The resulting ACC deami-
nase-producing transformed strain showed 35–40% increase in both nodule
numbers and biomass in alfalfa (M. sativa) plants, likely by reducing ethylene
production in the host plants. Furthermore, the genetically modified ACC deami-
nase-producing S. meliloti strain was more competitive in nodulation than the wild-
type strain, and perhaps utilizes ACC as a nutrient within the infection threads (Ma
et al. 2004). Thus, even if some rhizobial strains lack the ability to decrease
ethylene levels in host legumes, the introduction of genes coding for ACC deami-
nase into these rhizobia may enhance the symbiotic interactions with the cognate
host legumes and eventually reduce the ethylene level. This strategy of ethylene
reduction in legumes is an interesting area of research that could lead to facilitating
the productivity of legumes under stressed conditions. However, how the ACC
deaminase activity affects nodulation competitiveness is unclear. The plausible
explanation is that the ACC deaminase blocks ethylene biosynthesis locally at
the infection sites and cancels the effects of ethylene on nodulation during the
mitogenic process (Okazaki et al. 2004). Therefore, it has been suggested that the
rhizobia employ more than one strategies, i.e., the ACC deaminase and rhizobio-
toxine synthesis to reduce the extent of ethylene biosynthesis by the host
legumes and enhance nodule formation.
4.6 Performance of ACC Deaminase Containing Transgenic

Plants/Rhizobia
The exciting advances in molecular engineering facilitated the genetic manipula-

tion of organisms for achieving a defined set of goals and objectives. An approach
has been extended to understand the mechanism of reduction of ethylene levels in
plants through genetic manipulation in genetically engineered legumes or rhizobia,
for overexpression of ACC deaminase. Introduction of genes responsible for
expression of ACC deaminase in plants has received more attention than rhizobia
due to several reasons such as: (1) the ACC deaminase activity is widely reported
among autochthonous PGPR species including rhizobia; (2) the degree of success in
transforming the ACC deaminase genes into plants has been more successful; and
(3) engineered organisms after inoculation face severe competition from indige-
nous ones, and therefore the chances of survival and proliferation of genetically
modified transgenic bacteria in natural soil environments is quite often reduced.
Because of all these factors, the plant molecular biologists opt for developing
transgenic plants that express the ACC deaminase genes.
Many plant species have been genetically engineered with ACC deaminase
expression to protect them against multiple biotic and abiotic stresses. For example,
Grichko et al. (2000) expressed bacterial ACC deaminase in tomato (Lycopersicum
esculentum) cv. Heinz 902 under the transcriptional control of two tandem 35S
cauliflower mosaic virus promoters (constitutive expression), the rolD promoter
from Agrobacterium rhizogenes (root-specific expression) or the pathogenesis-
related prb-1b promoter from tobacco. Transgenic tomato plants expressing ACC
deaminase particularly controlled by the prb-1b promoter accumulate larger
amounts of metals within the plant tissues. However, because the tomato plants
are unlikely to be used in the phytoremediation of contaminated sites, Nie et al.
(2002) expressed ACC deaminase genes in canola (Brassica napus) plants and
tested their potential to grow in the presence of high levels of arsenate in the soil for
metal accumulation in plant tissues. Also, the ability of the plant growth-promoting
bacterium E. cloacae CAL2 has been tested to facilitate the growth of both non-
transformed and ACC deaminase-expressing canola plants for developing a suc-
cessful phytoremediation strategy. In all cases, transgenic canola expressing ACC
deaminase genes accumulated larger amounts of arsenate from the contaminated
soil than nontransformed canola plants. Similarly, the expression of Pseudomonas
sp. 6G5 acdS gene under the control of a 35S promoter in tomato plants led to
decreased ethylene synthesis and delayed fruit ripening (Klee et al. 1991). Such
heterologous expression studies provide a functional demonstration of the role of
AcdS to lower ethylene level in plants. In addition, these transgenic plants showed
higher tolerance to flooding, heavy metal stress and pathogen attack (Klee et al.
1991; Klee 1993; Stearns and Glick 2003), as expected from the involvement of
ethylene in stress responses.
Ethylene inhibits the establishment of symbiosis between rhizobia and legumes.
To examine how and when endogenous ethylene inhibits rhizobial infection and
nodulation, transgenic, L. japonicus carrying the mutated melon ethylene receptor

gene Cm-ERS1/H70A that confers ethylene insensitivity and fixes the transgene in
the T3 generation were produced. The resultant transgenic plants showed reduced
ethylene sensitivity because of ACC resistance and increased flowering duration,
probably due to a dominant negative mechanism. When inoculated with M. loti,
transgenic plants showed markedly higher numbers of infection threads and nodule
primordia on their roots than did either wild-type or azygous plants during the early
stage of cultivation period as well as during later stages, when the number of mature
nodules had reached a steady state. In addition, transcripts of NIN, a gene governing
infection thread formation, increased in the inoculated transgenic plants as com-
pared to the wild-type plants. The infection responses of transgenic plants were
similar to those of wild-type plants treated with ethylene inhibitors. These results
suggest that the endogenous ethylene in L. japonicus roots inhibits rhizobial
infection at the primary nodulation, probably via NIN gene, and suggest that
ethylene perception assists negative feedback regulation of secondary nodule
initiation (Nukui et al. 2004). Furthermore, in order to examine the regulation of
the acdS gene encoding ACC deaminase in M. loti MAFF303099 during symbiosis
with the host legume L. japonicus, Nukui et al. (2006) introduced the b-glucuronidase
(GUS) gene into acdS so that GUS was expressed under control of the acdS
promoter. The histochemical GUS assay demonstrated the exclusive expression
of acdS in mature root nodules. Two homologous nifA genes, mll5857 and
mll5837, were found in the symbiosis island of M. loti and were designated
nifA1 and nifA2, respectively. Quantitative reverse transcription-PCR demon-
strated that nifA2 disruption resulted in considerably diminished expression of
acdS, nifH, and nifA1 in bacteroid cells. In contrast, nifA1 disruption slightly
enhanced the expression of acdS transcripts and suppressed nifH to some extent.
These observations indicate that the acdS gene and other symbiotic genes are
positively regulated by the NifA2 protein, but not by the NifA1 protein, in
M. loti. The mode of gene expression suggests that M. loti acdS participates in
the establishment and/or maintenance of mature nodules by interfering with
the production of ethylene, which induces negative regulation of nodulation
(Nukui et al. 2006). Recently, the role of ACC deaminase of symbionts in
nodulation and growth of Leucaena leucocephala has been studied (Tittabutr
et al. 2008). The acdS genes encoding ACC deaminase were cloned from
Rhizobium sp. strain TAL1145 and Sinorhizobium sp. BL3 in multicopy plasmids,
and transferred to TAL1145. The BL3-acdS gene greatly enhanced ACC deaminase
activity in TAL1145 compared to the native acdS gene. The transconjugants
of TAL1145 containing the native or BL3 acdS gene could grow in minimal
media containing 1.5 mM ACC, whereas BL3 could tolerate up to 3 mM ACC.
The TAL1145 acdS gene is inducible by mimosine and not by ACC, while the
BL3 acdS gene was highly inducible by ACC and not by mimosine. The
transconjugants of TAL1145 containing the native- and BL3-acdS genes formed
nodules in greater numbers and sizes, and produced higher root mass on
L. leucocephala than did TAL1145. Introduction of multiple copies of the
acdS gene increased ACC deaminase activities of TAL1145 and enhanced its
symbiotic efficiency on L. leucocephala (Tittabutr et al. 2008). The acdS::gus

fusions in strains TAL1145:pUHR353::gus expressed in Leucaena nodules,
although at low levels., which suggests the possibility of increasing ACC deaminase
activity in the nodule through multiple copies of acdS in the symbiont. Thus, the
inoculation with TAL1145 carrying multiple copies of acdS could increase nodule
number, nodule dry weight and root dry weight of Leucaena seedlings after 16
weeks. The increases in nodule number and nodule dry weight due to inoculation
with TAL1145:pUHR353 and TAL1145:pUHR354 were detectable even when the
plants were harvested and analyzed after 8 weeks. Larger differences in the root dry
weights due to high ACC deaminase activity in the symbionts may be observed
when the plants are grown for longer periods of time under field conditions. It is
difficult to assess the effects of increased ACC deaminase activities on nodule
senescence and maintenance using Leucaena, which forms indeterminate type of
nodules that continue to grow longitudinally producing new meristematic and
bacteroid zones. The effects of increased symbiotic ACC deaminase activities
may be more visible on plants like beans and soybeans, which produce determinate
type of nodules that senesce entirely after a fixed period of nitrogen fixation. It is
expected that increased ACC deaminase activities may delay senescence and pro-
long the nitrogen-fixing period in such nodules, leading to increased growth
and yield of the plants. Thus, enhanced ACC deaminase activities in Rhizobium
bacteroids inside Leucaena nodules reduce ethylene biosynthesis and consequently
promote nodule development. Reducing ethylene synthesis may also help in nodule
maintenance by delaying senescence. These properties, together with enhanced
ACC deaminase activities in the nodule, are likely to result in increased N2 fixation
and higher growth of the plant. Besides synthesizing ACC deaminase, rhizobia
also facilitate legume growth by providing the plants with other benefits such
as siderophores (Wani et al. 2008), phytohormones (Wani et al. 2007; Ahmad
et al. 2008; Bajgiran et al. 2008), P solubilization (Sridevi et al. 2007), and metal
detoxification (Wani et al. 2007, 2008). Thus, by genetically engineering rhizobial
strains to produce ACC deaminase, there is a real possibility of obtaining better
strains for use as field inocula to increase the yield of leguminous crops.
4.7 Conclusion
Symbiotic nitrogen fixation process plays a significant role in improving the soil
fertility and productivity of low-N soils. Hence, the symbiosis can relieve the
requirements for added nitrogenous fertilizer during the growth of leguminous
crops. Symbiotic nitrogen fixers in addition to nitrogen fixation, utilize a variety
of mechanisms, both direct and indirect, to stimulate the growth of plants and/or to
compete in nodulation. Ethylene is a gaseous phytohormone produced by the plants
including legumes during normal growth conditions. However, under biotic and
abiotic stresses, the synthesis of ethylene increases and adversely affects the
legume–Rhizobium symbiosis. The rhizobia possessed with ACC deaminase
activity lowers the level of ethylene and consequently improves the overall perfor-
mance of legumes. Interestingly, the rhizobia that do not contain ACC deaminase
are unable to nodulate their cognate legumes to the same extent that they might be
able to if they possessed this enzyme. Moreover, it is possible to genetically
engineer strains that normally lack ACC deaminase with the gene encoding this
enzyme, with the expectation that the transformed strain will nodulate its cognate
legume to a greater extent than the nontransformed strain. In this regard, the studies
demonstrated that Rhizobium (e.g., Sinorhizobium meliloti Rm1021), which does
not have ACC deaminase activity, can nodulate its host legume (alfalfa) more
efficiently when it is transformed with the acdS and lrpL genes from R. legumino-
sarum bv. viciae 128C53K. Thus, by genetically engineering rhizobial strains to
produce ACC deaminase, there is a real possibility of obtaining better strains for use
as field inocula to increase the yield of leguminous crops. Furthermore, ethylene
production can be induced during many environmental stresses, such as infection
by pathogens, flooding, heavy metal poisoning, and mechanical wounding. The
ACC deaminase containing PGPR including symbiotic nitrogen fixers have been
found to be able to help the plants resist the detrimental effects of stress ethylene on
plant growth imposed by both biotic and abiotic stresses, besides providing other
benefits to legumes. Thus, the ethylene decreasing ability of nodule forming
bacteria is interesting and suggestive for further understanding of legume–rhizobia
interactions and signaling events of Rhizobium–legume symbiosis.
References
Abd-Alla MH (1994) Solubilization of rock phosphates by Rhizobium and Bradyrhizobium. Folia

Abeles FB, Morgan PW, Saltveit ME Jr, Abeles FB, Morgan PW, Saltveit ME Jr (1992) Regula-
tion of ethylene production by internal, environmental and stress factors. Ethylene in plant
biology, 2nd edn. Academic, San Diego, pp 56–119
Alikhani HA, Saleh-Rastin N, Antoun H (2006) Phosphate solubilization activity of rhizobia
native to Iranian soils. Plant Soil 287:35–41
Arshad M, Shaharoona B, Mahmood T (2008) Inoculation with Pseudomonas spp. containing
ACC-deaminase partially eliminates the effects of drought stress on growth, yield, and ripening
of pea (Pisum sativum L.). Pedosphere 18:611–620
Bajgiran AR, Lakzian A, Rastin NS (2008) Elongation of shoot and root in wheat by ACC
deaminase of Rhizobium Spp. indigenous to soils of Iran. Int J Agri Biol 10:481–486
Bartsev A, Kobayashi H, Broughton WJ (2004) Rhizobial signals convert pathogens to symbionts
at the legume interface. In: Gillings M, Holmes A (eds) Plant microbiology. Garland Science,
Abingdon, pp 19–31
Belimov AA, Hontzeas N, Safronova VI, Demchinskaya SV, Piluzza G, Bullitta S, Glick BR
(2005) Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian
mustard (Brassica juncea L. Czern.). Soil Biol Biochem 37:241–250
Belimov AA, Dodd IC, Safronova VI, Hontzeas N, Davies WJ (2007) Pseudomonas brassica-
cearum strain Am3 containing 1-aminocyclopropane-1-carboxylate deaminase can show
both pathogenic and growth promoting properties in its interaction with tomato. J Exp Bot
58:1485–1495
Blaha D, Prigent-Combaret C, Mirza MS, Moenne-Loccoz Y (2006) Phylogeny of the 1-amino-
cyclopropane-1-carboxylic acid deaminase-encoding gene acdS in phytobeneficial and patho-
genic Proteobacteria and relation with strain biogeography. FEMS Microbiol Ecol 56:455–470
Bleecker A (1999) Ethylene perception and signaling: an evolutionary perspective. Trends Plant
Sci 4:269–274
Bras CP, Joda MA, Wijfjes AAM, Hartveld M, Stuurman N, Thomas-Oates JE, Spaink HP (2000)
A Lotus japonicus nodulation system based on heterologous expression of the fucosyl trans-
ferase NodZ and the acetyl transferase NoIL in Rhizobium leguminosarum. Mol Plant Microbe
Interact 13:475–479
Cheng Z, Park E, Glick BR (2007) 1-aminocyclopropane-1-carboxylate deaminase from Pseudo-
monas putida UW4 facilitates the growth of canola in the presence of salt. Can J Microbiol
53:912–918
Cheng Z, Duncker BP, McConkey BJ, Glick BR (2008) Transcriptional regulation of ACC
deaminase gene expression in Pseudomonas putida UW4. Can J Microbiol 54:128–136
Contesto C, Desbrosses G, Lefoulon C, Bena G, Borel F, Galland M, Gamet L, Varoquaux F,
Touraine B (2008) Effects of rhizobacterial ACC deaminase activity on Arabidopsis indicate
that ethylene mediates local root responses to plant growth-promoting rhizobacteria. Plant Sci
175:178–189
Duan J, Müller KM, Charles TC, Vesely S, Br G (2008) 1-Aminocyclopropane-1-carboxylate
(ACC) deaminase genes in rhizobia from southern Saskatchewan. Microb Ecol . doi:10.1007/
s00248-008-9407-6
Duodu S, Bhuvaneswari TV, Stokkermans TJW, Peters NK (1999) A positive role for rhizobitox-
ine in Rhizobium-legume symbiosis. Mol Plant Microbe Interact 12:1082–1089
Ecker JR (1995) The ethylene signal transduction pathway in plants. Science 268:667–675
Esashi Y, Esashi Y (1991) Ethylene and seed germination. In: Matoo AK, Shuttle JC (eds) The
plant hormone ethylene. CRC, Boca Raton, Florida, pp 133–157
Farwell AJ, Vesely S, Nero V, Rodriguez H, McCormack K, Shah S, Dixon GD, Glick BR (2007)
Tolerance of transgenic canola plants (Brassica napus) amended with plant growth-
promoting bacteria to flooding stress at a metal-contaminated field site. Environ Pollut
147:540–545
Fernandez-Lopez M, Goormachtig S, Gao M, D’Haeze W, Van Montagu M, Holsters M (1998)
Ethylene-mediated phenotypic plasticity in root nodule development on Sesbania rostrata.
Proc Natl Acad Sci USA 95:12724–12728
Frankenberger WTJ, Arshad M (1995) Phytohormones in soil. Microbial production and function.
Marcel Dekker, New York, p 503
Gage DJ, Bobo T, Long SR (1996) Use of green fluorescent protein to visualize the early events
of symbiosis between Rhizobium meliloti and alfalfa (Medicago sativa). J Bacteriol 178:
7159–7166
41:109–117
Glick BR (2005) Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase.
FEMS Microbiol Lett 251:1–7
Glick BR, Jacobson CB, Schwarze MMK, Pasternak JJ (1994) Does the enzyme 1-aminocyclo-
propane-lcarboaylate deaminase play a role in plant growth promotion by Pseudomonas putida
GR12–2? In: Ryder MH, Stephens PM, Bowen GD (eds) Improving plant productivity with
rhizosphere bacteria. CSIRO, Adelaide, pp 150–152
Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentrations by
plant growth-promoting bacteria. J Theor Biol 190:63–68
Glick BR, Patten CL, Holguin G, Penrose DM (1999) Biochemical and genetic mechanisms used
by plant growth promoting bacteria. Imperial College Press, London, p 270
Glick BR, Cheng Z, Czarny J, Duan J (2007a) Promotion of plant growth by ACC deaminase-
producing soil bacteria. Eur J Plant Pathol 119:329–339
Glick BR, Todorovic B, Czarny J, Cheng Z, Duan J, McConkey B (2007b) Promotion of plant
growth by bacterial ACC deaminase. Crit Rev Plant Sci 26:227–242
Goodlass G, Smith KA (1979) Effects of ethylene on root extension and nodulation of pea (Pisum
sativum L.) and white clover (Trifolium repens L.). Plant Soil 51:387–395
Grichko VP, Glick BR (2001) Amelioration of flooding stress by ACC deaminase-containing plant
growth-promoting bacteria. Plant Physiol Biochem 39:11–17
Grichko VP, Filby B, Glick BR (2000) Increased ability of transgenic plants expressing the
bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn. J Bacteriol
81:45–53
Grobbelaar N, Clarke B, Hough MC, Grobbelaar N, Clarke B, Hough MC (1971) The nodulation
and nitrogen fixation of isolated roots of Phaseolus vulgaris L. III. The effect of carbon dioxide
and ethylene. Plant Soil Spl Vol:216–223
Guinel FC, Geil RD (2002) A model for the development of the rhizobial and arbuscular
mycorrhizal symbioses in legumes and its use to understand the roles of ethylene in the
establishment of these two symbioses. Can J Bot 80:695–720
Guinel FC, Sloetjes LL (2000) Ethylene is involved in the nodulation phenotype of Pisum sativum
R50 (sym 16). An pleiotropic mutant that nodulated poorly and has pale green leaves. J Exp
Bot 51:885–894
Hall J, Peirson AD, Ghosh S, Glick BR (1996) Root elongation in various agronomic crops by
the plant growth promoting rhizobacterium Pseudomonas putida GR12–2. Israel J Plant Sci
44:37–42
Hameeda B, Rupela OP, Reddy G, Satyavani K (2006) Application of plant growth-promoting
bacteria associated with composts and macrofauna for growth promotion of Pearl millet
(Pennisetum glaucum L.). Biol Fertil Soil 43:221–227
Heidstra RW, Yang WC, Yalcin Y, Peck S, Emons AM, van Kammen A, Bisseling T (1997)
Ethylene provides positional information on cortical cell division but is not involved in
Nod factor-induced root hair tip growth in Rhizobium-legume interaction. Development
124:1781–1787
Herder JD, Goormachtig S, Holsters M (2006) Ethylene in the Rhizobium-legume symbiosis.
In: Khan NA (ed) Ethylene action in plants. Springer , Berlin, pp 119–134
Hirsch AM, Fang Y (1994) Plant hormones and nodulation: what’s the connection? Plant Mol Biol
26:5–9
Hoa LTP, Nomura M, Tajima S (2004) Characterization of bacteroid proteins in soybean nodules
formed with Bradyrhizobium japonicum USDA110. Microb Environ 19:71–75
Honma M (1993) Stereospecific reaction of 1-aminocyclopropane-1-carboxylate deaminase.
In: Pech JC, Latche A, Balague C (eds) Cellular and molecular aspects of the plant hormone
ethylene. Kluwer, Dordrecht, The Netherlands, pp 111–116
Honma M, Shimomura T (1978) Metabolism of 1-aminocyclopropane- 1-carboxylic acid. Agric
Biol Chem 42:1825–1831
Hontzeas N, Richardson AO, Belimov A, Safronova V, Abu-Omar MM, Glick BR (2005)
Evidence for horizontal transfer of 1- aminocyclopropane-1-carboxylate deaminase genes.
Jackson MB (1991) Ethylene in root growth and development. In: Matto AK, Suttle JC (eds) The
plant hormone ethylene. CRC, Boca Raton, Florida, pp 159–181
Jalili F, Kazem Khavazi K, Pazira E, Nejati A, Rahmani HA, Sadaghiani HR, Miransari M (2008)
Isolation and characterization of ACC deaminase producing fluorescent pseudomonads, to
alleviate salinity stress on canola (Brassica napus L.) growth. J Plant Physiol . doi:doi:10.1016/
j.jplph.2008.08.004
Jeroen DH, Sofie G, Marcelle H (2006) Ethylene in the Rhizobium-legume symbiosis. In: Khan
NA, Khan NA (eds) Ethylene action in plants. Springer, Berlin Heidelberg, pp 119–134
Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K,

Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M,
Matsuno A, Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C,
Yamada M, Tabeta S (2000) Complete genome structure of the nitrogen-fixing symbiotic
bacterium Mesorhizobium loti. DNA Res 7:331–338
Kaneko T, Nakamura Y, Sato S, Minamisawa K, Uchiumi T, Sasamoto S, Watanabe A, Idesawa
K, Iriguchi M, Kawashima K, Kohara M, Matsumoto M, Shimpo S, Tsuruoka H, Wada T,
Yamada M, Tabata S (2002) Complete genomic sequence of nitrogen-fixing symbiotic bacte-
rium Bradyrhizobium japonicum USDA110. DNA Res 9:189–197
Klee HJ (1993) Ripening physiology of fruit from transgenic tomato (Lycopersicon esculentum)
plants with reduced ethylene synthesis. Plant Physiol 102:911–916
Klee HJ, Hayford MB, Kretzmer KA, Barry GF, Kishore GM (1991) Control of ethylene synthesis
by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3:1187–1193
Kloepper JW, Leong J, Teintze M, Schroth MN (1980) Pseudomonas siderophores: a mechanism
explaining disease suppressive soils. Curr Microbiol 4:317–320
Krishnan HB, Kang BR, Krishnan AH, Kim KY, Young Cheol Kim YC (2007) Rhizobium etli
USDA9032 engineered to produce a phenazine antibiotic inhibits the growth of fungal patho-
gens but is impaired in symbiotic performance. Appl Environ Microbiol 73:327–330
Kucey RM, Hynes MF (1989) Populations of Rhizobium leguminosarum bv. phaseoli and viciae
in fields of bean and pea in rotation with nonlegumes. Can J Microbiol 35:661–667
Lee KH, LaRue TA (1992) Exogenous ethylene inhibits nodulation of Pisum sativum L. cv.
Sparkle. Plant Physiol 100:1759–1763
Ligero F, Liuch C, Olivares J (1986) Evolution of ethylene from roots of Medicago sativa plants
inoculated with Rhizobium meliloti. J Plant Physiol 125:361–365
Ligero F, Caba JM, Lluch C, Olivares J (1991) Nitrate inhibition of nodulation can be overcome by
the ethylene inhibitor aminoethoxyvinylglycine. Plant Physiol 97:1221–1225
Long SR (1989) Rhizobium–legume nodulation: life together in the underground. Cell 56:203–214
Long SR, Staskawicz BJ (1993) Prokaryotic plant parasites. Cell 73:921–935
Lorteau MA, Ferguson BJ, Guinel FC (2001) Effects of cytokinin on ethylene production and
nodulation in pea (Pisum sativum) cv Sparkle. Physiol Plant 112:421–428
Lynch JM, Whipps JM (1991) Substrate flow in the rhizosphere. In: Keister DL, Cregan PB (eds)
The rhizosphere and plant growth. Kluwer, Dordrecht, The Netherlands, pp 15–24
Ma W, Guinel FC, Glick BR (2003a) The Rhizobium leguminosarum bv. viciae ACC deaminase
protein promotes the nodulation of pea plants. Appl Environ Microbiol 69:4396–4402
Ma W, Sebestianova S, Sebestian J, Burd GI, Guinel F, Glick BR (2003b) Prevalence of
1-aminocyclopropaqne-1-carboxylate in deaminase in Rhizobia spp. Anton Van Leeuwenhoek
83:285–291
Ma W, Charles TC, Glick BR (2004) Expression of an exogenous 1-aminocyclopropane-
1-carboxylate deaminase gene in Sinorhizobium meliloti increases its ability to nodulate
alfalfa. Appl Environ Microbiol 70:5891–5897
Mandal SM, Mondal KC, Dey S, Pati BR (2007) Optimization of cultural and nutritional condi-
tions for indole-3-acetic acid (IAA) production by a Rhizobium sp. isolated from root nodules
of Vigna mungo (L.) Hepper. Res J Microbiol 2:239–246
Mayak S, Tivosh T, Glick BR (1999) Effect of wild type and mutant plant growth promoting
rhizobacteria on the rooting of mungbeen cuttings. J Plant Growth Regul 18:49–53
Minami R, Uchiyama K, Murakami T, Kawai J, Mikami K, Yamada T, Yokoi D, Ito H, Matsui H,
Honma M (1998) Properties, sequence, and synthesis in Escherichia coli of 1- aminocyclo-
propane-1-carboxylate deaminase from Hansenula saturnus. J Biochem 123:1112–1118
Naveed M, Za Z, Khalid M, Asghar HN, Akhtar MJ, Arshad M (2008) Rhizobacteria containing
Acc-deaminase for improving growth and yield of wheat under fertilized conditions. Pak J Bot
40:1231–1241
Nelson LM (2004) Plant growth promoting rhizobacteria (PGPR): prospects for new inoculants.
Plant Manage Netw . doi:10.1094/CM-2004-0301-05-RV
Nie L, Shah S, Burd GI, Dixon DG, Glick BR (2002) Phytoremediation of arsenate contaminated
soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae
CAL2. Plant Physiol Biochem 40:355–361
Nukui N, Ezura H, Yuhashi K, Yasuta T, Minamisawa K (2000) Effects of ethylene precursor and
inhibitors for ethylene biosynthesis and perception on nodulation in Lotus japonicus and
Macroptilium atropurpureum. Plant Cell Physiol 41:893–897
Nukui N, Ezura H, Minamisawa K (2004) Transgenic Lotus japonicus with an ethylene receptor
gene Cm-ERS1/H70A enhances formation of infection threads and nodule primodia. Plant Cell
Physiol 45:427–435
Nukui N, Minamisawa K, Ayabe SI, Aoki T (2006) Expression of the 1-aminocyclopropane-
1-carboxylic acid deaminase gene requires symbiotic nitrogen-fixing regulator gene nifA2 in
Mesorhizobium loti MAFF303099. Appl Environ Microbiol 72:4964–4969
Okazaki S, Yuhashi K, Minamisawa K (2003) Quantitative and time-course evaluation of nodula-
tion competitiveness of rhizobitoxine-producing Bradyrhizobium elkanii. FEMS Microbiol
Ecol 45:155–160
Okazaki S, Nukui N, Sugawara M, Minamisawa K (2004) Rhizobial strategies to enhance
symbiotic interactions: rhizobiotoxine and 1-aminocyclopropane-1-carboxylate deaminase.
Microb Environ 19:99–111
Okazaki S, Sugawara M, Yuhashi KI, Minamisawa K (2007) Rhizobitoxine-induced chlorosis
occurs in coincidence with methionine deficiency in soybeans. Ann Bot 100:55–59
Oldroyd GE, Engstrom EM, Long SR (2001) Ethylene inhibits the Nod factor signal transduction
pathway of Medicago truncatula. Plant cell 13:1835–1849
Owens LD (1973) Herbicidal potential of rhizobitoxine. Weed Sci 21:63–66
Owens LD, Wright DA (1965) Rhizobial-induced chlorosis in soybeans: isolation, production in
nodules, and varietal specificity of the toxin. Plant Physiol 40:927–930
Owens LD, Thompson JF, Fennessy PV (1972) Dihydrorhizobitoxine, a new ether amino-acid
from Rhizobium japonicum. J Chem Soc Chem Commun 1972:715
Pawlowski K, Bisseling T (1996) Rhizobial and actiorhizal symbioses: what are the shared
featrues? Plant Cell 8:1899–1913
Penmetsa RV, Cook DR (1997) A legume ethylene-insensitive mutant hyper infected by its
rhizobial symbiont. Science 275:527–530
exudates and extracts of canola seeds treated with plant growth-promoting bacteria. Can J
Perret X, Staehelin C, Broughton WJ (2000) Molecular basis of symbiotic promiscuity. Microbiol
Mol Biol Rev 64:180–201
Peters NK, Crist-Estes DK (1989) Nodule formation is stimulated by the ethylene inhibitor
aminoethoxyvinylglycine. Plant Physiol 91:690–693
Pitts RJ, Cernac A, Estelle M (1998) Auxin and ethylene promote root hair elongation in
Arabidopsis. Plant J 16:553–560
Prayitno J, Rolfe BG, Mathesius U (2006) The ethylene insensitive sickle mutant of Medicago
truncatula shows altered auxin transport regulation during nodulation. Plant Physiol 142:
168–180
Reed MLE, Glick BR (2005) Growth of canola (Brassica napus) in the presence of plant growth-
promoting bacteria and either copper or polycyclic aromatic hydrocarbons. Can J Microbiol
51:1061–1069
Safronova VI, Stepanok VV, Engqvist GL, Alekseyev YV, Belimov AA (2006) Root-associated
bacteria containing 1-aminocyclopropane-1-carboxylate deaminase improve growth and nutri-
ent uptake by pea genotypes cultivated in cadmium supplemented soil. Biol Fertil Soils
42:267–272
Schmidt JS, Harper JE, Hoffman TK, Bent AF (1999) Regulation of soybean nodulation indepen-
dent of ethylene signaling. Plant Physiol 119:951–959
Shah S, Li J, Moffat BA, Glick BR (1998) Isolation and characterization of ACC deaminase genes
from two different plant growth promoting rhizobacteria. Can J Microbiol 44:833–843
Shaharoona B, Jamro GM, Zahir ZA, Arshad M, Memon KS (2007) Effectiveness of various
Pseudomonas spp. and Burkholderia caryophylli containing ACC-deaminase for improving
growth and yield of wheat (Triticum aestivum L.). J Microbiol Biotechnol 17:1300–1307
Spaink HP (1997) Ethylene as a regulator of Rhizobium infection. Trends Plant Sci 2:203–204
Sridevi M, Mallaiah KV (2007) Production of indole-3-acetic acid by Rhizobium isolates from
Sesbania species. Afr J Microbiol Res 1:125–128
Stearns J, Glick BR (2003) Transgenic plants with altered ethylene biosynthesis or perception.
Biotechnol Adv 21:193–210
Storey EP, Boghozian R, Little James L, Lowman DW, Chakraborty R (2006) Characterization of
’Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum
LARI 917. Biometals 19:637–649
Sturz AV, Christie BR, Nowak J (2000) Bacterial endophytes: potential role in developing
sustainable systems of crop production. Crit Rev Plant Sci 19:1–30
Suganuma N, Yamauchi H, Yamamoto K (1995) Enhanced production of ethylene by soybean
roots after inoculation with Bradyrhizobium elkanii. Plant Sci 111:163–168
Sugawara M, Shin O, Noriyuki N, Hiroshi E, Hisayuki M, Kiwamu M (2006) Rhizobitoxine
modulates plant–microbe interactions by ethylene inhibition. Biotech Adv 24:384–388
Sullivan JT, Trzebiatowski JR, Cruickshank RW, Guozy J, Brown SD, Elliot RM, Fleetwood DJ,
McCallum NG, Rossbach U, Stuart GS, Weaver JE, Webby RJ, DeBruijn FJ, Ronson CW
(2002) Comparative sequence analysis of the symbiosis island of Mesorhizobium loti strain
R7A. J Bacteriol 184:3086–3095
Tamimi SM, Timko MP (2003) Effects of ehtylene and inhibitors of ethylene synthesis and action
on nodulation in common bean (Phaseolus vulgaris L.). Plant Soil 257:125–131
Tanimoto M, Roberts K, Dolan L (1995) Ethylene is a positive regulator of root hair development
in Arabidopsis thaliana. Plant J 8:943–948
Timmers ACJ, Auriac M-C, Truchet G (1999) Refined analysis of early symbiotic steps of the
Rhizobium-Medicago interaction in relationship with microtubular cytoskeletion re- arrange-
ments. Development 126:3617–3628
Tittabutr P, Jonathan D, Awaya QXL, Dulal B (2008) The cloned 1-aminocyclopropane-
1-carboxylate (ACC) deaminase gene from Sinorhizobium sp. strain BL3 in Rhizobium sp.
strain TAL1145 promotes nodulation and growth of Leucaena leucocephala. Syst Appl
Uchiumi T, Ohwada T, Itakura M, Mitsui H, Nukui N, Dawadi P, Kaneko T, Tabata S,
Yokoyama T, Tejima K, Saeki K, Omori H, Hayashi M, Maekawa T, Sriprang R, Murooka
Y, Sioya K, Abe M, Minamisawa K (2004) Expression islands clustered on the symbiosis
island of the Mesorhizobium loti genome. J Bacteriol 186:2439–2448
van Spronsen PC, van Brussel AAN, Kijne JW (1995) Nod factors produced by Rhizobium
leguminosarum biovar viciae induce ethylene-related changes in root cortical cells of Vicia
sativa ssp. nigra. Eur J Cell Biol 68:463–469
VanLoon LC, Glick BR (2004) Increased plant fitness by rhizobacteria. In: Sandermann H (ed)
Molecular ecotoxicology of plants. Springer, Berlin, pp 177–205
VanLoon LC, Geraats PBJ, Linthorst HJM (2006) Ethylene as a modulator of disease resistance in
plants. Trends Plant Sci 11:184–190
Venkadasamy G, Senthilkumar M, Kishore G, Kannepalli A (2008) Isolation and characterization
of ACC deaminase gene from two plant growth-promoting rhizobacteria. Curr Microbiol
57:312–317
Vernié T, Sandra M, de Françoise B, Julie P, Jean-Philippe C, Christian Rogers GO, Florian FAN,
Pascal G (2008) EFD is an ERF transcription factor involved in the control of nodule number
and differentiation in Medicago truncatula. Plant Cell 20:2696–2713
Wang C, Knill E, Glick BR, Défago G (2000) Effect of transferring 1-aminocyclopropane-1-

carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its
gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities. Can J
Wani PA, Khan MS, Zaidi A (2007) Synergistic effects of the inoculation with nitrogen fixing and
phosphate solubilizing rhizobacteria on the performance of field grown chickpea. J Plant Nutr
Soil Sci 170:283–287
Wani PA, Khan MS, Zaidi A (2008) Chromium reducing and plant growth promoting
Mesorhizobium improves chickpea growth in chromium amended soil. Biotechnol Lett
30:159–163
Xie ZP, Staehelin C, Wiemken A, Boller T (1996) Ethylene responsiveness of soybean cultivars
characterized by leaf senescence, chitinase induction and nodulation. J. Plant Physiol 149:
690–694
Yang J, Kloepper JW, Ryu CM (2008) Rhizosphere bacteria help plants tolerate abiotic stress.
Trends Plant Sci . doi:10.1016/j.tplants.2008.10.004
Yuhashi K, Ichikawa N, Ezura H, Akao S, Minakawa Y, Nukui N, Yasuta T, Minamisawa K
(2000) Rhizobitoxine production by Bradyrhizobium elkanii enhances nodulation and compet-
itiveness on Macroptilium atropurpureum. Appl Environ Microbiol 66:2658–2663
Zahir ZA, Arshad M, Frankenberger WT Jr (2004) Plant growth promoting rhizobacteria: applica-
tions and perspectives in agriculture. Adv Agron 81:97–168
Zahir ZA, Munir A, Asghar HN, Shaharoona B, Arshad M (2008) Effectiveness of rhizobacteria
containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought
conditions. J Microbiol Biotechnol 18:958–963
Zhang Y, Zhao L, Wang Y, Yang B, Chen S (2008) Enhancement of heavy metal accumulation by
tissue specific co-expression of iaaM and ACC deaminase genes in plants. Chemosphere
72:564–571
Chapter 5
Strategies for Crop Improvement
in Contaminated Soils Using Metal-Tolerant
Bioinoculants
Anju Rani and Reeta Goel
Abstract Heavy metal contamination due to natural and anthropogenic sources is a

global environmental concern. Release of heavy metals without proper treatment
poses a serious threat to public health because of its persistence, biomagnification
and accumulation in food chain. Nonbiodegradability and sludge production are the
two major constraints of metal treatment. The bioremediation of soil, sludge,
sediments and wastes polluted with heavy metals generally involves the active
microbiological processes of biosorption, bioaccumulation, sequestration and
efflux. Bioremediation using microbes well adapted to diverse physiological
conditions could be utilized for remediation of heavy metal-contaminated sites.
The application of proteomics in environmental bioremediation program provides a
global view of the protein compositions of the microbial cells and offers a
promising approach to understand the molecular mechanisms of bioremediation.
In this chapter, attention is paid to highlighting the strategies for crop improvement
using metal-tolerant microbes in soils contaminated with heavy metals.
5.1 Introduction
Metals have been an important constituent of the earth’s crust from the time it was
evolved. Thus, even the early life has arisen in the presence of abundance of metals.
Over the ages, all living systems have evolved to use some metals as vital con-
stituents, while they have learned to grapple with some others which are toxic
(Choudhury and Srivastava 2001). Environmental pollution by metals became
evident when mining and industrial activities increased in the late nineteenth and
twentieth centuries. Potentially hazardous levels of heavy metals are being
R. Goel (*) and A. Rani

Department of Microbiology, Govind Ballabh Pant University of Agriculture and Technology,
Pantnagar, Uttaranchal, 263145, India
e‐mail: rg55@rediffmail.com

86 A. Rani and R. Goel
dispersed into subsurface sediments and groundwater in a number of metal-

contaminated sites and represent a challenge for environment restoration.
5.2 Heavy Metals
The term “heavy metals” refers to metals and metalloids having densities greater
than 5 g cm3 and is usually associated with pollution and toxicity, although some
of these elements (essential metals) are required by organisms at low concentrations
(Adriano 2001). For example, zinc (Zn) is the component of a variety of enzymes
(dehydrogenases, proteinases, peptidases) but is also involved in the metabolism of
carbohydrates, proteins, phosphate, auxins, in RNA and ribosome formation in
plants (Kabata-Pendias and Pendias 2001). Copper (Cu) contributes to several
physiological processes (photosynthesis, respiration, carbohydrate distribution,
nitrogen and cell wall metabolism, seed production) in plants including disease
resistance (Kabata-Pendias and Pendias 2001). The effective functioning of the
metabolisms of humans and bacteria is also dependent on these metals (Adriano
2001; Blencowe and Morby 2003; Cavet et al. 2003). However, at high concentra-
tions, these metals exhibit toxic effects on cells (Baker and Walker 1989). In
contrast, cadmium (Cd) is not involved in any known biological processes (nones-
sential metal) and may be quite toxic as it is accumulated by organisms. It is known
to: (1) disturb enzyme activities, (2) inhibit the DNA-mediated transformation in
microorganisms, (3) interfere in the symbiosis between microbes and plants, and (4)
increase plant predisposition to fungal invasion (Kabata-Pendias and Pendias
2001). In humans, it may promote several disorders in the metabolism of Ca and
vitamin D leading to bone degeneration and kidney damage (itai-itai disease)
(Adriano 2001). The excessive uptake of heavy metals by animals and humans is
the result of the successive accumulation of these elements in the food chain, the
starting point being the contamination of the soil.
5.3 Origin of the Contamination in Soils
The main problem with heavy metals (such as Cu, Zn and Cd) in soils is that, unlike
organic pollutants, they cannot be biologically degraded and, therefore, persist in
the environment for longer periods of time. Their presence in soils may be from
natural or anthropogenic origins. Natural sources include atmospheric emissions
from volcanoes, the transport of continental dusts, and the weathering of metal-
enriched rocks (Ernst 1998). However, the major source of contamination is from
anthropogenic origin: the exploitation of mines and smelters, the application of
metal-based pesticides and metal-enriched sewage sludges in agriculture, combus-
tion of fossil fuel, metallurgical industries and electronics (manufacture, use and
disposal), military training, etc., contribute to an increased input of heavy metals in
soils (Alloway 1995). Whereas the industrial emissions of metals may be controlled
5 Strategies for Crop Improvement 87
by the installation of adequate air filters, the main source of contamination for
humans remains the ingestion of plants growing in derelict soils. The use of
intensive farm management practices, like application of phosphatic fertilizers,
sewage sludge input and pesticide treatment, are responsible for the pollution of
conventional agricultural soils. Although these practices significantly increase the
yields by protecting plants from deleterious pathogens and providing them with all
the nutrients necessary for a rapid and sustained growth, they may also add large
amounts of heavy metals and organic pollutants to soil which, in turn, may
accumulate in plants. For instance, Hamon et al. (1998) have shown that the
addition of phosphatic fertilizers increased Cd uptake of wheat (Triticum aestivum).
However, the risk emerging from heavy metals largely depends on their bioavail-
ability (Adriano 2001).
Mineral rock weathering and anthropogenic sources provide two of the main
types of metal to soils. According to Ross (1994), the anthropogenic sources of
metal contamination can be divided to five main groups: (1) metalliferous mining
and smelting (e.g., arsenic, cadmium, lead, mercury), (2) industry (e.g., arsenic,
cadmium, chromium, cobalt, copper, mercury, nickel, zinc), (3) atmospheric depo-
sition (arsenic, cadmium, chromium, copper, lead, mercury, uranium), (4) agricul-
ture (e.g., arsenic, cadmium, copper, lead, selenium, uranium, zinc), and (5) waste
disposal (e.g., arsenic, cadmium, chromium, copper, lead, mercury, zinc).
5.4 Heavy Metal Toxicity to Plants
Exposure of plants even to minute concentrations of toxic heavy metals may lead to
the alteration of many cellular processes and structures (Hall 2000). One of the
characteristic effects of metal poisoning, observable at an early stage, is a reduction
in cell proliferation and growth (Schützendübel et al. 2001). It has also been
associated with the appearance of oxidative stress (Schützendübel and Polle
2002). Accumulation of reactive oxygen species (ROS), leading to an oxidative
burst, is thought to increase cellular damage through oxidation of several macro-
molecules (Hall 2000), such as lipids (Sandalio et al. 2001) and proteins (Romero-
Puertas et al. 2002). Some metals, such as Fe2+ and Cu,+ might induce oxidative cell
damage coupled to their autooxidation, through Fenton-type reactions. However,
this type of reaction has not been described for either Cd2+ or Hg2+ in plants
(Schützendübel and Polle 2002), despite the evidence of oxidative stress induction
in different plants after exposure to Cd (Lozano-Rodriguez et al. 1997; Dixit et al.
2001) and to Hg (Cho and Park 2000). Other cellular responses observed after
addition of heavy metals are changes in thiol-peptide metabolism (Rauser 1991).
Although lead has no defined biological function, it can be accumulated in plant
organs (such as roots and shoots) and exhibit toxicity leading to a decrease in
biomass and inhibition of chlorophyll biosynthesis (Koeppe 1981). Heavy metals
disrupt the physiological process by binding to protein sulfhydryl groups or cause
deficiency/substitution of essential metals (Van Assche and Clijsters 1990). Lead,
a strong reactant to protein N and S ligands, was shown to inhibit chlorophyll

synthesis (Sengar and Pandey 1996; Tripathi et al. 2005; Rani et al. 2008) as well as
electron transport and Rubisco activity (Stiborova et al. 1986) in vitro.
The ability of plants to bioaccumulate metals and possibly other contaminants
varies with both the genotypes of plant and the nature of metal contaminants (Naidu
et al. 2003). For instance, cadmium, when taken up in excess by plants, inhibits
directly or indirectly the physiological processes, such as respiration, photosynthe-
sis, cell elongation, plant water relationships, nitrogen metabolism and mineral
nutrition, resulting in poor growth and low biomass (Di et al. 1999; Rani et al.
2008). In follow-up studies with other metals, the higher concentrations of different
metals (e.g., cadmium, copper, zinc, lead, etc.), when used either alone or as
mixtures, have been shown to significantly reduce the dry matter accumulation,
symbiosis and yield of greengram (Vigna radiata (L.) Wilczek) (Wani et al. 2007a),
chickpea (Cicer arietinum) (Wani et al. 2007b) and pea (Pisum sativum)
(Wani et al. 2008a), grown in metal-treated sandy clay loam soils.
5.5 Mechanisms of Metal Toxicity in Microorganisms
Metal species exert toxicity through numerous biochemical pathways that can be
divided into five mechanistic categories. First, toxic metal species can bind to
proteins in lieu of essential inorganic ions, thereby altering the biological function
of the target molecule. An example is the replacement of Ni for Mg in some redox-
active metalloproteins or in DNA, which destroys their function and/or may lead to
DNA damage, respectively. Second, toxic metal species can participate in an array
of reactions with thiols and disulphides, thereby destroying the biological function
of proteins that contain sensitive S groups (Stohs and Bagchi 1995; Zannoni et al.
2007). These reactions frequently require and produce ROS, which are by-products
of normal metabolism (Fig. 5.1). Thiol groups are often involved in the binding of
Fig. 5.1 Biochemical mechanisms of microbiological metal toxicity

substrates to specific carriers whose transport mechanism can be impaired by toxic

metal species. The destruction of sensitive thiol groups on nascent proteins by
metals may also impair protein folding or binding of apoenzymes by cofactors,
thereby destructing the normal biological activity of the protein (Harrison et al.
2007). Third, certain transition metals can participate in catalytic reactions, known
as Fenton-type reactions, that produce ROS. Collectively, these reactions place the
cell in a state of oxidative stress, and increased levels of ROS damage DNA, lipids
and proteins through a range of biochemical routes (Geslin et al. 2001). Fourth,
toxic metal species must gain entry into cells through transporters or by binding
lipophilic carriers, as cell membranes are nonpermeable to these compounds. The
transporter-mediated uptake of toxic metals might interfere with the normal trans-
port of essential substrates owing to competitive inhibition. This transport process
gains energy from the proton motive force (PMF) or ATP pool (Foulkes 1998).
Fifth some metal oxy-anions are reduced by the oxidoreductase DsbB, which draws
electrons from the bacterial transport chain through the quinone pool (Borsetti et al.
2007). In effect, certain toxic metal species starve microbial cells by indirectly
siphoning electrons from the respiratory chain (Lohmeier-Vogel et al. 2004). The
formation of ROS that damage DNA, proteins and lipids also occur in normal
metabolic processes; however, the production of ROS is enhanced during metal
poisoning which may mediate additional cellular damage.
5.6 Influence of Bacteria on Heavy Metal Bioavailability
Overall toxic effects of heavy metals to soil microorganisms depend on their

bioavailability. Although heavy metal bioavailability is mainly dependent on the
soil properties (pH and organic matter), bacteria can also directly influence the
solubility of heavy metals by altering their chemical properties (Fig. 5.2).
Microbes
3 4 2 3 4
Abiotic
Immobile phase Mobile phase
components
2 2
2
1 1
Metal
Fig. 5.2 Microbial roles in the environmental mobility of metals

Microorganisms have evolved several mechanisms which can immobilize, mobi-

lize or transform heavy metals. The exploitation of these bacterial properties for the
remediation of heavy metal-contaminated sites has been shown to be a promising
bioremediation alternative (Lovely and Coates 1997; Lloyd and Lovley 2001).
5.7 Heavy Metal Resistance Systems in Bacteria
Bacteria have developed several efficient systems to detoxify the metals. These
mechanisms include: (1) intracellular sequestration, (2) export, (3) reduced perme-
ability, (4) extracellular sequestration, and (5) extracellular detoxification (Rough
et al. 1995). Almost all known bacterial resistance mechanisms are encoded on
plasmids and transposons (Silver and Walderhaug 1992), and it is probably by gene
transfer or spontaneous mutation that bacteria acquire their resistance to heavy
metals (Osborn et al. 1997). In Gram-negative bacteria (e.g., Ralstonia eutropha),
the czc system is responsible for the resistance to Cd, Zn and Co. The czc-genes
encode for a cation–proton antiporter (CzcABC) which exports Cd, Zn and Co
(Nies 1995). A similar mechanism, called ncc system, has been found in Alcali-
genes xylosoxidans which is resistant to Ni, Cd and Co. In contrast, the Cd
resistance mechanism in Gram-positive bacteria (e.g., Staphylococcus, Bacillus or
Listeria) is a Cd-efflux ATPase. The two most well-studied Cu resistance systems
are cop from Pseudomonas syringae pv. tomato and pco from Escherichia coli. The
cop genes encode for different Cu-binding proteins which allow the sequestration of
Cu in the periplasm or in the outer membrane. In contrast, the pco system is
expected to be an ion-dependent Cu antiporter (Kunito et al. 1998). The bacterial
resistance properties can be used for different purposes: in the case of mercury
pollution, the insertion of the microbial mercury reductase in a transgenic plant
significantly improved the phytoextraction process (Heaton et al. 1998). Another
example was the inoculation of heavy metal-resistant bacteria in a contaminated
soil which seemed to protect the plants from metal toxicity.
5.8 Heavy Metal Remediation
The contamination of agricultural land and groundwater by heavy metals is essen-

tially linked to human activities. Depending on the extension, depth and kind of the
contamination, different remediation approaches have been proposed (Mulligan
et al. 2001). In general, three strategies are possible: the containment of the
contaminants, their removal from the environment, or their in situ stabilization.
Physical containment is the least expensive approach but this leaves the contami-
nant in place without treatment. As ex situ techniques are expensive, environmen-
tally invasive and labor intensive, in situ approaches are generally preferred. One of
these in situ techniques, phytoremediation, uses plants to remove pollutants from
cover
Attenuation dilution
mixing clean soil
Polluted soil
ex-situ soil washing
remediation
remedial action
fixation
in-situ
remediation flushing
bio-remediation
Fig. 5.3 Option for remediation for polluted sites
the environment or to render them harmless (Salt et al. 1995; Flathman and Lanza
1998). These processes either “decontaminate” the soil, or “stabilize” the pollutant
within it (Fig. 5.3). Decontamination reduces the amount of pollutants within the
soil by removing them, while stabilization does not reduce the quantity of pollutant
at a site, but makes use of soil amendments to alter the soil chemistry and sequester
or absorb the pollutant into the matrix so as to reduce or eliminate environmental
risks (Cunningham et al. 1995). Furthermore, these traditional techniques present
significant disadvantages, such as energy requirements, and are very expensive.
5.9 Bioremediation
Many novel approaches to environmental clean-up are being developed or are

already in use as alternatives to costly physical methods, such as incineration.
Bio-remediation in its different forms is perceived as a relatively economical and
nonintrusive approach to remediate polluted environments. The biological treat-
ment utilizes the natural reactions of microorganisms living in the environments
and, hence, provides a crucial key technology in the remediation of heavy metal-
contaminated soils and waters (Khan et al. 2009). In addition, recent developments
in molecular biology have provided insight for enhancing the microorganism’s
natural remediation capability as well as improving the current biological treatment
(Fig. 5.4).
Several key microbial processes may affect mobilization or immobilization of
toxic elements by one or more of the following mechanisms: (1) chelation of
elements by metabolites, (2) oxidation–reduction of metals which affect the solu-
bility or their valency, (3) changes in pH which affect the ionic state, (4) biosorption
by functional groups on the cell surface, (5) bioaccumulation by an energy-dependent
Fig. 5.4 Metal processing mechanisms of microorganisms
Table 5.1 Examples of toxic Organism Element

heavy metals accumulating Citrobacter sp. Lead, cadmium
microorganisms Thiobacillus ferrooxidans Silver
Bacillus cereus Cadmium
Bacillus subtilis Chromium
Pseudomonas aeruginosa Uranium
Micrococcus luteus Strontium
Rhizopus arrhizus Mercury
Aspergillus niger Thorium
Saccharomyces cerevisiae Uranium
transport system, (6) immobilization due to formation of stable materials, (7)

biomethylation, and (8) biodegradation of organic complex of metals. A wide
variety of microorganisms including plant growth-promoting rhizobacteria, fungi,
yeast, and algae are known that can interact with metals employing several
mechanisms and can transform them to nontoxic or less toxic forms (Poole and
Gadd 1989; Wani et al. 2007c, 2008b) (Table 5.1).
5.9.1 Biosorption
Biosorption of toxic metals is based on nonenzymatic processes such as adsorption.

Adsorption is due to the nonspecific binding of ionic species to cell surface-
associated or extracellular polysaccharides and proteins (Mullen et al. 1989;
Volesky 1990). Bacterial cell walls and envelopes, and the walls of fungi, yeasts,
and algae, are efficient metal biosorbents that bind charged groups. The cell walls of
Gram-positive bacteria bind larger quantities of toxic metals than the envelopes of
the Gram-negative bacteria. Biosorbents may be regenerated by treatment with acid
or with certain chelating agents. Besides bacteria, waste fungal biomass derived
from several industrial fermentations may also provide an economical source of
biosorptive materials. Many species have high cell wall chitin contents which act as
an effective biosorbent as do the chitosan and glucans.
Biosorption is defined as a property of certain inactive or dead microbial
biomass to bind and concentrate heavy metals from even very dilute aqueous
solutions (Vasudevan et al. 2001). The uptake of metal could be active, passive
or both in nature. Passive uptake is independent of cellular metabolisms, and metal
binds to polyionic cell walls through ion exchange as the process is not affected by
physical conditions such as pH and ionic strength. It is a rapid process taking only
5–10 min to complete. The process is also reversible and can involve both living
and dead cells. The active process on the other hand is slow and depends on cellular
metabolism. It is affected by metabolic inhibitors, uncouplers and temperature. In
the active process, the metal complex with specific proteins like metallothionins are
contained in vacuole. Both the active and passive mode may occur simultaneously.
The passive process is relatively nonspecific with respect to the metal (Pandey
et al. 2001).
5.9.1.1 Biosorption by Fungi
Polysaccharides in association with lipids and proteins form the main constituent of
fungal cell wall. In filamentous fungi, the outer cell wall layers mainly contain
natural polysaccharides (glycans and mannans) while the inner layers contain more
of glucosamines (chitin and chitosan) in a microfibrillar structure, which are found
to be associated with metal-binding (Vasudevan et al. 2001). Mahesh et al. (2001)
investigated Cu2+ sorption behavior of three isolated native forms of fungi in a
laboratory-scale batch reactor. Fusarium solani fungal species tend to remove 90%
Cu2+ from solution at alkaline pH (8.0–10.0). Metal uptake capacities of some fungi
have also been observed by Bagdwal et al. (2003).
5.9.1.2 Biosorption by Algae
Special polysaccharides are present in the algal cell wall. The number and nature of
binding sites depends on the chemical composition of the cell wall. In pheophycean
members, algin is present which contributes significantly to metal-binding.
5.9.1.3 Biosorption by Bacteria
The anionic nature of bacterial surface enables them to bind metal cations through
electrostatic interactions. Because of their thickness and anionic character (which is
mainly due to peptidoglycan, teichoic acid and teichuronic acids), the cell walls of
Gram-positive bacteria have a high capacity for metal-binding. It has been reported
by Beveridge and Murray (1980) that peptidoglycan is the major cell wall compo-
nent responsible for metal-binding by Bacillus subtilis. In contrast, metal-binding
by Bacillus licheniformis is predominantly due to teichuronic acid. Three strains of
thermotolerant polymer-producing bacteria, B. subtilis WD90, B. subtilis SH29,
and Enterobacter agglomerans 5M 38, as well as their bioflocculants, were capable
of nickel and cadmium removal. However, the bioflocculant of the three isolates
gave higher metal adsorption than the cells due to synthesis of extracellular
polysaccharide and other cell wall components (Kaewchai and Praseptsan 2002).
In another study, Pseudomonas aeruginosa (CW-96-1) tolerated cadmium up to a
concentration of 5 mM, and, 140 h after inoculation, metal-tolerant strain (CW-96-1)
removed >99% of the cadmium from solution. Electron micrographs and energy
dispersive microanalysis indicated that cadmium was bound to the cell wall as a
sulfur complex with a 1:1 stoichiometry (Wang et al. 1997). Pseudomonas putida
actively accumulates cadmium from the medium, and the resistance mechanism
involves both polyphosphate and a series of low molecular weight cysteine-rich
cadmium proteins that are induced during different growth phases. Nuclear mag-
netic resonance (NMR) study on the major native cadmium protein (CdBP1)
establishes a definite relationship to cadmium metallothioneins (Higham
et al. 1984).
5.9.2 Bioaccumulation
Bioaccumulation has been described for such metals as mercury, lead, silver,
cadmium and nickel. Intracellular accumulation of toxic elements is carried out
by an energy-dependent transport system (Gadd 1988). Potential mechanisms of
toxic metal flux across membranes can be ion pumps, ion channels, carrier mediated
transport, endocytosis, complex permeation, and lipid permeation. Permeabiliza-
tion of cell membranes to toxic elements can result in further exposure of intracel-
lular metal-binding sites and increase passive accumulation. Assessment of heavy
metal accumulation in the microbial cells can be done by transmission electron
microscopy (TEM). For example, TEM analysis of P. putida 62BN demonstrated
intracellular and periplasmic accumulation of cadmium (Rani et al. 2009). A high
metal concentration may lead to intracellular precipitation of metal (Hughes and
Poole 1989). Additionally, cadmium transport via the Mn2+ transport system has
been reported in many bacteria (Laddaga and Silver 1985; Perry and Silver 1982)
and may also contribute to cadmium transport in 62BN. The intracellular and
periplasmic cadmium accumulation in P.putida 62BN suggested the presence of
metal-binding and/or efflux mechanisms inside the cells mediating resistance
against metal toxicity. Cytoplasmic (Yoshida et al. 2002) and periplasmic
(Naz et al. 2005; Pazirandeh et al. 1998) accumulation of heavy metal ions as a
result of metallothioneins expression has been reported in E. coli.
Microbial biofilms, natural or engineered, could be used to remediate heavy

metal pollution by biochemical modification and/or the accumulation of toxic metal
ions (Munoz et al. 2006; Chang et al. 2006). An understanding of metal toxicity in
biofilms is crucial to the successful design of bioreactors that are used for bio-
mining (Rawlings and Johnson 2007), as well as those reactors that are used for
biodegrading organic contaminants that are frequently intermingled with metals
(Singh et al. 2006). Moreover, this information might provide insights into the
observed vulnerabilities, physiological shifts and species changes of natural aquatic
biofilm communities that have been exposed to toxic heavy metals (Lawrence et al.
2004; Vilchez et al. 2007). Multimetal resistance and tolerance in microbial
biofilms is a multifactorial property of the adherent population. In general, the
decreased susceptibility of biofilms to toxic metal species can be viewed as an
emergent property that arises from several interrelated physiological and chemical
parameters. Many of these parameters arise from the natural process of phenotypic
diversification that is ongoing during biofilm growth. Biofilm formation is a process
that gives rise to multiple cell types – each with a range of expression patterns that
correspond to the diversity of biofilm microniches – which allows the population to
withstand a diverse range of environmental stresses, including metal toxicity.
5.9.3 Siderophores
When microorganisms are grown in an iron-deficient medium, they produce

specific iron chelators, so-called siderophores, in the medium. They play an
important role in the complexation of toxic metals and increase their solubility
(Neilands 1983). Siderophores are compounds that possess catecholate, phenolate
or hydroxamate as their binding groups. Over the past few years, many siderophore
or siderophore-like compounds have been identified from various biological
systems. Although siderophores are primarily specific for Fe (III), they can also
complex other metals and radionuclides. One way to relieve heavy metals toxicity
to plants might involve the use of growth-promoting bacteria. Free-living rhizo-
bacteria exert some beneficial effects on plant development when they are either
applied to seeds or incorporated into the soil (Kloepper et al. 1989; Glick et al.
1999). Of the several mechanisms used to facilitate plant growth, siderophore
synthesized by microbes including rhizobia (Wani et al. 2007d, 2008c) and species
of Bacillus, Pseudomonas and Azotobacter (Wani et al. 2007c; Ahmad et al. 2008)
is well documented due to their iron sequestration ability from the soil. Microbial
iron siderophore complex can be taken up by plants as an iron source (Wang et al.
1993). The most appropriate approach to prevent plant chlorosis due to high levels
of heavy metals should be to provide them with an associated siderophore-producing
bacterium that supplements sufficient amounts of iron to the plant (Tripathi et al.
2005; Wani et al. 2008c).
5.10 Tracking the Insights of Bioremediation Using

Proteomics/ Genomics
Environmental pollutants in the soil are a major concern worldwide. Bioremediation

mediated by microorganisms is a highly promising technology as it is environmen-
tally friendly, safe, effective and inexpensive. However, incomplete biological
information regarding the cellular responses in many microbial communities
restricts progress in the site-specific mineralization process. The application of
proteomics in environmental bioremediation research provides a global view
of the protein compositions of the microbial cells and offers a promising approach
to understanding the molecular basis of bioremediation. With the combination of
proteomics, functional genomics provide an insight into global metabolic and
regulatory networks that can enhance the understanding of gene functions (Fig. 5.5).
5.10.1 Proteomic Studies for the Cellular Responses

to Cd2+ in Microorganism
Compared with the molecular/cellular biological studies of individual genes or

proteins one at a time, as has traditionally been done, the global analysis either at
Fig. 5.5 Post-genomic technologies using a systematic biology approach to track the insights of
bioremediation
a genomic or at a proteomic level allows for a systematic overview of thousands of

genes or their products in a species at the same time (Tyers and Mann 2003; Pandey
and Mann 2000; Dutt and Lee 2000; Humphery-Smith et al. 1997). Proteomics can
produce more accurate and comprehensive information than genomic studies can
provide because protein expressions are regulated not only at transcriptional but
also at translational levels, resulting in more details about mature proteins and their
interactions than genome-based predictions (Humphery-Smith et al. 1997). There-
fore, significant discrepancies between mRNA and protein levels have also been
found in several studies (Gygi et al. 1999; Chen et al. 2002; Griffin et al. 2002). It is
inevitable that study of the cellular responses to different stresses at the proteomic
level will inform us what gene products are actually expressed and their changes
(Abram et al. 2008). In this regard, proteomics complements other functional
genomics approaches such as microarray-based expression profiles, systematic
phenotypic profiles at the cell and organism level, and small-molecule-based arrays.
An understanding of the growth conditions governing the expression of the proteome
(for example, enzymes and regulatory proteins of heavy metal resistance, energy
generation pathways, transport and stress-related proteins) in a specific environ-
ment is essential for developing rational strategies for successful bioremediation.
Cadmium (Cd2+) is one of the well-known toxic heavy metal ions. To gain a
global understanding of how Cd2+ affects cells at the molecular level, various
studies were performed. Proteome-wide investigation unequivocally identified
1133 Shizosaccharomyces pombe proteins, of which the AACT-based quantitative
analysis revealed 106 upregulated and 55 downregulated proteins on the Cd2+
exposure. In the most prevalent functional class in the upregulated proteins, 28%
of proteins were involved in protein biosynthesis, showing a time-dependent
biphasic expression pattern characteristic with rapid initial induction and later
repression. Most significantly, 27 proteins, functionally classified as cell rescue
and defense, were upregulated for oxygen and radical detoxification, heat-shock
response, and other stress responses (Bae and Chen 2004).
Moreover, Pukárová et al. (2001) reported that during cadmium-induced growth
arrest of E. coli cells, DNA and RNA synthesis is rapidly inhibited while the rates of
protein synthesis and proteolysis increase transiently (Ferianc et al. 2000). The
protein synthesis includes de novo synthesis of cadmium-induced proteins (CDPs)
(Ferianc et al. 2000), which together make up the cadmium stress stimulon (Ferianc
et al. 1998). Some CDPs belong to global regulatory networks, which include the
SOS, oxidative stress, heat-shock and stringent response networks (Ferianc et al.
1998; VanBogelen et al. 1987). However, only a limited number of the proteins in
these regulons are induced during cadmium exposure and the synthesis of these
CDPs constitutes a minor fraction of the overall cellular response (VanBogelen
et al. 1987). In addition, these general stress responses are only transiently activated
during cadmium-induced growth inhibition. When proliferation resumes, these
regulons are downregulated and expression reaches a new steady-state level
(Ferianc et al. 1998). Other CDPs, however, are specific to cadmium stress. These
CDPs retain an elevated production level, relative to the production level of general
stress proteins, even in accommodated cells that have resumed growth during
prolonged cadmium exposure. One of these proteins has been identified as the
product of ORF o216 (Ferianc et al. 1998), later renamed yodA, a 216 amino acid
residue protein, identified on two-dimensional polyacrylamide gels (VanBogelen
et al. 1996; Ferianc et al. 1998). N-terminal sequencing has demonstrated that the
protein is processed and contains a 24 amino acid signal sequence, suggesting that
the mature product is exported from the cytoplasm. YodA, together with two other
putative proteins, YrpE of B. subtilis (Sorokin et al. 1997) and pXO1-130 of
Bacillus anthracis (Okinaka et al. 1999), may constitute a new family of stress
proteins based on sequence similarity (44.6% identity) and size (YodA, 216; YrpE,
251; pXO1-130, 237 aa). These three proteins were found to also exhibit sequence
similarity with the 200 aa residue C-terminal part of the streptococcal adhesin,
AdcA (Pukárová et al. 2001), which is a lipoprotein containing a putative metal-
binding site (Dintilhac and Claverys 1997). In addition, YodA exhibits weak sequ-
ence similarity with the N-terminal part (about 200 aa) of the copper-binding protein
amine oxidase, encoded by maoA (Ferianc et al. 1998), of both E. coli (Azakami
et al. 1994) and Klebsiella aerogenes (Sugino et al. 1992).
Further, Rab et al. (2006) showed that the bacterium Rhodobacter capsulatus
B10 in the presence of 150mM CdCl2 induced heat-shock proteins (GroEL and
Dnak), S-adenosylmethinine synthetase, ribosomal protein S1, aspartate amino-
transferase, and phosphoglycerate kinase. Ribosomal protein S1 appeared to be
involve in the repair of cadmium-mediated cellular damage. Five cadmium binding
proteins, including 2-methylcitrate dehydratase, phosphate periplasmic binding
protein, inosine-50 - monophosphate dehydrogenase/guanosine-50 - monophosphate
reductase, inositol monophosphatase, and lytic murein transglycosylase, were also
identified.
5.10.2 Differential Gene Expression Under Cadmium Stress
In Caulobacter crescentus (6M cadmium sulfate), 144 genes were upregulated by

at least twofold under cadmium stress. Several groups of annotated genes are given
in Table 5.2 (Hu et al. 2005).
5.11 Conclusion
Generally, the concentration of heavy metals in soil has increased during the last
few years posing a serious threat to the environment and human health world-
wide. However, due to the fact that large areas of land contaminated with metals
cannot be economically decontaminated by applying conventional chemical
approaches, microbe/plant-mediated approaches can prove to be the best viable
and inexpensive alternative for remediation of heavy metal-polluted sites.
Remediation of metal-polluted soils using biological systems (both plants and
Table 5.2 Selected genes (and description) upregulated under cadmium stress
Gene Fold change Annotation
Efflux pumps (Cluster I)
CC2721 21.3 Outer membrane efflux protein
CC2722 36 Metal ion efflux membrane fusion protein, contains HlyD
domain
CC2723 20 Hypothetical protein
CC2724 22.8 Homologous to nccA and czcA
CC2725 8 Conserved hypothetical protein
CC2726 12.4 Cation transporting P-type ATPase
CC2727 6 Conserved hypothetical protein
Efflux pumps (Cluster II)
CC3195 3.4 Outer membrane efflux protein
CC3196 2.6 Contains HlyD domain
CC3197 3 Cation/multidrug efflux pump, with AcrB/ AcrD/ AcrF
domain
Protect against oxidative stress
CC1777 18.9 Superoxide dismutase (cofactor, Mn2+) (sod A)
CC3557 2.2 Superoxide dismutase (cofactor, Fe2+) (sod B)
CC1316 3.2 Glutathione S-transferase
CC2434 2.2 Glutathione S-transferase
CC0062 2.4 Thioredoxin-like protein
CC0110 2 Thioredoxin
CC3539 2.3 Thioredoxin
CC2505 2.3 Glutaredoxin-related protein
CC0994 2.5 Peptide methinine sulfoxide reductase
CC1039 2.3 Peptide methinine sulfoxide reductase
CC0141 2.3 Glutathione synthetase
CC0885 3.6 Riboflavin biosynthesis protein (rib D)
CC0886 4.3 Riboflavin synthase, alpha subunit (rib E)
CC0887 4 GTP cyclohydrolase II (rib AB)
CC0888 3.6 Riboflavin synthase, alpha subunit (rib E)
CC0459 4.1 GTP cyclohydrolase I (tetrahydrofolate biosynthesis
pathway)
Arsenic resistance
CC1503 4.8 Arsenic reductase (arsC)
CC1504 4.4 Transmembrane channel protein
CC1505 4.4 Transcriptional regulator (arsR)
CC1506 9.9 Arsenic resistant protein
DNA Repair
CC1428 6 Deoxyribodipyrimidine photolyase, removes cyclobutane –
type pyrimidine dimmers in DNA
CC2590 2 Excinuclease ABC, subunit A
Others
CC0260 2.7 Ribonucleotide reductase, alpha subunit
CC3492 2.2 Ribonucleotide reductase, beta subunit
CC2129 4.5 NADH: flavin oxidoreductase
microbes) is an emerging area of interest and has shown a substantial progress in

situ, which needs to be further consolidated through field trials under different
agro-climatic zones of the world. Furthermore, with progress in genomics and
proteomic studies in many laboratories around the world, we may expect this
research area to develop and expand further in the near future, delivering more
robust technologies for the bioremediation of metal-contaminated soils. And,
hence, the molecular engineering of both microbes and plants with desired
genes is likely to help immensely to enhance the efficiency of microbe-mediated
or plant-based remediation of contaminated soils. However, to make remediation
a successful option for detoxifying contaminated sites, some of the problems, like
how the remediation effects will change under field conditions, and how mobili-
zation and transfer of metal to different organs of plants is effected, need to be
critically addressed by the scientists before the potential of bioremediation in
decontaminating polluted sites or reducing the toxicity of metals can be appre-
ciated.
References
Abram F, Su WL, Wiedmann M, Boor KJ, Coote P, Botting C, Karatzas KAJ, O’ Bryne CP (2008)
Proteomic analysis of a Listeria monocytogenes mutant lacking sB identify new components of
the sB regulon and highlight a role for sB in the utilization of Glycerol. Appl Environ
Adriano DC (2001) Trace elements in terrestrial environments: biochemistry, bioavailability and
risks of metals. Springer, New York
Alloway BJ (1995) Soil processes and the behaviour of heavy metals. In: Alloway B (ed) Heavy
metals in soils. Chapman and Hall, New York, pp 11–37
Azakami H, Yamashita M, Roh JH, Suzuki H, Kumagai H, Murooka Y (1994) Nucleotide
sequence of the gene for monoamine oxidase (maoA) from Escherichia coli. J Ferment Bioeng
77:315–319
Bae W, Chen X (2004) Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces
pombe through amino acid-coded mass tagging and liquid chromatography tandem mass
spectrometry. Mol Cell Proteomics 3(6):596–607
Bagdwal N, Gupta A, Goel R (2003) Metal resistant growth promotry fluorescent Pseudomonads.
In: Trivedi PC (ed) Microbial biotechnology. Aavishkar, Jaipur, India, pp 126–161
Baker AJM, Walker PL (1989) Ecophysiology of metal uptake by tolerant plants. In: Shaw A (ed)
Heavy metal tolerance in plants - evolutionary aspects. CRC, Boca Raton, pp 155–177
Beveridge TJ, Murray RGE (1980) Sites of metal deposition in the cell wall of Bacillus subtilis.
J Bacteriol 141:876–887
Blencowe DK, Morby AP (2003) Zn(II) metabolism in prokaryotes. FEMS Microbiol Rev
27:291–311
Borsetti F, Francia F, Turner RJ, Zannoni D (2007) The thiol:disulfide oxidoreductase DsbB
mediates the oxidizing effects of the toxic metalloid tellurite (TeO3 2–) on the plasma
membrane redox system of the facultative phototroph Rhodobacter capsulatus. J Bacteriol
189:851–857
Cavet JS, Borrelly GPM, Robinson NJ (2003) Zn, Cu and Co in cyanobacteria: selective control of
metal availability. FEMS Microbiol Rev 27:165–181
Chang WC, Hsu GS, Chiang SM, Su MC (2006) Heavy metal removal from aqueous solution by
wasted biomass from a combined AS-biofilm process. Bioresource Technol 97:1503–1508
Chen G, Gharib TG, Huang CC, Taylor JM, Misek DE, Kardia SL, Giordano TJ, Iannettoni MD,
Orringer MB, Hanash SM, Beer DG (2002) Discordant protein and mRNA expression in
lungadenocarcinomas. Mol Cell Proteomics 1:304–313
Cho UH, Park JO (2000) Mercury-induced oxidative stress in tomato seedlings. Plant Sci 156:1–9
Choudhury R, Srivastava S (2001) Zinc resistance mechanism in bacteria. Curr Sci 81:768–775
Cunningham SD, Berti WR, Huang JW (1995) Phytoremediation of contaminated soils. Trends
Biotechnol 13:393–397
Di S, Toppi L, Gabbrielli R (1999) Response to cadmium in higher plants. Environ Exp Bot
41:105–130
Dintilhac A, Claverys JP (1997) The adc locus, which affects competence for genetic transforma-
tion in Streptococcus pneumoniae, encodes an ABC transporter with a putative lipoprotein
homologous to a family of streptococcal adhesins. Res Microbiol 148:119–131
Dixit V, Pandey V, Shyam R (2001) Differential antioxidative responses to cadmium in roots and
leaves of pea (Pisum sativum L. cv. Azad). J Exp Bot 52:1101–1109
Dutt MJ, Lee KH (2000) Proteomic analysis. Curr Opin Biotechnol 11:176–179
Ernst WHO (1998) The origin and ecology of contaminated, stabilized and non-pristine soils.
In: Vangronsveld J, Cunningham SD (eds) Metal-contaminated soil. Springer, New York,
pp 17–29
Ferianc P, Farewell A, Nyström T (1998) The cadmium-stress stimulon of Escherichia coli K-12.
Microbiology 144:1045–1050
Ferianc P, Pukárová A, Godoı́ková J, Polek B, Tóth D (2000) The effect of cadmium on
culturability, macromolecule synthesis and protein degradation in a marine Vibrio sp. Biologia
55:653–659
Flathman PE, Lanza GR (1998) Phytoremediation: current views on an emerging green technology.
J Soil Contam 7:415–432
Foulkes EC (1998) Biological membranes in toxicology. Taylor and Francis, Philadelphia
Gadd GM (1988) Accumulation of metals by microorganisms and algae. In: Rehm HJ (ed)
Biotechnology: a comprehensive treatise. VCH Verlagsgesellschaft, Weinheim, pp 401–433
Geslin C, Llanos J, Prieur D, Jeanthon C (2001) The manganese and iron superoxide dismutases
protect Escherichia coli from heavy metal toxicity. Res Microbiol 152:901–905
Glick BR, Pattern CL, Holguin G, Penrose DM (1999) Biochemical and genetic mechanism used
by plant growth promoting bacteria. Imperial College Press, London
Griffin TJ, Gygi SP, Ideker T, Rist B, Eng J, Hood L, Aebersold R (2002) Complementary
profiling of gene expression at the transcriptome and proteome levels in Saccharomyces
cerevisiae. Mol Cell Proteomics 1:323–333
Gygi SP, Rochon Y, Franza BR, Aebersold R (1999) Correlation between protein and mRNA
abundance in yeast. Mol Cell Biol 19:1720–1730
Hall JL (2000) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot
53:1–11
Hamon RE, Mclaughlin MJ, Naidu R, Correll R (1998) Long-term changes in cadmium bioavail-
ability in soil. Envion Sci Technol 32:3699–3703
Harrison JJ, Ceri H, Turner RJ (2007) Multimetal resistance and tolerance in microbial biofilms.
Nature 5:928–938
Heaton ACP, Rugh CL, Wang NJ, Meagher RB (1998) Phytoremediation of mercury- and
methylmercury-polluted soils using genetically engineered plants. J Soil Contam 7:497–509
Higham DP, Sadler PJ, Scawen MD (1984) Cadmium resistance in Pseudomonas putida: growth
and uptake of cadmium. J Gen Microbiol 131:2539–2544
Hu P, Brodie EL, Sujuki Y, McAdams HH, Anderson GL (2005) Whole-genome transcriptional
analysis of heavy metal stresses in Caulobacter crescentus. J Bacteriol 187:8437–8449
Hughes MN, Poole RK (1989) Metals and micro-organisms. Chapman and Hall, New York, NY,
p 290
Humphery-Smith I, Cordwell SJ, Blackstock WP (1997) Proteome research: complementarity and
limitations with respect to the RNA and DNA worlds. Electrophoresis 18:1217–1242
Kabata-Pendias A, Pendias H (2001) Trace elements in soils and plants. CRC, London
Kaewchai S, Praseptsan P (2002) Biosorption of heavy metal by thermotolerant polymer producing
bacterial cells and the bioflocculant. Songklanakarin J Sci Technol 24:421–430
remediation of metal contaminated soils. Environ Chem lett 7:1–19
Kloepper JW, Litshitz R, Zablotowicz RM (1989) Free living bacterial inocula for enhancing crop
producitivity. Trends Biotechnol 7:39–43
Koeppe DE (1981) Lead: understanding the minimal toxicity of lead in plants. In: Lepp NW (ed)
Effect of heavy metal pollution on plants, vol 2. Applied Science, London and New Jersey, pp
55–76
Kunito T, Oyaizu H, Matsumoto S (1998) Ecology of soil heavy metal-resistant bacteria and
perspective of bioremediation of heavy metal-contaminated soils. Rec Res Dev Agric Biol
Chem 2:185–206
Laddaga RA, Silver S (1985) Cadmium uptake in Escherichia coli K-12. J Bacteriol 162:
1100–1105
Lawrence JR, Chenies MR, Roy R, Beaumier D, Fortin N, Swerhone GDW, Neu TR, Greer CW
(2004) Microscale and molecular assessment of impacts of nickel, nutrients, and oxygen level
on structure and function of river biofilm communities. Appl Environ Microbiol 70:4326–4339
Lloyd JR, Lovley DR (2001) Microbial detoxification of metals and radionuclides. Curr Opin
Lohmeier-Vogel EM, Ung S, Turner RJ (2004) In vivo 31P nuclear magnetic resonance investiga-
tion of tellurite toxicity in Escherichia coli. Appl Environ Microbiol 70:7342–7347
Lovely DR, Coates JD (1997) Bioremediation of metal contamination. Curr Opin Biotechnol
8:285–289
Lozano-Rodriguez E, Hernandez LE, Bonay P, Carpena-Ruiz RO (1997) Distribution of cadmium
in shoot and root tissues of maize and pea plants: physiological disturbances. J Exp Bot
306:123–128
Mahesh S, Ramesh HS, Sudhir HS, Kumar BR (2001) Microbial scavengers-fungi for Cu+2
removal from industrial waste water. J Ind Pollut Control 17:135–140
Mullen MD, Wolf DC, Ferris FC, Beveridge TJ, Flemming CA, Bailey FW (1989) Bacterial
sorption of heavy metals. Appl Environ Microbiol 55:3143–3149
Mulligan CN, Yong RN, Gibbs BF (2001) Remediation technologies for metal contaminated soils
and groundwater: an evaluation. Eng Geol 60:193–207
Munoz R, Alvarez MT, Munoz A, Terrazas E, Guieysse B, Mattiasson B (2006) Sequential
removal of heavy metal ions and organic pollutants using an algal-bacterial consortium.
Naidu R, Oliver D, McConnell S (2003) Heavy metal phytoxicity in soils. In: Proceedings of the
Fifth National Workshop on the assessment of site contamination. Adelaide, May 2002
Naz N, Young HK, Ahmed N, Gadd GM (2005) Cadmium accumulation and DNA homology with
metal resistance genes in sulfate-reducing bacteria. Appl Environ Microbiol 71:4610–4618
Neilands JB (1983) Siderophores. In: Eichhorn L, Marzilli LG (eds) Advances in inorganic
biochemistry, vol 5. Elsevier, Amsterdam, pp 137–166
Nies DH (1995) The cobalt, zinc, and cadmium efflux system CzcABC from Alcaligenes eutro-
phus functions as a cation-proton antiporter in Escherichia coli. J Bacteriol 177:2707–2712
Okinaka RT, Cloud K, Hampton O et al (1999) Sequence and organization of pXO1, the large
Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol 181:6509–6515
Osborn AM, Bruce KD, Strike P, Ritchie DA (1997) Distribution, diversity and evolution of the
bacterial mercury resistance (mer) operon). FEMS Microbiol Rev 19:239–262
Pandey A, Mann M (2000) Proteomics to study genes and genomes. Nature 405:837–846
Pandey A, Nigam P, Singh D (2001) Biotechnological treatment of pollutants. Chem Ind Digest
14:93–95
Pazirandeh M, Wells BM, Ryan RL (1998) Development of bacterium-based heavy metal biosor-
bents: enhanced uptake of cadmium and mercury by Escherichia coli expressing a metal
binding motif. Appl Environ Microbiol 64:4068–4072
Perry RD, Silver S (1982) Cadmium and manganese transport in Staphylococcus aureus
membrane vesicles. J Bacteriol 150:973–976
Poole RK, Gadd GM (1989) Metal-microbe interaction. IRL, Oxford
Pukárová A, Janeek S, Ferianc P, Polek B (2001) Putative Cd-stress proteins YodA, YrpE and
pXO1–130 share sequence similarity with adhesin AdcA. Biologia 56:337–339
Rab MFGS, Abdel FSA, Fukumori Y (2006) Effects of cadmium stress on growth, morphology
and protein expression in Rhodobacter capsulatus B10. pp. Biosci Biotechnol Biochem
70:2394–2402
Rani A, Shouche Y, Goel R (2008) Declination of copper toxicity in pigeon pea and soil system by
growth promoting Proteus vulgaris KNP3 strain. Curr Microbiol 57:78–82
Rani A, Shouche Y, Goel R (2009) Comparative assessment for in situ bioremediation potential of
cadmium resistant acidophilic Pseudomonas putida 62BN and alkalophilic Pseudomonas
monteilli 97AN strains on soybean. Int J Biodet Biodegrad 63:62–66
Rauser WE (1991) Cadmium-binding peptides from plants. Methods Enzymol 205:319–333
Rawlings DE, Johnson DB (2007) The microbiology of biomining: development and optimization
of mineral oxidizing microbial consortia. Microbiology 153:315–324
Romero-Puertas MC, Palma JM, Gomez M, Del Ro LA, Sandalio LM (2002) Cadmium causes the
oxidative modification of proteins in pea plants. Plant Cell Environ 25:677–686
Ross S (1994) Toxic metals in soil-plant systems. Wiley, Chichester, UK
Rough DA, Lee BTO, Morby AP (1995) Understanding celllular responses to toxic agents: a
model for mechanism-choice in bacterial metal resistance. J Ind Microbiol 14:132–141
Salt DE, Blaylock M, Kumar NPBA, Dushenkov V, Ensley BD, Chet I, Raskin I (1995) Phytor-
emediation: a novel strategy for the removal of toxic metals from the environment using plants.
Biotechnology 13:468–474
Sandalio LM, Dalurzo HC, Gomez M, Romero-Puertas MC, Del Ro LA (2001) Cadmium-induced
changes in the growth and oxidative metabolism of pea plants. J Exp Bot 52:2115–2126
Schützendübel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxida-
tive stress and protection by mycorrhization. J Exp Bot 53:1351–1365
Schützendübel A, Schwanz P, Teichmann T, Gross K, Langenfeld-Heyser R, Goldbold DL, Polle
A (2001) Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and
differentiation in Scots pine roots. Plant Physiol 127:887–898
Sengar RS, Pandey M (1996) Inhibition of chlorophyll biosynthesis by lead in greening Pisum
sativum leaf segments. Biol Plant 38:459–462
Silver S, Walderhaug M (1992) Gene-regulation of plasmid-determined and chromosome-deter-
mined inorganic ion transport in bacteria. Microbiol Rev 56:195–228
Singh R, Paul D, Jain R (2006) Biofilms: implications in bioremediation. Trends Microbiol
14:389–397
Sorokin A, Bolotin A, Purnelle B, Hilbert H, Lauber J, Dusterhoft A, Ehrlich SD (1997) Sequence
of the Bacillus subtilis genome region in the vicinity of the lev operon reveals two new
extracytoplasmic function RNA polymerase sigma factors SigV and SigZ. Microbiology
143:2939–2943
Stiborova M, Doubravova M, Leblova SC (1986) Comparative study of the effect of heavy metal
ions on ribulose-1, 1-biphosphate carboxylase and phosphoenolpyruvete carboxylase. Bio-
chem Physiol Pflanzen 181:373–379
Stohs SJ, Bagchi D (1995) Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol
Med 18:321–336
Sugino H, Sasaki M, Azakami H, Yamashita M, Murooka Y (1992) A monoamine-regulated

Klebsiella aerogenes operon containing the monoamine oxidase structural gene (maoA) and
the maoC gene. J Bacteriol 174:2485–2492
Tripathi M, Munot HP, Shouche Y, Meyer JM, Goel R (2005) Isolation and functional characteri-
zation of siderophore producing lead and cadmium resistant Pseudomonas putida KNP9. Curr
Tyers M, Mann M (2003) From genomics to proteomics. Nature 422:193–197
Van Assche F, Clijsters H (1990) Effects of metals on enzyme activity in plants. Plant Cell Environ
13:19–206
VanBogelen RA, Kelley PM, Neidhardt FC (1987) Differential induction of heat shock, SOS, and
oxidation stress regulons and accumulation of nucleotides in Escherichia coli. J Bacteriol
169:26–32
VanBogelen RA, Abshire KZ, Pertsemlidis A, Clark RL, Neidhardt FC (1996) Gene-protein
database of Escherichia coli K-12. In: Neidhardt FC (ed) Escherichia coli and Salmonella.
American Society for Microbiology, Washington, DC, pp 2067–2117
Vasudevan P, Padmavathy V, Tewari N, Dhingra SC (2001) Biosorption of heavy metal ions. J Sci
Ind Res 60:112–120
Vilchez R, Pozo C, Gomez MA, Rodelas B, Gonzalez-Lopez J (2007) Dominance of sphingomo-
nads in a copper exposed biofilm community for groundwater treatment. Microbiology
153:325–337
Volesky B (1990) Biosorption of heavy metals. CRC, Boca Raton
Wang V, Brown HN, Crowley DE, Szaniszlo PJ (1993) Evidence for direct utilization of side-
rophore, ferroxamine B in axenically grown cucumber. Plant Cell Environ 16:579–585
Wang CL, Michel PC, Dawson SC, Kitisakkul S, Baross JA, Keasling JD, Clark DS (1997)
Cadmium removal by a new strain of Pseudomonas aeruginosa in aerobic culture. Appl
Environ Microbiol 63:4075–4078
Wani PA, Khan MS, Zaidi A (2007a) Cadmium, chromium and copper in greengram plants. Agron
Sustain Dev 27:145–153
Wani PA, Khan MS, Zaidi A (2007b) Impact of heavy metal toxicity on plant growth, symbiosis,
seed yield and nitrogen and metal uptake in chickpea. Aust J Exp Agric 47:712–720
Wani PA, Khan MS, Zaidi A (2007c) Chromium reduction, plant growth-promoting potentials,
and metal solubilization by Bacillus sp. isolated from alluvial soil. Curr Microbiol 54:237–243
Wani PA, Khan MS, Zaidi A (2007d) Effect of metal tolerant plant growth promoting Bradyrhi-
zobium sp. (vigna) on growth, symbiosis, seed yield and metal uptake by greengram plants.
Wani PA, Khan MS, Zaidi A (2008a) Effects of heavy metal toxicity on growth, symbiosis, seed
yield and metal uptake in pea grown in metal amended soil. Bull Environ Contam Toxicol
81:152–158
Wani PA, Khan MS, Zaidi A (2008b) Chromium reducing and plant growth promoting Mesorhi-
zobium improves chickpea growth in chromium amended soil. Biotechnol Lett 30:159–163
Wani PA, Khan MS, Zaidi A (2008c) Effect of metal tolerant plant growth promoting Rhizobium
on the performance of pea grown in metal amended soil. Arch Environ Contam Toxicol 55:
33–42
Yoshida N, Kato T, Yoshida T, Ogawa K, Yamashita M, Murooka Y (2002) Bacterium-based
heavy metal biosorbents: enhanced uptake of cadmium by Escherichia coli expressing a
metallothionein fused to b-galactosidase. Biotechniques 32:551–558
Zannoni D, Borsetti F, Harrison JJ, Turner RJ (2007) The bacterial response to the chalcogen
metalloids Se and Te. Adv Microb Physiol 53:1–71
Chapter 6
Functional Diversity Among Plant
Growth-Promoting Rhizobacteria:
Current Status
Mohammad Saghir Khan, Almas Zaidi, P. A. Wani,

Munees Ahemad, and Mohammad Oves
Abstract Root-colonizing bacteria (rhizobacteria) that exert beneficial effects on

plant development via direct or indirect mechanisms have been defined as plant
growth-promoting rhizobacteria (PGPR). These natural bioresources provide
essential nutrients to plants and improve growth, competitiveness, and responses to
external stress factors by an array of mechanisms under different agro-ecosystems.
The PGPR facilitate plant growth by synthesizing or altering: the concentration of
phytohormones: asymbiotic and symbiotic N2 fixation, antagonism against phyto-
pathogenic microorganisms by producing siderophores, antibiotics and cyanide;
solubilization of mineral phosphates and other nutrients; and by synthesizing
1-aminocyclopropane-1-carboxylate (ACC) deaminase, which helps to reduce the
inhibitory effects of ethylene on plants. And hence, use of such PGPR may be a viable
alternative to chemical fertilizers for increasing the productivity of various crops.
However, despite their proven ability of growth promotion, PGPR have yet to fulfil
their promise and potential as commercial bioinoculants. Understanding functional
diversity of PGPR is vital for low-input sustainable production. Recent progress
focusing on the principles and mechanisms of action of PGPR is reviewed and
discussed.
6.1 Introduction
Growth of plants is influenced by a myriad of abiotic and biotic factors. In agricul-

tural practices, in order to manage the soil environment and consequently to
improve crop yields, growers routinely adopt physical and chemical approaches.
However, these approaches are not only expensive but their excessive and repeated
M.S. Khan (*), A. Zaidi, P.A. Wani, M. Ahemad, and M. Oves

Faculty of Agricultural Sciences, Department of Agricultural Microbiology, Aligarh Muslim
University, Aligarh, U.P., India
106 M.S. Khan et al.
use may also deplete soil fertility. An alternative to these physico-chemical methods
is the exploitation of naturally occurring microbes whose application, however, is
inexpensive but less common. These microbes thrive well in the region around the
root (the rhizosphere) which is relatively rich in nutrients, due to the accumulation
of plant photosynthates, released from the roots of different plants. These photo-
synthates support microbial communities of soils which may exhibit beneficial,
neutral, or deleterious effects on plant growth. Beneficial rhizobacteria capable of
aggressively colonizing the rhizosphere and facilitating plant growth are often
termed plant growth-promoting rhizobacteria (PGPR) (Kloepper and Schroth
1978) or plant health-promoting rhizobacteria (PHPR) according to their mode of
action (Sikora 1992). In contrast, deleterious rhizobacteria are presumed to ad-
versely affect plant growth and development through the production of undesirable
metabolites (phytotoxins) or through competition for nutrients or inhibition of the
other beneficial effects (Sturz and Christie 2003). In order to exert their effect on
plants, PGPR must (1) be able to colonize the root, (2) survive and multiply in the
rhizosphere or within plant tissues, and (3) facilitate plant growth (Barea et al.
2005). These PGPR have been found to facilitate plant growth in laboratory and
greenhouse environments, but responses have been variable in the field. Although
many studies have been conducted to identify the specific traits by which PGPR
promote plant growth, the majority of them have focused on one or two of these
traits. Moreover, the presence of a PGPR trait in vitro does not guarantee that a
particular isolate is a PGPR. The mechanisms by which PGPR enhance plant
growth are not fully understood, but are believed to include: the ability to produce
or change the concentration of the plant hormones, such as indoleacetic acid
(Ahmad et al. 2008), gibberellic acid (Mahmoud et al. 1984), and cytokinins
(Frankenberger and Arshad 1995) and ethylene (Glick 1995; Garcia de Salamon
et al. 2001); N2 fixation (Zaidi 1999; Wani et al. 2007a); antagonism against
phytopathogenic microorganisms (Khan et al. 2002) by production of siderophores
(Wani et al. 2007b), b-1,3-glucanase (Flaishman et al. 1996), chitinases (Renwick
et al. 1991), antibiotics (Shanahan et al. 1992), and cyanide (Wani et al. 2007a); and
solubilization of inorganic phosphates and toxic metals (Wani et al. 2007b, 2007c).
Due to these properties, these beneficial microbes have become a potential compo-
nent in management practices to achieve the optimum yield. This chapter surveys
the developments in the functional diversity among PGPR that could help to
develop a bioinoculant possessing multifaceted activity for use in diverse agro-
ecological regions of the world.
6.2 Rhizosphere and Plant Growth Promoting Rhizobacteria
The rhizosphere can be defined as any volume of soil specifically influenced by

plant roots and/or in association with root hairs and plant-produced materials
(Bringhurst et al. 2001). This space includes soil bound by plant roots, often
extending a few millimeters from the root surface, and can include the plant root
6 Functional Diversity Among Plant Growth-Promoting Rhizobacteria: Current Status 107
epidermal layer (Mahaffee and Kloepper 1997). Plant exudates in the rhizosphere,
such as amino acids and sugars, provide a rich source of energy and nutrients for
rhizosphere microbes including PGPR, resulting in bacterial populations greater in
this area than outside the rhizosphere. Broadly, PGPR can be divided into two
major groups according to their relationship with the host plants: (1) symbiotic
bacteria, and (2) free-living rhizobacteria (Khan 2005). Somers et al. (2004) have,
however, classified PGPR into the following functional groups depending on their
inherent activities: (1) biofertilizers (capable of accelerating the accessibility of
nutrients to the plant); (2) phytostimulators (capable of facilitating the plant growth
usually by synthesizing phytohormones); (3) rhizoremediators (involved in the
degradation of organic pollutants); and (4) biopesticides (capable of managing
plant diseases by the production of antimicrobial metabolites). Furthermore,
based on their localization, PGPR can be: (1) intracellular PGPR (iPGPR) which
are bacteria residing inside plant cells, producing nodules and being localized
inside those specialized structures (e.g., nodules); and (2) extracellular PGPR
(ePGPR) which are those bacteria living outside plant cells and not producing
nodules, but enhancing plant growth through production of signal compounds that
directly stimulate growth, improve disease resistance, or nutrient status of soil. The
ePGPR has further been subdivided into three types, based on the degree of
association with plant roots: (1) those living near, but not in contact with the
roots; (2) those colonizing the root surface; and (3) those living in the spaces
between cells of the root cortex. Of these PGPR, iPGPR are mostly Gram-negative
and rod-shaped, with a few bacterial populations being Gram-positive rods, cocci
and pleomorphic forms. Generally, iPGPR include the members of rhizobiace,
capable of forming nodules on the root systems of leguminous plants. In contrast,
some of the agronomically important ePGPR include genera such as Bacillus
(Ryder et al. 1999), Pseudomonas (De Freitas and Germida 1991) Erwinia (Nelson
1998), Enterobacter (Tannii et al. 1990), Caulobacter, Serratia (Zhang et al.
1996), Flavobacterium (Tannii et al. 1990), Actinobacter sp. (Tannii et al. 1990),
Aeromonas (Inbar and Chet 1991), Agrobacterium (Ryder and Jones 1990),
Alcaligenes sp. (Yuen et al. 1985), Phyllobacterium sp. (Lambert et al. 1990), and
Bacillus (Bai et al. 2002), Hyphomycrobium, Azotobacter, Azospirillum, and
Acetobacter (Prithiviraj et al. 2003).
6.3 Search for Plant Growth-Promoting Rhizobacteria
Microbial communities in general, and PGPR in particular, form an important

component of soil and help in predicting the changes in soils, as they affect the
physico-chemical properties of soil. These microbes can be identified using physio-
logical and biochemical tests, which require a regular subculturing. The commu-
nity structure of PGPR in general can be characterized using phenotypic and
genotypic approaches. Of these, phenotypic methods include standard plating
methods on selective media, community level physiological profiles (CLPP)
using the BIOLOG system (Garland 1996), and phospholipid fatty acid (PLFA) and
fatty acid methyl ester (FAME) profiling (Germida et al. 1998). Moreover, the
culture-independent molecular techniques that are based on direct extraction of
DNA from soil, and 16S-rRNA gene sequence analysis, bacterial artificial chromo-
some, or expression cloning systems, (Rondon et al. 1999) have allowed the easier
identification and determination of the potentials they possess. These approaches
can also be used to determine the impact of inoculation of PGPR on the rhizosphere
community (Steddom et al. 2002). Yet one of the challenges in developing PGPR
for commercial application is ensuring its effective selection and screening proce-
dure, so that the most promising PGPR with multiple traits are identified and raised.
However, no efficient high-throughput assays to select the best PGPR are currently
available. Various approaches for initial selection and screening of rhizobacterial
isolates include host plant specificity, adaptation to a particular soil, and screening
assays (Bowen and Rovira 1999). Furthermore, the functional properties associated
with PGPR, like root colonization, synthesis of IAA, solubilization of insoluble P,
synthesis of ACC deaminase, and antibiotics and siderophores, have also been used
to characterize these PGPR. Moreover, the impact of pollutants including heavy
metals on these rhizosphere microbes in metal-stressed soils can be assessed using
the signature biomarkers such as nucleic acid and fatty acids. The development of
methods for direct extraction of nucleic acids and fatty acids from both contami-
nated and nonpolluted soil samples in combination with recombinant DNA and
molecular phylogeny methods have provided a new insight into the identification of
specific bacterial strains.
6.4 Mechanism of Growth Promotion by Plant

Growth-Promoting Rhizobacteria
PGPR can affect plant growth either indirectly or directly (Glick et al. 1999; Antoun
and Prévost 2006). The indirect promotion of plant growth occurs when PGPR
lessen or prevent the deleterious effects of one or more phytopathogenic organisms
by: synthesizing antibiotics; depletion of iron from the rhizosphere; induced sys-
temic resistance; synthesis of antifungal metabolites; production of fungal cell wall
lysing enzymes; competition for sites on the root; stimulation of other beneficial
symbioses; and by decreasing the toxicity of hazardous substances in contaminated
soils. While using direct mechanisms, PGPR promotes the growth of plants by
either providing plants with a compound synthesized by the bacterium or faci-
litating the uptake of certain nutrients from the environment, iron sequestration
by siderophores, the production of bacterial volatiles and phytohormones, and
lowering the ethylene level in plants. Thus, PGPR functions in three different
ways: (1) synthesizing particular compounds for uptake by plants (Zaidi et al.
2003; Zaidi and Khan 2006; Khan et al. 2007); (2) facilitating the uptake of certain
nutrients from environment (Lucas Garcı́a et al. 2004; Çakmakçi et al. 2006); and
(3) protecting plants from diseases (Guo et al. 2004; Pandey et al. 2006; Trivedi
et al. 2008). Regardless of the mechanisms of plant growth promotion, PGPR must
colonize the rhizosphere around the roots, the rhizoplane or the root itself (Glick
1995). In general, PGPR improves plant growth by synthesizing phytohormones
precursors (Wani et al. 2007a, b; Ahmad et al. 2008), vitamins, enzymes, and
siderophores (Wani et al. 2008a), as well as antibiotics (Burd et al. 2000; Glick
2001), and by inhibiting ethylene synthesis (Glick et al. 2007), in addition to their
ability to fix atmospheric N (N2 fixers) and to solubilize inorganic P (Khan et al.
2007), and to make these elements accessible to plants (Perveen et al. 2002;
Wani et al. 2007a, b; Khan and Zaidi 2007), to mineralize organic phosphate
(Ponmurugan and Gopi 2006), and to improve plant stress tolerance to drought,
salinity and metal toxicity (Wani et al. 2008b). Biochemical and molecular
approaches are providing new insights into the genetic basis of these traits, the
biosynthetic pathways involved, their regulation, and their importance for bio-
logical control in laboratory and field studies. An overview of the plant growth
promotion by PGPR is presented in Fig.6.1, while the growth-promoting substances
synthesized by various PGPR are summarized in Table 6.1.
Use of such microbes possessing multiple traits, including their role in metal
resistance/reduction and ability to promote plant growth in metal-contaminated
soils also make them one of the most suitable choices for bioremediation. Among
other PGPR, the symbiotic nitrogen fixers enhance the growth of legumes by: (1)
biological N2 fixation, (2) increasing the availability of nutrients in the rhizosphere,
(3) inducing increases in root surface area, (4) enhancing other beneficial symbioses
of the host, (5) reducing or preventing the deleterious effects of phytopathogenic
Phytohormones Siderophores
Symbiotic
N2 fixers
Pseudomonas tolaasii
ACC Antibiotics
deaminase HCN/NH4+
Bradyrhizobium
Mesorhizobium
Pseudomonas
Azospirillum
Rhizobium
Insoluble P
N2 fixation
Mechanism of growth Bacteria/Fungi P-Solubilization

promotion by PGPR Actinomycetes
Microbacterium
Sphingomonas
Azotobacter
Clostridium
Organic P
ASymbiotic
N2 fixers
Nutrients
Metal detoxification mineralization
Reduction Adsorption Solubilization
Fig. 6.1 Functional diversity among plant growth-promoting rhizobacteria

Table 6.1 Growth-promoting substances produced by plant growth-promoting rhizobacteria

Organisms Growth regulators Reference
Azotobacter, fluorescent IAA, siderophore, ammonia, Ahmad et al. (2008)
Pseudomonas, Bacillus HCN, P-solubilization
Pantoea dispers strain 1A P-solubilization, IAA, Selvakumar et al. (2008)
siderophores, HCN
Bacillus spp. IAA, siderophore, HCN Wani et al. (2007b)
Pseudomonas, Bacillus Siderophores, IAA, Rajkumar et al. (2006)
P-solubilization
Brevibacillus sp. IAA Vivas et al. (2006)
Xanthomonas sp. RJ3, IAA Sheng and Xia (2006)
Azomonas sp. RJ4,
Pseudomonas sp. RJ10,
Bacillus sp. RJ31
Bacillus sp. P-solubilization Canbolat et al. (2006)
Brevibacterium sp. Siderophore Noordman et al. (2006)
Bacillus subtilis IAA, P-solubilization Zaidi et al. (2006)
Variovorax paradoxus, IAA, siderophores Belimov et al. (2005)
Rhodococcus sp. and
Flavobacterium (Cd tolerant)
Pseudomonas fluorescens IAA, siderophore, Gupta et al. (2005)
P-solubilization
Pseudomonas putida Siderophore Tripathi et al. (2005)
Azotobacter, Pseudomonas IAA Ahmad et al. (2005)
fluorescens
Bacillus, Azospirillum sp. IAA, P-solubilization Yasmin et al. (2004)
Pseudomonas aeruginosa IAA, siderophore, HCN Bano and Musarrat (2003)
Bacillus, Pseudomons, P-solubilization, IAA Tank and Saraf (2003)
Azotobacter,
Azospirillum
Pseudomonas sp. Siderophore Sharma et al. (2003)
Pseudomonas sp. IAA, siderophore, Gupta et al. (2002)
P-solubilization
Pseudomonas fluorescens Siderophore Khan et al. (2002)
Azotobacter chroococcum Gibberellin, kinetin, IAA Verma et al. (2001)
Kluyvera ascorbata Siderophore Burd et al. (2000)
organisms (Khan et al. 2002), and (6) combination of modes of action. As an

example of plant growth promoter, IAA, phytohormone of the auxin series pro-
duced by many rhizobia (Abd-Alla 1994; Wani et al., 2007a, 2007b, 2008a, 2008b,
2008c), and its metabolically related precursor, anthranilic acid, can reductively
solubilize soil Fe (III), and increase its availability via a mechanism different from
that involving siderophores. Further plant growth-promoting substances, sidero-
phores, are specific Fe (III)-chelating agents that make the chelated iron unavailable
to pathogenic organisms thereby leading to an increase in plant health. Microbial
siderophores are known to regulate the availability of Fe in the plant rhizosphere,
and it has been found that competition for iron in the rhizosphere is controlled
by the affinity of the siderophores for iron. Interestingly, the binding affinity of
phyto-siderophores for iron is less than the affinity of microbial siderophores, but
plants require a lower iron concentration for normal growth than do microbes.
Although several rhizobial species are known to produce growth-promoting sub-
stances under metal-free environment, the syntheses of these compounds by metal-
tolerant rhizobia have been limited. Nevertheless, there has been certain evidence
where metals at lower concentrations exert no harmful effect and rather stimulate
plant growth promoting activities of rhizobia. For instance, Bradyrhizobium
(RM8), tolerant to nickel and zinc, Rhizobium sp. (RL9), tolerant to zinc and
Rhizobium sp. (RP5), tolerant to zinc and nickel, produced substantial amounts of
IAA (Wani et al. 2008a, c) under metal-stressed conditions. The production of
growth promoting substances by metal-tolerant and natural rhizobial strains are
presented in Table 6.2.
6.4.1 Plant Growth Regulators
Plant growth regulators (PGRs) are the substances that influence physiological
processes of plant at very low concentrations and modify or control one or more
specific metabolic events of a plant. According to the Environmental Protection
Agency (EPA), the plant regulators have been defined as “any substance or mixture
of substances intended, through physiological action, to accelerate or retard the rate
of growth or maturation, or otherwise alter the behavior of plants or their produce.”
Such compounds produced by the plant or by PGPR are called plant hormones
Table 6.2 Plant growth-promoting substances synthesized by symbiotic nitrogen fixers

Symbiotic N2 fixers Plant growth-promoting substances References
Mesorhizobium IAA, siderophore, Ahmad et al. (2008)
ammonia, HCN,
P-solubilization
Bradyrhizobium IAA, siderophores, HCN Wani et al. (2008a)
Mesorhizobium IAA, siderophores Wani et al. (2008c)
Rhizobium spp. IAA, siderophores Sridevi et al. (2008a, b)
Rhizobium spp. P-solubilization Sridevi et al. (2007)
Bradyrhizobium japonicum IAA Shaharoona et al. (2006)
Rhizobium HCN, siderophore Deshwal et al. (2003)
Bradyrhizobium (Arachis) Siderophore, IAA, Deshwal et al. (2003)
P-solubilization
Rhizobium P-solubilization, IAA Tank and Saraf (2003)
Mesorhizobium, Siderophore Khan et al. (2002)
Bradyrhizobium sp. (vigna)
Rhizobium meliloti Siderophore Arora et al. (2001)
Bradyrhizobium, Rhizobium IAA, HCN, siderophore Antoun et al. (1998)
Bradyrhizobium, Rhizobium Siderophore Duhan et al. (1998)
Rhizobium ciceri Siderophore Berraho et al. (1997)
Bradyrhizobium japonicum Siderophore Wittenberg et al. (1996)
Rhizobium, Bradyrhizobium P-solubilization Abd-Alla (1994)
(Davies 1995; Karadeniz et al. 2006). The phytohormones and other compounds
synthesized by PGPR are now reviewed and discussed.
6.4.1.1 Phytohormones
Indoleacetic acid is commonly produced by PGPR by using the rich supplies of

substrates exuded from the roots and it releases auxin in the rhizospheres as
secondary metabolites. Among the PGPR, N2-fixing bacteria in general are
known exclusively for their N2-fixing ability, yet they are also reported to produce
IAA (Table 6.3). For example, species of Bradyrhizobium, Rhizobium and Mesor-
hizobium produced a substantial amount of IAA under in vitro conditions (Antoun
et al. 1998; Wani et al. 2008a, 2008b, 2008c; Ahmad et al. 2008). Among other
PGPR strains, Pseudomonas, Bacillus, Agrobacterium sp., Alcaligenes piechaudii
and two strains of Comamonas acidovorans secreted IAA at lower levels as
compared to deleterious bacteria (Barazani and Friedman 1999; Rajkumar et al.
2006). Bacteria associated with the roots of greenhouse tropical orchids have also
been shown to produce IAA as demonstrated by thin layer chromatopraphy (TLC)
and by biotests (Tsavkelova et al. 2005). In another study, numerous bacterial
isolates recovered from wheat (Triticum aestivum) rhizosphere demonstrated the
production of auxins (ranging from 1.1 to 12.1 mg l 1) under in vitro conditions.
However, when the medium was supplemented exogenously with tryptophan, it
significantly enhanced the auxin biosynthesis which was confirmed by high perfor-
mance liquid chromatography (HPLC) analysis (Khalid et al. 2004).
6.4.1.2 Biosynthesis of Indole Acetic Acid
Indole-3-acetic acid and its analog are the primary active auxin in most plants. They
are synthesized from tryptophan, primarily in leaf primordial, young leaves and
developing seeds. Auxin plays an important role in the development of roots
including root initiation, cell enlargement and cell division (Fig.6.2). It has been
shown that free IAA is easily converted into esterified IAA with sugar or amide-
linked IAA, and that such conjugated forms are the forms in which IAA is stored in
plants. Two kinds of genes that are involved in the formulation of conjugated IAA
and the hydrolysis of IAA have been identified (Bartel and Fink 1995). However,
the biosynthetic process of IAA in plants at the molecular level have not yet been
characterized due to several reasons: (1) levels of IAA in intact cells are low, (2)
indole compounds are nonenzymatically degraded, (3) bacterial contamination can
complicate assays of enzymatic activity, and (4) compartmentalization of cells is
disrupted under in vitro conditions. Indoleacetic acid is also important for the
microbes that interact with plants, and the biosynthesis of IAA has been assayed
mainly for plant-associated bacteria. At the molecular level, two pathways of IAA
production have been identified: (1) the indole-3-pyruvic acid pathway, reported in
PGPR, Enterobacter cloacae, Rhizobium and Bradyrhizobium, and the (2) indole
acetamide (IAM) pathway, which is often found in tumor-forming bacteria, such as

Pseudomonas syringae pv. savastanoi and Agrobacterium, for which genes are
plasmid-borne.
E. cloacae, isolated from the cucumber (Cucumis sativus) rhizosphere, has
shown the secretion of IAA in the culture medium by the indole-3-pyruvic acid
pathway (Koga et al. 1991). Interestingly, the cloned E. coli possessing IAA genes
of E. cloacae produced large amounts of IAA, even though three different enzymes
in the indole-3-pyruvic acid pathway are involved. Moreover, the gene (ipdc)
transformed from E. cloacae did not encode tryptophan aminotransferase, regarded
as the enzyme that catalyzes the rate-limiting step in the IAA synthesis. The gene
encoded indole pyruvate decarboxylase, whose detection has been very difficult.
The IAM pathway was first reported in P. syringae pv. savastanoi, which induces
the production of tumorous outgrowths on olive (Olea europaea) and oleander
(Nerium oleander) plants. The pathway depends on the products of two genes, iaaM
and iaaH. The iaaM gene encodes tryptophan 2-monooxygenase, which catalyzes
the conversion of L-tryptophan to IAM, while iaaH encodes IAM hydrolase, which
catalyzes the conversion of IAM to IAA. Induction of tumor formation by
P. syringae pv. savastanoi on its host plants requires the overproduction of IAA.
This pathway has also been reported for other tumor-forming bacteria and Erwinia
herbicola pv. gypsophilae (Clark et al. 1993).
6.4.2 Siderophores
Iron plays an important role in various biochemical and physiological processes,

such as respiration, photosynthetic transport, nitrate reduction, chlorophyll synthesis,
and N2 fixation (Robinson and Postgate 1980). Iron also acts as a cofactor or is
required for proper functioning of enzymes and proteins (e.g., peroxidase, POX,
superoxide dismutase, nitrogenase, glutamate synthase, ribonucleotide, diphos-
phate reductase, aconitase, cytochromes, ferrodoxin, and flavoproteins) that facili-
tate electron transport, oxygen transport and other life-sustaining process. It exists
in aerobic soil and water environments in the Fe3+ state, most insoluble at physio-
logical pH (Crowley et al. 1987). A level of at least 1M iron is required for optimum
growth, and if greater than 1M, it is an iron-stressed condition (Ownley et al. 2003).
The limitation of iron can inhibit growth, decrease genetic materials and inhibit
sporulation, and can also change the cell morphology. The environmental restric-
tions and biological imperatives, therefore, require that microorganisms form this
specific nutrient, which is, though, abundant but essentially unavailable (Leeman
et al. 1996). Generally, all aerobic and facultative anaerobic prokaryotes and some
plants produce low molecular weight compounds to provide themselves with iron.
The low molecular mass (0.5–1.5 kDa) ferric-specific iron-chelator compounds,
often called siderophores (iron bearers) (Nielsen and Sorensen 2003), are pro-
duced by PGPR. More than 500 different siderophores have been identified from
microorganisms, and some bacteria produce more than one type of siderophores.
Table 6.3 Plant growth-promoting rhizobacteria used in bioremediation

Bacteria Plant Heavy Conditions Role of References
metals PGPR
Methylobacterium Lycopersicon Ni, Cd Gnotobiotic Madhaiyan
oryzae, esculentom and pot et al.
Berknolderia sp. culture (2007)
experiments
Bacillus megaterium Brassica Pb, Zn Experiments in Protected plant Wu et al.
HKP-1 Juncea greenhouse from metal (2006a)
toxicity
Bacillus subtilis Brassica Ni Experiments Facilitated Ni Zaidi et al.
SJ-101 Juncea in growth accumulation (2006)
chamber
Xanthomonas sp. Brassica Cd Experiment in Stimulated plant Sheng and
RJ3, Azomonas napus pots growth and Xia
sp. RJ4, increased (2006)
Pseudomonas sp. cadmium
RJ10, accumulation
Bacillus sp. RJ31
Pseudomonas sp, Mustard Cr (VI) Pot experiment Stimulated plant Rajkumar
Bacillus sp. growth and et al.
decreased Cr (2006)
(VI) content
Ochrobactrum, Mungbean Cr (VI) Experiment Lowers the Faisal and
Bacillus cereus in pots toxicity of Hasnain
chromium to (2006)
seedlings by
reducing Cr
(VI) to Cr
(III)
Brevibacillus Trifolium Zn Pot experiment Enhanced plant Vivas et al.
repens growth and (2006)
nutrition of
plants and
decreased
zinc
concentration
in plant
tissues
Variovox paradoxus, Brassica Cd Experiment in Stimulating root Belimov
Rhodococcus sp, juncea Petri dishes elongation et al.
Flavobacterium (2005)
Pseudomas Soybean Hg Experiment in Increased plant Gupta et al.
fluorescens greenhouse growth (2005)
Ochrobactrum Sunflower Cr (VI) Experiment in Increased plant Faisal and
intermedium pots growth and Hasnain
decreased Cr (2005)
(VI) uptake
Kluyvera ascorbata Indian Ni, Pb, Experiments Both strains Burd et al.
SUD165 mustard Zn in growth decresed (2000)
K. ascorbata Canola, chamber some plant
SUD165 tomato growth
inhibition by
(continued)
Table 6.3 (continued)

Bacteria Plant Heavy Conditions Role of References
metals PGPR
heavy metals,
No increase of
metal uptake
with either
strain over
noninoculated
plants
Brevundimonas None Cd Culture Sequestered Robinson
Kro13 media cadmium et al.
directly from (2001)
solution
Pseudomonas sp. Soybean, Ni, Cd, Experiment Promotes Gupta et al.
mungbean, Cr in pots growth (2002)
wheat of plants
(continued)
Elongation of primary Formation of lateral

Biosynthesis of various roots and adventitious roots
metabolites
Root initiation
Apical dominance Cell enlargement
Role of IAA in
Fluorescence plants Cell division
Phosphate
solubilizing bacteria
Uptake by CH2COOH
plant N
H
Indole–3–acetic acid
Pigments formation Tissue differentiation
Increase rate of
Fructification of plants Stimulation of nitrogen xylem formation
fixation Resistance to stress
factors
Fig. 6.2 Role of IAA in plant growth promotion
When grown under iron-deficient conditions, many microbes will synthesize and
excrete siderophores in excess of their own dry cell weight to sequester and
solubilize iron. Most of the siderophores are water-soluble and can be secreted
extracellularly or produced inside (intracellularly). Generally, most siderophore
transport systems are highly specific for certain siderophores, although some
broad-range siderophore-recognition systems have been described based on ligand-
exchange mechanisms (Bultreys et al. 2003).
6.4.2.1 Siderophore Production by Microorganisms
The siderophore production in iron-stress conditions confers upon these organisms

an added advantage, resulting in exclusion of pathogens due to iron starvation.
Siderophore production by rhizobial strains has been considered as a potential way
to improve nodulation and N2 fixation in iron-deficient conditions (O’ Hara et al.
1988; Khan et al. 2002), and, hence, favors the persistence of rhizobia in iron-
deficient soils (Lesueur et al. 1993). In a study, strains of Mesorhizobium, showed
the production of siderophores in Chrome Azurol S (CAS) agar medium while the
supernatants of this strain yielded salicylic acid and 2,3-dihydroxybenzoic acid
(DHBA) as phenolate-type siderophores. Addition of ferric iron to the culture
medium, though, increased growth yield, but decreased the synthesis of sidero-
phores (Berraho et al. 1997). Similarly, other bradyrhizobial and rhizobial strains
infecting greengram (Vigna radiata L. Wilczek), pigeonpea (Cajanus cajans) and
pea (Pisum sativum) have shown the production of siderophores using CAS agar
plate and CAS solution assay (Wani et al. 2008a, 2008b). The predominant form
of siderophores secreted by these strains included hydroxymate and catechol.
Similarly, nitrogen-fixing iron-stressed A. vinelandii in continuous culture formed
DHBA, 2-N,6-N-di-(2,3-dihydroxybenzoyl)-L-lysine (DHBL) and a chromophoric
yellow-green fluorescent peptide (YGFP) (Fekete et al. 1983).
6.4.2.2 Chemical and Biological Properties of Siderophores
Broadly, siderophores have been classified into four groups: (1) hydroxamate, (2)
phenol catecholates, (3) carboxylate, and (4) salicylic acid (2-hydroxy benzoic
acid). Generally, PGPR produce both hydroxamate and catecholate siderophores
(Witter and Luther 1998). Different types of siderophores produced by PGPR
strains are presented in Fig.6.3. Of these, hydroxamate siderophores are generally
referred to as pseudobactin- (Fig.6.4) or pyoverdine-type siderophores (Meyer et al.
1997). Each pyoverdin has three Fe binding ligands, one of which is always a
a-dihydroxy aromatic group derived from quinoline located in the chromophore.
The other two are located in the peptide chain and are hydroxamic acids derived
from ornithine either acylated N-hydroxyornithine or cyclized N-hydroxyornithine,
or one hydroxamic acid derived from ornithine plus a b-hydroxyaspartic acid
residual. A catecholate siderophore complex consists of three catecholamide groups
ligating the metal ion by six oxygen atoms. The catecholamide groups are linked to
a trilactone ring or they are connected by a backbone of alkyl chain beginning at a
tertiary carbon or a nitrogen atom. The oxygen atoms possess a high electron
density, which exhibit a high affinity for protons when deprotonated at pH values
above 6.5. In addition, because Fe (III) is a strong Lewis acid it readily donates
protons to other atoms, such as the polarizable oxygen atoms of the catechol
moiety. This electrostatic interaction gives catecholate siderophores a greater
affinity for Fe (III) compared to their hydroxamate counterparts.
Fig. 6.3 Types of siderophores
Even though the major function of siderophores is to obtain iron from insoluble
hydroxides or from iron adsorbed onto solid surfaces, they can also extract iron
from various other soluble and insoluble iron compounds. For instance, they can
extract iron from ferric citrate, ferric phosphate, Fe-transferrin, ferritin or iron
bound to sugars, plant flavone pigments and glycosides, or even from artificial
chelators like EDTA and nitrilotriacetate by Fe (III)/ligand-exchange reactions.
Siderophores are thus not only directly involved in iron solubilization, but can
indirectly make iron available to both microbes and plants. The efficiency of
siderophores in microbial metabolism is based mainly on three facts: (1) side-
rophores consisting of hydroxamate, catecholate or a-hydroxycarboxylate ligands
contain the most efficient iron-binding ligand types in nature and satisfy the six
coordination sites on ferric ions; siderophores also increase the stability due to its
chelating effects; (2) regulation of siderophore biosynthesis is an economic means
of spending metabolic energy, but it also allows the production of high local
concentrations of siderophores in the vicinity of microbial cells during iron limita-
tion, while over-production of siderophores by host-adapted bacterial strains leads
to increased virulence; and (3) besides their ability to solubilize iron and to function
as external iron carriers, siderophores exhibit structural and conformational speci-
ficities to fit into membrane receptors and/or transporters (Stintzi et al. 2000) and
are involved in various biological processes (Fig.6.5). Moreover, the siderophore-
producing strains stimulate the N2-fixing efficiency of the rhizobial strains (Duhan
Fig. 6.4 Representative siderophores
et al. 1998). Furthermore, of the 12 isolates of Rhizobium meliloti isolated from the
medicinal plant, Mucuna pruriens, only two isolates (RMP3 and RMP5) inhibited
the growth of phytopathogens (Macrophomonia phaseolina). Further, a marked
enhancement in percentage seed germination, seedling biomass, nodule number
and nodule weight of M. phaseolina-infected groundnut (Arachis hypogaea) plants
inoculated with the strains RMP3 and RMP was observed, suggesting the growth
promoting activities of siderophores (Arora et al. 2001). Among other PGPR,
Pseudomonas aeruginosa (GRC1), isolated from potato (Solanum tuberosum)
rhizosphere, produced several plant growth-promoting substances, including a side-
rophore. The siderophore was identified as hydroxymate and, when P. aeruginosa
was used in field trials, enhanced growth and yield of Indian mustard (Brassica
juncea) (Pandey et al. 2005).
6.4.3 Mineral Phosphate Solubilizing Activity
Phosphorus (P) is an essential plant nutrient whose deficiency restricts crop yields
severely. Most tropical and some subtropical soils are acidic, and strong P-sorption
combined with low inherent P stocks lead to widespread P deficiency (Gaume
2000). Even where inorganic and organic P-forms are abundant in soils, their
Fig. 6.5 Biological functions of siderophores
concentration in the soil solution is in the micromolar range (Frossard et al. 2000).
These low levels of P are mainly due to high reactivity of soluble P soil elements
(Lindsay et al. 1989). Therefore, substantial amounts of manufactured water-solu-
ble P (WSP) fertilizers such as superphosphate are commonly applied to correct P
deficiencies. Most developing countries import these fertilizers, which are often in
limited supply and represent a major outlay for resource-poor farmers. It is,
therefore, imperative to explore alternative P sources. In this context, the PGPR-
possessing mineral phosphate-solubilizing (MPS) activity provides an inexpensive
and sustainable alternative to chemical P fertilizers (Pradhan and Sukla 2005; Khan
et al. 2007). The microbial solubilization of soil P in liquid medium has often been
due to the excretion of organic acids (Maliha et al. 2004) which can either directly
dissolve the mineral P or can chelate both Fe and Al ions associated with P (Omar
1998). However, no definite correlation between the acids produced by PGPR and
amounts of P solubilized are reported (Asea et al. 1988). Such PGPR strains
possessed with mps activity, when used either alone or as composite cultures,
have shown a substantial increase by supplying both P and other essential nutrients
to plants.
6.4.4 Growth Modulation Enzyme
The PGPR also increase the growth of plants through the synthesis of specific
enzyme, 1-aminocyclopropane- 1-carboxylate (ACC) deaminase, which induce
physiological changes in plants. Ethylene is a plant hormone that is involved in the

regulation of many physiological processes, such as leaf senescence, leaf abscis-
sion, epinasty, and fruit ripening (Arshad and Frankenberger 2002). Also, ethylene
regulates nod factor signaling and nodule formation and has primary functions in
plant defense systems. Besides its physiological role in different developmental
stages of plants, ethylene is also considered as a stress hormone, whose synthesis in
plants is increased substantially by a number of biotic and abiotic stresses. At
higher concentrations, ethylene, however, inhibits growth and development of
plants (Grichko and Glick 2001). However, ACC deaminase synthesized by PGPR
(Belimov et al. 2005; Safronova et al. 2006; Madhaiyan et al. 2006; Rajkumar et al.
2006; Mellado et al. 2007) alleviates the stress induced by ethylene-mediated
impact on plants by hydrolyzing ACC, the immediate precursor of ethylene in
plants to NH3 and a-ketobutyrate (Glick et al. 1998; Penrose and Glick 2001; Reed
et al. 2005; Safronova et al. 2006), and consequently reduce the ethylene levels in
plants. The bacteria utilize the NH3 evolved from ACC as a source of N and thereby
restrict the accumulation of ethylene within the plant, which otherwise inhibits
plant growth (Belimov et al. 2002). Thus, the decreased levels of ethylene in turn
allow the plants to grow better (Madhaiyan et al. 2007; Zahir et al. 2008). It has
been observed that plants that are inoculated with PGPR containing ACC deami-
nase are dramatically more resistant to the deleterious effects of stress ethylene that
is synthesized as a consequence of stressful conditions such as flooding (Grichko
and Glick 2001), heavy metals (Burd et al. 1998; Grichko et al. 2000), presence of
phytopathogens (Wang et al. 2000), drought, and high salt contents (Mayak et al.
2004a, b). In most of these cases, it has been reported that the PGPR-containing
ACC deaminase significantly lowered the level of ACC in the stressed plants,
thereby limiting the amount of stress ethylene synthesis and hence damage to the
plant. Therefore, the use of such plant growth-promoting bacteria containing ACC
deaminase may prove useful in developing strategies to facilitate plant growth in
stressed soil environments. And, hence, it may be possible to productively cultivate
a variety of crop plants under stressed conditions without genetically manipulating
plants, provided these plants are grown in the presence of a suitable PGPR.
6.4.5 Antibiotics Production by Plant Growth Promoting

Rhizobacteria
PGPR also promotes the growth of plants by secreting antimicrobial compounds,

induction of systemic resistance (ISR), and production of pathogen-related (PR)
proteins (Compant et al. 2005). Antibiotic production by biocontrol PGPR is
perhaps the most powerful mechanism against phytopathogens (Bashan and
de-Bashan 2005), and the first clear-cut experiment demonstrating the role of
PGPR in suppression of plant disease through antibiotic production was reported
by Tomashow and Weller (1988). These antibiotics may be antitumor, antiviral,
antimicrobial, antihelmenthic, and cytotoxic (Fernando et al. 2005). The antibiotics
can also contribute to microbial competitiveness besides their role in suppressing

the growth of plant root pathogens. The PGPR strains that produce these
compounds are, therefore, of considerable interest as biological control agents
(Thomshow et al. 2003) and provide an alternative to chemical pesticides. Several
antimicrobial compounds belonging to polypeptides, heterocyclic nitrogenous
compounds, and lipopeptides groups active against phytopathogens have been
reported (Thomshaw and Webler 1995). In addition, plants can acquire local and
systemic resistance to diseases through various biological agents, including necro-
tizing pathogens, nonpathogens, and soil-borne rhizosphere bacteria and fungi (Van
Loon et al. 1998). This type of resistance, known as induced systemic resistance, is
mediated by a jasmonate/ethylene sensitive pathway (Van Loon et al. 1998).
Induction of systemic resistance has been established as a new mechanism by
which plants defend themselves against pathogen attachment. Various reports
confirm the induction of systemic resistance by PGPR. For instance, PGPR strains,
i.e., P. putida (strain 89B-27), S. marcescens (strain 90-166), Flavomonas oryziha-
bitans strain (INR-5), and Bacillus pumilus (strain INR-7), have significantly
reduced populations of the striped cucumber (Zehnder et al. 1997). Furthermore,
the combined inoculation of PGPR (Bacillus and Pseudomonas) and Rhizobium sp.
increased the production of defense-related enzymes, i.e., L-phenylalanine ammo-
nia lyase (PAL), POX and polyphenol oxidase (PPO), in coinoculated pigeonpea
plants which in turn decreased the dry weight of mycelium and fusaric acid
production by fusarial wilt of pigeonpea, suggesting that the combined use of
PGPR and rhizobia for induction of systemic resistance against fusarial wilt in
pigeon pea (Dutta et al. 2008).
Antibiosis and antagonistic activities of PGPR recovered from wheat
(T. aestivum) and rice (Oryza sativa) seeds, corn (Zea mays) plants, and potato
have been suggested as possible mechanisms of growth inhibition of various
phytopathogens (Lodewyckx et al. 2002; Rosenblueth and Martı́nez-Romero
2006). For instance, P. fluorescens capable of synthesizing 2,4-diacetyl phloro-
glucinol (DAPG) has shown the production of antimicrobial compounds in planta
conditions. Similarly, production of antibiotics phenazine, pyocyanine and DAPG
by Pseudomonas spp. associated with induced systemic resistance (ISR) activity
in sugarcane (Saccharum officinarum) against red rot disease has been reported
(Viswanathan and Samiyappan 2004). However, the bacterial strains varied in
their capability to produce the metabolites. The purified compounds tested for
their antifungal activity completely arrested the conidial germination and myce-
lial growth of red rot pathogen (Colletotrichum falcatum), suggesting that the
metabolites played an important role in antagonism/ISR. Moreover, the suppres-
sive ability of PGPR even against nematodes is reported (Sturz and Kimpinski
2004). Similarly, Bacillus lentimorbus and Bacillus cereus isolated from coffee
(Coffea canephora) demonstrated inhibitory effects against coffee rust pathogen
(Hemileia vastatrix) and significantly enhanced the coffee production. The patho-
gen suppression was suggested to be due possibly to the synthesis of a significant
amount of fungal cell wall lysing enzymes, antibiosis, competition, and ISR in host
(Shiomi et al. 2006). Recently, PGPR bioformulations (Pseudomonas and Bacillus)
were tested for their efficacy against blister blight (Exobasidium vexans) disease in
tea (Camellia sinensis) under field conditions for two seasons. Among the biofor-
mulations tested, foliar application of Pseudomonas fluorescens Pf1 at 7-day inter-
vals consistently reduced the disease incidence of blister blight for two seasons,
almost comparable with that of chemical fungicide. In addition to disease control, it
also increased tea yield significantly compared to the untreated control. Defense
enzymes, such as peroxidase, polyphenol oxidase, phenylalanine ammonia lyase,
chitinase, b-1,3-glucanase, and phenolics were found more in P. fluorescens Pf1-
treated plants compared to control. This finding revealed the probable influence of
plant growth promotion and induced systemic resistance (ISR) in enhancing the
disease resistance in tea plants against blister disease by PGPR bioformulations
(Saravanakumar et al. 2007).
6.4.6 Hydrogen Cyanide Production
Cyanide is yet another secondary metabolite produced during the early stationary
growth phase (Knowles and Bunch 1986) by several PGPR, notably Pseudomonas
spp. and Bacillus (Wani et al. 2007b; Ahmad et al. 2008), Chromobacterium
(Faramarzi and Brand 2006), and Rhizobium spp. (Wani et al. 2008a, 2008b,
2008c) by oxidative decarboxylation pathway using glycine, glutamate, or methio-
nine as precursors (Castric 1977; Curl and Truelove 1985). The cyanide so released
by microbial communities in solution acts as a secondary metabolite and confers a
selective advantage on the producer strains (Vining 1990). Although cyanide is a
phytotoxic agent capable of disrupting enzyme activity involved in major metabolic
processes, its role as a biocontrol substance is overwhelming (Voisard et al. 1989;
Devi et al. 2007). Hydrogen cyanide (HCN) effectively blocks the cytochrome
oxidase pathway and is highly toxic to all aerobic microorganisms at picomolar
concentrations. However, producer microbes, mainly pseudomonads, are reported
to be resistant (Bashan and de-Bashan 2005).
6.4.7 Production of Lytic Enzymes
A variety of other microbial compounds are involved in the suppression of

phytopathogenic growth leading thereby to the reduction in damage to plants.
These microbially synthesized compounds include defense enzymes, such
as chitinase, b-1,3-glucanase, peroxidase, protease, and lipase (Bashan and
de-Bashan 2005; Karthikeyan et al. 2006). Chitinase and b-1,3-glucanase degrade
the fungal cell wall and cause lysis of fungal cell. Furthermore, chitin and glucan
oligomers released during degradation of the fungal cell wall by the action of
lytic enzymes act as elicitors that elicit various defense mechanisms in plants
(Karthikeyan et al. 2005). Such enzymes produced by Pseudomonas stutzeri have
demonstrated the lysis of the pathogen Fusarium sp. (Bashan and de-Bashan
2005). Peroxidase represents another component of an early response in plants
to pathogen attack and plays a key role in the biosynthesis of lignin which limits
the extent of pathogen spread (Bruce and West 1989). In bean, rhizosphere
colonized by various bacteria induced PO activity (Zdor and Anderson 1992).
In a study, a rapid increase in PO activity was recorded in coconut (Cocos nucifera
L.) treated with a mixture of P. fluorescens, T. viride and chitin which contributed to
induced resistance against invasion by Ganoderma lucidum, the causal agent of
Ganoderma disease (Karthikeyan et al. 2006). These findings suggest that PGPR
possessing the ability to synthesize hydrolytic enzymes can effectively be utilized
for managing the plant diseases and can help to reduce the pesticide usage.
6.5 Performance of Plant Growth-Promoting Rhizobacteria

in Metal-Contaminated Soils
The PGPR have largely been used as growth-promoting agents in conventional

agronomic practices; substantial emphasis is being placed on them in order to also
exploit their bioremediation potential. Contaminated soils are often nutrient poor or
sometimes nutrient deficient, due to the loss of beneficial microbes. However, such
soils can be made nutrient rich by applying metal tolerant microbes, especially the
PGPR, which would provide not only the essential nutrients to the plants growing in
the derelict soils but also play a major role in detoxifying heavy metals (Mayak
et al. 2004a) and thus helping plants to grow better (Glick 2003; Wani et al. 2008a,
2008b, 2008c). For example, PGPR Kluyvera ascorbata SUD165 isolated from
metal-contaminated wetland near Sudbury, Ontario, Canada, when applied to soils
amended with nickel, zinc, lead, and chromate, have shown to increase the growth
of canola (Brasica rapa) while protecting the plants from nickel toxicity (Burd et al.
1998). Similarly, nickel-resistant K. ascorbata protected tomato (Lycopersicon
esculentum L.), Indian mustard (Brassica campestris) and canola plants when
grown in soils supplemented with nickel, lead and zinc (Burd et al. 2000). More-
over, the growth-promoting rhizobacteria, Variovorax paradoxus, Rhodococcus sp.
and Flavobacterium sp., stimulated root elongation of Indian mustard seedlings
either in the presence or absence of toxic cadmium (Belimov et al. 2005), suggest-
ing that these bacterial strains could be developed as inoculants to improve growth
of the metal-accumulating Indian mustard in the presence of toxic cadmium
concentration, and for the development of plant inoculant systems useful for
phytoremediation of polluted soils. Similarly, the canola plants inoculated with
Enterobacter cloacae, when grown in the presence of arsenates, grew to a signifi-
cantly greater extent than nontransformed canola plants (Nie et al. 2002). In yet
other studies, Ochrobacterium intermedium and Bacillus cereus protected green-
gram plants against chromium toxicity (Faisal and Hasnain 2006), while inoculation
of Ochrobacterium intermedium improved the overall growth of sunflower
(Helianthus annus), when grown in metal-amended soils (Faisal and Hasnain
2005) as also reported for other crops (Chaudri et al. 2000; Wani et al. 2008b).
The increase in the growth of plants grown in derelict soils by applying metal-
tolerant rhizobacteria was attributed to the ability of rhizobacterial strains to
mitigate the toxic effects of metals, besides providing plants with sufficient
amounts of growth-promoting substances. Recent examples of PGPR affecting
growth and development of plants in metal-affected soils are presented in
Table 6.3. The remediation of heavy metal-contaminated sites using rhizobac-
teria is an exciting area of research, since these organisms can easily and
inexpensively be mass produced. Therefore, the molecular engineering of both
PGPR and plants with desired genes would help immensely to enhance the
efficiency of growth-promoting rhizobacteria mediation or plant-based remedi-
ation of contaminated soils and, consequently, could lead to restoration of
polluted soils for cultivation.
6.6 Conclusion and Future Prospects
Form the above discussion, it is evident that PGPR offer an environmentally sound
and sustainable approach to increase soil fertility and, in turn, the crop productivity.
In recent times, the understanding of the complex environment of the rhizosphere,
functional diversity among PGPR, mechanisms of their action, and, of course,
methods of development of the inoculants and their formulation and delivery system
has increased considerably. We therefore hope to see new and exciting PGPR
products with multiple traits in the commercial markets for ultimate transfer to the
agrarian communities. However, there are several limitations to the use of PGPR for
commercial use. Chief among them is inconsistent performance of PGPR under field
conditions. Researchers, therefore, need to develop the PGPR inoculants with
persistent plant growth-promoting activities and should also suggest ways as to
how variations in soil type, management practices (e.g., agrochemical use, rota-
tions), and indeed the effect of weather on the efficacy of PGPR could be minimized.
With the advancement in the understanding of the mechanisms adopted by PGPR,
it will become possible to enhance their capacity to stimulate plant growth by
modifying/manipulating promising traits of PGPR by introducing genes responsible
for the biosynthesis of desirable metabolites into other microbial communities. Such
genetically engineered PGPR endowed with multiple growth-promoting traits could
lead to improve colonization and growth-promoting efficiency, and in turn the
sustainable plant productivity, while maintaining soil health and reducing the
environmental pollution caused by the use of agro-chemicals. Further work focusing
on the functional diversity, precise mode of action, and ecophysiology of these
agronomically beneficial microbes would assist in unleashing their full promise as
potential bio-inoculants for maintaining soil fertility and, consequently, the sustain-
ability of crops in diverse agro-ecosystems.
References
Abd-Alla MH (1994) Solubilization of rock phosphates by Rhizobium and Bradyrhizobium. Folia

Ahmad F, Ahmad I, Khan MS (2005) Indole acetic acid production by the indigenous isolates of
Azotobacter and fluorescent Pseudomonas in the presence and absence of tryptophan. Turk
J Biol 29:29–34
Antoun H, Prévost D (2006) Ecology of plant growth promoting rhizobacteria. In: Siddiqui ZA
(ed) PGPR: biocontrol and biofertilization. Springer, Heidelberg, pp 1–38
Antoun H, Beauchamp CJ, Goussard N, Chabot R, Llande R (1998) Potential of Rhizobium and
Bradyrhizobioum species as plant growth promoting rhizobacteria on non-legumes: effect on
radishes. Plant Soil 204:57–67
Arora NK, Kang SC, Maheshwari DK (2001) Isolation of siderophore producing strains of
Rhizobium meliloti and their biocontrol potential against Macrophomina phaseolina that
causes charcoal rot of groundnut. Curr Sci 81:673–677
Arshad M, Frankenberger WT Jr (2002) Ethylene: agricultural sources and applications. Kluwer,
New York, p 342
Asea PEA, Kucey RMN, Stewart JWB (1988) Inorganic phosphate solubilization by two Penicil-
lium species in solution culture and soil. Soil Biol Biochem 20:459–464
Bai Y, Souleimanov A, Smith DL (2002) An inducible activator produced by Serratia proteama-
culans strain and its soybean growth promoting activity under greenhouse conditions. J Exp
Bot 53:1495–1502
Bano N, Musarrat J (2003) Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a
potential biocontrol agent. Curr Microbiol 46:324–328
Barazani OZ, Friedman J (1999) Is IAA the major root growth factor secreted from plant growth
mediating bacteria. J Chem Ecol 25:2397–2407
Barea JM, Pozo M, Azcón R, Azcón-Aguilar C (2005) Microbial co-operation in the rhizosphere.
J Exp Bot 56:1761–1778
Bartel B, Fink GR (1995) Molecular cloning of the gene for indole pyruvate decarboxylase from
Enterobacter cloacae. Science 268:1745–1748
Bashan Y, de-Bashan LE (2005) Bacteria. In: Hillel D, Hillel D (eds) Encyclopaedia of soils in the
environment, vol 1. Elsevier, Oxford, UK, pp 103–115
Belimov AA, Safranova VI, Mimura T (2002) Response of spring rape (Brassica napus) to
inoculation with PGPR containing ACC-deaminase depends on nutrient status of plant. Can
J Microbiol 48:189–199
Belimov AA, Kunakova AM, Safronova VI, Stepanok VV, Yudkin LY, Alekseev YV,
Kozhemyakov AP (2004) Employment of rhizobacteria for the inoculation of barley plants
cultivated in soil contaminated with lead and cadmium. Microbiology 73:99–106
(2005) Cadmium-tolerant plant growth-promoting rhizobacteria associated with the roots of
Indian mustard (Brassica juncea L. Czern.). Soil Biol Biochem 37:241–250
Berraho EL, Lesueur D, Diem HG, Sasson A (1997) Iron requirement and siderophore production
in Rhizobium ciceri during growth on an iron-deficient medium. World J Microbiol Biotechnol
13:501–510
Bowen GD, Rovira AD (1999) The rhizosphere and its management to improve plant growth. Adv
Agron 66:1–102
Bringhurst RM, Cardon ZG, Gage DJ (2001) Galactosides in the rhizosphere: utilization by
Sinorhizobium meliloti and development of a biosensor. Proc Natl Acad Sci USA 98:4540–4545
Bruce RJ, West CA (1989) Elicitation of lignin biosynthesis and isoperoxidase activity by pectic
fragments in suspension cultures of cluster bean. Plant Physiol 91:889–897
Bultreys A, Gheysen I, Wathelet B, Maraite H, de Hoffman E (2003) High-performance

liquid chromatography analyticalyses of pyoverdine siderophores differentiate among phyto-
pathogenic fluorescent Pseudomonas species. Appl Environ Microbiol 69:1143–1153
Burd GI, Dixon DG, Glick BR (1998) A plant growth-promoting bacterium that decreases nickel
toxicity in seedlings. Appl Environ Microbiol 64:3663–3668
Burd GI, Dixon DG, Glick BR (2000) Plant growth-promoting bacteria that decrease heavy metal
toxicity in plants. Can J Microbiol 46:237–245
Çakmakçi R, Dönmez F, Aydm A, Şahin F (2006) Growth promotion of plants by plant growth-
promoting rhizobacteria under greenhouse and two different field soil conditions. Soil Biol
Biochem 38:1482–1487
Canbolat MY, Bilen S, Cakmakci R, Sahin F, Aydin A (2006) Effect of plant growth promoting
bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties and
Castric PA (1977) Glycine metabolism by Pseudomonas aeruginosa: hydrogen cyanide biosyn-
thesis. J Bacteriol 130:826–831
Chaudri AM, Allain CM, Barbosa-Jefferson VL, Nicholson FA, Chambers BJ, McGrath SP (2000)
A study of the impacts of Zn and Cu on two rhizobial species in soils of a long term field
experiment. Plant Soil 22:167–179
Clark E, Manulis S, Ophir Y, Barash I, Gafni Y (1993) ILRI, an amidohydrolase that releases
active indole-3-acetic acid from conjugates. Phytopathology 83:234–240
Compant S, Reiter B, Sessitsch A, Nowak J, Clément C, Barka EA (2005) Endophytic colonization
of Vitis vinifera L. by plant growth-promoting bacterium Burkholderia sp. strain PsJN. Appl
Crowley DE, Reidd CPP, Szaniszlo PJ (1987) Mirobial siderophores as iron sources for plants. In:
Winkelmann G, Van der Helm D, Neilands JB (eds) Iron transport in animals, plants and
microorganisms. VCH Chemie, Weinheim, Germany
Curl EA, Truelove B (1985) The rhizosphere. Springer, Berlin
Davies PJ (1995) Plant hormones. Kluwer, Dorderecht
De Freitas JR, Germida JJ (1991) Pseudomonas cepacia and Pseudomonas putida as winter wheat
inoculants for biocontrol of Rhizobium solani. Can J Microbiol 37:780–789
Deshwal VK, Pandey P, Kang SC, Maheshwari DK (2003) Rhizobia as a biological control agent
against soil borne plant pathogenic fungi. Ind J Exp Biol 41:1160–1164
Devi K, Nidhi S, Shalini K, David K (2007) Hydrogen cyanide producing rhizobacteria kill
subterranean termite Odontotermes obesus (Rambur) by cyanide poisoning under in vitro
conditions. Curr Microbiol 54:74–78
Duhan JS, Dudeja SS, Khurana AL (1998) Siderophore production in relation to N2 fixation and
iron uptake in pigeon pea–Rhizobium symbiosis. Folia Microbiol 43:421–426
Dutta S, Mishra AK, Dileep Kumar BS (2008) Induction of systemic resistance against fusarial
wilt in pigeon pea through interaction of plant growth promoting rhizobacteria and rhizobia.
Soil Biol Biochem 40:452–461
Faisal M, Hasnain S (2005) Bacterial Cr (VI) reduction concurrently improves sunflower
(Helianthus annuus L. ) growth. Biotechnol Lett 27:943–947
Faisal M, Hasnain S (2006) Growth stimulatory effect of Ochrobactrum intermedium and Bacillus
cereus on Vigna radiata plants. Lett Appl Microbiol 43:461–466
Faramarzi MA, Brand H (2006) Formation of water-soluble metal cyanide complexes from solid
minerals by Pseudomonas plecoglossicida. FEMS Microbiol Lett 259:47–52
Fekete FA, Spence JT, Emery T (1983) Siderophore produced by nitrogen-fixing Azotobacter
vinelandii OP in iron-limited continuous culture. Appl Environ Microbiol 46:1297–1300
Fernando WGD, Nakkeeran S, Zhang Y (2005) Biosynthesis of antibiotics by PGPR and its
relation in biocontrol of plant diseases. In: Siddiqui ZA (ed) PGPR: biocontrol and biofertiliza-
tion. Springer, Dordrecht, The Netherlands, pp 111–142
Flaishman MA, Eyal Z, Zilberstein A, Voisard C, Hass D (1996) Suppression of Septoria tritici
blotch and leaf rust of wheat by recombinant cyanide-producing strains of Pseudomonas
putida. Mol Plant Microbe Interact 9:642–645
Frankenberger WT Jr, Arshad M (1995) Phytohormones in soil: microbial production and func-
tion. Marcel Dekker, New York
Frossard E, Condron LM, Oberson A, Sena JS, Fardeu JC (2000) Progress governing phosphorus
availability in temperate soils. J Environ Qual 29:12–53
Garcia de Salamon IE, Hynes RK, Nelson LM (2001) Cytokinin production by plant growth
promoting rhizobacteria and selected mutants. Can J Microbiol 47:404–411
Garland JL (1996) Patterns of potential C source utilization by rhizosphere communities. Soil Biol
Biochem 28:223–230
Gaume A (2000) Low P tolerance of various maize cultivars; the contribution of the root
exudation. PhD dissertation, Swiss Federal institute of Technology, Zurich, Switzerland
Germida JJ, Siciliano SD, de Freitas JR, Seib AM (1998) Diversity of root-associated bacteria
associated with field-grown canola (Brassica napus L.) and wheat (Triticum aestivum). FEMS
Microbiol Ecol 26:43–50
41:109–117
Glick BR (2001) Phytoremediation: synergistic use of plants and bacteria to clean up the
environment. Biotechnol Adv 21:383–395
Glick BR, Penrose DM, Li J (1998) A model for the lowering of plant ethylene concentration by
plant growth promoting bacteria. J Theor Biol 190:63–68
by plant growth promoting bacteria. Imperial College Press, London
Glick BR, Cheng Z, Czarn YJ, Duan J (2007) Promotion of plant growth by ACC-deaminase
producing soil bacteria. Eur J Plant Pathol 119:329–339
Grichko VP, Glick BR (2001) Amelioration of flooding stress by ACC deaminase containing plant
growth-promoting bacteria. Plant Physiol Biochem 39:11–17
Grichko VP, Filby B, Glick BR (2000) Increased ability of transgenic plants expressing the
bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn. J Biotechnol
81:45–53
Guo JH, Qi HY, Guo YH, Ge HL, Gong LY, Zhang LX (2004) Biocontrol of tomato wilt by plant
growth-promoting rhizobacteria. Biol Control 29:66–72
Gupta A, Meyer JM, Goel R (2002) Development of heavy metal resistant mutants of
phosphate solubilizing Pseudomonas sp. NBRI4014 and their characterization. Curr
Gupta A, Rai V, Bagdwal N, Goel R (2005) In situ characterization of mercury resistant growth
promoting fluorescent Pseudomonads. Microbiol Res 160:385–388
Inbar J, Chet I (1991) Evidence that chitinase produced by Aeromonas caviae is involved in the
biological control of soil-borne plant pathogens by bacterium. Soil Biol Biochem 23:974–978
Karadeniz A, Topeuoglu SF, Inan S (2006) Auxin, gibberellin, cytokinin and abscisic acid
production in some bacteria. World J Microbiol Biotechnol 22:1061–1064
Karthikeyan M, Jayakumar V, Radhika K, Bhaskaran R, Velazhahan R, Alice D (2005) Induction
of resistance in host against the infection of leaf blight pathogen (Alternaria palandui) in onion
(Allium cepa var aggricatum). Indian J Biochem Biophys 42:371–377
Karthikeyan M, Radhika K, Mathiyazhagan S, Bhaskaran R, Samiyappan R, Velazhahan R (2006)
Induction of phenolics and defense-related enzymes in coconut (Cocos nucifera L.) roots
treated with biocontrol agents. Brazil J Plant Physiol 18:367–377
Khalid A, Arshad M, Zahir ZA (2004) Screening of plant growth promoting rhizobacteria for
improving growth and yield of wheat. J Appl Microbiol 96:473–480
Khan AG (2005) Role of soil microbes in the rhizospheres of plants growing on trace metal
contaminated soils in phytoremediation. J Trace Elem Med Biol 18:355–364
Khan MS, Zaidi A (2007) Synergicstic effects of the inoculation with plant growth promoting
rhizobacteria and an arbuscular mycorrhizal fungus on the performance of wheat. Turk J Agric
For 31:355–362
Khan MS, Zaidi A, Aamil M (2002) Biocontrol of fungal pathogens by the use of plant growth
promoting rhizobacteria and nitrogen fixing microorganisms. Indian J Bot Soc 81:255–263
Khan MS, Zaidi A, Wani PA (2007) Role of phosphate solubilizing microorganisms in sustainable
agriculture-A review. Agron Sustain Dev 27:29–43
Kloepper JW, Schroth MN (1978) Plant growth-promoting rhizobacteria on radishes. Fourth
International Conference on Plant Pathogen Bacteria, Angers, France, vol 2, pp 879–882
Knowles CJ, Bunch AW (1986) Microbial cyanide metabolism. Adv Microb Physiol 27:73–111
Koga J, Adachi T, Hidaka H (1991) The cloning and characterization of iaaM and iaaH from
Erwinia herbicola pathovar gypsophilae. Mol Gen Genet 226:10–16
Lambert B, Joos H, Dierick S, Vantomme R, Swings J, Kerters K, Van Montagu M (1990)
Identification and plant interaction of Phyllobacterium sp., a predominant rhizobacterium of
young sugar beat. Appl Environ Microbiol 56:1093–1102
Leeman M, Den OFM, Van PJA, Dirkx FPM, Steijl H, Bakker PAHM, Schippers B (1996) Iron
availability affects induction of systemic resistance to Fusarium wilt of radish by Pseudomo-
nas fluorescens. Phytopathology 86:149–155
Lesueur D, Diem HG, Meyer JM (1993) Iron requirement and siderophore production in
Bradyrhizobium strains isolated from Acacia mangium. J Appl Bacteriol 74:675–682
Lindsay WL, Vlek PLG, Chien SH (1989) In: Dixon JB, Weed SB (eds) Phosphate minerals, soil
environment, 2nd edn. Soil Science Society of America, Madison, pp 1089–1130
Lodewyckx CJ, Vangronsveld F, Porteous ERB, Moore ST, Mezgeay M, van der Lelie D (2002)
Endophytic bacteria and their potential applications. Crit Rev Plant Sci 21:583–606
Lucas Garcı́a JA, Domenech J, Santamarı́a C, Camacho M, Daza A, Gutierrez Mañero FJ (2004)
Growth of forest plants (pine and holm-oak) inoculated with rhizobacteria: relationship with
microbial community structure and biological activity of its rhizosphere. Environ Exp Bot
52:239–251
Madhaiyan M, Poonguzhali S, Ryu JH, Sa T (2006) Regulation of ethylene levels in canola
(Brassica campestris) by 1-aminocyclopropane-1-carbxylate deaminase containing Methylo-
bacterium fujisawaense. Planta 224:268–278
Madhaiyan M, Poonguzhali S, Sa T (2007) Metal tolerating methylotrophic bacteria
reduces nickel and cadmium toxicity and promotes plant growth of tomato (Lycopersicon
esculentum L.). Chemosphere 69:220–228
Mahaffee WF, Kloepper JW (1997) Temporal changes in the bacterial communities of soil,
rhizosphere, and endorhiza associated with fieldgrown cucumber (Cucumis sativus L.). Microb
Ecol 34:210–223
Mahmoud SAZ, Ramadan EM, Thabet FM, Khater T (1984) Production of plant growth promoting
substances by rhizosphere microorganisms. Zbl Mikrobiol 139:227–232
Maliha R, Samina K, Najma A, Sadia A, Farooq L (2004) Organic acid production and phosphate
solubilisation by phosphate solubilising microorganisms under in vitro conditions. Pak J Biol
Sci 7:187–196
Mayak S, Tirosh S, Glick BR (2004a) Plant growth promoting bacteria that confer resistance to
water stress in tomatoes and peppers. Plant Physiol 166:525–530
Mayak S, Tirosh T, Glick BR (2004b) Plant growth promoting bacteria confer resistance in tomato
plants to salt stress. Plant Physiol Biochem 42:565–572
Mayak S, Tirosh T, Glick BR (2004c) Plant growth promoting bacteria that confer resistance to
water stress in tomato and peppers. J Plant Sci 166:525–530
Bakker PAHM, Corné MJP L, van Loon LC (2007) Induced systemic resistance by fluorescent
Pseudomonas spp. Phytopathology 97:239–243
Mellado JC, Onofre-Lemus J, Santos PE, Martinez-Aguilar L (2007) The tomato rhizosphere, an
environment rich in nitrogen fixing Burkholderia species with capabilities of interest for
agriculture and bioremediation. Appl Environ Microbiol 73:5308–5319
Meyer JM, Stintzi A, De Vos D, Cornelis P, Tappe R, Taraz K, Budzikiewicz H (1997) Use of
siderohpores to type pseudomonads, three Pseudomonas aereuginosa pyoverdine systems.
Microbiology 143:35–43
Nelson EB (1998) Biological control of Pythium seed rot and preemergence damping-off of cotton
with Enterobacter cloacae and Ervinis herbicola applied as seed treatments. Plant Dis 72:140–142
Nie L, Shah S, Dixon BGI, DG GBR (2002) Phytoremediation of arsenate contaminated soil by
transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2.
Plant Physiol Biochem 40:355–361
Nielsen TH, Sorensen J (2003) Production of cyclic lipopeptides by Pseudomonas fluorescens
strains in bulk soil and in the sugarbeet rhizosphere. Appl Environ Microbiol 69:861–868
Noordman WH, Reissbrodt R, Bongers RS, Rademaker ILW, Bockelmann W, Smit G (2006)
Growth stimulation of Brevibacterium sp. by siderophores. J Appl Microbiol 101:637–646
O’ Hara GW, Dilworth MI, Boonkerd N, Parkpian P (1988) Iron deficiency specifically limits
nodule development in pea nut inoculated with Bradyrhizobium sp. New Phytol 108:51–57
Omar SA (1998) The role of rock phosphate solubilizing fungi and vesicular arbuscular mycorrhi-
za (VAM) in growth of wheat plants fertilized with rock phosphate. World J Microbiol
Ownley BH, Duffy BK, Weller DM (2003) Identification and manipulation of soil properties to
improve the biological control performance of phenazine-producing Pseudomonas fluorescens.
Pandey P, Kang SC, Gupta CP, Maheshwari DK (2005) Rhizosphere competent Pseudomonas
aeruginosa GRC1 produces characteristic siderophore and enhances growth of Indian mustard
(Brassica campestris). Curr Microbiol 51:303–309
Pandey A, Trivedi P, Kumar B, Palni LMS (2006) Characterization of a phosphate solubilizing and
antagonistic strain of Pseudomonas putida (B0) isolated from a sub-alpine location in the
Indian Central Himalaya. Curr Microbiol 53:102–107
exudates and extracts of canola seeds treated with plant growth promoting bacteria. Can
Persello-Cartieaux F, Nussaume L, Robaglia C (2003) Tales from the underground: molecular
plant-rhizobacteria interactions. Plant Cell Environ 26:189–199
Perveen S, Khan MS, Zaidi A (2002) Effect of rhizospheric microorganisms on growth and yield
of greengram (Phaseolus radiatus). Ind J Agric Sci 72:421–423
Ponmurugan P, Gopi C (2006) In vitro production of growth regulators and phosphatase activity
by phosphate solubilizing bacteria. Afr J Biotechnol 5:340–350
Pradhan N, Sukla LB (2005) Solubilization of inorganic phosphate by fungi isolated from
agriculture soil. Afr J Biotechnol 5:850–854
Prithiviraj B, Zhou X, Souleimanov A, Kahn W, Smith DL (2003) A host-specific bacteria to-plant
signal molecule (Nod factor) enhances germination and early growth of diverse crop plants.
Planta 216:437–445
Rajkumar M, Nagendran R, Kui Jae L, Wang Hyu L, Sung Zoo K (2006) Influence of plant growth
promoting bacteria and Cr (vi) on the growth of Indian mustard. Chemosphere 62:741–748
Reed MLE, Warner B, Glick BR (2005) Plant growth-promoting bacteria facilitate the growth of
the common reed Phragmites australis in the presence of copper or polycyclic aromatic
hydrocarbons. Curr Microbiol 51:425–429
Renwick A, Campbell R, Coe S (1991) Assessment of in vivo screening systems for potential
biocontrol agents of Gaeumannomyces graminis. Plant Pathol 40:524–532
Robinson RL, Postgate JR (1980) Oxygen and nitrogen in biological nitrogen fixation. Annu Rev
Robinson B, Russell C, Hedley CB (2001) Cadmium adsorption by rhizobacteria: implications for
New Zealand pastureland. Agric Ecosyst Environ 87:315–321
Rondon MR, Goodman RM, Handelsman J (1999) The earth’s bounty: assessing and accessing
soil microbial diversity. Trends Biotechnol 17:403–409
Rosenblueth M, Martı́nez-Romero E (2006) Bacterial endophytes and their interactions with hosts.
Mol Plant Microbe Interact 19:827–837
Ryder MH, Jones DA (1990) Biological control of crown gall. In: Hornby D, Cook RJ, Henis Y
(eds) Biological control of soil- borne plant pathogens. CAB International, Oxford, UK,
pp 45–63
Ryder MH, Yan Z, Terrace TE, Rovira AD, Tang W, Correll RL (1999) Use of strains of Bacillus
isolated in China to suppress take-all and rhizoctonia root rot, and promote seedling growth of
glasshouse grown wheat in Australian soils. Soil Biol Biochem 31:19–29
Safronova VI, Stepanok VV, Engqvist GL, Alekseyev YV, Belimov AA (2006) Root associated
bacteria containing1-aminocyclopropane-1-carboxylate deaminase improve growth and nutri-
42:267–272
Saravanakumar D, Vijayakumar C, Kumar N, Samiyappan R (2007) PGPR-induced defense
responses in the tea plant against blister blight disease. Crop Prot 26:556–565
Selvakumar G, Kundu S, Joshi P, Nazim S, Gupta AD, Mishra PK, Gupta HS (2008) Characteri-
zation of a cold-tolerant plant growth-promoting bacterium Pantoea dispersa 1A isolated from
a sub-alpine soil in the North Western Indian Himalayas. World J Microbiol Biotechnol
24:955–960
Shaharoona B, Arshad M, Zahir ZA (2006) Effect of plant growth promoting rhizobacteria
containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on
nodulation in mung bean. Lett Appl Microbiol 42:155–159
Shanahan P, O’Sullivan DJ, Simpson P, Glennon JD, O’Gara F (1992) Isolation of 2, 4-
Diacetylphloroglucinol from a fluorescent pseudomonad and investigation of physiological
parameters influencing its production. Appl Environ Microbiol 58:353–358
Sharma A, Johri BN, Sharma AK, Glick BR (2003) Plant growth-promoting bacterium Pseudo-
monas sp. strain GRP3 influences iron acquisition in mung bean (Vigna radiata L. Wilzeck).
Sheng XF, Xia JJ (2006) Improvement of rape (Brassica napus) plant growth and cadmium uptake
by cadmium-resistant bacteria. Chemosphere 64:1036–1042
Shiomi HF, Silva HAS, de Melo IS, Nunes FV, Bettiol W (2006) Bioprospecting endophytic
bacteria for biological control of coffee leaf rust. Sci Agric 63:32–39
Sikora RA (1992) Management of the antagonistic potential in agricultural ecosystems for the
biological control of plant parasitic nematodes. Annu Rev Phytopathol 30:245–270
Somers E, Vanderleyden J, Srinivasan M (2004) Rhizosphere bacterial signalling: a love parade
beneath our feet. Crit Rev Microbiol 30:205–240
Sridevi M, Yadav NCS, Mallaiah KV (2008a) Production of Indole-acetic-acid by Rhizobium
isolates from Crotalaria species. Res J Microbiol 3:276–281
Sridevi M, Kumar KG, Mallaiah KV (2008b) Production of catechol-type of siderophores by
Rhizobium sp. isolated from stem nodules of Sesbania procumbens (Roxb.) W and A. Res
Steddom K, Menge JA, Crowley D, Borneman J (2002) Effect of repetititve applications of the
biocontrol bacterium Pseudomonas putida 06909-rif/nal on citrus soil microbial communities.
Phytopathology 92:857–862
Stintzi A, Barnes C, Xu J, Raymond KN (2000) Microbial iron transport via a siderophore shuttle:
a membrane ion transport paradigm. Proc Natl Acad Sci USA 97:10691–10696
Sturz AV, Christie BR (2003) Beneficial microbial allelopathies in the root zone: the management
of soil quality and plant disease with rhizobacteria. Soil Tillage Res 72:107–123
Sturz A, Kimpinski J (2004) Endo-root bacteria derived from marigolds (Tagetes spp.) can
decrease soil population densities of root lesion nematodes in the potato root zone. Plant Soil
262:241–249
Tank N, Saraf M (2003) Phosphate solubilization, exopolysaccharide production and indole
acetic acid secretion by rhizobacteria isolated from Trigonella graecum. Indian J Micro-
biol 43:37–40
Tannii A, Takeuchi T, Horita H (1990) Biological control of scab, black scruff and soft rot of
potato by seed bacterization. In: Hornby D, Cook RJ, Henis Y (eds) Biological control of soil-
borne plant pathogens. CAB International, Oxford UK, pp 143–146
Thomashow LS, Weller DM (1988) Role of a phenazine antibiotic from Pseudomonas
fluorescens in biological control of Gaeumannomyces graminis var. tritici. J Bacteriol
170:3499–3508
Thomashow LS, Bonsal RF, Weller DM (2003) Detection of antibiotics production by soil and
rhizosphere microbes in situ. Thomashow Lab Methods:1–13
Thomshaw LS, Webler DM (1995) Current concepts in the use of introduced bacteria
for biological disease control of Gaeumamomyces graminis var. tritici. J Bacteriol
170:3499–3508
Tripathi M, Munot HP, Shouche Y, Meyer JM, Goel R (2005) Isolation and functional characteri-
zation of siderophore producing lead and cadmium resistant Pseudomonas putida KNP9. Curr
Trivedi P, Pandey A, Palni LMS (2008) In vitro evaluation of antagonistic properties of Pseudo-
monas corrugata. Microbiol Res 163:329–336
Tsavkelova EA, Cherdyntseva TA, Netrusov AI (2005) Auxin production by bacteria associated
with orchid roots. Microbiology 74:46–53
Van Loon LC, Bakker PAHM, Pieterse CMJ (1998) Systemic resistance induced by rhizosphere
bacteria. Annu Rev Phytopathol 36:453–483
Verma A, Kukreja K, Pathak DV, Suneja S, Narula N (2001) In vitro production of plant growth
regulators (PGRs) by Azorobacter chroococcum. Indian J Microbiol 41:305–307
Vining LC (1990) Functions of secondary metabolites. Annu Rev Microbiol 44:395–427
Vivas A, Biro B, Ruiz-Lozano JM, Barea JM, Azcon R (2006) Two bacterial strains isolated from
a Zn-polluted soil enhance plant growth and mycorrhizal efficiency under Zn toxicity. Chemo-
sphere 52:1523–1533
Voisard C, Keel C, Haas D, Defago G (1989) Cyanide production by Pseudomonas fluorescens
helps suppress black root of tobacco under gnotobiotic conditions. EMBO J 8:351–358
Wang C, Knill E, Glick BR, Defago G (2000) Effect of transferring 1-aminocyclopropane-l-
carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHAO and its
gac A derivative CHA96 on their growth promoting and disease-suppressive capacities. Can J
Wani PA, Khan MS, Zaidi A (2007a) Synergistic effects of the inoculation with nitrogen fixing
and phosphate-solubilizing rhizobacteria on the performance of field grown chickpea. J Plant
Nutr Soil Sci 170:283–287
Wani PA, Khan MS, Zaidi A (2007b) Chromium reduction, plant growth promoting potentials and
metal solubilization by Bacillus sp. isolated from alluvial soil. Curr Microbiol 54:237–243
Wani PA, Khan MS, Zaidi A (2007c) Co inoculation of nitrogen fixing and phosphate
solubilizing bacteria to promote growth, yield and nutrient uptake in chickpea. Acta
Agron Hung 55:315–323
Wani PA, Khan MS, Zaidi A (2008a) Chromium reducing and plant growth promoting Mesorhi-
Wani PA, Khan MS, Zaidi A (2008b) Effect of metal tolerant plant growth promoting Rhizobium
33–42
Wani PA, Khan MS, Zaidi A (2008c) Impact of zinc-tolerant plant growth promoting rhizobacteria
on lentil grown in zinc-amended soil. Agron Sustain Dev 28:449–455
Wittenberg JB, Wittenberg BA, Day DA, Udvardi MK, Appleby CA (1996) Siderophore bound
iron in the peribacteroid space of soybean root nodules. Plant Soil 178:161–169
Witter AE, Luther WG (1998) Variation in Fe-organic complexation with depth in the Northwest-
ern Atlantic Ocean as determined using a kinetic approach. Marine Chem 62:241–258
Wu CH, Wood TK, Mulchandani A, chen W (2006a) Engineering plant microbe symbioses for
rhizoremediation of heavy metals. Appl Environ microbial 72:1129–1134
Yasmin S, Rahman M, Hafeez FY (2004) Isolation, characterization and beneficial effects of

rice associated plant growth promoting bacteria from Zanzibar soils. J Basic Microbiol
44:241–252
Yuen GY, Schroth MN, McCain AH (1985) Reduction of Fusarium wilt of carnation with
suppressive soils and antagonistic bacteria. Plant Dis 69:1071–1075
Zahir ZA, Munir A, Asghar HN, Shaharoona B, Arshad M (2008) Effectiveness of rhizobacteria
containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought
conditions. J Microb Biotechnol 18:958–963
Zaidi A (1999) Synergistic interactions of nitrogen fixing microorganisms with phosphate mobi-
lizing microorganisms. PhD Thesis, Aligarh Muslim University, Aligarh
Zaidi A, Khan MS (2006) Co-inoculation effects of phosphate solubilizing microorganisms and
Glomus fasciculatum on greengram-Bradyrhizobium symbiosis. Turk J Agric For 30:223–230
Zaidi A, Khan MS, Amil M (2003) Interactive effect of rhizotrophic microorganisms on yield and
nutrient uptake of chickpea (Cicer arietinum L.). Eur J Agron 19:15–21
Zaidi S, Usmani S, Singh BR, Musarrat J (2006) Significance of Bacillus subtilis strain SJ 101 as a
bioinoculant for concurrent plant growth promotion and nickel accumulation in Brassica
juncea. Chemosphere 64:991–997
Zdor RE, Anderson AJ (1992) Influence of root colonizing bacteria on the defense responses in
bean. Plant Soil 140:99–107
Zehnder G, Klopper J, Yao C, Wei G (1997) Induction of systemic resistance in cucumber against
cucumber beetles (Coleoptera Chrysomelidae) by plant growth promoting rhizobacteria.
J Econ Entomol 90:391–396
Zhang F, Dhasti N, Hynes R, Smith DL (1996) Plant growth promoting rhizobacteria and soybean
[Glycine max (L.) Merr.] nodulation and nitrogen fixation at sub-optimal root zone tempera-
tures. Ann Bot 77:453–459
Chapter 7
Plant Growth Promoting Rhizobacteria
and Sustainable Agriculture
Azeem Khalid, Muhammad Arshad, Baby Shaharoona, and Tariq Mahmood
Abstract The diverse groups of bacteria in close association with roots and capable
of stimulating plant growth by any mechanism(s) of action are referred to as plant
growth-promoting rhizobacteria (PGPR). They affect plant growth and develop-
ment directly or indirectly either by releasing plant growth regulators (PGRs) or
other biologically active substances, altering endogenous levels of PGRs, enhanc-
ing availability and uptake of nutrients through fixation and mobilization, reducing
harmful effects of pathogenic microorganisms on plants and/or by employing
multiple mechanisms of action. Recently, PGPR have received more attention for
use as a biofertilizer for the sustainability of agro-ecosystems. Selection of efficient
PGPR strains based on well-defined mechanism(s) for the formulation of bio-
fertilizers is vital for achieving consistent and reproducible results under field condi-
tions. Numerous studies have suggested that PGPR-based biofertilizers could be used
as effective supplements to chemical fertilizers to promote crop yields on sustainable
basis. Various aspects of PGPR biotechnology are reviewed and discussed.
7.1 Introduction
Sustainability in agricultural systems without compromising the environmental

quality and conservation is one of the major concerns of today’s world. The
excessive use of agro-chemicals (fertilizers and pesticides) is posing serious threats
M. Arshad (*) and B. Shaharoona

Institute of Soil and Environmental Sciences, University of Agriculture, Faisalabad 38040,
Pakistan
e-mail: arshad_ises@yahoo.com; shaha_ss@yahoo.com
A. Khalid and T. Mahmood
Department of Environmental Sciences, PMAS Arid Agriculture University, Rawalpindi 46300,
Pakistan
e-mail: azeemuaf@yahoo.com; qiratm@yahoo.com
134 A. Khalid et al.
to the environment. The gradual reduction in the use of chemicals in agriculture

without affecting yield or quality of the crop produce can only be possible with
a new generation of technologies. Furthermore, the long-term sustainability of
agricultural systems depends most likely on effective handling of the internal/
indigenous resources of agro-ecosystems. During the last couple of decades, the
new biotechnologies have opened new vistas for enhancement of agriculture
productivity in a sustainable manner. Advances in understanding of soil microbiol-
ogy and biotechnology have made possible exploitation of soil microorganisms for
improving crop productivity and, in turn, have offered an economically attractive
and ecologically viable supplement to reduce external inputs to some extent.
The plant rhizosphere is a remarkable ecological environment as myriad micro-
organisms colonize in, on, and around the roots of growing plants. Distinct commu-
nities of beneficial soil microorganisms are associated with the root systems of all
higher plants (Khalid et al. 2006). These rhizobacteria are considered as efficient
microbial competitors in the root zone, and the net effect of plant–microbe associa-
tions on plant growth could be positive, neutral, or negative. Such bacteria inhabiting
plant roots and influencing plant growth positively are often referred to as
plant growth-promoting rhizobacteria (PGPR) (Kloepper et al. 1986; Arshad and
Frankenberger 1998; Zahir et al. 2004). These bacteria, after inoculation, rapidly
colonize onto seeds and roots in response to exudation and, thus, affect plant growth.
A complex matrix of organic and inorganic constituents of soil, particularly
rhizosphere, creates a unique and dynamic environment for the microorganisms
which affect plants and other associative microorganisms. Many microorganisms
are highly dependent for their survival on preformed substrates exuded by plant
roots (Frankenberger and Arshad 1995; Glick et al. 1998; Khalid et al. 2006). In
turn, the soil microflora inhabiting the rhizosphere can cause dramatic changes in
plant growth and development by producing plant growth regulators (PGRs) or
biologically active substances, or by altering endogenous levels of PGRs, and/or by
facilitating the supply and uptake of nutrients and providing other benefits.
A diverse array of bacteria, including species of Rhizobium, Bradyrhizobium,
Pseudomonas, Azospirillum, Azotobacter, Bacillus, Klebsiella, Enterobacter,
Xanthomonas, Serratia and many others, have been shown to facilitate plant growth
by various mechanisms.
Over the years, the PGPR have received worldwide importance and acceptance
for agricultural benefits. These microorganisms are the potential tools for sustain-
able agriculture because they not only ensure the availability of essential nutrients
to plants but also enhance the nutrient use efficiency. Several authors have reported
significant increases in growth and yield of agricultural crops in response to
microbial (PGPR) inoculants both under greenhouse and field conditions (Kennedy
et al., 2004; Khalid et al. 2004a, 2006; Gravel et al. 2007; Kumar et al. 2007;
Zhuang et al. 2007; Arshad et al. 2008; Banchio et al. 2008; Contesto et al. 2008;
Figueiredo et al. 2008; Mubeen et al. 2008; Naiman et al. 2009). Beside promoting
plant growth, PGPR also enhance efficiency of fertilizers, mitigate abiotic stresses,
manage plant pathogens, and cause the degradation of xenobiotic compounds
(Glick 2003, 2004; Huang et al. 2004; Khan 2005; Arshad et al. 2007; Saleem
7 Plant Growth Promoting Rhizobacteria 135
et al. 2007; Zhuang et al. 2007; Ahmad et al. 2008; Amor et al., 2008; Dell’Amico
et al. 2008; Lebeau et al., 2008; Masoud and Abbas 2009; Kohler et al. 2009).
Many promising microorganisms have been isolated and marketed as biofertili-
zers; however, their effects on crop yields fluctuate from crop to crop, place to
place, and from season to season, depending on the survival of the introduced
microorganisms on seed, roots, and in soil (Poi and Kabi 1979; Chanway and Holl
1992; Nowak 1998; Khalid et al. 2004a; Hafeez et al. 2006). To make effective
utilization of microbial inoculants, accurate and reliable methods for monitoring the
fate of applied PGPR in the rhizosphere/rhizoplane are required to enhance their
efficacy under field conditions. Scientists have used multidisciplinary approaches to
understand the adaptation of PGPR to the rhizosphere, including their mechanisms of
action and root colonization, production of determinants, and biodiversity, etc. They
are trying to manipulate the rhizosphere so that PGPR perform better and help to
increase food production for mankind on a sustainable basis. To achieve this suc-
cessfully, we need to know the players and to understand their interactions with each
other and with the growth substrate, in addition to abiotic factors which otherwise
have drastic effects on PGPR as well as on plant growth under diverse field condi-
tions. In this chapter, the potential mechanisms of action of PGPR, reasons for
inconsistency in their performance, and formulation of effective biofertilizers to be
used as the supplement to chemical fertilizers, are critically reviewed and discussed.
7.2 Mechanisms of Action
PGPR affect growth and development of plants by direct or indirect mechanisms

(Table 7.1). The direct mechanisms include N2-fixation, mobilization of nutrients
via production of phosphatases, siderophores, or organic acids, and production of
phytohormones and enzymes (Lucy et al. 2004; Khalid et al. 2004a, b; Gray and
Smith 2005; Çakmakçi et al. 2006; Tsavkelova et al. 2007). Indirectly, the bacteria
may exert a positive influence on plant growth by lessening certain deleterious
effects of a pathogenic organism by inducing host resistance to the pathogen or by
knocking out the pathogen from root surfaces or by producing chitinases or other
pathogen-suppressing substances (Raj et al. 2003; Guo et al. 2004; Van Loon and
Glick 2004; Van Loon 2007). Although scientists have reported both direct and
indirect methods of growth stimulation by PGPR, but there is no clear separation
between these two mechanisms. Certain bacteria posses multiple traits to affect
plant growth where one trait may dominate the other one (Shaharoona et al. 2006a,
Shaharoona et al. 2008; Hafeez et al. 2006). A bacterium influencing plant growth
by releasing PGRs can also play a role in controlling plant pathogens and diseases,
and vice versa. So, plant response to PGPR is a complex phenomenon, and recent
advances in research at the molecular level have provided a sufficient basis to
understand these mechanisms more precisely. The major mechanisms of PGPR
action involved in the improvement of plant growth and development are discussed
in the following sections.
t1:1 Table 7.1 Possible mechanism(s) of action of PGPR for plant growth promotion
t1:2 Plants PGPR Suggested References
mechanism(s)
of action
Lactuca sativa Pseudomonas mendocina ACC deaminase Kohler et al.
t1:3 L. cv. Tafalla Palleroni activity (2009)
Oryza sativa Methylobacterium sp. strain Indole-3-acetic Senthilkumar
t1:4 NPFM-SB3 acid, cytokinins et al. (2009)
Solanum Bacillus sp. Auxins Ahmed and
tuberosum Hasnain
t1:5 (2008)
Arabidopsis Phyllobacterium brassicacearum ACC deaminase Contesto et al.
t1:6 thaliana STM196, Pseudomonas putida activity (2008)
UW4, Rhizobium leguminosarum
bv. viciae 128C53K,
Mesorhizobium loti MAFF303099
Phaseolus Rhizobiumtropici (CIAT899), Indole acetic acid, Figueiredo et al.
t1:7 vulgaris L. Paenibacillus polymyxa (DSM 36), cytokinin, (2008)
Rhizobium, P. polymyxa strain N2-fixation
Loutit (L), Bacillus sp.
Triticum Pseudomonas spp., Burkholderia ACC deaminase Shaharoona
aestivum L. caryophylli activity, et al.
chitinase (2007b,
t1:8 2008)
Cicer Serratia oderifera (J118), ACC Shahzad et al
t1:9 arietinum L. Pantoea dispersa (J112) deaminase, P- (2008)
and Enterobactor gergoviae solubilization
(J107)
Malus domestica PGPR strains (OSU-142, OSU-7, Indole acetic Aslantas et al.
t1:10 Borkh BA-8 and M-3) acid, cytokinin (2007)
Brassica rapa Pseudomonas putida UW4 ACC deaminase Cheng et al.
t1:11 activity (2007)
Lycopersicon Pseudomonas fluorescens, P. Indole acetic Gravel et al.
t1:12 esculentum fluorescens subgroup G strain 2, acid (2007)
P. marginalis, P. putida subgroup
B strain 1 and P. syringae strain1
Apios americana Pseudomonas fluorescens TDK1 ACC deaminase Saravanakumar
activity and
Samiyappan
t1:13 (2007)
Pisum sativum Pseudomonas putida biotype A, ACC deaminase, Shaharoona
A7’ Acinetobacter calcoaceticus, ethylene et al.
t1:14 M9, P. fluorescens, AM3 (2007a)
Triticum Bacillus pumilus Auxin, siderophore Hafeez et al.
t1:15 aestivum L. (2006)
Rubus niveus Bacillus sp. N2-fixation, Orhan et al.
t1:16 P-solubilization (2006)
Zea mays L., Pseudomonas spp. ACC deaminase Shaharoona
Vigna radiata activity, et al.
t1:17 chitinase (2006a, b)
activity
(continued)
Table 7.1 (continued) t1:18

Plants PGPR Suggested References t1:19
mechanism(s)
of action
Zea mays L. Azotobacter chroococcum, N2-fixation, Wu et al. (2005) t1:20
Bacillus megaterium, P-
Bacillus mucilaginous solubilization,
K-
solubilization
Triticum PGPR Indole-3-acetic Khalid et al.
aestivum L. acid (2004a, b) t1:21
Lycopersicon PGPR Auxins Garci’a et al.
esculentum, (2003) t1:22
Capsicum
annuum
Brassica rapa, P. putida GR12-2 and Indole-3-acetic Patten and
Vigna radiata an IAA-deficient mutant acid Glick (2002) t1:23
Hordeum vulgare Arthrobacter mysorens 7, Indole-3-acetic Pishchik et al.
Flavobacterium sp. L30, acid, ethylene (2002) t1:24
Klebsiella mobilis CIAM880
Pinus pinea Bacillus spp. Gibberellins Probanza et al.
(2002) t1:25
Oryza sativa L. Rhizobium, Azospirillum Indole-3-acetic Biswas et al.
acid (2000) t1:26
Oryza sativa L. Rhizobium leguminosarum Indole-3-acetic Dazzo et al.
(strain E11) acid (2000) t1:27
Vigna radiata P. putida GR 12-2 (wild type), Indole-3-acetic Mayak et al.
GR12-2/acd36 (ACC acid, ethylene (1999) t1:28
deaminase minus mutant),
GR 12-2/aux1 (IAA over
producers)
Vigna radiata Pseudomonas sp. Biocontrol Sindhu et al.
(1999) t1:29
– Paenibacillus polymyxa cytokinins Timmusk et al.
(1999) t1:30
Cucumis sativus P. putida, Serratia Biocontrol Wei et al.
marcescens (1996) t1:31
7.2.1 Fixation, Mobilization and Uptake of Nutrients
Nutrients are one of the extremely important factors which influence growth, yield,
and quality of different crops. Soil microorganisms can provide nutrients to plants
either through the fixation of atmospheric N2 or by enhancing nutrient mobilization/
uptake through their biological activities, such as mineralization and through side-
rophore, organic acid and phosphatase production, etc.
Biological N2 fixation by rhizobia and associative diazotrophic bacteria is a
spontaneous process and one of the widely studied mechanisms by which plants
benefit from the interacting partners. The bacteria benefit the plants by fixing N2 in
exchange for fixed carbon either provided directly to the bacteria or indirectly by
releasing carbon as root exudates. A range of bacteria participates in interactions

with different plants, significantly increasing their vegetative growth and grain
yields. However, obtaining maximum benefits on farms from diazotroph PGPR
biofertilizer requires a systematic strategy designed to fully utilize all these benefi-
cial factors, allowing crop yields to be maintained or even increased while fertilizer
applications are reduced (Kennedy et al. 2004). Numerous studies have shown that
different species of bacteria fix atmospheric N2 and consequently affect growth and
yield of various crops (Sindhu et al. 2002; Bai et al. 2003; Orhan et al. 2006; Afzal
and Bano 2008; Khalequzaman and Hossain 2008; Figueiredo et al. 2008). In
addition to biological N2-fixation, PGPR are also known to affect the nutrient
availability to the plant through acidification and redox changes or by producing
iron chelators and siderophores, and/or mobilizing the metal phosphates (Burd et al.
2000; Römkens et al. 2002; Abou-Shanab et al. 2003). Several reports have
suggested that PGPR can stimulate plant growth through their P-solubilizing
activity (Khan et al. 2007; Wani et al. 2007; Afzal and Bano 2008). Furthermore,
Wu et al. (2005) reported increased assimilation of nutrients, such as N, P, and K, in
plants, in response to inoculation with P-solubilizer (Bacillus megaterium) and
K-solubilizer (Bacillus mucilaginous). Likewise, Orhan et al. (2006) reported that
inoculation with a phosphate-solubilizing Bacillus strain M3 significantly improved
P, Fe, and Mn contents of the leaves of raspberry (Rubus idaeus), suggesting that
Bacillus M3 alone or in combination with some other strains had the potential to
increase the nutrition of raspberry plants, in addition to growth and yield.
7.2.2 Production of Plant Growth-Regulating Substances
Plant growth-regulating substances are naturally occurring organic compounds that

influence various physiological processes in plants, such as cell elongation and cell
division. They perform these functions at concentrations far below the levels at
which nutrients and vitamins normally affect plant processes. It is now well
established that the majority of soil microorganisms can produce plant growth-
regulating substances, including phytohormones (auxins, gibberellins, cytokinins,
ethylene, and abscisic acid) and enzymes (Frankenberger and Arshad 1995; Glick
1995; Khalid et al. 2006), which is considered as one of the major mechanisms of
plant growth promotion by PGPR. Production of PGRs by microorganisms is
affected by the presence of suitable substrate(s)/precursor(s) as well as type and
concentration of exudates. The inocula in the presence of a specific physiological
precursor of a PGR and/or inocula that produce physiologically-active concentra-
tions of a phytohormone can be highly effective in promoting plant growth and
enhancing consistency and reproducibility (Frankenberger and Arshad 1995;
Arshad and Frankenberger 1998, 2002).
Microflora capable of producing PGRs in vitro predominates in the rhizosphere
of plants. However, the type and amount of growth-regulating substances released
by such microorganisms are variable. The quantitative or qualitative variations in
plant growth-promoting substances in turn lead to the differences in plant responses

to the PGPR inocula. Several studies have reported the ability of various PGPR to
produce auxins in vitro and in vivo (Almonacid et al. 2000; Khalid et al. 2004a, b;
Gravel et al. 2007). Similarly, plant-associated phototrophic purple bacterium
(Serdyuk et al. 1995) and Methylobacterium sp. (Senthilkumar et al. 2009) have
been reported to be capable of producing cytokinins in vitro. Different bacterial
species such as Proteus mirabilis, P. vulgaris, Klebsiella pneumoniae, Bacillus
megaterium, B. cereus, Escherichia coli and many more have been reported to
synthesize plant growth-promoting substances, including auxin, gibberellin, cyto-
kinin and abscisic acid (Tuomi and Rosenquist 1995; Karadeniz et al. 2006;
Tsavkelova et al. 2007). Soil microbiota is also known to produce the gaseous
phytohormone ethylene in vitro and in vivo (Weingart et al. 1999; Akhtar et al.
2005).
Some PGPR can influence plant growth by altering the synthesis of endogenous
phytohormones through the production of specific enzymes. Among these enzymes,
bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase plays a significant
role in the regulation of a plant hormone, ethylene, and thus the modification of the
growth and development of plants (Arshad and Frankenberger 2002; Glick 2005).
Bacterial strains with ACC deaminase can at least partially eliminate the stress-
induced ethylene-mediated negative impact on plants by converting the germinat-
ing seed/root’s ACC into a-ketobutyrate and ammonia (Glick et al. 1998). Since
then, numerous species of Gram-negative and Gram-positive bacteria have been
reported to produce ACC deaminase (Belimov et al. 2005; Pandey et al. 2005;
Sessitsch et al. 2005; Blaha et al. 2006; Madhaiyan et al. 2006; Stiens et al. 2006;
Shaharoona et al. 2006a, 2006b, 2007a, 2007b, 2008; Shahzad et al. 2008).
7.2.3 Biological Control
Soil-borne pathogens are one of the major limiting factors for low crop productivity
due to their inhibitory effects on plant health. In modern agronomic practices, a
huge amount of chemicals (insecticide/fungicide) are used to offset various patho-
gens inflicting severe losses to crop yields. Although these chemicals are vital for
controlling the pathogens on the one hand, on the other hand they can drastically
affect the microbial diversity and functional properties of natural microbial com-
munities of soils, leading thereby to imbalanced agro-ecosystems (Singh et al.
2005; Vig et al. 2006; Mubeen et al. 2008). It is therefore important to discover
the most viable and economical means for effective disease management in an
environment-friendly manner. Currently, biopesticides are receiving worldwide
attention and considered important for the sustainability of the agricultural system.
Furthermore, the WTO guideline that suggests that only residue-free agricultural
produce can be exported has further created a great interest and demand for the use
of biopesticides in crop protection systems. In recent times, PGPR have emerged as
potential candidates with wide scope for inducing systemic resistance in crop plants
against many pathogens (Jeun et al. 2004; Nakkeeran et al. 2005; Khalequzaman
and Hossain 2008; Mirik et al. 2008; Masoud and Abbas 2009). Various species of
bacteria including Pseudomonas fluorescens, P. putida, P. cepacia, P. aeruginosa,
Bacillus spp., Rhizobium and many other PGPR exhibit biological control activity
and inhibit pathogens by synthesizing chitinase, and by production of hydrogen
cyanide, protease, siderophores, and cellulase, and/or indirectly by promoting plant
growth and health through any mode of action (Zahir et al. 2004; Hafeez et al. 2006;
Narayanasamy 2008).
7.2.4 Multiple Mechanisms of Action
Some PGPR can stimulate plant growth through multifarious activities. Although
PGPR have been reported to influence plant growth by an array of mechanisms, the
specific traits by which PGPR facilitate plant growth, yield, and nutrient uptake
were limited to the expression of one or more of the traits expressed simultaneously
in a given environment of plant–microbe interaction. A PGPR can promote plant
growth by improving plant nutrition, modifying root growth architecture, and by
plant responses to external stress factors, simultaneously (Egamberdiyeva and
Hflich 2004; Dey et al. 2004; Saleem et al. 2007; Shaharoona et al. 2006a, b,
2007a, b, 2008). However, one trait may dominate the other one of the same PGPR
when exposed to certain environmental conditions. For instance, Dey et al. (2004)
reported more than one mechanism of PGPR responsible for growth promotion.
They suggested that, besides ACC deaminase activity, expression of one or more of
the traits such as suppression of phytopathogens, solubilization of tricalcium
phosphate, production of siderophore and/or nodulation promotion by the PGPR
might have simultaneously contributed to the enhancement of growth, yield, and
nutrient uptake of peanut (Arachis hypogaea). Similarly, Shaharoona et al. (2006a, b)
reported plant growth promotion by PGPR employing multiple mechanisms,
such as ACC deaminase activity, chitinase activity, and root colonization. Further-
more, Hafeez et al. (2006) attributed increase in plant growth to multiple traits (such
as production of IAA, and siderophores and P-solubilization) of PGPR. We have
also found that PGPR expressing dual traits, such as ACC deaminase and nitroge-
nase activity or ACC deaminase with phosphatase activity performed better at
enhancing growth and yield of wheat (Triticum aestivum) and maize (Zea mays)
than PGPR possessing a single trait (Arshad et al., unpublished data).
7.3 Application of PGPR in Agriculture
PGPR are commonly used as inoculants for improving the growth and yield of
agricultural crops. They represent an essential and large component of biofertilizer
technology to improve the productivity of agricultural systems in the long run
(Zahir et al. 2004; Khalid et al. 2006; Naiman et al. 2009). Many PGPR have shown
great promise as potential inoculants for agriculture uses and environmental pro-
tection, and can play very critical roles in maintaining the sustainability of agro-
ecosystems. However, the current use of PGPR in agriculture is poor despite
numerous reports on their fair performance under laboratory conditions.
PGPR possess the ability to colonize and establish an ongoing relationship with
plants, resulting in better root growth, more biomass, and a substantial increase in
crop yields. In this context, significant effects of PGPR have been observed on
various agricultural crops, including legumes, cereals, and noncereals, and other
environmentally important plant species. Furthermore, the impact of PGPR on root
development with the consequent advantage on increasing water and nutrient use
efficiencies has also been observed (Zahir et al. 2005; Ahmad et al. 2008; Arshad
et al. 2008). The potential uses and benefits of PGPR in the improvement of overall
performance of plants are discussed in the following sections.
7.3.1 Effect of PGPR on Plant Growth
The potential of PGPR for improving growth and yields of various crops has been
extensively documented (Table 7.2). However, most of the studies have been
conducted under controlled environments rather than under natural field conditions.
Results of these studies have demonstrated clearly that PGPR carry abundant
potentials to enhance growth and yield of target crops. However, the selection of
a functionally effective PGPR strain is very critical, and the plant responses are
often variable depending upon the bacterial strain, plant genotypes, and experimen-
tal sites. It has also been claimed that the PGPR isolated from a particular crop or
ecological zone are more effective in producing consistent results if reapplied to the
same crop and reused in the same ecological zone (Chanway and Holl 1992; Nowak
1998). This might be due to a greater adaptability of the introduced PGPR in the
given rhizosphere, while inconsistency in the responses of same crop to same PGPR
could be attributed to (1) the poor quality of inocula, (2) short shelf life of PGPR,
(3) lack of standard delivery systems, and/or (4) failure in maintaining a required
density of PGPR onto seeds or roots. Moreover, the nature and composition of the
material used as a carrier for a PGPR also plays a significant role in producing its
impact on the inoculated plants.
Another important aspect of these trials is that the effects of PGPR have been
investigated under different fertilizer doses, which has been shown to affect the
efficiency of PGPR, leading to inconsistent performance under different agro-
ecosystems (Shaharoona et al. 2008). Considering the acute demand for food
supply, it is wise to make efforts to improve crop production by using PGPR,
over and above what is achievable with optimum chemical fertilizers. It is also
pertinent that most of the investigations have been focused on diazotrophs, which
were tested under different N application rates. It could be very useful if selected
PGPR were tested under different rates of all the three major nutrients (N, P, and K)
t2:1 Table 7.2 Plant responses to inoculation with PGPR
142
t2:2 PGPR Plants Comments References

t2:3 Azospirillum brasilense Oryza sativa L The inoculation increased aerial and root biomass and Naiman et al. (2009)
Az1 and Az2, P. grain yield by 12, 40 and 16%, respectively, over
fluorescens Pf uninoculated control
t2:4 A. brasilense, Pantoea Capsicum annuum Inoculation increased the concentration of citric, Amor et al. (2008)
dispersa ascorbic and succinic acids in green fruit of
sweet pepper compared to noninoculated control
t2:5 Bacillus sp. Solanum tuberosum Bacterial inoculation caused increment in the growth Ahmed and Hasnain (2008)
of the plants compared to the noninoculated
treatments
t2:6 P. fluorescens, B. subtilis, Origanum majorana L. Only P. fluorescens and Bradyrhizobium sp. showed Banchio et al. (2008)
Sinorhizobium meliloti, significant increases in shoot length, shoot weight,
Bradyrhizobium sp. number of leaves and node, and root dry weight,
in comparison to control plants or plants treated with
other PGPR. Essential oil yield was also significantly
increased relative to noninoculated plants, without
alteration of oil composition
t2:7 Phyllobacterium Arabidopsis thaliana Root hairs of seedlings inoculated with the ACC Contesto et al. (2008)
brassicacearum STM196, deaminase strains were significantly longer
P. putida UW4, R.
leguminosarum
bv. viciae 128C53K,
Mesorhizobium loti
t2:8 Rhizobiumtropici (CIAT899), Phaseolus vulgaris L. Beans coinoculated with R. tropici (CIAT899) and Figueiredo et al. (2008)
Paenibacillus P. polymyxa (DSM 36) had higher leghemoglobin
polymyxa (DSM 36), concentrations, nitrogenase activity and N2 fixation efficiency
Rhizobium, and thereby formed associations of
P. polymyxa strain Loutit greater symbiotic efficiency. Inoculation with
(L), Rhizobium and P. polymyxa strain Loutit (L)
Paenibacillus, Bacillus stimulated nodulation.
sp. PGPR also stimulated specific-nodulation
(number of nodules per gram of root dry weight) and increased
A. Khalid et al.
accumulated N
t2:9 Bacillus strains Capsicum annuum Stem diameter, root elongation, root dry weight, shoot Mirik et al. (2008)
dry weight and yield were increased in response to
inoculation in the field experiment by 7.0–20.5, 7.0–17.0,
4.5–23.5, 16.5–38.5, and 11.0–33.0%, respectively
t2:10 Pseudomonas spp. Triticum aestivum L. Inoculation significantly increased growth, yield and Shaharoona et al. (2008)
nutrient use efficiency of wheat
t2:11 Serratia oderifera (J118), Cicer The PGPR in the presence of P-enriched compost resulted Shahzad et al. (2008)
Pantoea dispersa (J112), arietinum L. in a highly significant increase in fresh biomass (84%),
Enterobactor gergoviae number of pods plant 1 (97%), grain yield (79%) and
(J107) number of nodules plant 1 (87%) compared to
uninoculated control
t2:12 PGPR strains OSU-142, Malus domestica Borkh) Inoculation with OSU-142, OSU-7, BA-8 and M-3 PGPR Aslantas et al. (2007)
OSU-7, BA-8 and M-3) increased average shoot length by 59.2, 18.3, 7.0 and
14.3% relative to the control and fruit yield by 116.4,
7 Plant Growth Promoting Rhizobacteria
88.2, 137.5 and 73.7%, respectively. Bacterial inoculation

increased shoot diameter from 7.0 to 16.3% compared to control
t2:13 P. fluorescens, P. fluorescens Lycopersicon esculentum Pseudomonas putida was shown to improve fruit yields Gravel et al. (2007)
subgroup G strain 2, in rockwool and in organic medium.
P. marginalis, P. putida The production of IAA was shown as a
subgroup B possible mechanism for plant growth
strain 1 and P. syringae stimulation by the bacterium. In addition, roots of tomato
strain 1) seedlings grown in the presence of increasing concentrations of
IAA were significantly longer when seeds were treated with P.
putida
t2:14 B. megaterium, B. subtilis, Zea mays L. All three bacterial inoculants resulted in an increment Kumar et al. (2007)
Pseudomonas corrugate in grain yield of maize up to 122, 135 and 194%, respectively,
compared to respective control.
The overall beneficial effects of bacterial
inoculations contributed to the colonization and
survival of the introduced bacteria, and to
stimulation of the indigenous microflora in the rhizosphere
(continued)
143
t2:15 Table 7.2 (continued)
144

B. subtilis BEB-lSbs (BS13) Lycopersicon esculentum Yield per plant, fruit weight and length were increased Mena-Violante and Olalde-
t2:17 significantly by the Bacillus subtilis BEB-lSbs (BS13) treatment Portugal (2007)
when compared to the control
Pseudomonas sp., Triticum aestivum L. Both PGPR containing ACC deaminase positively Shaharoona
t2:18 Burkholderia caryophylli influenced growth and yield of wheat et al. (2007b)
t2:19 B. pumilus 8 N-4 Inoculation of the wheat variety Orkhon with PGPR Hafeez et al. (2006)
B. pumilus 8 N-4 (originated from Mongolia) resulted
in the maximum increase in plant biomass, root length,
and total N and P contents in plants. The isolate was
also capable of producing auxin and siderophore
t2:20 Cyanobacterial strains Oryza sativa L. Significant increases in grain and straw yield were Jha and Prasad (2006)
observed when rice seedlings were inoculated with
four cyanobacterial strains either applied alone or
in combination with chemical fertilizer. In addition,
a saving of 25 kg Nha 1 was attained through cyanobacterial
fertilization
Pseudomonas sp. Zea mays L., Vigna Significant increases in plant height, root weight and Shaharoona
t2:21 radiata total biomass were observed in response to et al. (2006a, b)
inoculation with PGPR containing ACC deaminase. Similarly,
inoculation significantly improved grain
yield of maize in the presence of nitrogenous fertilizers.
Effect of PGPR was also positive on nodulation of mung
bean (Vigna radiata)
t2:22 Pseudomonas, Azotobacter, Triticum aestivum L. Significant positive effects of inoculation on germination and Shaukat et al. (2006)
Azospirillum growth of wheat were observed
t2:23 A. chroococcum, Zea mays L. The application of PGPR significantly increased the plant Wu et al. (2005)
B. megaterium, growth and resulted in the highest biomass and seedling
B. mucilaginous height. Inoculation not only increased the nutritional
assimilation of plant (total N, P, and K), but also improved soil
properties, such as organic matter content and total N in soil
A. Khalid et al.
t2:24 Pseudomonas spp. Arachis hypogaea L. Seed inoculation with PGPR containing ACC deaminase resulted in Dey et al. (2004)
a significantly higher pod yield than the control, in pots, during
rainy and post-rainy seasons. PGPR also significantly enhanced
pod yield (23–26,
24–28, and 18–24%, respectively), haulm yield and nodule dry
weight over the control under field
conditions
t2:25 B. licheniformis CECT 5106, Quercus ilex ssp. ballota Only B. licheniformis promoted the growth of Q. ilex seedlings. Domenech et al. (2004)
B. pumilus CECT 5105 Furthermore, B. licheniformis inhibited
fungal growth as revealed by ergosterol/chitin analysis
t2:26 PGPR isolates Triticum aestivum L. Peat-based seed inoculation with selected PGPR strains capable of Khalid et al. (2004a)
producing auxins exhibited stimulatory
effects on grain yields of tested wheat cv. in pot
(up to 14.7% increase over control) and field
experiments (up to 27.5% increase over control); however, the

response varied with cv. and PGPR
strains. It was concluded that the strain which
produced the highest amount of auxins in nonsterilized
rhizosphere soil also caused maximum increase in
growth and yield of both the wheat cultivars
t2:27 PGPR Quercus ilex ssp. ballota, All strains significantly increased stem length, neck Garci´a et al. (2003)
Pinus pinea diameter and shoot dry weight of the inoculated plants
t2:28 Enterobacter cloacae, P. Brassica rapa Inoculation significantly enhanced root elongation Penrose and Glick (2003)
putida, of canola under gnotobiotic conditions
P. fluorescens
t2:29 Rhizobacteria Brassica A significant increase in growth was observed in the inoculated Asghar et al. (2002)
juncea seedlings. The PGPR were also capable of producing IAA
t2:30 P. putida Am2, P. putida Brassica Significant increase in root elongation of phosphorus-sufficient Belimov et al. (2002)
Bm3, juncea L. seedlings of rapeseed in a growth-pouch
Alcaligenes xylosoxidans culture experiment was observed in response to inoculation
cm4,
Pseudomonas sp. Dp2
145
(continued)
t2:31 Table 7.2 (continued)
146

t2:33 P. putida GR12-2 and an Brassica rapa, Primary roots of canola seeds treated with wild-type strain Patten and Glick (2002)
IAA-deficient mutant Vigna radiata were 35–50% longer than the roots from seeds treated with the
IAA-deficient mutant and the roots from uninoculated seeds.
Exposing mung bean cuttings to
high levels of IAA by soaking in a suspension of the
wild-type strain stimulated formation of many adventitious roots
t2:34 Arthrobacter mysorens 7, Hordeum vulgare All the PGPR actively colonized barley root system Pishchik et al. (2002)
Flavobacterium sp. L30, and rhizosphere, and significantly stimulated root
Klebsiella mobilis elongation up to 25%
CIAM880
B. licheniformis CECT 5106, Pinus pinea Both Bacillus strains promoted the growth of Probanza
t2:35 B. pumilus CECT 5105 P. pinea seedlings et al. (2002)
t2:36 Rhizobium, Azospirillum Oryza sativa L. Inoculation with diazotrophs had significant Biswas et al. (2000)
growth-promoting effects on rice seedlings
t2:37 R. leguminosarum (strain Oryza sativa L. Growth-promoting effects of inoculation on rice seedlings Dazzo et al. (2000)
E11) were observed under axenic conditions
Azotobacter Zea mays L. Inoculation with strains efficient in IAA production had Zahir et al.
t2:38 significant growth-promoting effects on maize seedlings (2000)
t2:39 P. putida GR 12-2 (wild- Vigna radiata Only the wild-type strain produced longer roots Mayak et al. (1999)
type),
GR12-2/acd36 (ACC
deaminase
minus mutant), GR 12-2/
aux1
(IAA over producers)
A. Khalid et al.
instead of N only. Moreover, the soil fertility status should also be considered while
using PGPR along with specific doses of N, P, and K fertilizers. Therefore, the
proper understanding of mechanism(s) of action of chosen PGPR can substantially
help in obtaining consistent responses in terms of improved growth and yield of
bioprimed plants/crops. If all these factors are considered properly, the agriculture
industry can draw significant benefits from the PGPR-based biotechnological
approaches. Another aspect which requires urgent attention is the quality improve-
ment of the agricultural produce following PGPR inoculation. Since PGPR exert
their influence through different mechanism(s), it is very likely that they can affect
quality of the produce; unfortunately, most of the studies have reported the effects of
PGPR on growth and yield of inoculated plants in quantitative terms, but very little is
known about the quality parameters of produce modified by the PGPR inoculations.
Future efforts should, hence, be directed to assess how the quality of food produce is
influenced by PGPR, besides their role in growth and development of plants.
7.3.2 PGPR in Stress Agriculture
Agricultural crops are exposed to many stresses that are induced by both biotic and
abiotic factors. These stresses invariably affect plant growth and yield of crops
depending on the type and intensity of stress. Under stress conditions, such as
salinity, drought, waterlogging, heavy metals and pathogenicity, the production of
ethylene in plants at substantially accelerated rates is a very common feature, which
adversely affects the root growth and, consequently, the development of the plants.
As described earlier (see Sect. 2.2), certain PGPR lower ethylene synthesis by
metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher
plants) into a-ketobutyrate and ammonia, and, thus, mitigate the negative impact
of both biotic and abiotic stresses on plants (Saleem et al. 2007). Recently, several
authors have documented profound effects of inoculation with PGPR containing
ACC deaminase on plant growth under stress conditions (Table 7.3).
In the present scenario of several biotic and abiotic stresses to which agriculture
is confronted, the role of PGPR containing ACC deaminase could be crucial for
sustainable crop production. However, some beneficial aspects of these PGPR
under salinity, drought, waterlogging, biocontrol, temperature, and nutritional
stresses, and in the cut-flower industry and in nodulation in legumes have not been
thoroughly exploited. Glick (2006) reported that transgenic tomato (Lycopersicon
lycopersicum), canola (Brassica napus), and tobacco (Nicotiana tabacum) plants
that express ACC deaminase exclusively in their roots behaved physiologically
similarly to nontransformed plants treated with ACC deaminase-containing
PGPR, both in the presence and absence of various stresses. Consequently, there is
no apparent advantage to the use of ACC deaminase transgenic plants compared to
treating the roots of the plants with ACC deaminase-containing PGPR. However,
genetic modification of all plant species is not possible due to many limitations,
such as proprietary rights and international trade agreements on genetically
148
t3:1 Table. 7.3 Effect of PGPR on plant growth under various abiotic and biotic stresses
t3:2 Plant species PGPR Plant responses under stress References
t3:3 Lactuca sativa Pseudomonas mendocina The inoculated plants had significantly greater shoot biomass Kohler et al. (2009)
L. cv. Tafalla Palleroni than
the control plants at low and high salinity levels. At the
highest salinity level, the water content was greater in leaves
of plants
treated with P. mendocina. The plants also showed higher
concentrations of foliar K and lower concentrations of foliar
Na under high salt conditions
Sorghum bicolor, Pseudomonas spp. Inoculation with PGPR containing ACC deaminase significantly Arshad and
t3:4 Zea mays L. improved fresh biomass under water-deficient field conditions Khalid (2008)
Pisum sativum L Pseudomonas spp. The inoculation partially eliminated the effects of water stress on Arshad et al. (2008),
t3:5 growth, yield and ripening of P. sativum L., both in pot and Zahir et al. (2008)
field trials
t3:6 Brassica rapa P. putida UW4 Induced salt tolerance of plants by lowering the synthesis of Cheng et al. (2007)
salt-induced stress ethylene and promoted the growth of
canola
in a saline environment
Apios americana P. fluorescens TDK1 The PGPR strain enhanced the saline resistance in the plants and Saravanakumar and
t3:7 increased yield as compared to strains lacking ACC Samiyappan (2007)
deaminase activity
Zea mays L. Unidentified PGPR Significantly increased plant growth under salinity stress Nadeem et al. (2006,
t3:8 conditions 2007)
t3:9 Vitis vinifera L. Burkholderia Inoculation enhanced plant growth and physiological activity at Barka et al. (2006)
phytofirmans PsJN both ambient (26 C) and low (4 C) temperatures. Inoculation
also increased root growth and plantlet biomass. Moreover,
the bacterium significantly improved plantlet cold tolerance
compared to that of the nonbacterized control, which was
more sensitive to exposure to low temperatures
A. Khalid et al.
t3:10 Solanum tuberosum PGPR PGPR were capable of antagonizing at least one of the two potato Rasche et al. (2006)
pathogens Ralstonia solanacearum and Rhizoctonia solani
Pisum sativum Pseudomonas sp. Inoculation with bacteria counteracted the Cd-induced inhibition Safronova
t3:11 of nutrient uptake by roots et al (2006)
t3:12 Brassica napus PGPR Increases (up to 31%) in root elongation of inoculated rape Sheng and Xia (2006)
seedlings compared to the control plants were observed.
Inoculation with the isolates was found to increase root dry
weight (ranging from 8 to 20%) and shoot dry weight (ranging
from 6 to 25%) of rape in cadmium-amended soil in pot
experiments. The bacterial isolates were also able to colonize
and develop in the rhizosphere soil of rape after root
inoculation
t3:13 Brassica juncea L. Variovorax paradoxus, Plant growth was improved in Cd2+-supplemented media in Belimov et al. (2005)
Rhodococcus sp. response to inoculation

t3:14 Pisum sativum Variovorax paradoxus 5C-2 Inoculated plants gave more seed yield (25–41%), seed number Dodd et al. (2005)
and seed nitrogen accumulation than uninoculated plants
under moisture stress and watering conditions
Chamaecytisus proliferus P. fluorescens The bacterium showed positive effect in antagonizing the growth Donate-Correa et al.
t3:15 of Fusarium oxysporum and Fusarium proliferatum in (2005)
growth medium.
t3:16 Mimosa pudica Burkholderia sp. The bacterium exhibited antagonistic activity against Rhizoctonia Pandey et al. (2005)
solani and Sclerotinia sclerotiorum
t3:17 Phragmites australis Pseudomonas asplenii AC Inoculation resulted in normal plant growth under high levels Reed et al. (2005)
of Cu2+ and creosote
t3:18 Lycopersicon esculentum Achromobacter piechaudii Inoculation significantly increased the fresh and dry weights of Mayak et al. (2004a, b)
tomato seedlings grown in the presence of NaCl salt
concentration up to 172 mM. The bacterium also significantly
increased the fresh and dry weights of both tomato and pepper
seedlings exposed to transient water stress
(continued)
149
t3:19 Table. 7.3 (continued)
150
t3:20 Plant species PGPR Plant responses under stress References

t3:21 Glycine max B. subtilis NEB4, NEB5, B. Coinoculation exhibited consistent and significant increases in Bai et al. (2003)
thuringiensis NEB17, nodule number, nodule weight, shoot weight, root weight,
Bradyrhizobium japonicum total biomass, total nitrogen, and grain yield at low (25, 17,
and
15 C) root zone temperatures
Brassica juncea L. Pseudomonas sp., Alcaligenes The bacteria were tolerant to Cd2+ toxicity and stimulated Belimov
t3:22 sp., Variovorax paradoxus, root elongation of rape seedlings in the presence et al. (2001)
B. pumilus, Rhodococcus sp. of 300mM CdCl2 in the nutrient solution
Lycopersicon esculentum P. putida UW4, Enterobacter Inoculated plants showed substantial tolerance to flooding Grichko and
t3:23 cloacae CAL2, P. Putida stress Glick (2001)
t3:24 Brassica juncea L., Kluyvera ascorbata Toxic effects of heavy metals (Ni2+, Pb2+ and Zn2+) were not Burd et al. (2000)
Lycopersicum esculentum pronounced in inoculated plants
Mill
t3:25 Cucumis sativus P. putida UW4 The bacterial strains were effective in biocontrol of Wang et al. (2000)
Pythium ultimum
t3:26 Solanum tuberosum Burkholderia phytofirmans PsJN PGPR helped potato plants in maintaining normal Bensalim et al. (1998)
growth under heat stress
A. Khalid et al.
modified crops and restrictions in the use of genetically modified microorgan-

isms or plants. The use of PGPR containing ACC deaminase activity along with
other traits, such as the ability to synthesize nitrogenases, phosphatases, and
chitinases could prove to be a cost-effective and environment-friendly strategy
to ensure sustainable agriculture.
7.3.3 PGPR for Bioremediation
Recently, the application of PGPR in association with plants has been expanded to
remediate contaminated soils (Khan et al. 2009). The addition of PGPR increases
the removal of pollutant most likely by enhancing germination, and by stimulating
plant growth including root biomass and survival of plants, in soils that are heavily
contaminated (Huang et al. 2004; Reed et al. 2005; Safronova et al. 2006; Arshad
et al. 2007). Some rhizobacteria can enhance phytoremdiation by promoting plant
growth through the synthesis of siderophores, phytohormones, enzymes, and anti-
biotics (Pattern and Glick 1996; Burd et al. 2000; Khalid et al. 2006; Arshad et al.
2007; Wani et al. 2008a), and/or through stimulation of certain metabolic pathways,
such as nitrogen fixation and the uptake of N, P, S, Mg, Ca, and other nutrients
(Belimov and Dietz 2000). Similarly, PGPR can increase the tolerance of plants to
contaminants; the PGPR–plant system cannot survive in comparatively extreme
environments such as with high concentrations of heavy metals (Wani et al. 2008a).
Although microbial communities in polluted soils have been studied, little is known
about the composition of microbial community in the plant rhizosphere growing on
highly polluted soils. Usually, rhizosphere soil is more conducive to remediation
due to high concentrations of nutrients exuded from the roots and dense bacterial
populations. Many workers have reported bioremediation of both organic
(Narasimhan et al. 2003; Huang et al. 2004, 2005; Villacieros et al. 2005; Muratova
et al. 2005) and inorganic contaminants in the environment by using PGPR
(Hallberg and Johnson 2005; Kao et al. 2006; Umrania 2006; Burd et al. 2000;
Kamnev et al. 2005; Abou-Shanab et al. 2006; Wu et al. 2006; Zaidi et al. 2006;
Sheng and Xia 2006; Wani et al. 2008b).
7.4 Formulations of Effective Biofertilizers
A number of steps are involved in developing effective PGPR-based biofertilizers

for achieving consistent results in terms of crop productivity under field conditions
(Fig. 7.1). The most critical steps involved in the development of biofertilizers
include isolation of bacterial strains from the same habitat and/or crop followed by
screening of PGPR under axenic conditions by conducting repeated trials. The
strains showing better results under controlled conditions should be tested further
for their performance under natural conditions by conducting pot and field trials.
Fig. 7.1 Steps involved in the

Isolation of bacteria Screening
development of an effective
biofertilizer product
Trials under axenic conditions
Pot trials
Trials under field conditions
Commercial biofertilizer Standardization

(Quality control)
Finally, the PGPR strain selected for biofertilizer formulation should be investi-
gated thoroughly to maintain its quality. Quality of biofertilizers is one of the most
critical factors which determine their success or failure and acceptance or rejection
by end users, the farmers. The functionality of selected PGPR must be defined well
before using it as a candidate for biofertilizer formulation, which could be achieved
by employing biochemical and molecular tests in the laboratory and then determin-
ing correlations between PGPR traits with the growth promotion of inoculated
plants under axenic and natural conditions. However, there is no established basis
for acceptance of a formulation as an effective biofertilizer. Since PGPR based
biofertilizers contain living entities, which are very sensitive to environmental
conditions, the consistency in effectiveness cannot be as good as observed in the
case of chemical fertilizers. However, maintaining a particular population of a
selected PGPR strain could help in enhancing the consistency of biofertilizer
effects. A strict control over quality is the only answer to avoid failure of biofer-
tilizers in different agronomic regions of the world.
The production of biofertilizer and its acceptance by farming communities are
closely linked. For their use to expand globally at the farmers’ end, quality
management is essential and must be performed consistently in order to supply
contaminant-free bioproducts to the users. For this, skilled personnel are required
who know how to work with these materials and be able to respond to the modern
conditions of agricultural production. In addition, they should be well aware of the
sustainability and environmental protection measures. Furthermore, proper guide-
lines for the production and commercialization of biofertilizers should be framed in
order to popularize the use of such bioagents for maintaining the sustainability of
agro-ecosystems across the globe.
7.5 Conclusion
Enhancement in the use of PGPR is one of the newly emerging options for meeting
agricultural challenges imposed by the still-growing aggregate demand for food.
Moreover, this biotechnology is also likely to ensure conservation of our environments.
However, before PGPR can contribute to such benefits, scientists must learn
more about them and explore ways and means for their better utilization in the
farmers’ fields.
Future research should focus on managing plant–microbe interactions, particu-
larly with respect to their mode of actions and adaptability to conditions under
extreme environments for the benefit of plants. Furthermore, scientists need to
address certain issues, like how to improve the efficacy of biofertilizers, what
should be an ideal and universal delivery system, how to stabilize these microbes
in soil systems, and how nutritional and root exudation aspects could be controlled
in order to get maximum benefits from PGPR application. Biotechnological and
molecular approaches could possibly develop more understanding about PGPR
mode of actions that could lead to more successful plant–microbe interaction.
Efforts should also be directed towards the use of PGPR to reduce pesticide
applications. In brief, PGPR biotechnology provides an excellent opportunity to
develop environment-friendly biofertilizer to be used as supplements and/or alter-
natives to chemical fertilizers.
References
Abou-Shanab RI, Angle JS, Chaney RL (2006) Bacterial inoculants affecting nickel uptake by
Alyssum murale from low, moderate and high Ni soils. Soil Biol Biochem 38:2882–2889
Abou-Shanab RI, Angle JS, Delorme TA, Chaney RL, van Berkum P, Moawad H, Ghanem K,
Ghozlan HA (2003) Rhizobacterial effects on nickel extraction from soil and uptake by
Alyssum murale. New Phytol 158:219–224
Afzal A, Bano A (2008) Rhizobium and phosphate solubilizing bacteria improve the yield and
phosphorus uptake in wheat (Triticum aestivum). Int J Agri Biol 10:85–88
Ahmad R, Arshad M, Khalid A, Zahir ZA (2008) Effectiveness of organic-/bio-fertilizer supple-
mented with chemical fertilizers for improving soil water retention, aggregate stability, growth
and nutrients uptake of maize (Zea mays L.). J Sust Agri 31:57–77
Ahmed A, Hasnain S (2008) Auxin producing Bacillus sp.: auxin quantification and effect on the
growth of Solanum tuberosum. J Biotechnol 136:766–767
Akhtar MJ, Arshad M, Khalid A, Mehmood MH (2005) Substrate-dependent biosynthesis of
ethylene by rhizosphere soil fungi and its influence on etiolated pea seedlings. Pedobiologia
49:211–219
Almonacid S, Quintero N, Martinez M, Vela M (2000) Determination of quality parameters of
bacterial inocula based on liquid formulation elaborated with strains producing indole acetic
acid (IAA). Auburn university websitehttp://www.ag.auburn.edu/argentina/pdfmanuscript/
almonacid.pdf
Amor FMD, Martı́nez AS, Fortea MI, Legua P, Delicado EN (2008) The effect of plant-associative
bacteria (Azospirillum and Pantoea) on the fruit quality of sweet pepper under limited nitrogen
supply. Sci Hortic 117:191–196
Arshad M, Frankenberger WT Jr (1998) Plant growth regulating substances in the rhizosphere:
microbial production and functions. Adv Agron 62:146–151
Arshad M, Frankenberger WT Jr (2002) Ethylene: agricultural sources and applications. Kluwer
Academic, New York
Arshad M, Saleem M, Hussain S (2007) Perspectives of bacterial ACC deaminase in phytoreme-
diation. Trends Biotechnol 25:356–362
Arshad M, Khalid A (2008) Annual report. University of Agriculture, Faisalabad, Pakistan

Arshad M, Shaharoona B, Mahmood T (2008) Inoculation with plant growth promoting rhizo-
bacteria containing ACC-deaminase partially eliminates the effects of water stress on growth,
yield and ripening of Pisum sativum L. Pedosphere 18:611–620
Asghar HN, Zahir ZA, Arshad M, Khaliq A (2002) Relationship between in vitro production of
auxins by rhizobacteria and their growth-promoting activities in Brassica juncea L. Biol Fertil
Soils 35:231–237
Aslantas R, Cakmakci R, Sahin F (2007) Effect of plant growth promoting rhizobacteria on young
apple tree growth and fruit yield under orchard conditions. Scientia Hort 111:371–377
Bai Y, Zhou X, Smith DL (2003) Enhanced soybean plant growth resulting from coinoculation of
Bacillus strains with Bradyrhizobium japonicum. Crop Sci 43:1774–1781
Banchio E, Bogino PC, Zygadlo J, Giordano W (2008) Plant growth promoting rhizobacteria
improve growth and essential oil yield in Origanum majorana L. Biochem Syst Ecol 36:
766–771
Barka EA, Nowak J, Clément C (2006) Enhancement of chilling resistance of inoculated grapevine
plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans Strain
PsJN. Appl Environ Microbiol 72:7246–7252
Belimov AA, Dietz KJ (2000) Effect of associative bacteria on element composition of barley
seedlings grown in solution culture at toxic cadmium concentrations. Microbiol Res 155:
113–21
(2005) Cadmium-tolerant plant growth-promoting bacteria associated with the roots of Indian
mustard (Brassica juncea L. Czern.). Soil Biol Biochem 37:241–250
Belimov AA, Safronova VI, Mimura T (2002) Response of spring rape (Brassica napus var.
oleifera L.) to inoculation with plant growth promoting rhizobacteria containing 1-aminocy-
clopropane-1-carboxylate deaminase depends on nutrient status of the plant. Can J Microbiol
48:189–199
Belimov AA, Safronova VI, Sergeyeva TA, Egorova TN, Matveyeva VA, Tsyganov VE, Borisov
AY, Tikhonovich IA, Kluge C, Preisfeld A, Dietz KJ, Stepanok VV (2001) Characterization of
plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocy-
clopropane-1-carboxylate deaminase. Can J Microbiol 47:242–252
Bensalim S, Nowak J, Asiedu SK (1998) A plant growth promoting rhizobacterium and tempera-
ture effects on performance of 18 clones of potato. J Potato Res 75:145–152
Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG (2000) Rhizobial inoculation influences
seedling vigor and yield of rice. Agron J 92:880–886
Blaha D, Prigent-Combaret C, Mirza MS, Moenne-Loccoz Y (2006) Phylogeny of the 1-amino-
cyclopropane-1-carboxylic acid deaminaseencoding gene acdS in phytobeneficial and patho-
genic Proteobacteria and relation with strain biogeography. FEMS Microbiol Ecol 56:
455–470
Burd GI, Dixon DG, Glick BR (2000) Plant growth-promoting bacteria that decrease heavy metal
toxicity in plants. Can J Microbiol 46:237–245
Çakmakçi R, Dönmez F, Aydm A, Şahin F (2006) Growth promotion of plants by plant growth-
promoting rhizobacteria under greenhouse and two different field soil conditions. Soil Biol
Biochem 38:1482–1487
Chanway CP, Holl FB (1992) Influence of soil biota on Douglas fir (Pseudotsuga menziesii)
seedling growth: the role of rhizosphere bacteria. Can J Bot 70:1025–1031
Cheng Z, Park E, Glick BR (2007) 1-Aminocyclopropane-1-carboxylate (ACC) deaminase from
Pseudomonas putida UW4 facilitates the growth of canola in the presence of salt. Can
Contesto C, Desbrosses G, Lefoulon C, Bena G, Borel F, Galland M, Gamet L, Varoquaux F,
Touraine B (2008) Effects of rhizobacterial ACC deaminase activity on Arabidopsis indicate
that ethylene mediates local root responses to plant growth-promoting rhizobacteria. Plant Sci
175:178–189
Dazzo FB, Yanni YG, Rizk R, DeBruijn FJ, Rademaker J, Squartini A, Corich V, Mateos P,
Martinez-Molina E (2000) Progress in multinational colaboraive studies on the beneficial
association between Rhizobium leguminoserum bv. Trifolii and rice. In: Ladha JK, Reddy
PM (eds) The quest for nitrogen fixation in rice. IRRI, Los Banos, pp 167–189
Dell’Amico E, Cavalca L, Andreoni V (2008) Improvement of Brassica napus growth under
cadmium stress by cadmium-resistant rhizobacteria. Soil Biol Biochem 40:74–84
Dey R, Pal KK, Bhatt DM, Chauhan SM (2004) Growth promotion and yield enhancement of
peanut (Arachis hypogaea L.) by application of plant growth-promoting rhizobacteria. Micro-
biol Res 159:371–394
Dodd IC, Belimov AA, Sobeih WY, Safronova VI, Grierson D, Davies WJ (2005) Will modifying
plant ethylene status improve plant productivity in water-limited environments? Fourth inter-
national crop science congresshttp://www.cropscience.org.au/icsc2004/poster/1/3/4/510_dod-
dicref.htm. Accessed 17 June 2007
Domenech J, Solano BR, Probanza A, Garca JAL, Colon JJ, Gutierrez-Manero FJ (2004)
Bacillus spp. and Pisolithus tinctorius effects on Quercus ilex ssp. ballota: a study on tree
growth, rhizosphere community structure and mycorrhizal infection. Forest Ecol Manage
194:293–303
Donate-Correa J, Leon-Barrios M, Perez-Galdona R (2005) Screening for plant growth-promoting
rhizobacteria in Chamaecytisus proliferus (tagasaste), a forage tree-shrub legume endemic to
the Canary Islands. Plant Soil 266:261–272
Egamberdiyeva D, Hflich G (2004) Effect of plant growth-promoting bacteria on growth and
nutrient uptake of cotton and pea in a semiarid region of Uzbekistan. J Arid Environ 56:
293–301
Figueiredo MVB, Martinez CR, Burity HA, Chanway CP (2008) Plant growth-promoting rhizo-
bacteria for improving nodulation and nitrogen fixation in the common bean (Phaseolus
vulgaris L.). World J Microbiol Biotechnol 24:1187–1193
Frankenberger WT Jr, Arshad M (1995) Phytohormones in soils: microbial production and
function. Marcel Dekker, New York
Garci’a JL, Probanza A, Ramos B, Manero FG (2003) Effects of three plant growth-promoting
rhizobacteria on the growth of seedlings of tomato and pepper in two different sterilized and
nonsterilized peats. Arch Agron Soil Sci 49:119–127
Glick BR (1995) The enhancement of plant growth by free living bacteria. Can J Microbiol
41:109–117
Glick BR (2004) Teamwork in phytoremediation. Nature Biotechnol 22:526–527
Glick BR (2005) Modulation of plant ethylene levels by the bacterial enzyme ACC deaminase.
FEMS Microbiol Lett 251:1–7
Glick BR (2006) Plant responses to ACC deaminase-containing PGPR. 7th international workshop
on plant growth promoting rhizobacteria, 28 May–2 June 2006. Noordwijkerhout, The Nether-
lands, p 30
Glick BR, Penrose DM, Li J (1998) A model for lowering plant ethylene concentration by plant
growth promoting rhizobacteria. J Theor Biol 190:63–68
Gravel V, Antoun H, Tweddell RJ (2007) Growth stimulation and fruit yield improvement of
greenhouse tomato plants by inoculation with Pseudomonas putida or Trichoderma atroviride:
possible role of indole acetic acid (IAA). Soil Biol Biochem 39:1968–1977
Gray EJ, Smith DL (2005) Intracellular and extracellular PGPR: commonalities and distinctions in
the plant–bacterium signaling processes. Soil Biol Biochem 37:395–412
Grichko VP, Glick BR (2001) Flooding tolerance of transgenic tomato plants expressing the
bacterial enzyme ACC deaminase controlled by the 35 S, rolD or PRB-1b promoter. Plant
Physiol Biochem 39:19–25
Guo JH, Qi HY, Guo YH, Ge HL, Gong LY, Zhang LX, Sun PH (2004) Biocontrol of tomato wilt
by plant growth-promoting rhizobacteria. Biol Control 29:66–72
Hafeez FY, Yasmin S, Ariani D, Mehboob-ur-Rahman ZY, Malik KA (2006) Plant growth-
promoting bacteria as biofertilizer. Agron Sust Dev 26:143–150
Hallberg KB, Johnson DB (2005) Microbiology of a wetland ecosystem constructed to remediate
mine drainage from a heavy metal mine. Sci Total Environ 338:53–66
Huang XD, El-Alawi Y, Penrose DM, Glick BR, Greenberg BM (2004) Responses of three grass
species to creosote during phytoremediation. Environ Pollut 130:453–63
Huang XD, El-Alawi Y, Penrose DM, Glick BR, Greenberg BM (2005) Responses of hydro-
carbons (TPHs) from soils. Microchem J 81:139–47
Jeun YC, Park KS, Kim CH, Fowler WD, Kloepper JW (2004) Cytological observations
of cucumber plants during induced resistance elicited by rhizobacteria. Biol Control
29:34–42
Jha MN, Prasad AN (2006) Efficacy of new inexpensive cyanobacterial biofertilizer including its
shelf-life. World J Microbiol Biotechnol 22:73–79
Kamnev AA, Tugarova AV, Antonyuk LP, Tarantilis PA, Polissiou MG, Gardiner PHE (2005)
Effects of heavy metals on plant-associated rhizobacteria: comparison of endophytic and non-
endophytic strains of Azospirillum brasilense. J Trace Elem Med Biol 19:91–5
Kao PH, Huang CC, Hseu ZY (2006) Response of microbial activities to heavy metals in a neutral
loamy soil treated with biosolid. Chemosphere 64:63–70
Karadeniz A, Topcuoglu SF, Inan S (2006) Auxin, gibberellin, cytokinin and abscisic acid
production in some bacteria. World J Microbiol Biotechnol 22:1061–1064
Kennedy IR, Choudhury ATMA, Kecskés ML (2004) Non-symbiotic bacterial diazotrophs in
crop-farming systems: can their potential for plant growth promotion be better exploited? Soil
Khalequzaman KM, Hossain I (2008) Effect of seed treatment with Rhizobium strains and
biofertilizers on foot/root rot and yield of bushbean in Fusarium oxysporum infested soil.
J Agric Res 46:55–64
Khalid A, Arshad M, Zahir ZA (2004a) Screening plant growth-promoting rhizobacteria for
improving growth and yield of wheat. J Appl Microbiol 96:473–480
Khalid A, Tahir S, Arshad M, Zahir ZA (2004b) Relative efficiency of rhizobacteria for auxin
biosynthesis in rhizosphere vs. non-rhizosphere soil. Aust J Soil Res 42:921–926
Khalid A, Arshad M, Zahir ZA (2006) Phytohormones: microbial production and applications. In:
Uphoff N et al (eds) Biological approaches to sustainable soil systems. Taylor & Francis, Boca
Raton, Florida, pp 207–220
Khan AG (2005) Role of soil microbes in the rhizospheres of plants growing on trace metal
contaminated soils in phytoremediation. J Trace Elem Med Biol 18:355–364
Khan MS, Zaidi A, Wani PA (2007) Role of phosphate solubilizing microorganisms in sustainable
agriculture: a review. Agron Sustain Dev 27:29–43
remediation of metal contaminated soils. Environ Chem Lett 7:1–19
Kloepper JW, Scher FM, Tripping B (1986) Emergence promoting rhizobacteria: description and
implication for agriculture. In: Swinburne TR (ed) Iron, siderophores and plant diseases.
Plenum, New York, pp 155–164
Kohler J, Hernández JA, Caravaca F, Roldán A (2009) Induction of antioxidant enzymes is
involved in the greater effectiveness of a PGPR versus AM fungi with respect to increasing
the tolerance of lettuce to severe salt stress. Environ Exp Bot 65:245–252
Kumar B, Trivedi P, Pandey A (2007) Pseudomonas corrugata: a suitable bacterial inoculant
for maize grown under rainfed conditions of Himalayan region. Soil Biol Biochem
39:3093–3100
Ladha JK, Reddy PM (2000) The quest for nitrogen fixation in rice. IRRI, Los Banos
Lebeau T, Braud A, Jézéquel K (2008) Performance of bioaugmentation-assisted phytoextraction
applied to metal contaminated soils: a review. Environ Pollut 153:497–522
Lucy M, Reed E, Glick BR (2004) Applications of free living plant growth-promoting rhizobac-
teria. Antonie Van Leeuwenhoek 86:1–25
Madhaiyan M, Suresh Reddy BV, Anandham R, Senthilkumar M, Poonguzhali S, Sundaram SP,

Sa TM (2006) Plant growth promoting Methylobacterium induces defense responses in
groundnut (Arachis hypogaea L.) compared to rot pathogens. Curr Microbiol 53:270–276
Masoud A, Abbas ST (2009) Evaluation of fluorescent pseudomonads for plant growth promotion,
antifungal activity against Rhizoctonia solani on common bean, and biocontrol potential. Biol
Control 48:101–107
Mayak S, Tirosh T, Glick BR (2004a) Plant growth-promoting bacteria that confer resistance to
water stress in tomato and pepper. Plant Sci 166:525–530
Mayak S, Tirosh T, Glick BR (2004b) Plant growth-promoting bacteria confer resistance in tomato
plants to salt stress. Plant Physiol Biochem 42:565–572
Mayak S, Tivosh T, Glick BR (1999) Effect of wild type and mutant plant growth-promoting
rhizobacteria on the rooting of mungbeen cuttings. J Plant Growth Regul 18:49–53
Mena-Violante HG, Olalde-Portugal V (2007) Alteration of tomato fruit quality by root inocula-
tion with plant growth-promoting rhizobacteria (PGPR): Bacillus subtilis BEB-13bs. Sci
Hortic 113:103–106
Mirik M, Aysan Y, Cinar O (2008) Biological control of bacterial spot disease of pepper with
Bacillus strains. Turk J Agri For 32:381–390
Mubeen F, Aslam A, Radl V, Schloter M, Malik KA, Hafeez FY (2008) Role of nature’s fertility
partners with crop protectants for sustainable agriculture. In: Dakora FD et al (eds) Biological
nitrogen fixation: towards poverty alleviation through sustainable agriculture. Springer, The
Netherlands, pp 153–154
Muratova AY, Turkovskaya OV, Antonyuk LP, Makarov OE, Pozdnyakova LI, Ignatov VV
(2005) Oil-oxidizing potential of associative rhizobacteria of the genus Azospirillum. Micro-
biol 74:210–215
Nadeem SM, Zahir ZA, Naveed M, Arshad M, Shahzad SM (2006) Variation in growth hizobac-
teand ion uptake of maize due to inoculation with plant growth promoting rria under salt stress.
Soil Environ 25:78–84
Nadeem SM, Zahir ZA, Naveed M, Arshad M (2007) Preliminary investigations on inducing salt
tolerance in maize through inoculation with rhizobacteria containing ACC-deaminase activity.
Can J Microbiol 53:1141–1149
Naiman AD, Latronico A, Garca de Salamone IE (2009) Inoculation of wheat with Azospirillum
brasilense and Pseudomonas fluorescens: impact on the production and culturable rhizosphere
microflora. Eur J Soil Biol 45:44–51
Nakkeeran S, Fernando WGD, Siddiqui A (2005) Plant growth promoting rhizobacteria
formulations and its scope in commercialization for the management of pests and diseases.
In: Siddiqui ZA (ed) PGPR: biocontrol and biofertilization. Springer, The Netherlands,
pp 257–296
Narasimhan K, Basheer C, Bajic VB, Swarup S (2003) Enhancement of plant-microbe interactions
using a rhizosphere metabolomics-driven approach and its application in the removal of
polychlorinated biphenyls. Plant Physiol 132:146–53
Narayanasamy P (2008) Molecular biology in plant pathogenesis and disease management.
Springer, The Netherlands
Nowak J (1998) Benefits of in vitro ‘biotization’ of plant tissue cultures with microbial inoculants.
In Vitro Cell Dev Biol Plant 34:122–130
Orhan E, Esitken A, Ercisli S, Turan M, Sahin F (2006) Effects of plant growth promoting
rhizobacteria (PGPR) on yield, growth and nutrient contents in organically growing raspberry.
Sci Hortic 111:38–43
Pandey P, Kang SC, Maheshwari DK (2005) Isolation of endophytic plant growth promoting
Burkholderia sp. MSSP from root nodules of Mimosa pudica. Curr Sci 89:170–180
Patten CL, Glick BR (1996) Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol
42:207–220
Patten CL, Glick BR (2002) Role of Pseudomonas putida indoleacetic acid in development of the
host plant root system. Appl Environ Microbiol 68:3795–3801
Penrose DM, Glick BR (2003) Methods for isolating and characterizing ACC deaminase-contain-
ing plant growth-promoting rhizobacteria. Physiol Plant 118:10–15
Pishchik VN, Vorobyev NI, Chernyaeva II, Timofeeva SV, Kozhemyakov AP, Alexeev YV,
Lukin SM (2002) Experimental and mathematical simulation of plant growth promoting
rhizobacteria and plant interaction under cadmium stress. Plant Soil 243:173–186
Poi SC, Kabi MC (1979) Effect of Azotobacter inoculation on growth and yield of jute and wheat.
Indian J Agri Sci 49:478–480
Probanza A, Garca JAL, Palomino MR, Ramos B, Mañero FJG (2002) Pinus pinea L. seedling
growth and bacterial rhizosphere structure after inoculation with PGPR Bacillus (B. licheniformis
CECT 5,106 and B. pumilus CECT 5,105). Appl Soil Ecol 20:75–84
Raj SN, Deepak SA, Basavaraju P, Shetty HS, Reddy MS, Kloepper JW (2003) Comparative
performance of formulations of plant growth promoting rhizobacteria in growth promotion and
suppression of downy mildew in pearl millet. Crop Prot 22:579–88
Rasche F, Velvis H, Zachow C, Berg G, Van Elsas JD, Sessitsch A (2006) Impact of transgenic
potatoes expressing anti-bacterial agents on bacterial endophytes is comparable with the
effects of plant genotype, soil type and pathogen infection. J Appl Ecol 43:555–566
Reed MLE, Warner BG, Glick BR (2005) Plant growth-promoting bacteria facilitate the growth of
the common reed Phragmites australis in the presence of copper or polycyclic aromatic
hydrocarbons. Curr Microbiol 51:425–429
Römkens P, Bouwman L, Japenga J, Draaisma C (2002) Potentials and drawbacks of chelate-
enhanced phytoremediation of soils. Environ Pollut 116:109–121
Safronova VI, Stepanok VV, Engqvist GL, Alekseyev YV, Belimov AA (2006) Root-associated
bacteria containing 1-aminocyclopropane-1-carboxylate deaminase improve growth and nutri-
42:267–272
Saleem M, Arshad M, Hussain S, Bhatti AS (2007) Perspectives of plant growth promoting
rhizobacteria (PGPR) containing ACC deaminase in stress agriculture. J Indust Microbiol
Saravanakumar D, Samiyappan R (2007) ACC deaminase from Pseudomonas fluorescens
mediated saline resistance in groundnut (Arachis hypogea) plants. J Appl Microbiol
102:1283–1292
Senthilkumar M, Madhaiyan M, Sundaram SP, Kannaiyan S (2009) Intercellular colonization and
growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza
sativa L. Cv CO-43). Microbiol Res 164:92–104
Serdyuk OP, Smolygina LD, Muzafarov EN, Adanin VM, Arinbasarov MU (1995) 4-Hydroxy-
phenethyl alcohol – a new cytokinin-like substance from the phototrophic purple bacterium
Rhodospirillum rubrum 1R. FEBS Lett 365:10–12
Sessitsch A, Coenye T, Sturz AV, Vandamme P, Barka E, Wang-Pruski G, Faure D, Reiter B,
Glick BR, Nowak J (2005) Burkholderia phytofirmins sp. Nov., a novel plant-associated
bacterium with plant beneficial properties. Int J Syst Evol Microbiol 55:1187–1192
Shaharoona B, Arshad M, Zahir ZA, Khalid A (2006a) Performance of Pseudomonas spp.
containing ACC-deaminase for improving growth and yield of maize (Zea mays L.) in the
presence of nitrogenous fertilizer. Soil Biol Biochem 38:2971–2975
Shaharoona B, Arshad M, Zahir ZA (2006b) Effect of plant growth promoting rhizobacteria
containing ACC-deaminase on maize (Zea mays L.) growth under axenic conditions and on
nodulation of mungbean (Vigna radiata). Lett Appl Microbiol 42:155–159
Shaharoona B, Arshad M, Khalid A (2007a) Differential response of etiolated pea seedling to 1-
aminocyclopropane-1-carboxylate and/or L-methionine utilizing rhizobacteria. J Microbiol
45:15–20
Shaharoona B, Jamro GM, Zahir ZA, Arshad M, Memon KS (2007b) Effectiveness of
various Pseudomonas spp. and Burkholderia caryophylli containing ACC-deaminase for
improving growth and yield of wheat (Triticum aestivum L.). J Microbiol Biotechnol
17:1300–1307
Shaharoona B, Naveed M, Arshad M, Zahir ZA (2008) Fertilizer dependent efficiency of Pseu-

domonads containing ACC-deaminase for improving growth, yield and nutrient use efficiency
of wheat (Triticum aestivum L.). Appl Microbiol Biotechnol 79:147–155
Shahzad MS, Khalid A, Arshad M, Khalid M, Mehboob I (2008) Integrated use of plant growth
promoting bacteria and P-enriched compost for improving growth, yield and nodulation of
chickpea. Pak J Bot 40:1735–1744
Shaukat K, Affrasayab S, Hasnain S (2006) Growth responses of Triticum aestivum to plant growth
promoting rhizobacteria used as a biofertilizer. Res J Microbiol 1:330–338
Sheng XF, Xia JJ (2006) Improvement of rape (Brassica napus) plant growth and cadmium uptake
by cadmium-resistant bacteria. Chemosphere 64:1036–1042
Sindhu SS, Gupta SK, Dadarwal KR (1999) Antagonistic effect of Pseudomonas spp. on patho-
genic fungi and enhancement of plant growth in green gram (Vigna radiata). Biol Fertil Soils
29:62–68
Sindhu SS, Suneja S, Goel AK, Parmar N, Dadarwal KR (2002) Plant growth promoting effects of
Pseudomonas sp. on coinoculation with Mesorhizobium sp. Cicer strain under sterile and “wilt
sick” soil conditions. Appl Soil Ecol 19:57–64
Singh B, Kahlon RS, Sahoo SK et al (2005) Residues of lindane and endosulfan in soil and their
effect on soil microbial population and dehydrogenase activity. Pesticide Res J 17:88–90
Stiens M, Schneiker S, Keller M, Kuhn S, Pühler A, Schlüter A (2006) Sequence analysis of
the 144-kilobase accessory plasmid psmesm11a, isolated from a dominant Sinorhizobium
meliloti strain identified during a long-term field release experiment. Appl Environ Microbiol
72:3662–3672
Timmusk S, Nicander B, Granhall U, Tillberg E (1999) Cytokinin production by Paenibacillus
polymyxa. Soil Biol Biochem 31:1847–1852
Tsavkelova EA, Cherdyntseva TA, Botina SG, Netrusov AL (2007) Bacteria associated with
orchid roots and microbial production of auxin. Microbiol Res 162:69–76
Tuomi T, Rosenquist H (1995) Detection of abscisic, gibberellic and indole-3-acetic acid from
plant and microbes. Plant Physiol Biochem 33:725–734
Umrania VV (2006) Bioremediation of toxic heavy metals using acidothermophilic autotrophes.
Bioresour Technol 97:1237–1242
Van Loon LC (2007) Plant response to plant growth-promoting rhizobacteria. Eur J Plant Pathol
119:243–254
Van Loon LC, Glick BR (2004) Increased plant fitness by rhizobacteria. In: Sandermann H (ed)
Molecular ecotoxicology of plants. Springer, Berlin, pp 178–205
Vig K, Singh DK, Sharma PK (2006) Endosulfan and quinalphos residues and toxicity to soil
microarthropods after repeated applications in a field investigation. J Environ Sci Health Part B
41:681–692
Villacieros M, Whelan C, Mackova M, Molgaard J, Sanchez-Contreras M, Lloret J (2005)
Polychlorinated biphenyl rhizoremediation by Pseudomonas fluorescens F113 derivatives,
using a Sinorhizobium meliloti nod system to drive bph gene expression. Appl Environ
Wang CKE, Glick BR, Defago G (20 00) Effect of transferring 1-aminocyclopropane-1-carboxylic
acid (ACC) deaminase genes into pseudomonas fluorescens strain CHA0 and its gacA deriva-
tive CHA96 on their growth-promoting and disease-suppressive capacities. Can J Microbiol
46:898–907
Wani PA, Khan MS, Zaidi A (2007) Co-inoculation of nitrogen fixing and phosphate solubilizing
bacteria to promote growth yield and nutrient uptake in chickpea. Acta Agron Hung 55:
315–323
Wani PA, Khan MS, Zaidi A (2008a) Effect of metal tolerant plant growth promoting Rhizobium
33–42
Wani PA, Khan MS, Zaidi A (2008b) Chromium reducing and plant growth promoting Mesorhi-
Wei G, Kloepper JW, Tuzun S (1996) Induced systemic resistance to cucumber diseases and
increased plant growth by plant growth promoting rhizobacteria under field conditions.
Phytopathol 86:221–224
Weingart H, Volksch B, Ullrich MS (1999) Comparison of ethylene production by Pseudomonas
syringae and Ralstonia aolanacearum. Phyopathology 89:360–365
Wu SC, Caob ZH, Lib ZG, Cheunga KC, Wonga MH (2005) Effects of biofertilizer containing N-
fixer, P and K solubilizers and AM fungi on maize growth: a greenhouse trial. Geoderma
125:155–166
Wu SC, Luo YM, Cheung KC, Wong MH (2006) Influence of bacteria on Pb and Zn speciation,
mobility and bioavailability in soil: a laboratory study. Environ Pollut 144:765–773
Zahir ZA, Abbas SA, Khalid A, Arshad M (2000) Substrate-dependent microbially derived plant
hormones for improving growth of maize seedlings. Pak J Biol Sci 3:289–291
Zahir ZA, Arshad M, Frankenberger WT (2004) Plant growth promoting rhizobacteria: applica-
tions and perspectives in agriculture. Adv Agron 81:96–168
Zahir ZA, Asghar HN, Akhtar MJ, Arshad M (2005) Precursor (L-tryptophan)-inoculum interac-
tion for improving yields and nitrogen uptake of maize. J Plant Nutr 28:805–817
Zahir ZA, Munir A, Asghar HN, Arshad M, Shaharoona B (2008) Effectiveness of rhizobacteria
containing ACC-deaminase for growth promotion of peas (Pisum sativum) under drought
conditions. J Microbiol Biotechnol 18:982–987
Zaidi S, Usmani S, Singh BR, Musarrat J (2006) Significance of Bacillus subtilis strain SJ-101 as a
bioinoculant for concurrent plant growth promotion and nickel accumulation in Brassica
juncea. Chemosphere 64:991–997
Zhuang X, Chen J, Shim H, Bai Z (2007) New advances in plant growth promoting rhizobacteria
for bioremediation. Environ Int 33:406–413
Chapter 8
Soil Health – A Precondition for Crop
Production
Niharendu Saha and Biswapati Mandal
Abstract Soil health is an assessment of ability of a soil to meet its range of

ecosystem functions appropriate to its environment. Soil health is generally used to
measure the competence of soil to sustain plant and animal productivity, determine
structural and functional diversity of microbes, maintain or enhance water and air
quality, and support human health and habitation. Direct assessment of such para-
meters is a herculean task. As biological attributes are indicators of soil health, they
are used for the diagnosis of soil heath. A comprehensive methodology integrating
those indicators for soil health diagnosis is discussed. Further, the impact of soil
health on crop production, quality improvement, production sustenance, and
human, animal and environmental health is reviewed and discussed. In addition,
the influence of soil management practices, like tillage, application of pesticides,
fertilization, organic amendments, etc., on soil health is discussed. Inclusion of
farmers and their experiences in the whole exercise of soil research are advocated
for development of handy and farmer-friendly soil diagnosis protocols.
8.1 Introduction
Soil is the vital nonrenewable natural resource base. It is under competitive demand
for increased crop production to feed the ever-growing population. This has led to
intensification of agriculture with extensive use of chemicals, exploitation of
surface and ground water for irrigation, and adoption of mechanization. The rapid
pace of development in agriculture during recent years has resulted in an overall
deterioration in soil-based ecosystems. The most important of such effects are
the depletion of soil organic matter (SOM) and loss of biodiversity in the soil.
N. Saha (*) and B. Mandal

Bidhan Chandra Krishi Viswavidyalaya, Kalyani, 741235, Nadia, West Bengal, India
e-mail: nihar_bckv@rediffmail.com
162 N. Saha and B. Mandal
This leads to an overall deterioration of soil health resulting in damage to essential

ecosystem functions, sustainability of agricultural production, and soil resilience
capacity (Buresh et al. 1997; Doran and Zeiss 2000). The attendant problems are
evident from the gradual decline in the rate of responses to external inputs and also
deterioration in the quality of the product, as often observed even with the best
possible management practices. The more the degree of intensification, the more is
the severity of the problem (Abrol et al. 2000). Soil degradation and concomitant
decline in soil health are often emphasized as constraints to crop productivity
(Askegaard et al. 2004; Dick and Gregorich 2004; Wang et al. 2003). As a result,
soil health has emerged as the key for assessing soil condition in the context of land
management and sustainable crop production (Doran 2002; Karlen et al. 2004;
Schjonning et al. 2004).
What does one mean by the term soil health? Does it include the soil with good
bioavailability of plant nutrients? Does it embody the soil with good aggregate,
proper aeration and high water-holding capacity? Or does it mean the empower-
ment of ecosystem processes leading to soil functioning for better crop production?
The concept of Doran et al. (1996) and Gregory et al. (2002) includes all the
attributes as mentioned and extends to ensure human, animal, plant and environ-
ment health. More than that, soil health is the specific condition of soil resulting
from soil quality. As the term ‘health’ is normally associated with living entities, in
this chapter, the soil’s biotic component, particularly microbiological attributes,
will be used for soil health diagnosis. The healthy condition of soil is vital both for
production and for environmental sustainability (Karlen et al. 2003a, 2003b; Carter
et al. 2004). Direct measurement of soil health is difficult, so indicators are used.
The attributes which are most sensitive to management are considered to be the best
indicators. Larson and Pierce (1991) proposed a minimum dataset (MDS) which
was later on modified by Doran and Parkin (1994), who included biological proper-
ties in it, which is also supported by Schjonning et al. (2004).
Soil health assessment using physical and chemical indicators is sometimes
misleading as it takes a longer time to exhibit a perceptible change in chemical
and physical properties owing to the great buffering capacity of soil (Norfleet et al.
2003). So, the measurement of the biological component of soils in the first instance
will be more suitable to assess soil health. Whenever there is any aberration in the
soil system, soil organisms receive the stresses and respond immediately, and
meaningfully, as they have a higher surface to volume ratio. In most cases, changes
in populations of soil organisms or their activities can be detected much earlier than
any change occurring in the physico-chemical properties of soil. This leads to being
able to show improvement or degradation of soil health (Pankhurst et al. 1997), but
there is inadequate available information on this aspect (Dick 1992; Heyer et al.
2003). Thus, many researchers have emphasized that biological indicators should
be used to assess soil health (Elliot et al. 1996). Some of the biological indicators
include microbial biomass C and N, dehydrogenase activity, acid and alkaline
phosphatase activity, urease activity, arylsulphatase activity, soil respiration and
ergosterol concentration, microbial diversity, and functional groups of soil fauna,
etc. (Karlen et al. 1992; Hseu et al. 1999). Indicators influencing soil biological
8 Soil Health – A Precondition for Crop Production 163
health, however, vary according to the locations and the production systems
involved (Karlen et al. 1994). Wylie (1994) concluded that it is not possible to
develop a single list of biological indicators that could be suitable for all purposes
and management practices. However, a few good biological indicators for assessing
soil health for different production systems have already been constructed and are
ready to use in soil health diagnosis (Anonymous 2005).
Microbe-mediated processes like decomposition, fixation, solubilization, filtra-
tion, remediation- and suppression of pathogens make the soil healthy and fertile.
Soils with rich microbial diversity also protect crop from losses due to unpredictable
climatic conditions of inundation, flood, and drought, etc. Experimental evidence
showed microbial indicators like diversity, biomass and enzyme activities supporting
higher biological yield and quality of crude protein and oil content (Anonymous
2005). So, identifying and quantifying the biotic component of soil for assessing
aggradation or degradation of soil health for different production systems and
subsequent mitigation options are a precondition for successful crop production.
8.2 Soil Health Concept
Soil is a wonderful gift of nature to humankind whose good health is essential for
societal existence. But the high demographic pressure including nonagricultural
operations and intensive cultivation is imposing tremendous stresses on soils. Some
of the common stresses affecting soils are presented in Table 8.1. These are
ultimately manifested in declined productivity of crops even under best possible
management practices, and make soil “sick”, so that it cannot respond efficiently to
fertilization and other inputs. To arrest this deterioration, the general recommenda-
tion is to measure the nutrient status of soil and subsequently correct the nutrient
deficiency, as well as control pest and disease incidents and involve other conser-
vative steps. Routine measurement of such attributes of soils and recommendations
for soil testing is not currently a problem, but on long-term basis it could be
misleading and result in decreased productivity. Mere tests of soil for a few
Table 8.1 Common soil stress and related degradation processes

Stress Degradation process
1. Heavy load due to vehicular traffic 1. Crusting, compacting, structural decline
2. Poor internal drainage, slow surface 2. Soil wetness and anaerobiosis
drainage
3. Intensive cropping 3. Chemical degradation, nutrient imbalance, soil
organic matter depletion, and habitat destruction
4. Intensive use of agrochemicals and 4. Biological degradation, acidification, reduction in
monoculture soil biodiversity
5. Monoculture 5. Reduction in belowground diversity, infestation of
soil-borne crop associated diseases, layer specific
nutrient depletion, structural deterioration, etc.
parameters inadequately address the problems of farmers, and, hence, the health of
soil (fertility) under intensive agriculture cannot be properly judged. So, to address
such problems associated with the deficiencies in agricultural practices, vital forces
and processes should be identified and quantified. The vital forces and the associa-
tive processes conferring good living conditions to soil leading to healthy crop
production may be termed as soil health.
Various researchers have expressed their views differently regarding soil health
(Kinyangi 2007). Keeping pace with social priorities and increasing understanding
of soil science, the concept of soil health has consistently changed (Warkentin
1995). Generally, the modern concept about soil health is mainly based on various
functions that soil performs in any ecosystem. Based on the functional approach,
Anderson and Gregorich (1984) defined soil health as “the sustained capability of a
soil to accept, store and recycle water, nutrient and energy”. Larson and Pierce
(1991) further defined soil health as “the capacity of a soil to function within its
ecosystem boundaries and interact positively with the environment external to that
ecosystem”. Later on, Acton and Gregorich (1995) stressed environmental concerns
by defining soil health as “the soil’s capacity or fitness to support crop growth
without resulting in soil degradation or otherwise, harming the environment”.
A more detailed definition was developed by Soil Science Society of America
(1995) as “the capacity of a specific kind of soil to function, within natural or
managed ecosystem boundaries, to sustain plant and animal productivity, maintain
environmental quality and promote plant, animal and human health”. This defini-
tion is similar to that of Doran et al. (1996), who defined soil quality as the
“capacity of a soil to function, within ecosystem and land use boundaries, to sustain
biological productivity, maintain environmental quality, and promote plant, animal
and human health”.
8.3 Soil Health Indicators and Criteria for Selection
Direct measurement of vital soil forces is next to impossible, so indicators are used
to attain the goals. These indicators may be defined as measurable soil properties
that influence the capacity of the soil to perform a specific function (Acton and
Padbury 1993; Andrews and Moorman 2002). Attributes that are most sensitive to
management and having low ecological redundancy are most desirable as indica-
tors. In general, appropriate indicators include ecosystem process, the soil’s physi-
cal, chemical and biological properties and processes, and a component of existing
soil databases. Such indicators should also be sensitive to variation in management
practices and climate, be easily measurable and reproducible, accessible to many
users and applicable to field conditions, able to measure changes in soil function
both at plot and landscape, and should lead to management decisions (Bouma 2002;
Menge 2003).
The productivity of agricultural soils is known to depend greatly upon the
activities of diverse microbial communities (Giller et al. 1997). But recently, soil
biodiversity is being threatened by agricultural intensification, particularly by land

use change which is projected to have the largest global impact on soil biodiversity
by the year 2100. This is more acute in tropical agriculture. Functional diversity, the
key to ecological strategy to bring sustainability to production, is largely affected
by long-term cultivation. Such cultivation leads to a decline in diversity and, hence,
to poor soil health. Similarly, genetic diversity, particularly the diversity among
microbes like mycorrhizal fungi (Boggs et al. 2003; Boddington and Dodd 2000),
symbiotic nitrogen-fixing rhizobia (McGarth et al. 1998; Coutinho et al. 1999), and
denitrifiers and nitrifiers (Cavigelli and Robartson 2000; Bruns et al. 1999) are also
affected by high input agricultural activities. As functional diversity provides
sustainability to agro-ecosystems and the genetic diversity imparts stability to
speces (Hooper et al. 2000), they create the environment around themselves in
such a way that other organisms can live only in the conditions thus created. These
parameters can be included as indicators in soil health studies (Pompili et al. 2006;
Saito 2007). However, high diversity is not always essential to run ecosystems
because sometimes microorganisms fail to perform a specific function. Moreover,
under unpredictable ecosystems (e.g., flood, drought, inundation, etc.), high micro-
bial diversity is deemed necessary for imparting resistance against those stresses
and resilience. In fact, some European countries are now including those para-
meters in routine soil monitoring programs (Nielsen and Winding 2002). For
smallholder farmers, these tools need to be simple measures of soil health, such
as consistency, color and workability. For extension and policy personnel, they
provide basic information needed to arrive at management decisions. For research-
ers, there is a need to conduct detailed tests while controlling variations in order to
develop meaningful assessments of soil fertility (Kang et al. 2005).
8.4 Significance of Soil Health Indicators
Soil biological parameters are early indicators of soil degradation and contamina-
tion (Visser and Parkinson 1992). A biological indicator is defined as “an organism,
part of an organism, product of organism (e.g., enzyme), collection of organisms or
biological processes which can be used to obtain information on the status of all or
part of the environment” (Pankhurst et al. 1997).
Agricultural practices such as tillage, crop rotation, fertilization, and irrigation
are generally known to have a significant effect on physico-chemical properties of
soil; less is known about the associated changes in soil organisms and soil bio-
chemical properties, and how such changes influence plant production and sustain-
ability (Heyer et al. 2003). The activity of soil biota brings out nutrient
transformation in soils and underpins a number of fundamental soil properties
such as fertility and structure (Filip 2002; Osinski et al. 2003). Therefore, changes
in soil organism activity are indicative of, and extremely sensitive to, changes in
soil health (Nielsen and Winding 2002; Anderson 2003). Despite their small
volume in the soil, microorganisms play a role in the cycling of elements by
decomposing organic residues. The organic residues are converted to biomass or

mineralized to CO2, H2O, mineral N, P, and other nutrients by these soil residents.
Mineral nutrients immobilized in microbial biomass are subsequently released by
mineralization. In addition to the effect on nutrient cycling, microorganisms also
affect the physical properties of soil. For example, production of extracellular
polysaccharides and cellular debris by microorganisms helps in improving soil
structure and stabilizing soil aggregates. Thereby, they also affect WHC, infiltration
rate, crusting, erodibility, and susceptibility to compaction. Thus, populations of
soil organisms and/or biochemical processes mediated by them are potentially
sensitive bioindicators of changes in soil health (Arshad and Martin 2002). Many
researchers have, therefore, stressed the importance of identifying biological indi-
cators (Doran and Parkin 1994; Elliot et al. 1996). In some instances, changes in an
organism’s populations or soil biotic activities could precede detectable changes in
the physico-chemical properties of soil, thereby providing an early sign of improve-
ment or warning of soil degradation. So, monitoring of soil quality includes the
assessment of biomass C and N, respiration measurement, mineralization rate, soil
enzyme activities, microbial diversity, and functional groups of soil fauna. Indica-
tors influencing soil health, however, vary according to the locations, production
system, and management practices adopted (Table 8.2). Therefore, it is not possible
to develop a single list of biological indicators which could be suitable for all
purposes and management practices (Wylie 1994). However, microbial biomass
carbon (MBC) is almost universally accepted as a biological indicator irrespective
of production system and management practice (Anonymous 2005). The microbial
biomass C is used as a sensitive indicator of changes in soil processes due to
changes in management practices and cropping systems, because it has a much
faster turnover rate than the total SOM and, hence, is the most labile C pool in soils
(Jenkinson 1981; Paul 1984). Moreover, nutrient availability and productivity of
agro-ecosystems depend on the size and activity of microbial populations; a strong
link between soil microbial biomass, soil fertility, and soil health is reported (Hart
and Brookes 1996; Kandeler and Eder 1993).
Microbial quotient (% Cmic/Corg) can be used as an indicator of changing soil
processes under different cropping sequences and management practices and is a
Table 8.2 Microbial/biochemical indicators identified for different soil types and cropping
systems (Adapted from Anonymous 2005)
Soil type Cropping system Identified indicators
Clay loam Rice–rice Dehydrogenase enzyme activity, organic carbon
Sandy loam Groundnut–redgram Organic carbon, microbial biomass carbon (MBC)
Sandy loam Rice–lentil Dehydrogenase enzyme activity, organic carbon
Sandy Sorghum–castor MBC
Sandy loam Jute–rice–wheat Dehydrogenase enzyme activity
Sandy clay Rice–rice Dehydrogenase enzyme activity and microbial
loam biomass nitrogen
Silty clay loam Rice–field pea MBC
Sandy loam Rice–wheat Organic carbon, alkaline phosphatse enzyme
activity, and potentially mineralizable nitrogen
more useful measure than either MBC or total carbon assessed individually (Dilly
and Munch 1998), and it also helps in comparing soils with different organic matter
contents. Entry et al. (1996) suggested that microbial biomass C and the Cmic:Corg
ratio are poor predictors of annual crop yield but may be an accurate indicator of
soil health and a good predictor of long-term crop yield. Furthermore, Insam and
Haselwandter (1989) reported that the respiratory quotient (mg CO2–C h1 mg1
MBC) could be used as an indicator of soil development, substrate quality, ecosys-
tem development, and ecosystem stress.
Enzyme activities are an important index of the biological activity of a soil
because they are involved in the dynamics of soil nutrient cycling and energy
transfer (Table 8.4). Enzymatic processes are closely associated with soil fertility
as they mediate the conversion of unavailable forms of nutrients to forms that are
readily assimilable by plants and microbes (Sarkar et al. 1989). Moreover, enzyme
activity of depends not only on nutrient status and availability but also on the
turnover of N, P, and other nutrients in soils. As enzymes do not react readily to
environmental changes as do the microbes, enzyme activity is considered a more
stable indicator of biological processes (Bandick and Dick 1999).
8.5 Approaches Used to Diagnose Soil Health
8.5.1 Methodology for Selection of Master Indicators
8.5.1.1 Data screening, Representative Variables and Redundancy
Data are reduced to meaningful manageable size, i.e., to a MDS of soil quality
indicators, through a series of uni- and multivariate statistical methods. Both
parametric (randomized block design) as well as nonparametric statistics (Kruskal–
Wallis w2) are used to identify indicators with significant treatment differences. Only
variables with significant differences between treatments are chosen for the next
step in MDS formation. To select a subset from a large dataset, principal component
analysis (PCA) for each statistically significant variable is performed, assuming that
principal components receiving high Eigen values and variables with high factor
loadings best represent system attributes, and examining only the principal compo-
nents that explain at least 5% of the variation in the data up to 85% of the cumulative
variation. Within each principal component, only highly weighted factors, i.e., those
with absolute values within 10% of the highest weight, are retained for the MDS. To
reduce redundancy and rule out spurious groupings among the highly weighted
variables within each principal component, multivariate correlation coefficients are
run to determine the strength of the relationships among variables. Well-correlated
variables are considered redundant and candidates for elimination from the dataset.
Conversely, any uncorrelated, highly weighted variables are considered important
and, therefore, retained in the MDS (Andrews et al. 2004).
8.5.1.2 MDS Validation and Indicator Transformation (Scoring)
Multiple regressions and hierarchical cluster analysis are run using the final MDS
components as the independent variables and each management-goal attribute (e.g.,
yield, sustainable yield index, protein yield, etc.) as a dependent variable. These
regressions and cluster analysis serve to check the MDS representation of manage-
ment system goals. After determining the variables for the MDS, every observation
of each MDS indicator is transformed for inclusion in the SHI methods (e.g., linear
scoring technique). In this technique, indicators are ranked in ascending or des-
cending order depending on whether a higher value is considered “good” or “bad”
in terms of soil function. For ‘more is better’ indicators, each observation is divided
by the highest observed value such that the highest observed value received a score
of 1. For ‘less is better’ indicators, the lowest observed value (in the numerator) is
divided by each observation (in the denominator) such that the lowest observed
value receives a score of 1. (Anonymous 2005).
8.5.1.3 Indicator Integration Into Indices
After transforming MDS into scoring, they are integrated into soil health indices
following additive SHI (ADD SHI) and a weighted, additive SHI (WTD SHI). The
additive index is a summation of the scores from the MDS indicators. From this
summed score, the ADD SQI treatment means and standard deviations are calcu-
lated. In the weighted additive index after transformation, the MDS variables for
each observation are weighted using the PCA results. The percentage, standardized
to unity, provides the weight for variables chosen under a given PC. The weighted
MDS variables scores for each observation are then summed and the treatment
means and standard deviations are calculated. For all the indexing methods, SHI
scores for the management treatment are compared using a two-way ANOVA.
Higher index scores are assumed to mean better soil quality.
8.6 Soil Health and Its Importance
Soil contains soil organisms which perform different functions and facilitate crop
production and value addition. In addition, they detoxify all the nuisance stuffs
dumped into soil to make the earth clean and fit for living organisms. The multifac-
eted activities of soils are discussed in the following section.
8.6.1 Crop Production
Healthy soil fosters fertility which in turn increases crop production (Table 8.3).
The health of soil can affect not only the productivity but also the quality of produce
Table 8.3 Yield of Kharif rice and sustainability yield index for 18 years of cultivation with
organic and inorganic sources of nutrients
Treatment Soil health index Yield (kg ha1) Sustainable yield index
(additive linear index)
Control 2.783 1,493b 0.234c
Na 2.688 2,000b 0.616d
e
NP 3.188 2,560 0.684f
NPK 3.628 3,240f 0.638b
NPK þ FYM 3.726 3,320 b
0.669e
Mean – 2,523 0.568
SE (m) – 169.34 –
a
Recommended dose of fertilizer (Adapted from Anonymous 2005)
b–f
Values indicate the mean of three replicates. Mean values followed by different letters are
significantly different within rows or column at P 0.05 according to Tukey test.
and the sustainability of land (Acton and Gregorich 1995; Doran 2002; Wander
et al. 2002; Carter et al. 2004). Under healthy soil conditions, numerous nutrient
cycles influenced by microbes operate simultaneously in soil. Soil thus contributes
nutrients uninterruptedly to the nutrient pool of soil ensuring balanced plant
nutrition. Moreover, healthy soil conserves nutrients and releases them more or
less synchronously with the demand of growing crops. Thus, nutrient use efficiency
and nutrient conversion ratio remain favorable for reasonable production. Well-
managed soils harbor a wide spectrum of microorganisms performing a variety of
activities including synthesis of phytohormones, antibiotics, growth regulators,
siderophores, etc., which directly or indirectly affect crop productivity. Under
good soil health, internal soil defensive mechanisms remain constantly strong and
vital to combat soil-borne diseases. Thus, crop loss due to diseases can be mini-
mized and consequently yield is increased.
8.6.2 Crop Quality
Lack of balanced nutrition is the most important reason for poor quality produce in
unhealthy soil. Under intensified agriculture practices where soil health is
in a miserable condition, the fundamental ratio of N:P:K has shifted from 2:1:1 to
different imbalance ratios in different regions. Thus, production may be increased
for a while but crop quality does not increase with the productivity. Production,
productivity and quality of agricultural produce largely depend on the state of soil,
microbial status and their specific functions leading to consistent soil health. The
influence on crop quality is triggered by the specific biochemical reaction of
microbes. For example, polyunsaturated fatty acid, Omega 3 fatty acid content in
oil, essential amino acid in proteins, inulin and fructan in carbohydrates (Saha et al.
2007), and antioxidant and antiaging agents like b-carotene, lycopene, etc., are the
quality attributes of agricultural produce. Experimental evidence showed
that microbial indicators like diversity, biomass and enzyme activities have
supported higher biological yield and certain qualities like protein and oil content
of crops (Anonymous 2005), and the quality under organically managed soil has
been found to be superior to conventionally managed soil.
8.6.3 Production Sustainability
The sustainability of any production system depends on the stability of that system.
Stability, on the other hand, of an ecosystem encompasses functional resistance,
i.e., the capacity of the system to endure stresses imposed and to carry out all the
essential ecosystem services during the entire period of stress leading to good
production and functional resilience (Seybold et al. 1997, 1999). The two basic
ecosystem attributes measure soil health and impart sustainability in agricultural
production system. Healthy soil with strong lifeline activities capable of resisting
all unusual situations emerging from indiscriminate agricultural practices maintains
a satisfactory level of production. The influence of soil health on sustainability of
agricultural production is more appropriate in unpredictable agro-ecosystems like
drought, flood, inundation, and contaminated soils. Narrow ecological associations,
such as legume–rhizobia sysbiosis and sustainability of legume production are
highly dependent on soil health (Table 8.3).
8.6.4 Human Health
The nutritional health and well being of humans are entirely dependent on plant
foods either directly or indirectly (Leo et al. 2002). Plant foods provide almost all
essential vitamins and minerals, carbohydrate, protein, fat, and a number of other
health-promoting phytochemicals like antioxidants and antiaging substances.
Healthy soil provides a conducive environment for plants to grow better so that
plants can synthesize all the basic molecules which, through the food chain enter
the human system and get metabolized for carrying out particular biochemical
reactions. Generally, micronutrient concentrations are low in cereals and fortification
of multi-micronutrients in food stuffs causes antagonistic effects on the micronu-
trients. Sustainable soil management leading to improving the bioavailability of
micronutrients in order to improve crop nutritional quality is the best alternative.
The clinical health of humans is also dependent on quality plant food to some
extent. The antioxidant and antiaging molecules like b-carotene and lycopene,
and cardiovascular disease-inducing agents like, triglycerides in oilseed crops,
etc., are largely influenced by balanced nutrition. Organically produced food
generally contains higher amounts of antioxidant and antiaging agents and lower
magnitude triglycerides, indicating that soil health under an organic system is
perfectly good.
8.6.5 Animal Health
The modern soil health concept encompasses sound animal health as one of its
components. Healthy soil is essential for producing nutrient-rich fodder for healthy
animals in any farming system. Under good soil conditions, a micronutrient like
iodine content in fodder crops increases, which might result in better growth of
animals, higher iodine content of animal products such as meat and milk, and
indirectly affect human health. Soil management practices has led to exhaustion
of micronutrients in a given area and, hence, water derived from this area may cause
micronutrient deficiency which could be reflected in animal health (Lee et al. 1999;
Ellison 2002; Vaarst et al. 2003).
8.6.6 Environmental Health
Soils via microbes perform myriads of ecosystem services including decomposi-

tion, transformation, detoxification, infiltration, bioremediation, fixation, emission,
solubilization, disinfection (antibiosis), commensalisms, protocooperation, symbi-
osis, predation, and resistance and resilience against different stresses; these col-
lectively represent the lifeline activities of healthy soil. If soil is healthy, lifeline
activities remain in resonance. Farming methods and activities sometimes disrupt
the rhythm and, thus, soil becomes sick. Unhealthy soil cannot sequester carbon but
rather triggers CO2, methane, and other greenhouse gas emissions which in turn
affects ecosystems. For instance, high concentrations of greenhouse gasses have
caused an increase in global temperature that has threatened both food security and
shelter. Changes in land use contribute about 14% of the total human-generated
emissions of greenhouse gases, and much of this land development is for agricul-
tural purposes (Rosenzweig and Hillel 1998). But healthy soil can prevent global
warming by sequestering carbon in the soil as a way to slow down the concentration
of CO2 in the air, thus slowing the rate of climate change. Indiscriminate use of
fossil fuel-based inputs may lead to a gradual decline in nutrients and WHC of soils.
Thus, a plant nutrient is either leached down to pollute groundwater or emitted out
to pollute air. Due to poor WHC along with poor carbon sequestration, desertifica-
tion is now a phenomenal development of unhealthy soils in different parts of the
globe, particularly in the sub-Saharan region of Africa. Reports suggest that soil
influences environmental quality and the overall functioning of the biosphere by
functioning as a living filter, through which water is cycled and xenobiotics are
altered (Karlen et al. 2001). Consequently, the manner in which soils are managed
has a tremendous impact on the environment and its inhabitants. This fact is
supported by archaeological evidence showing that soil degradation was responsible
for the extinction and collapse of the Harappa civilization in India, Mesopotamia in
Asia, and the Mayen culture in Central America (Olson 1981).
8.7 Problems in Agricultural Practices
8.7.1 Lack of Good Soil Husbandry
The rise and fall of many civilizations in the world was associated with the soil
husbandry followed by the citizens of the nations (Ponting 1992). Poor land care
by the inhabitants was one of the serious causes of ruin of the great Harappan
civilization of ancient Indian Subcontinent (Olson 1981). In contrast, the glorious
Egyptian Civilization was due to good stewardship of soils of the Nile basin. In
ancient times, the soil was treated as ‘Mother’ and any agricultural activities in
the soil were started with the worship of the land. Soil is now considered as the
machinery for crop production. Soils are extracted in all possible ways without
paying due care. Round the year cultivation resulting in soaring cropping intensity
allows little rest to the land to recover its deficits in its health status. Thus, soil
health further deteriorates. Previously, soil was routinely fed with organic matter
from diverse sources, but the overnight magical effect turns the farmers towards
proponents of chemical fertilizers in present day agriculture. The avoidance of
organics, particularly in the post-green revolution era, has damaged soil health
including structural aberrations leading to a hostile habitat for microbes, poor
aeration, WHC, and nutrient retention capacity. Soils, thus, become sick. The
health of soil, hence, requires greater attention as to how the fertility could be
restored in degraded lands.
8.7.2 Agricultural Intensification and Inappropriate

Cropping System
The increasing demands for food to satisfy the need of the ever-expanding human
population has led to intensification of agricultural practices with extensive use of
chemicals, exploitation of surface and ground water for irrigation, and adoption
of mechanization. These practices have resulted in severe adverse effects on the
physical, chemical, and biological properties of soil. The most important of such
effects is the depletion of SOM and loss of microbial diversity which together lead
to damage to the essential ecosystem functions, the sustainability of agricultural
production system and to soil resilience capacity (Buresh et al. 1997; Doran and
Zeiss 2000).
A particular cropping system in any area has its own history. After being tested
for a long time, it has been agronomically accepted not only for its good economic
return but also for its role in maintaining soil health. In a traditional cropping
system, legumes have been the farmers’ choice. But recently, with the introduction
of vegetables (particularly hybrid vegetables), the space for the legumes is shrinking,
leading to poor soil health. Vegetable cultivation round the year degrades soil
aggregates into talc-like powder which facilitates fertile topsoil erosion, higher
leaching of nutrients to ground water, as well as to eutrophication of water bodies.
And, hence, fertilizer use efficiency declines, production decreases, and human,
animal and environmental health are affected.
8.7.3 Blanket Application of Agrochemicals
From the very beginning of the green revolution, farmers have been desperately and
indiscriminately using fertilizers and pesticides. Moreover, new molecules of bio-
logically active metabolites, growth regulators, and hormones are being frequently
used in high input agriculture. All those chemicals are foreign bodies to soil
interacting differently with the soil’s inherent entities and creating hostile environ-
ments leading to poor soil health. In this context, genetically modified inputs like
seed and biocontrol agents are in the queue to join modern agricultural activities.
Though confusing, they may have a long-term soil health hazard problem directly
or indirectly (US National Research Council, 2000; Nordlee et al. 1996).
8.7.4 Reduction in Agricultural Biodiversity/Monoculture
Practicing a very few restricted cropping systems and/or monocultures over the
years results in decreases in aboveground plant biodiversity as well as a sharp
decline in belowground biodiversity including the organisms antagonist to plant
pathogens. The production system thus becomes vulnerable to harmful soil organ-
isms and the soil becomes sick. Emergence of nematode diseases as one of the
important problems in recent years is a fallout of such soil sickness.
8.7.5 Heavy Vehicle Traffic and Indiscriminate Use of

Sewage Sludge
Low energy-based conventional bullock-driven agricultural operations are almost

absent in modern agriculture. They have been substituted by the introduction of
energy-intensive heavy vehicles, like tractors, power tillers, combined harvesters,
etc. The operation of such machines in agricultural practices results in soil compac-
tion leading to destruction of microhabitats, reduction of soil biological entities,
decline in nutrient transformation, poor aeration, and poor drainage. The long-term
effects of such operations in agricultural soils causes soil health to deteriorate.
Due to the low capacity of sewage treatment plants, agricultural soils receive
raw effluent emerging from different industrial or agrochemical sources. Raw sewage
water contains organic and inorganic pollutants, such as heavy metals, as well as
human and plant pathogenic organisms. The discharge of heavy metals into the soil
or the use of sewage sludges in agronomic practices as a source of irrigation leads to
loss in soil fertility, disturbances in microbial equilibrium, and eventually makes
the soil unsuitable for cultivation (Khan et al. 2009).
8.7.6 Lack of Appropriate Soil Testing and

Diagnostic Protocol
Routine soil testing measures the status of soil nutrients and a few physical proper-
ties of the soils. Such analysis of soil is done to find out the nutrient deficiency in
soils which further help to design the nutrient strategy for the deficient soils. The
current concept of fertilizer application is directed to feed the plants but not the soil.
Thus, underestimating the contribution of soil and its role in consistently supplying
nutrients to plants is important. Moreover, even though soil microflora drives the
key processes of soil, no serious attempt has yet been made to assess the functional
diversity of microbes impacting on soil health. Furthermore, laboratory-based
indicators for assessing soil are not easily available to farmers and ready to use
soil health indicators at the field level are poorly developed.
8.7.7 Insufficient Awareness Among Farmers

About Soil Health
Farmers are the central figures of any soil health management program. They are
the stewards of soil health. But, presently there is a gap between skills and
knowledge acquired by individual farmers, while at the community level there is
inadequate awareness about modern scientific innovations. Effective participation
of farmers in combined learning and experimental research for soil health assess-
ment and management is still lacking. Approaches adopted to motivate farmers,
such as state of the art information and how to make maximum use of modern
agronomic tools are not sufficiently available to agrarian communities.
8.7.8 Lack of Proper Legislation and Contractual Farming
Unlike air, water and sound pollution, no systematic and proper guidelines for
assessing soil health is currently in action in many countries experiencing soil
deterioration. Although the European Union has initiated legislation to protect
soil health, no governmental initiative regarding soil health protection is in action
in developing countries. Furthermore, since no legal institutions are available in

almost all countries to police over the misuse and abuse of soil, no ethics are
followed in the use of soil for specific purposes. Licensees of contractual farms try
to extract all possible benefits from the leased land within the stipulated contract
period without considering soil as a nonrenewable resource. So, a piece of healthy
land turns sick by the time it is returned to the licensor. This is a common picture in
contractual farming systems. Moreover, farmers in some countries, like India, sell
the top soil to brick factories or to contractors for land fill and other nonagricultural
purposes after the crop harvest. Thus healthy soil, generated over a decade or more,
is destroyed with the stroke of a spade.
8.7.9 Government Policy
Government subsidies often help perpetuate unsustainable practices. Subsidies

often stimulate greater use of chemicals, despite their environmental and public
health risks. Rice farmers in Japan, Taiwan, and Korea use just over one-half of
all insecticides applied to rice worldwide yet produce only 2% of the world’s
crops. The reason is that large government price support makes it profitable to
increase insecticide use even when the resulting production gains are small
(Vorley and Keeney 1998). This is equally true for India which indiscriminately
uses highly subsidized nitrogenous fertilizer. Besides encouraging harmful prac-
tices, farm subsidy programs often fail to reward good stewardship. They tend to
emphasize a handful of major crops and put resource-conserving crop rotations at
a financial disadvantage (Faeth and Westra 1993). Farmers receive no govern-
ment incentives for sustainable practices such as growing alfalfa or dhaincha
(Sesbania rostata).
8.8 Soil Health Management
8.8.1 Crop Rotation and Organic Amendments
Crop rotation is a viable strategy for increasing SOM for the establishment of
healthy, fertile, and productive soil. Crop rotation fulfils nutrient demand from
different depth of soil, thus reducing nutrient mining and hardpan by root penetra-
tion (Corselius et al. 2001). For example, rotations that include cereals leave a
significant amount of leftover stubbles after harvest. By including these crops in
vegetable rotations, a grower can reduce the incidence of potentially devastating
soil-borne diseases and outbreaks of phytopathogenic nematodes (FAO 2008).
Rotations can also check weeds and insect pests that use the weeds as alternative
hosts for perpetuation. The practice of crop rotation with legumes can thus improve
Table 8.4 Soil health index (SHI) value for different management at various experimental sites
Treatment/site AAU ANGRAU BHU CRIDA CRIJAF CRRI OUAT BCKV
Control 2.27 0.92 1.63 0.95 1.04 2.77 0.31 2.78
Na 2.60 – 1.48 – 1.38 2.91 0.35 –
NP 2.59 – – 1.02 1.66 3.21 0.78 –
NPK 2.79 0.97 1.52 – 1.87 3.10 0.81 2.69
NPK þ FYM 2.84 2.00 1.87 1.27 2.10 4.00 1.13 3.63
a
Recommended dose of fertilizer (Adapted from Anonymous 2005)
soil quality, protect top fertile soil erosion, and improve SOM status, which in turn
improves productivity and consistency in production system,. Furthermore, the
appropriate land management, such as fertilization combined with crop rotation
and reducing 1-year-old fallows, would be useful ways to improve or maintain soil
fertility (Jia et al. 2005).
Deliberate and routine carbon sources are essential to achieving good soil health
in agricultural production systems (Majumder et al. 2008; Mitchell et al. 2000).
Special care is needed to select carbon sources that will ensure short-term produc-
tivity while building long-term soil health. Farm yard manure (FYM) is considered
as an organic amendment to improve soil health in different production systems. In
a case study, the soil health index was computed from a minimum dataset in an
integrated nutrient management system with FYM as sole organics: the soil health
index was superior to conventional farming with chemical fertilizers (Table 8.4).
However, as there is no definite protocol for FYM preparation, quality varies
tremendously. Thus, compost, phosphocompost, and vermicompost with relatively
long-lasting and strong persistence of carbon prepared under defined protocols have
fair scope to be used as organics for better soil health. Furthermore, concentrated
organics, like edible and nonedible oil cakes, rice bran, pulse bran, etc., can be used
for improving soil health. In situ stubble incorporation in combination with cellu-
lolytic microorganisms and green manuring are of great practical importance for
maintaining soil health under rice-based cropping systems (Bhattacharya 2004).
Such organic amendments add significant amounts of carbon to soil and are
generally associated with rich microbial diversity in protected and conserved
ecosystems for carrying out lifeline activities of soil.
8.9 Cover crops and Tillage Conservation
Cover cropping (also called green manuring) is a wise prescription for a soil health
management program (Leo et al. 2002). Cover crops can provide a practical and
economical means for supplying organic matter, enhancing soil fertility, suppres-
sing weed growth, attracting beneficial insects, spiders and predatory mites, and
reducing nitrate leaching looses to the groundwater (FAO 2008). Soils differing in
fertility as well as health properties routinely receive these inputs compared to
conventionally managed soil (Mitchell et al. 2000).
The beneficial effect of the tillage systems (no till and low till farming) are
based on the premise that minimizes the disturbance to the soil leading to an
increase in the retention of water, nutrients, and the topsoil itself (Pretty 1995;
Leo et al. 2002). In tillage conservation, crops are grown with minimal cultivation
of the soil. When the amount of tillage is reduced, the stubble or plant residues
are not completely incorporated, and most or all remain on top of the soil rather
than being ploughed or disked into the soil. The new crop is planted into this
stubble or into small strips of tilled soil. Weeds are controlled with cover crops or
herbicides rather than by cultivation. Fertilizer and lime are either incorporated
earlier in the production cycle or placed on top of the soil at planting (FAO
2008). The tillage encourages soil flora and fauna under the protected habitat to
bring out the biogeochemical processes leading to optimization of carbon turn-
over and bioavailability of nutrient elements in the soil (Mitchell et al. 2000).
Natural processes involved in conservation farming include insect tunnels made
by the movement of earthworms (drillosphere) and arthropods, and root tunnels
created after the decomposition of leftover root debris make the soil porous and a
loose fit for root ramification of the next crop with a good stand. Successful
cultivation of relay cropping of legumes and/or mustard with winter rice is an
example of no till farming in different parts of India.
8.9.1 Agricultural Diversification
There is a relationship between aboveground and belowground diversity. After the

green revolution, there has been a dramatic reduction in crop diversity which in turn
has had a negative impact on belowground microbial diversity. The soil foodweb
and its internal regulatory system have been badly disrupted. Furthermore, soil
becomes vulnerable to diseases and pests and, consequently, production declines.
This can be repaired by agricultural diversification. Growing a variety of crops will
ensure rich belowground diversity which in turn revitalizes soil health and also
provides a buffer against both ecological and economical problems. This can be
achieved by the following.
8.9.1.1 Selection of Right Cropping System
Cropping system as a whole has a great influence on soil health (Johnson et al.
2003). A production system causing deterioration in an agroecosystem is recom-
mended to be omitted to minimize further deterioration. A system which leaves
more residues and produce more exudates (rhizodeposition) can sequester higher
carbon in the soil. For example, a cereal-based cropping system which retains
stubble and huge root biomass and debris is preferred in degraded and fragile
ecosystems for restoration of soil health. In addition, double-cropping rice practice
also helps to maintain soil carbon and, thus, soil health (Mandal et al. 2008). In a
rice–rice system, kharif rice may be coupled with lentil as a relay crop, and winter
rice followed by a green manuring crop to revitalize the soil internal regulatory
system with a huge supply of easily accumulative carbon. Moreover, farmers can
harvest a third crop with minimal investment and disturbance to the soil.
8.9.1.2 Tailoring of Existing Cropping System
The cropping system may affect soil health by modifying soil microbial community
structure leading thereby to a negative impact on soil fertility and, hence, crop
yields (Kowalchuk et al. 2002; Mandal et al. 2008). Immediately after identifying
the crops severely damaging soil biological health, it must be eliminated and a
new cropping system be developed by incorporating suitable means for repairing
the damage. For this, the following options may be explored. In rice–potato–
sesame, sesame (Sesamum indicum L.) may be replaced by cowpea, whereas in
rice–vegetable–vegetable, late cauliflower may be replaced by bean. The basis of
replacement of sesame by cowpea is (1) sesame is susceptible to Macrophomina
spp. causing low yield, so (b) to arrest this, a new crop such as cowpea may be
introduced, as legumes have a special role in N-economy. However, in rice–
vegetable–vegetable, late cauliflower may be replaced by bean because (1) early
cauliflower followed by late cauliflower may impair the soil health by increasing
inocula density of cauliflower associated soilborne pathogens, and (2) a cauliflower–
cauliflower system causes seasonal glut in the market that hardly helps farmers
to earn a remunerative price.
8.9.1.3 Farming System Diversification
Crop diversification alone cannot bring stability to soil health. Rather, farming
system diversification is necessary for overall improvement of soil health. Integrat-
ing animal and plant production has been considered as a wise policy to maintain
a higher biotic diversity in sustainable farming. Agriculture and its allied activities,
i.e., horticulture, agroforestry, organic farming, animal husbandry, fishery, poultry,
etc., can benefit each other. Fish cum rice cultivation can be readopted in some
specific rice growing areas for sustainable rice production and farm income.
Resources can be properly recycled under diverse activities in mixed farming.
Thus, resource utilization becomes optimum under mixed farming systems which
in turn impart sustainability to any production system (Leo et al. 2002).
8.9.2 Organic Husbandry
This is a holistic production management system that promotes and enhances agro-
ecosystem health, including biodiversity, biological cycles, and soil biological
activities. Organic production systems are based on specific and precise standards
of production which aim to achieve optimal agro-ecosystems which are socially,
ecologically, and economically sustainable. The primary goal of organic agriculture
is to optimize the health and productivity of interdependent communities of soil
life, plants, animals, and people. Organic agriculture manages locally available
resources to optimize competition for food and space between different plant and
animal species. The manipulation of the temporal and spatial distribution of
biodiversity is the main productive input of organic farmers. Organic practices
such as crop rotation, cover crops, organic fertilizers, and minimum tillage increase
the diversity and richness of indigenous soil life which in turn impact essential
ecosystem services.
8.9.3 Soil-Based Fertilizer and Development of Farmer’s

Friendly Indicator
There is a conspicuous gap between nutrient demand and addition. This is mostly
due to nonconsideration of crop nutrition with crop demand. Thus, a net nutrient
deficit increases day by day. The nutrient imbalance in soil has, thus, caused a poor
growth in agricultural production necessitating soil testing and fertilizer recom-
mendation on the basis of the test values. Such nutritional analysis of soils will help
to design balance fertilization for various crops and will also help to take measures
against environmental pollution. Farmers have a vested interest in soil health, and,
hence, its maintenance has been the top priority for them. Farmer interest in soil
health may have been encouraged by their desire to examine and validate the
management practices they use in their own farm. Thus, they need familiar soil
characteristics easy to interpret by themselves as soil indicators. The working
knowledge of farmers obtained through experience will help researchers to articu-
late their experience with research findings to design farmer-friendly indicators.
The soil taxonomies and intricate methods for fertility management should be taken
into consideration while developing handy soil health indicators.
8.9.4 Legislation for Soil Health Protection
Attempts should be made to enact legislation for protecting soil against its abuse
and misuse. Selling of top soil for other nonagricultural activities should be treated
as a punishable offence. In addition, government-endorsed drafts of agreement
between licensor and license in regard to the status quo of soil health in contractual
farming systems should be followed. And, at the time of return of the land to the
licensor, if the soil health has deteriorated, the licensee should be liable to pay extra
at certain rates to rejuvenate it.
8.10 Conclusion
Food demand for rapidly increasing the global human population is expected to
expand substantially over the next decades, which in turn could make them food
insecure. The excessive inputs and unethical activities in agriculture in developing
countries are some of the reasons why fertile land is degrading rapidly and destroy-
ing ecosystems. Due to these problems, soil resource inventory and monitoring
have gained momentum. And, as a consequence, the concept of soil health has
generated awareness among agriculturists regarding the importance of soil in
maintaining plant productivity and environment quality. There is a need to better
understand relationships between the status of soil health indicators and fluctuating
soil functions. In this context, state of the art research is needed in order to find and
develop proper indicators, applicable at the farmer scale. The technology thus
developed would be likely to help in setting up effective and ready to use protocols
which may guarantee to generate the necessary baseline soil data in order to
develop appropriate MDS encompassing all aspects of agro-ecosystem perfor-
mance. In this regard, innovative use of geographical information systems and
remote sensing will significantly improve the acceptance of soil health information
by stakeholders. Laboratory-based indicators so far developed definitely have a
substantive role in the scientific and more precise monitoring of terrestrial ecology.
But sometimes such parameters lose their relevancy to the farmers and, hence,
there is an urgent need to develop handy, meaningful and farmer-friendly sets of
indicators.
References
Abrol IP, Bronson KF, Duxbury JM, Gupta RK (2000) Long-term fertility experiments in rice-
wheat cropping systems. Rice-wheat consortium paper series 6. Rice-wheat consortium for the
Indo-Gangetic Plains, New Delhi, India, p 171
Acton DF, Gregorich IJ (1995) Understanding soil health. In: DF Acton, IJ Gregorich (eds) The
health of our soils: towards sustainable agriculture in Canada. Centre for Land and Biological
Resource Research, Agricultural and Agri-Food, Ottawa, Ontario, Canada, pp 5–10
Acton DF, Padbury GA (1993) A conceptual framework for soil quality assessment and monitor-
ing. In: Acton DF (ed) A program to assess and monitor soil quality in Canada: soil quality
evaluation program summary (Interim). Centre for land and biological resources research.
Contribution No. 93–94. Research Branch, Agriculture Canada, Ottawa, pp 2–7
Anderson TH (2003) Microbial eco-physiological indicators to assess soil quality. Agric Ecosyst
Environ 98:285–293
Anderson DW, Gregorich FG (1984) Effect of soil erosion on soil quality and productivity. In: Soil
erosion and degradation. Proceedings of 2nd Annual Western Prov. Conference on National
Water Soil Research management, Saskatoon, Canada, pp 105–113
Andrews SS, Moorman TB (2002) Soil quality as an indicator of sustainable land management:
demonstrated successes and continued needs. Agron J 94:1–2
Andrews SS, Karlen DL, Cambardella CA (2004) The soil management assessment framework: a
quantitative soil quality evaluation method. Soil Sci Soc Am J 68:1945–1962
Anonymous (2005) Assessment and improvement of soil quality and resilience for rainfed
production system completion report code no RRPS-20. National Agricultural Technology
Project (NATP), CRIDA, Hyderabad, pp 10–12
Arshad MA, Martin S (2002) Identifying critical limits for soil quality indicators in agro-ecosys-
tems. Agric Ecosys Environ 88:153–160
Askegaard M, Eriksen J, Johnson AE, Schjonning P, Elmholt S, Christensen BT (2004) Sustain-
able management of potassium. Managing soil quality: challenges in modern agriculture. CAB
International, Wallingford, UK, pp 85–101
Bandick AK, Dick RP (1999) Field management effects on soil enzyme activities. Soil Biol
Biochem 31:1471–1479
Bhattacharya B (2004) In situ utilization of rice stubble in relation to soil fertility vis-à-vis
performance of wheat crop. PhD Dissertation, BCKV, Kalyani, India
Boddington CL, Dodd JC (2000) The effect of agricultural practices on the development of indigenous
arbuscular mycorrhizal fungi. I. Field studies in an Indonesian Ultisol. Plant Soil 218:137–144
Boggs LC, Stahl PD, Lindstrom MJ, Schumacher TE (2003) Soil microbial properties under
permanent grass, conventional tillage, and no-till management in South Dakota. Soil Till Res
71:15–23
Bouma J (2002) Land quality indicators of sustainable land management across scales. Agric
Ecosyst Environ 88:129–136
Bruns MA, Stephen JR, Kowalchuk GA, Prosser JI, Paul EA (1999) Comparative diversity of
ammonia-oxidizer 16 S rRNA gene sequences in native, tilled, and successional soils. Appl
Buresh RJ, Sanchez PA, Calhour F (1997) Replenishing Soil Fertility in Africa, vol 51. Soil
Science Society of America, Madison, USA
Carter MR, Andrews SS, Drinkwater LE (2004) Systems approaches for improving soil quality. In:
Schjonning P, Elmholt S, Christensen BT (eds) Managing soil quality: challenges in modern
agriculture. CAB International, Wallingford, UK, pp 261–281
Cavigelli MA, Robertson GP (2000) The functional significance of denitrifier community compo-
sition in the terrestrial ecosystem. Ecology 81:1402–1414
Corselius K, Wisniewski S, Ritchie M (2001) Sustainable agriculture: making money, making
sense. The Institute for Agriculture and Trade Policy, Washington, DC
Dick RP (1992) A review: long-term effects of agricultural systems on soil biochemical and
microbial parameters. Agric Ecosyst Environ 40:25–36
Dick WA, Gregorich EG (2004) Developing and maintaining soil organic matter levels. In:
Dilly O, Munch JC (1998) Ratios between estimates of microbial biomass content and microbial
activity in soils. Biol Fertil Soils 27:374–379
Doran JW (2002) Soil health and global sustainability: translating science into practice. Agric
Ecosyst Environ 88:119–127
Doran JW, Alice JJ (1996) Methods for assessing soil quality. Soil Sci Soc Am J 49:410
Doran JW, Parkin TB (1994) Defining and assessing soil quality. In: Doran JW, Coleman DC,
Bezdicek DF, Stewart BA (eds) Defining soil quality for sustainable environment, vol 35.
SSSA, Madison, Wisconsin, pp 3–21
Doran JW, Zeiss MR (2000) Soil health and sustainability: managing the biotic component of soil
quality. Appl Soil Ecol 15:3–11
Doran JW, Coleman DC, Bezdicek D, Stewart BA (1994) Defining soil quality for a sustainable
environment, vol 35. SSSA, Madison, Wisconsin, pp 3–21
Doran JW, Sarrantonio M, Liebig MA (1996) Soil health and sustainability. Adv Agron 56:1–54
Doran JW, Stamatiadis SI, Haberern J (2002) Soil health as indicator of sustainable management.
Agric Ecosyst Environ 88:107–110
Elliot LF, Lynch JM, Papendick RI (1996) The microbial component of soil quality. In: Stozky G
(ed) Soil biochemistry, vol 9. Marcel Dekker, New York, pp 1–21
Ellison RS (2002) Major trace elements limiting livestock performance in New Zealand. N Z Vet J
50:35–40
Entry IA, Mitchell CC, Backman CB (1996) Influence of management practices on soil organic
matter, microbial biomass and cotton yield in Alabama’s “Old Rotation”. Biol Fertil soils
23:353–358
Faeth P, Westra J (1993) Alternatives to corn and soybean production in two regions of the United
States. Agricultural policy and sustainability: case studies from India, Chile, the Philippines
and the United States. World Resources Institute, Washington, DC
FAO (2008) Conservation agriculture. http://www.fao.org/ag/ca
Filip Z (2002) International approaches to assess soil quality by ecologically-related biological
parameters. Agric Ecosyst Environ 88:169–174
Giller KE, Beare MH, Lavelle P, Izac AMN, Swift MJ (1997) Agricultural intensification, soil
biodiversity and agro-ecosystem function. Appl Soil Ecol 6:3–16
Gregory PJ, Ingram JSI, Anderson R, Betts RA, Brovkin V, Chase TN, Grace PR, Gray AJ,
Hamilton N, Hardy TB, Howden SM, Jenkins A, Meybeck M, Olssol M, Schulze RE, Thiem
M, Valentin C, Wilkinson MJ (2002) Environment consequences of alternative practices for
intensifying crop production. Agric Ecosyst Environ 88:279–290
Hart MR, Brookes PC (1996) Soil microbial biomass and mineralisation of soil organic matter
after 19 years of cumulative field applications of pesticides. Soil Biol Biochem 28:1641–1649
Heyer W, Hulsbergen KJ, Wittman CH, Papaja S, Christen O (2003) Field related organisms as
possible indicators for evaluation of land use intensity. Agric Ecosys Environ 98:453–461
Hooper DU, Bignell DE, Brown BK, Brussard L, Dangerfield JM, Wall DH, Wardle DA, Coleman
DC, Giller KE, Lavelle P, van der Putten WH, Ruiter PCD, Rusek J, Silver WI, Tiedje JM,
Wolters V (2000) Interactions between aboveground and belowground biodiversity in terres-
trial ecosystems: patterns, mechanisms, and feedbacks. Bioscience 50:1049–1061
Hseu ZY, Chen ZS, Tasi CC (1999) Selected indicators and conceptual framework for assessment
methods of soil quality in arable soils of Taiwan. Soil Enviorn 2:135–139
Insam H, Haselwandter K (1989) Metabolic quotient of the soil microflora in relation to plant
succession. Oecologia 79:174–178
Jenkinson DS (1981) Microbial biomass in soil: measurement and turnover. In: Paul EA, Ladd JN
(eds) Soil biochemistry. Marcel Dekker, New York, pp 415–471
Jia GM, Cao J, Wang G (2005) Influence of land management on soil nutrients and microbial
biomass in the central loess plateau, northwest China. Land Degrad Dev 16:455–462
Johnson MJ, Lee KY, Scow KM (2003) DNA fingerprinting reveals link among agricultural crops,
soil properties, and the composition of soil microbial communities. Geoderma 114:279–303
Kandeler E, Eder G (1993) Effect of cattle slurry in grassland on microbial biomass and on
activities of various enzymes. Biol Fetil Soils 16:249–254
Kang GS, Beri V, Sidhu BS, Rupela OP (2005) A new index to assess soil quality and sustain-
ability of wheat-based cropping systems. Biol Fertil Soils 41:389–398
Karlen DL, Eash NS, Unger PW (1992) Soil and crop management effects soil quality indicators.
Am J Alter Agric 7:48–55
Karlen DL, Wollenhaupt NC, Erbach DC, Berry EC, Swan JB, Eash NS, Jordhal NS (1994) Crop
residue effects on soil quality following 10 years of no-till corn. Soil Till Res 31:149–167
Karlen DL, Andrews SS, Doran JW (2001) Soil quality: current concepts and applications. Adv
Agron 74:1–40
Karlen DL, Andrews SS, Weinhold BJ, Doran JW (2003a) Soil quality: humankind’s foundation
for survival. J Soil Water Conserv 58:171–179
Karlen DL, Ditzler CA, Andrews SS (2003b) Soil quality: why and how? Geoderma 114:145–156
Karlen DL, Andrews SS, Weinhold BJ (2004) Soil quality, fertility and health historical context,
status and perspectives. In: Schjonning P, Elmholt S, Christensen BT (eds) Managing soil
quality: challenges in modern agriculture. CAB International, Wallingford, UK, pp 17–33
remediation of metal contaminated soils. Environ Chem Lett 7:1–19
Kinyangi J (2007) Soil health and soil quality: a review. Available on http://www.cornell.edu.org
Kowalchuk GA, Buma DS, de Boer W, Klinkhamer PGL, Van Veen JA (2002) Effects of above-
ground plant species composition and diversity on the diversity of soil-borne microorganisms.
Antonie van Leeuwenhoek 81:509–520
Larson WE, Pierce FJ (1991) Conservation and enhancement of soil quality. In: Dumanski J,
Pushparajah E, Latham M, Myers R (eds) Evaluation for sustainable land management in the
developing world, vol 2. Technical Papers, IBSRAM Proceedings No 12(2), Bangkok, pp 175–203
Lee J, Masters DG, White CL, Grace ND, Judson GJ (1999) Current issues in trace element
nutrition of grazing livestock in Australia and New Zealand. Aust J Agric Res 50:1341–1364
Leo H, Lawrence RS, Walker P (2002) How sustainable agriculture can address the environmental
and human health harms of industrial agriculture. Environ Health Perspect 110:445–456
Majumder B, Mandal B, Gangopadhyay A, Mani PK, Kundu AL, Mazumdar D (2008) Organic
amendments influence soil organic carbon pools and rice-wheat productivity. Soil Sci Soc Am
J 72:775–785
Mandal B, Majumder B, Adhya TK, Bandyopadhyay PK, Gangopadhyay A, Sarkar D, Kundu MC,
Gupta Choudhury S, Hazra GC, Kundu S, Samantaray RN, Mishra AK (2008) Potential of
double-cropped rice ecology to conserve organic carbon under subtropical climate. Glob
Chang Biol 14:1–13
Menge M (2003) Experiences with application, recordation and validation of agri-environmental
indicators in agricultural practices. Agric Ecosyst Environ 98:443–451
Mitchell J, Gaskell M, Smith R, Fouche C, Koike ST (2000) Soil management and soil quality for
organic crops. Organic vegetable production in California series, vol 7248. University of
California, USA
Nielsen MN, Winding A (2002) Microorganisms as indicators of soil health. NERI technical report
No. 388. Ministry of the Environment, National Environmental Research Institute, Denmark,
p 82
Nordlee JA, Taylor SL, Townsend JA, Thomas LA, Bush RK (1996) Identification of a Brazil-nut
allergen in transgenic soybeans. N Engl J Med 334:688–692
Norfleet ML, Ditzler CA, Puckett WE, Grossman RB, Shaw JN (2003) Soil quality and its
relationship to pedology. Soil Sci 168:149–155
Nortcliff S (2002) Standardization of soil quality attributes. Agric Ecosyst Environ 88:161–168
Olson GE (1981) Archaeology: lessons on future soil use. J Soil Water Conserv 36:261–264
Osinski E, Meier U, Buchs W, Weickel J, Matzdrof J (2003) Application of biotic indicators for
evaluation of sustainable land use–current procedure and future development. Agric Ecosyst
Environ 98:407–421
Pankhurst CE, Doube BM, Gupta VVSR (1997) Biological Indicators of soil health. CAB
International, Wallingford, UK, p 451
Paul EA (1984) Dynamick of organic matter in soils. Plant Soil 76:275–285
Pompili L, Mellina AS, Benedtti A (2006) Microbial indicators for evaluating soil quality in
differently managed soils. Geophysic Res Abst.8.06991
Ponting C (1992) A green history of the world. St. Martin, New York
Pretty JN (1995) Regenerating agriculture: policies and practice for sustainability and self-
reliance. Joseph Henry, Washington, DC
Saha N, Ulrich A, Becker R, Wirth S (2007) Assessing the changes in bacterial diversity in
rhizosphere and phyllosphere of transgenic and non-transgenic potato plants. Plant Tissue Cult
Saito M (2007) Can soil biodiversity be used for an indicator of soil health? Case studies in Japan.
www.webdomino1.oecd.org
Sarkar JM, Lenowiez P, Bollag JM (1989) Immobilization of enzymes on clay and soils. Soil Biol
Biochem 21:223–230
Schjonning P, Elmholt S, Christensen BT (2004) Soil quality management-synthesis. In:
Seybold CA, Mausbach MJ, Karlen DL, Rogers HH (1997) Quantification of soil quality. In: Lal
R, Kimble JM, Follett RF, Stewart BA (eds) Soil processes and the carbon cycle. CRC, Boca
Raton, New York, pp 387–404
Seybold CA, Herrick JE, Brejda JJ (1999) Soil resilience: a fundamental component of soil quality.
Soil Sci 164:224–234
US National Research Council (2000) Genetically modified pest-protected plants: science and
regulation. National Academy, Washington, DC
Vaarst M, Hovi M, Sundrum A, Younie D, Susanne P (2003) Sustaining animal health and food
safety in European organic livestock farming. Presentation at 54th annual EAAP Meeting,
Rome, 31 August 2003
Visser S, Parkinson D (1992) Soil biological criteria as indicator of soil quality: soil microorgan-
isms. Am J Alter Agric 7:33–37
Vorley W, Keeney D (1998) Bugs in the system: redesigning the pesticide industry for sustainable
agriculture. Earthscan, London
Wander MM, Walter GL, Nissen TM, Bollero GA, Andrews SS, Cavanaugh-Grant DA (2002) Soil
quality: science and process. Agron J 94:23–32
Wang Z, Chang AC, Wu L, Crowley D (2003) Assessing the soil quality of long-term reclaimed
wastewater-irrigated cropland. Geoderma 114:261–278
Warkentin BP (1995) The changing concept of soil quality. J Soil Water Conserv 5:226–228
Wylie P (1994) Indicators of sustainable cropping systems. In: Pankhurst CE, Doube BM, Gupta
VVSR, Grace PR (eds) Soil biota: management in sustainable farming systems. CSIRO, East
Melbourne, Australia, pp 224–229
Chapter 9
Recent Advances in Biopesticides
Parvez Qamar Rizvi, Rummana A. Choudhury, and Arshad Ali
Abstract Consistent and injudicious applications of pesticides leads to the develop-

ment of resistance in insects, destruction of beneficial organisms, and increases in
residual problems, thereby posing a threat to human health and its ecological
partners in the living biome. The need of the hour is to develop an eco-friendly
approach to combat insect pests that should be able to regulate pest populations by
exploring naturally occurring products, including extracts of plants and animals,
microbes, parasitic nematodes and insects, and certain minerals. This call for viable
alternatives has led the scientific community to engage in unveiling the potential of
biopesticides. Currently, some strains of Bacillus thuringiensis, nuclear polyhe-
drosis virus, fungi, and nematode parasites are commercially available. Exploiting
the benefits of biopesticides as biocontrol agents appears to be a more promising
approach, assuming that issues of phytopathogens and environmental problems
caused by synthetic pesticides can be resolved. This chapter emphasizes the experi-
ences and progress made in the potential and promise of biopesticides in the global
scenario.
9.1 Introduction
For many years, the use of synthetics and chemical pesticides has dominated the
scene of pest control. Their intensive use, misuse, and abuse have caused various
ecological and environmental problems, the resurgence of various insect and mites
pests, contaminated food commodities, have adversely affected non-target organi-
sms, and have also progressively increased occupational poisoning cases in develop-
ing countries. This tirade against their use has led agriculture to envision a new
P.Q. Rizvi (*), R.A. Choudhury, and A. Ali

Faculty of Agricultural Sciences, Department of Plant Protection, Aligarh Muslim University,
Aligarh, Uttar Pradesh, India
e-mail: pqrizvi@rediffmail.com
186 P.Q. Rizvi et al.
stride forward in the wake of biotechnological and other innovative developments

so as to open up new vistas of scientific advancement, which requires sustained
efforts and will certainly reap long-term benefits.
As a viable substitute to these conventional environmentally-unfriendly chemical
pesticides, eco-friendly alternatives of crop-pest control by either biological agents
or specifically designed synthetic anti-insect compounds are receiving greater atten-
tion. Though the initial acceptance of biopesticides was slow, owing to their non-
availability in sufficient quantities, poor shelf life, lack of standard products, and
collection/multiplication/application constraints, due to the organic food movement,
the development and standardization of biopesticides have accelerated dramatically.
Currently, the total world production of biopesticides is over 3,000 tons annually,
which is increasing at a rapid rate. India has a vast potential for biopesticides as it
utilizes more than 100,000 tons of pesticides each year. Most (80%) of the pesticides
are used on cotton (45%), rice (30%), and vegetables (5%), the remaining crops
receiving only 20% as their share (http://www.molecular-plant-biotechnology.info/
industrial-microbiology/advantages-and-limitations-of-biopesticides-and-chemical-
pesticides.htm).
Classical biological control involving Bacillus thuringiensis (Bt), nuclear poly-
hedral virus (NPV), Trichoderma harzianum, Saccharopolyspora spinzosa and
Pseudomonas species have proved effective in checking pest infestations, along
with pathogens like Metarhizium anisopliae, Beauveria bassiana and entomopatho-
genic nematodes such as Steinernema carpcapse, which has received much recog-
nition. Great success in the reduction of populations rather than their elimination is
also advocated in the cases of plant-feeding insects and pathogens as weed biocon-
trol agents (e.g., plant rusts) in rangelands, forests, and other natural habitats.
Furthermore, a very critical approach is necessary towards the use of genetic
engineering in agriculture in order to improve the properties of the biological
control agents, and towards engineering crop plants to be resistant to pests using
the recombinant DNA technology, which produces several transgenic crops with
built-in resistance to insect pest and weeds. There is also a need to develop stra-
tegies based on the bio-chemical and ecological behavior of the pests. Thus, to
complement and eventually substitute synthetic pesticides with biopesticides would
represent an economically beneficial and ecologically sound alternative.
9.2 Shade of Biopesticide
Biopesticides are certain types of pesticides derived from natural materials such as
animals, plants, bacteria, or some minerals. For example, garlic (Allium sativum L.),
mint (Mentha arvensis), canola (Brassica napus) oil, and baking soda all have pes-
ticidal activity and are considered biopesticides. However, as defined by Sudakin
(2003), the term “biopesticide” encompasses a broad array of microbial pesticides,
bio-chemicals derived from microorganisms and other natural sources, and pro-
cesses involving the genetic incorporation of DNA into agricultural commodities
that confer protection against pest damage (plant-incorporated protectants).
According to Zechendorf (1995), the scope of biopesticides includes substances
9 Recent Advances in Biopesticides 187
such as plant extracts, hormones, pheromones, and toxins of organic origin.

Copping and Menn (2000) defined the term biopesticides to encompass many
aspects of pest control such as microbial, entomophagous nematodes, plant-derived
pesticides, secondary metabolites from microorganisms, pheromones, and genes
used to transform the crops to express resistance to pests. More recently, the
encouragement of natural enemies (parasitoids, predators, microbes, etc.) and the
use of transgenic crop varieties, pheromones, growth regulators, and plant-derived
materials in pest management have been considered to constitute the biopesticide
umbrella. At the end of 1998, there were approximately 175 registered biopesticide
active ingredients and 700 products, which increased to 195 active ingredients and
780 products in 2001. Data on microbial biopesticide agents from Agriculture
and Agri-food Canada (Kabulak and Gazdik 2005) and the US Environmental
Protection Agency (EPA) indicate that more than 200 products are being sold in
the United States, compared to only 60 comparable products in the European
Union. In the UK, only 5 microbial products are currently being sold, compared
to 10 in Germany, and 15 each in France and the Netherlands.
9.3 Types of Biopesticides
9.3.1 Microbial Pesticides
Of the nearly 1 million known species of insects, about 15,000 are considered pests
and about 300 require some form of control. Fortunately, most insect pests have
pathogenic microorganisms associated with them. Entomopathogens have been high-
lighted as controlling agents of insect pests for over a century, and include species of
bacteria, fungi, viruses, algae, and protozoa as the active ingredient. These patho-
genic organisms are isolated from diseased insects during naturally occurring epi-
demics. Over 400 species of fungi and more than 90 species of bacteria which infect
insects have been reported. These microbial control agents have a range of properties
that make them desirable for integrated crop management (Hajek 2004). To achieve a
double pay-off of better systems of environmental risk evaluation, and an effective
and more sustainable microbial control, attention is required for the better under-
standing of phylogeny of microbial natural enemies (Rehner and Buckley 2005), their
biogeography and factors determining bio-diversification of gene flow (Bidochka and
Small 2005), and assessment of their background levels in agricultural and natural
ecosystems (Mensink and Sheepmaker 2007).
9.3.1.1 Bacteria
Among bioagents, bacteria are the most potent. On the basis of their pathogenecity,
entomopathogenic bacteria are divided into four groups: (1) obligate pathogens (e.g.,
Bacillus popilliae), (2) crystalliferous spore-formers (e.g., B. thuringiensis), (3) fac-
ultative pathogens (e.g., B. sphaericus), and (4) potential pathogens (e.g., Seerratia
marscens). Of these, B. thuringiensis is the most effective, with about 6,000 isolates
Table 9.1 Molecular organization of the genes coding d – endotoxin with target host insect
Delta endotoxin gene M.W. (kDa) Insect host target
CrylA (a) Lepidoptera
CrylA (b) 130 a 133 Lepidoptera
CrylA (c) 138 Lepidoptera
CrylB 135 Lepidoptera
CrylC 130 a 134 Lepidoptera
CrylD,E,F
CryllA 71 Lepidoptera/diptera
CryllB 71 Lepidoptera
CryllC 70 Lepidoptera
CrylllA 73 Coleoptera
CrylllB 74 Coleoptera
CrylllC 129 Coleoptera
CrylllD 73 Coleoptera
CrylVA, CrylVB 134 a 128 Diptera
CrylVC 78 Diptera
CrylVD 72 Diptera
CryVA 81 Lepidopera/coleoptera
Adapted from Nasrine Moazami. Biopesticide Production, Biotechnology (EOLSS) manuscript
(http://www.eolss.net)
stored in various repositories throughout the world. This bacterium was first recorded
in 1901 as the cause of the damaging “Sotto” disease in silkworms in Japan (Ishikawa
1936), and was isolated again in 1927 by Maltes in Germany and given the name
B. thuringiensis. It occurs naturally in insect-rich locations, including soil, plant
surfaces, and grain stores. So far, more than 40,000 strains of Bacillus thuringiensis
have been isolated and identified as belonging to 39 serotypes. The Bt strains are
classified according to their H antigens into 27 groups and 7 subgroups, and according
to structure and molecular organization of the genes coding for the parasporal
delta-endotoxins. Only a few strains are used commercially as bio-control agents.
Chief among them includes Bacillus thuringiensis, B. sphaericus and B. popilliae.
They are active against insect pests belonging mainly to the orders lepidoptera,
diptera, or coleoptera (Table 9.1). B. thuringiensis produces three types of entomo-
cidal toxins: alpha exotoxin, beta exotoxin, and delta endotoxin (insecticidal crystal
protein, ICP). When ingested, the crystals paralyze the digestive tracts of insects,
often killing them within 24–48 h. The stability of the Bt formulations, however,
depends on the biological properties of insects, such as age and maturity, presence
of the degenerative enzymes, pH, residual nutrient availability, ionic strength and
osmotic pressure, type of preservatives used, type of surfactants used, and the
temperature.
Mode of Action
The toxic crystal Bt proteins in commercial formulations are effective only when
eaten by insects with a specific (usually alkaline) gut pH and the specific gut
membrane structures required to bind the toxin. So, when (1) the insect has the
correct physiology, (2) been at a susceptible stage of development, and (3) the
bacterium has been eaten in sufficient quantity, only then does the toxic protein
damage the gut lining, leading eventually to gut paralysis of the infected insect. As
a consequence, the insect stops feeding and dies from the combined effects of
starvation and tissue damage. However, B. thuringiensis spores do not usually
spread to other insects or cause disease outbreaks on their own as occurs with
many other pathogens (Whalon and McGaughey 1998). Formulation plays a
significant role in determining the final efficacy of a Bacillus-based product. As a
dry powder, B. thuringiensis is quite stable, whereas flowable formulations are
difficult to prepare and stabilize, and are quite sensitive to heat and, hence, must be
protected during storage. Formulations of Bt var. tenebrionis and sandiego have
been registered for use against Colorado potato beetle larvae and elm leaf beetle
adults and larvae. B. thuringiensis subsp. israelensis is marketed for use against
black flies and mosquitoes, while Bt var. aizawai is used to control fungus gnats,
wax moth larvae in bee hives and various caterpillars (Duan et al. 2004; Zehnder
and Gelernter 1989).
9.3.1.2 Virus
The efficacy of the insect-specific viruses can be seen among 1,600 viruses that
have been recorded from more than 1,000 species of insects, particularly caterpillar
pests. Different strains of naturally occurring nuclear polyhedrosis virus (NPV) and
granulosis virus are present at low levels in many insect populations. No threat to
humans or wildlife is posed by these insect viruses. A family of viruses called
baculoviruses is the most popular choice for microbial control, which have been
used regularly for pest control since the 1950s, particularly in forestry where they
have been highly effective at controlling sawflies. They are usually small (less than
0.001 mm across), and are composed primarily of double-stranded DNA that codes
for genes needed for virus establishment and reproduction. Because this genetic
material is easily destroyed by exposure to sunlight or by conditions in the host gut,
an infective baculovirus particle (virion) is protected by a protein coat called a
polyhedron. Most insect baculoviruses must be eaten by the host to produce an
infection, which is typically fatal to the insect. The majority of baculoviruses used as
biological control agents belong to genus Nucleopolyhedrovirus. These viruses are
considered suitable candidates for species-specific, narrow spectrum insecticidal
applications, have no negative impacts on plants, mammals, birds, fish, or even
on non-target insects, and are especially desirable when beneficial insects are being
conserved to aid in an overall EPM program (Ramkrishnan 1992). They have a
characteristic shiny-oily appearance, and are seen hanging limply from vegetation.
They are extremely fragile to the touch, rupturing to release fluid filled with
infective virus particles. This tendency to remain attached to foliage and then
rupture is an important feature of the virus life-cycle. As discussed above, infection
of other insects will occur only if they eat foliage that has been contaminated by
virus-killed larvae. Baculoviruses infect their hosts through ingestion. Virus parti-
cles invade the cells of the gut before colonizing the rest of the body. Infection
reduces mobility and feeding, and insects are killed in 5–8 days.
Mass production of baculoviruses can be done only in insects, but this is
economically viable for larger hosts such as caterpillars, and formulation and
application are straightforward. At present, there are approximately 16 products
available for use or under development, mostly for control of caterpillar pests.
Commercial products are available in Switzerland, Germany, and Spain for the
control of codling moth and the summer fruit tortrix. Based on their safety and
potential to replace chemical pesticides, five baculoviruses have been registered as
pesticides: Helicoverpa zea nuclear polyhedrosis virus (HzSNPV) in 1975, Orgyia
pseudotsugata (Ot) MNPV in 1976, and Lymantria dispar (Ld) MNPV in 1993.
However, the only privately produced and commercially available viral pesticide
comes from the USA and is the SeMNPV (Spod-XTM).
Mode of Action
Viruses invade an insect’s body via the gut. They replicate in tissues and can disrupt
components of an insect’s physiology, interfering with feeding, egg laying, and
movement. Different viruses cause different symptoms. NPV-infected larvae may
initially turn white and granular or very dark. Some may climb to the top of the crop
canopy, stop feeding, become limp, and hang from the upper leaves or stems, hence
the common name “caterpillar wilt” or “tree top” disease. Victims of a granulosis
virus may turn milky white and stop feeding, or contents of the dead larvae are
liquefied and the cuticle ruptures easily to release infectious viral particles. Death
from a virus infection usually occurs within 3–8 days. Their killing efficiency may
be augmented by genetic modification of its genome with genes of other natural
pathogens (Szewczyk et al. 2006).
9.3.1.3 Entomopathogenic Fungi
Over 750 species of fungi kill insects. Entomopathogenic fungi are widely distributed
throughout the fungal kingdom, although the majority occur in the Deuteromycotina
and Zygomycotina families. Some insect-pathogenic fungi have restricted host
ranges, for example, Aschersonia aleyrodis infects only scale insects and whiteflies,
while other fungal species have a wide host range, with individual isolates being more
specific, for example, Metarhizium anisopliae and Beauveria bassiana. The potential
of fungal pathogens in the control of insect pests has been recognized since the latter
part of the nineteenth century, when M. anisopliae was tested against the wheat
cockchafer, Anisoplia austriaca and sugar beet curculionid, Cleonus punctuventris.
Over the past century, there have been many attempts to exploit Verticillium lecanii,
A. aleyrodis, B. bassiana, Nomuraea rileyi, M. anisopliae, and some species of
Entomophthorales for pest control. At present, fungi are being used for pest control
on a moderate scale in China (Nasrine Moazami, Biopesticide Production, Biotech-

nology (EOLSS) manuscript http://www.eolss.net).
Entomopathogenic fungi invade their hosts using spores that grow through the
cuticle, and hence they are particularly suited for control of pests with piercing
mouthparts, such as aphids and whiteflies, which are unlikely to acquire pathogens
through feeding. Infection requires high humidity at the insect surface, but this can
be circumvented using oil-based formulations. Beauveria bassiana are becoming
available for the control of a range of glasshouse pests. Many entomopathogenic
fungi, especially those in the entomophthorales, are responsible for epizootics that
often successfully regulate pest insect populations. Although inoculation of insect
populations with entomopathogenic fungi has provided classical biological control
of some pests, most notably against the gypsy moth, the most common method
of employing fungi for insect control is through inundatory means. Development of
effective methods for production of resting spores and competent mycelia of
entomophthoralean species will ultimately increase the utility of these fungi.
Fungi often become attenuated (lose virulence or antagonistic characteristics)
when maintained on artificial media. Cultural conditions must be identified which
retain virulence without increasing production costs. At present, little progress has
been made in this area partly because the underlying mechanisms for attenuation
have not been elucidated. While 750 species of entomopathogenic fungi are known,
less than 20 have received serious attention as control agents of insect pests
(Hawksworth et al. 1995; Copping 2004). Among fungal pesticides, five have
been introduced since 1979, and three in 1981. Many countries with centrally
planned economies have been using fungal pesticides successfully for many
years. Nowadays, about 20 products are available worldwide for managing sap-
feeding insects, beetles, caterpillars, flies, and locusts.
Mode of Action
Entomopathogenic fungi invade their hosts by direct penetration of the host exo-
skeleton or cuticle. As occurs in many plant-pathogenic fungi, conidia germinate on
the host surface and often differentiate to form an appressorium. An infected hypha
penetrates down through the host cuticle and eventually emerges into the haemo-
coel of the insect. As with plant pathogens, entry into the host involves both
enzymic degradation and mechanical pressure. A range of extracellular enzymes
that can degrade the major components of insect cuticle is produced when
M. anisopliae is grown in vitro with the cuticle as the sole carbon and nitrogen source.
Growth of fungi in the haemolymph of insects may be as yeast-like blastospores,
hyphal bodies, or protoplasts, rather than in the form of a mycelium (from which
toxic compounds can be extracted). Death is caused by tissue destruction and,
occasionally, by toxins produced by the fungus. The fungus frequently emerges
from the insect’s body to produce spores that, when spread by wind, rain, or contact
with other insects, can spread infection. Destruxins (DTXs), a group of cyclic
depsipeptides (peptides containing ester linkages) produced by M. anisopliae,
have received most attention, and are responsible for causing insect mortality by the
fungus (Zimmermann 2007). DTXs are insecticidal by injection and, in some cases,
when ingested by mouth; toxicity is most acute among lepidopteran larvae and adult
Diptera.
9.3.1.4 Nematodes
Entomopathogenic nematode worms are just visible to the naked eye, being about
0.5 mm in length. The entomopathogenic nematodes are a nematode–bacterium
complex forming a strong mutualistic relationship. The nematode may appear as
little more than a biological syringe for its bacterial partner. The bacteria break
down the insect body, which provides food for the nematodes. After the insect dies,
the juvenile nematodes develop to adults and reproduce. A new generation of
infective juveniles emerges 8–14 days after infection. Entomopathogenic nema-
todes are extraordinarily lethal to many important soil insect pests, yet are safe for
plants and animals. This high degree of safety means that, unlike chemicals, or
even Bacillus thuringiensis, nematode applications do not require other safety
equipment; and re-entry time, residues, groundwater contamination, chemical
trespass, and pollinators are not major issues. Like other biological control agents,
nematodes are constrained by being living organisms that require specific condi-
tions to be effective. Thus, desiccation or ultraviolet light rapidly inactivates
insecticidal nematodes; chemical insecticides are less constrained. Similarly,
nematodes are effective within a narrower temperature range than chemicals, and
are more impacted by suboptimal soil type, depth, and irrigation frequency. In
addition, unlike other entomopathogens, nematodes are exempted from registration
and so have been popular choices for commercialization. Over 60 products
prepared from nematodes are available in Europe. Nematodes require moist con-
ditions to operate and have been marketed predominantly against soil pests, such as
vine weevil and sciarid fly larvae. However, they may also control foliar pests, for
example Nemasys (Becker Underwood) which can be used to control western
flower thrips.
Mode of Action
Juvenile nematodes parasitize their hosts directly by penetrating the cuticle through
natural openings and introducing symbiotic bacteria, which multiply rapidly and
cause death by septicaemia, often within 48 h. Nematode growth and reproduction
depend upon conditions established in the host cadaver by the bacterium. The
bacterium further contributes anti-immune proteins to assist the nematode in over-
coming host defences, and anti-microbials that suppress colonization of the cadaver
by competing secondary invaders. Conversely, the bacterium lacks invasive powers
and is dependent upon the nematode to locate and penetrate suitable hosts.
9.3.1.5 Entomopathogenic Protozoa and Microsporida
Protozoan diseases of insects are ubiquitous and perform an important regulatory

role in insect populations. They are generally host specific and slow acting, most
often producing chronic infections. They develop only in living hosts and many
species require an intermediate host. Species in the Microsporida are among the
most commonly observed. Their main advantages are persistence and recycling in
host populations and their debilitating effect on reproduction and overall fitness of
target insects. As inundatively-applied microbial control agents, only a few species
have been moderately successful. The main disadvantages of the protozoa as
inundatively-applied microbial control agents are the requirement for in vivo pro-
duction and low levels on immediate mortality. Nosema locustae is the only
commercially available species of microsporidium, marketed under several labels
for the control of grasshoppers and crickets. It is applied with insect-attractant bait.
Because of its slow mode of action, this product is better suited to long-term
management of rangeland pests than to the more intensive demands of commercial
crop or even home garden production.
9.3.2 Plant Incorporated Protectants
9.3.2.1 Botanicals
Every native plant is evolved with a specific recipe of natural controls. The rich
plant bio-diversity of India which continues to be a major untapped source of a
diverse range of bio-active molecules needs to be explored for safe crop protection.
A range of secondary metabolites, like alkaloids, flavonoids, phenols, glycosides,
sitosterols, and tannins, have a record of protecting plants against their herbivorous
pests (Ahmad 2007). However, greater emphasis needs to be laid on developing
innovative formulations with good storage ability and persistence. There is also a
need to improve the knock-down characters in natural pesticides which are
endowed with slow acting behavioral and physiological modes of action. Histori-
cally, plant materials have been in use longer than any other chemicals (with the
exception of sulphur) but after the Second World War, they lost their importance
with the introduction of synthetic organic chemicals. Tobacco (Nicotiana taba-
cum), pyrethrum (Chrysanthemum cinerariifolium), derris (Derris elliptica), helle-
bore (Helleborus niger), quassia (Picrasma excels), camphor (Cinnamonum
camphora), and turpentine (Syncarpia glomulifera) were some of the important
plant products in use before the organized and systematic search began in the 1940s.
In the US, the use of botanical pesticides like pyrethrum, nicotine, and rotenone
peaked during 1966, but due to some limitations like photosensitivity, degradation,
and availability, their use declined thereafter. Chief among the adverse effects are
structural modifications and structure–activity relationships which led to the dis-
covery of synthetic pyrethrin-like materials called synthetic pyrethroids which are
stable in light and act even at very low concentrations. Similarly, nicotinoids
(previously referred to as nitro-quanidines, neonicotinyls, neonicotinoids, and
chloronicotines, and more recently referred as chloronicotinyls) are considered
the siblings of structurally modified pesticides. Therefore, there is an urgent need
to explore these virgin plants for their bioactivities. However, the major bottlenecks
in the commercialisation of biopesticides are: (1) resource availability, (2) pro-
blems in chemical standardization and delivery systems, (3) less effective field
reports than laboratory results, (4) lack of novel, effective, stable and economical
formulations of botanical products, and (5) lack of general awareness among the
end users which discourage product realization. Despite all these constraints, the
Energy and Resources Institute (TERI) under the patronage of the Department of
Biotechnology, Ministry of Science and Technology, Government of India, has
developed a plant extract-based biopesticide formulation “Bollcure”, that could be
used to control cotton bollworm (Helicoverpa armigera), the most challenging
larvae capable of inflicting severe losses to the yield of cotton (Gossypium hirsu-
tum) crops.
9.3.3 Biochemical Pesticides
9.3.3.1 Allellochemicals
Plant biodiversity provides a vast repository of biologically active compounds that

find application in traditional medicines and crop protection systems. Among the
estimated over a half a million species of the existing plant kingdom, nearly 2,500
species belonging to 235 plant families have exhibited measurable anti-pest proper-
ties (Saxena 1998). Phytochemicals have a wide range of activity including hor-
monal, neurological, nutritional, and enzymatic, many of which still need to be
explored. Recent research in plant resistance to insect pests of Nicotiana spp. and
Solanum spp. have demonstrated that leaf exudates produced by them play a
significant role in determining resistance or susceptibility to infestation to insects
in such plants (Severson et al. 1985). These exudates produce a protective waxy
layer comprising numerous secondary metabolites such as glycolipids and glycer-
olipids as well as free fatty acids/esters and terpenes. The bioactive glycerolipids
consisting of 1,2-diacylglycerol, 1,3-diacylglycerol and 1,2,3- triglycerol provide
in-built plant resistance to invading pests and are active against certain phytopha-
gous insects and pathogens. High levels of a-amaryl alkanoate and cycloartenyl
alkanoate in epicular waxes from aphid-resistant raspberry (Rubus spp.) plants is
responsible for its resistance. Moreover, some light-activated phytotoxins, like
substituted acetylenes, thiophenes, acetylenic thiophenes, quinines, furanocoumar-
ins and related compounds, have also shown significant pest control properties.
Allelochemicals from microbes include spinosyns derived from aerobic fermenta-
tion of the soil-inhabiting actinomycetes Saccharopolyspora spinosa (Thompson
et al. 2000), while avermectin and milbemycin are derived from a family of
macrocyclic lactones produced from Streptomycin avermitilis (Campbell 1989).
9.3.3.2 Sex Pheromones
Pheromones specifically disrupt the reproductive cycle of harmful insects. In this

way, farmers can reduce the amount of insecticide they need; spraying only when
insects are in a vulnerable stage or when their numbers exceed certain levels. There
is no alteration to the natural biological and ecological cycle, hence ensuring that
there is no environmental or health hazard. They are portable, less expensive, and a
more natural form of crop-protecting agent. Pheromones lure and trap is an insect-
trapping apparatus which essentially works by using the sex pheromones generated
by female insects to attract their male counterparts. Pheromones as monitoring tools
provide cost-effective and simple techniques to time application of biological
control agents and bio-pesticides in integrated pest management (IPM). In 1987,
Pest Control of India (PCI) became the first company in India to commercially
introduce pheromone technology for agricultural use by launching sex pheromone
lures and traps for monitoring Helicoverpa armigera and Spodoptera litura. Since
then, PCI has introduced commercial pheromone lures for monitoring a range of
pests, including cotton bollworms, tobacco caterpillar, rice yellow stem borer,
sugarcane borers, diamond back moth, brinjal shoot and fruit borer, and fruit
flies. Furthermore, PCI has also been regularly introducing suitable traps for use
with these pheromone lures, and today has in its trap range funnel traps, delta traps,
McPhail traps, cross-vane traps, water traps, and bucket traps.
9.4 Potentials and Constraints of Biopesticides
All these biopesticide strategies employed to control insect pests have distinct
advantages and disadvantages under specific conditions. Although there has been
an extensive programme for mass rearing and release of beneficial insects such as
Trichogramma species, perhaps the greatest potential for successful biological
control through augmentation of natural enemies exists with pathogenic microor-
ganisms. The utility of these agents has, however, been limited by difficulties in the
production and formulations of products, particularly with viruses and fungi, and
has limited spectra of activity against pest complexes in comparison with chemical
insecticides. However, recent research has indicated that the efficacy of bacteria
and viruses have improved through recombinant DNA technology. The advantages
of microbial insecticides over chemical insecticides in terms of reduced threats to
nontarget species and limited potential for environmental degradation are compel-
ling reasons for increasing efforts to improve these agents and increase their utility
for IPM programmes (Cuperus et al. 2004).
Although many compounds have been isolated, characterized, and evaluated as

anti-insect compounds from plants, not much headway has been achieved in the
commercialization of such products. The standardization of plant based anti-insect
preparations has been the biggest constraint and has subsequently hindered their
potential marketability compared with conventional pesticides. Generally, natural
plant products tend to be rather slow acting, of modest toxicity, and rapidly degrade
in the environment. The variation in efficacy of the compounds between test species
is probably the greatest barrier to their commercialization. However, the concept of
phytosynergistic strategy and assessing the toxicity of co-occurring toxins seems to
have potential and could lead to further advancement in the development of
biopesticides (Koul and Dhaliwal 2001).
9.4.1 Advantages of Using Biopesticides
The potential advantages of bioinsecticides include that they: (1) are usually
inherently less harmful than conventional pesticides, (2) do not leave harmful
residues, (3) are designed to affect just the specific target organism in contrast to
broad spectrum of conventional ones, (d) are effective in small quantities and often
disperse quickly (Ware 1994), thereby resulting in lower exposures and largely
avoiding the pollution problems caused by conventional pesticides, (e) as a compo-
nent of IPM, greatly reduce the use of conventional synthetics while crop yields
remain high, and (f) are often cheaper than chemical pesticides. The introduction of
transgenic technology has opened up new vistas in crop protection management.
Although investment in agricultural biotechnology is concentrated in industrialized
countries, many developing countries like India, China, and Brazil have committed
significant levels of public funding to biotechnology. Recent studies show that
small-scale farmers in developing countries stand to benefit more from transgenic
technology than commercial farmers in industrial countries, due to limited access to
agrochemicals and greater losses associated with insect pests in tropical and
subtropical climates. The best example of such progress in developing countries
is the use of Bt cotton containing Cry 1ac gene from Baccillus thuringiensis which
is commercially grown in India and China. Multi-level endocrine systems
controlling a wide range of physiological processes (e.g., moulting, metamorphosis,
diapause, reproduction, etc.) advocating multiple functional capacities of insect
neuropeptides, also provide opportunities for new insect control strategies. As a
biopesticide, they induce adult sterility, physical body change, water loss, and
premature death in insects (Muraleedharan and Devi 1992). Their chief disadvan-
tages are: (1) very high specificity, which will require an accurate identification of
the pest/pathogen and may require multiple pesticides to be used, and (2) often
variable efficacy due to the influences of various biotic and abiotic factors (since
biopesticides are usually living organisms, which bring about pest/pathogen control
by multiplying within the target insect pest/pathogen).
9.4.2 Constraints in the Promotion of Biopesticides
9.4.2.1 Technological
These includes (1) lack of technological advancement relating to the transfer of

technology from pilot point scale to mega-scale, (2) lack of mass rearing facilities,
(3) inconsistent field results, (4) limited or short shelf life (say 6 months), and (e)
existence of very crude formulations.
9.4.2.2 Financial
This includes (1) huge rates of tax, (2) benefit of economy of scale rather limited,
and (3) lack of subsidy from Government bodies.
9.4.2.3 Extension Related
l Existing mindset of farmers to compare biological vis-à-vis chemical pesticides

l Lack of field demonstration and training programmes
9.4.2.4 Regulatory/Statutory
(1) Need for a separate body for registering biological compounds, (2) need to
liberalize expensive and time-consuming registration processes, (3) require strict
quality control and standardised formulations, and (d) lack of large number of nodal
biopesticide testing laboratories.
9.5 Biopesticides Global Scenario
During the 1970s, research institutions around the globe had started opting for
biopesticides as a new and sustainable alternatives to conventional pesticides. The
tactics of using biopesticides seems ideal for IPM if they could spare the pests’
natural enemies, as they are narrowly selective and pose few problems to the
nontarget ones. Except for Bacillus thuringiensis, the predicted demand for com-
mercial biopesticides has not been realised. Estimated world sales of all biopesti-
cides amount to 0.5% of the total world pesticide market accounting for more than
90% of the total sales of Bt products (Rodgers 1993). Among the best examples of
its demand in developing countries, Brazilian farmers have obtained exceptional
control of the velvetbean caterpillar, Anticarsia gemmatalis, by spraying NPV onto
soyabean, Glycine max (Moscardi and Sosa-Gomez 1996). Chinese farmers applied
Beauveria bassiana to the soil surface to infect the larvae dropping from the plants
to overwinter (Kogan and Turnipseed 1987) but, like the Brazilians, they used it as
an IPM tool and did not rely merely on these biopesticides. Among the plant
pathologists, at least 30 different biological control organisms are presently avail-
able as commercial formulations to manage plant diseases (Lumsden et al. 1995).
For example, Agrobacterium tumefaciens (strain K84), which is able to synthesize
antibiotic agrocin 84, has been used to prevent crown gall (Copping 2004;
Kabulak and Gazdik 2005). The control of the coconut rhinocerous beetle, Oryctes
rhinoceros, on coconut (Cocos nucifera) in Asia and the Pacific Islands is a good
example of how entomopathogens could provide benefits to such plants
(Waterhouse and Norris 1987).
9.6 Biopesticide Production
9.6.1 Use of Genetic-Engineering Technology
Major breakthroughs in molecular biology and biotechnology since the early 1980s
indicate that rapid improvements in the competitive ability of biological control
methods are possible, and that biopesticides can play a major role in crop protection
in the future. It has become possible to improve some of the critical properties that
earlier hampered the usefulness of many biocontrol agents. Valuable genes from
completely unrelated organisms can now be utilized for biological control pur-
poses. Biological control using recombinant DNA (genetic engineering) technology
can be achieved in several different ways: control agents may be improved; crop
plants can be engineered to carry better resistance genes; or organisms associated
with the plant may be modified to provide protection. All these approaches have
successfully been used experimentally in several different ways. Product develop-
ment has been very active in the area of incorporating resistant genes – mainly from
Bt directly into plants. Successes include potato (Solanum tuberosum), tomato
(Lycopersicon lycopersicum), tobacco, and cotton. General root-colonizing bacteria
of plants have also been engineered to produce insecticidal toxins, which protect
against pests such as the corn rootworm.
9.6.2 Engineering Biological Control Agents
The genetic improvement of biological agents is a relatively new concept. For this,
a great deal must be known about the biology, ecology, and behavior of the
organism.
9.6.3 Engineering Crop Plants
The first gene encoding the Bt toxin was cloned by Schnepf and Whiteley (1981),
and Bt gene regulation was known by 1986 (Whiteley et al. 1987). The crop plants
were tobacco and tomato, producing the delta endotoxin of Bacillus thuringiensis to
make them resistant against caterpillars. The uses of genetically modified crops that
express insecticidal genes (e.g., Bt crops) also provide an important tool to control
Lepidopterous pests (Shelton et al. 2002). Similar strategies have been employed
to develop mosquitocidal species of algae. Through genetic engineering tech-
niques, the Autographa californica multinucleocapsid nucleopolyhedrosis virus
(AcMNPV) was engineered to kill insects more quickly by expressing either
enzymes or toxins soon after host invasion. Of particular interest is the possibility
of making viruses produce insect neurohormones, which can cause rapid physio-
logical disruptions in minutely defined target hosts. Parasexual recombination not
only facilitates genetic analysis in asexually reproducing fungi, but also provides an
important tool in strain improvement of bioprotectant fungi.
9.7 Biopesticide Regulations
Integrated pest management is being redefined as BIPM (biointensive integrated

pest management) by the advent of biologically-based alternatives, which are
environment-friendly and ensure food safety and security, apart from maintaining
the ecological balance of the living biota. In India, all pesticides (including biopes-
ticides) are regulated under the Insecticidal Act 1968 and require mandatory
registration from the Central Insecticidal Board and Registration Committee (CIB
and RC). This body has streamlined the registration guidelines and brought the
quantum of data generation to a lower scale, granting the registration of the
biopesticide on a priority basis.
9.8 Biopesticide Commercialization
Biopesticides have not been adequately evaluated in terms of their costs relative to
their benefits to farmers and others. Examples from both developed countries
(Benbrook 1996) and developing countries (Oka 1996) show that farmers commonly
reduce insecticide use by 50–100% without any loss in crop yield after switching
to IPM. In addition, the synthetic pesticides market is expected to show a declining
trend at the rate of 1.5% per annum. At the same time, the biopesticide market is
growing and is expected to reach more than a billion dollars in the next 5 years
(Table 9.2 and Fig. 9.1). Key developments expected in the coming years are more
R&D in biopesticides, an increase in genetically modified crops, the application of
Table 9.2 The global market is rapidly expanding with 150–300% increase over next 5 years
($ millions)
Europe NAFTA Latin Africa ASIA Oceania Total
America
Macrobials 70 100 15 8 30 20 243
Microbials
Bacteria 15 80 10 5 20 30 160
Virus 10 15 10 2 5 10 42
Fungi 25 45 20 3 15 20 128
Total 50 140 40 10 40 60 330
Biorationals
Natural 30 70 25 10 40 15 180
Semisynthetic 40 80 20 10 30 20 200
Total 70 150 45 20 70 35 390
TOTAL 190 390 100 38 140 115 973
Source: International Biocontrol Manufacturer’s Association (2004)
Bacillus thuringiensis Bacterial Virus Fungal Nematode
15
37
18
159
32
Fig. 9.1 $260MM estimated 2005 sales projected to grow to $350–400MM by 2015
Source: Nasrine Moazami. Biopesticide Production, Biotechnology (EOLSS) manuscript (http://
www.eolss.net)
integrated pest management concepts, and a widening of organic farming. Biopes-

ticides today represent about 2.5% of the overall pesticides market, and are
expected to grow to about 4.2% by 2010 (Fig. 9.2). Of all the crops, orchard
crops hold the largest share of biopesticides use at 55%.
The high cost of labor has also slowed down the development of other bio-
pesticides. The spiralling cost, coupled with regulatory constraints and problems
with formulations and marketing, have led to serious setbacks to the application of
biopesticides. To apprehend a slow acting biological control agent (the essence of a
biopesticide) to challenge an impressive and rapidly acting synthetic chemical is
quixotic. Ecosystems are highly variable and unpredictable and to assume that
either their resident pests or biocontrol agents will respond constantly over space
and time is unrealistic. The inconsistent performance may merely be alluding to the
variable nature of their crop ecosystem and are not flaws in the biopesticide per se,
and the state of affairs will continue if we treat them like conventional pesticides
30,000
25,000
20,000
$ Millions
15,000
10,000
5,000
0
2003 2004 2005 2010
Biopesticides Synthetic pesticides
Fig. 9.2 Global biopesticide market

Report ID: CHM029B, Published (adapted from Thakore 2006)
and expect them to perform evenly across a range of situations and apply them in an
ecological abyss.
The successful commercialization of insect-pathogenic viruses has been limited.
Thus far, NPV strains have only been mass produced in living insects, a costly
procedure. Viral insecticide development is further hindered by the fact that the
viruses are specific to one species or genus, ensuring a relatively small market.
Genetic engineering offers a potential solution to these shortcomings. A foreign
pesticidal gene can be inserted into the viral genome, and expression of the
pesticidal gene product during replication will allow the virus to kill insects faster
or cause rapid cessation of feeding. Foreign genes which have been inserted into
baculoviruses for this purpose include the Buthus eupeus insect toxin-1, the Man-
duca sexta diuretic hormone, the Bacillus thuringiensis ssp. kurstaki HD-73 delta-
endotoxin, the Heliothis virescens juvenile hormone esterase, the Pyemotes tritici
TxP-I toxin, Androctonus australis neurotoxin, Dol m V gene and T-urf 13 genes
[Nasrine Moazami.Biopesticide Production, Biotechnology (EOLSS) manuscript
(http://www.eolss.net).]
9.9 Conclusion
Although chemicals characteristically have good storage life, relatively wide spec-
trum of activity, fast speed of kill, and relatively short persistence, frequent and
injudicious applications leads to environmental hazards and poses a serious toxico-
logical threat. In contrast, biological control agents tend to have relatively poor
storage life, high target specificity, slow speed of kill, potentially long persistence
through secondary cycling, and consequently lower frequency of application. They
are environmentally friendly and have a low degree of hazard for humans and
livestock. The need of the hour is for a biological agent that possesses the desirable
properties of a chemical pesticide, is highly toxic to the target organism, is able to

be mass-produced on an industrial scale, has a long shelf life, can be safely
transported, and is highly effective both in its capacity to kill and to reproduce on
pests (compounding its killing action). It is important that microbial control of
insect pests be further developed and that entomologists along with microbiologists
should be able to quantify and make contributions to the regulation of insect
populations by naturally occurring pathogens.
Biological control uses a different approach to pest management, focusing on
natural enemies of plant pests and diseases to manage their populations. The
strategies rely on detailed knowledge of the ecology, the life cycles, and the food
chains in each system, thus developing highly target-specific control strategies that
leave the nontarget plants, insects, or other animals unharmed. Researchers should
employ a range of strategies, often looking for means to strengthen local, natural
enemies or to produce them as biopesticides. Fungi, insect viruses, and competing
but harmless strains of the same pest need to be tried so as to develop “safer
pesticides”.
References
Ahmad M (2007) Insecticide resistance mechanisms and their management in Helicoverpa

armigera (Hubner). Rev J Agric Res 45:319–335
Benbrook CM (1996) Pest management at the crossroads. Consumers Union, Yonkers, NY, p 272
Bidochka MJ, Small CL (2005) Phylogeography of Metarhizium, an insect pathogenic fungus. In:
Vegaand FE, Blackwell M (eds) Insect fungal associations: ecology and evolution. Oxford
University Press, Oxford, UK, pp 28–50
Campbell WC (1989) Ivermectin and abamectin. Springer, New York, p 363
Copping LG (2004) The manual of biocontrol agents. British Crop Protection Council, Farnham,
UK, p 752
Copping LG, Menn JJ (2000) Biopesticides: a review of their action, applications and efficacy.
Pest Manag Sci 56:651–676
Cuperus GW, Berberet RC, Noyes RT (2004) The essential role of IPM in promoting sustainability
of agricultural production systems for future generations. In: Koul O, Dhaliwal GS, Cuperus GW
(eds) Integrated pest management: potential, constraints and challenges. CAB International,
Wallingford, UK, pp 265–280
Duan JJ, Head G, Jensen A, Reed G (2004) Effects of transgenic Bacillus thuringiensis potato and
conventional insecticides for Colorado potato beetle (Coleoptera: Chrysomelidae) manage-
ment on the abundance of ground-dwelling arthropods in Oregon potato ecosystems. Environ
Entomol 33:275–281
Hajek A (2004) Natural enemies: an introduction to biological control. Cambridge University
Press, Cambridge, UK
Hawksworth DL, Kirk PM, Sutton BC, Pegler DN (1995) Ainsworth and Bisby’s dictionary of
fungi, 8th edn. CAB International, Wallingford, UK, p 616
Ishikawa K (1936) Pathology of the silkworm. Meibundo, Tokyo, p 512
Kabulak T, Gazdik K (2005) Directory of microbial pesticides for agricultural crops in OECD
countries. Agriculture and Agri-food Canada, http.//www.Agr.Gr.Ca/env/pdf/cat_e_pdf.
Accessed 28.06.07
Kogan M, Turnipseed SG (1987) Ecology and management of soybean arthropods. Ann Rev
Entomol 32:507–538
Koul O, Dhaliwal GS (2001) Phytochemical biopesticides. Harwood Academic, ISBN:90-5823-

089-9.ISSN:1563-6712
Lumsden RD, Lewis JA, Fravel DR (1995) Formulation and delivery of biocontrol agents for use
against soil-borne pathogens (unpubl. Report cited in National Research Council 1996).
Ecologically based pest management: new solutions for a new century. Natl Acad Press,
Washington, DC, p 144
Mensink BJWG, Sheepmaker JWA (2007) How to evaluate the environmental safety of micro-
bialplant protection products: a proposal. Biocontrol Sci Technol 17:3–20
Moazami N (1996) Biopesticide production, biotechnology (EOLSS) manuscript (http://www.
eolss.net)
Moscardi F, Sosa-Gomez DR (1996) Soybean in Brazil. In: Persley GJ (ed) Biotecnology and
integrated pest management. CAB Int, Wellingford, UK, pp 98–112
Muraleedharan D, Devi DS (1992) Endocrine manipulations as an insect pest management
strategy. Agric Zool Rev 5:253–272
Oka IN (1996) Integrated crop pest management: one way to empower farmers to develop efficient
and environmentally sound agricultural practices. IARD J 18:1–12
Ramkrishnan N (1992) Nuclear polyhedrosis virus of Spodoptera litura : an approach for its
efficient use. In: Ananthakrishnan TN (ed) Emerging trends in biological control of phytopha-
gous insects. Oxford and IBH Pub. Co. Pvt. Ltd, New Delhi, pp 159–164
Rehner SA, Buckley E (2005) A Beauveria phylogeny inferred from nuclear ITS and EF1-a sequences:
evidence for cryptic diversification and links to Cordyceps telemorphs. Mycologia 97:84–98
Rodgers PB (1993) Potential of biopesticides in agriculture. Pesticide Sci 39:117–129
Saxena RC (1998) Green revolutions without blues: botanicals for pest management. In:
Dhaliwal GS, Randhawa NS, Arora R, Dhawan AK (eds) Ecological agriculture and sustain-
able development, vol 2. Indian Ecological Society and Centre for Research in Rural and
Industrial Development, Chandigarh, pp 111–127
Schnepf HE, Whiteley HR (1981) Cloning and expression of the Bacillus thuringiensis crystal
protein gene in Escherichia coli. Proc Natl Acad Sci USA 78:2893–2897
Shelton AM, Zhao JH, Roush R (2002) Economic, ecological, food safety and social consequences
of the deployment of Bt transgenic plants. Ann Rev Entomol 47:845–881
Sudakin DL (2003) Biopesticides. Toxicol Rev 22:83–90
Szewczyk B, Hoyos-Carvajal L, Paluszek M, Skrzecz I, Lobo de Souza M (2006) Baculoviruses
emerging biopesticides. Biotechnol Adv 24:143–160
Thakore Y (2006) The biopesticide market for global agricultural use. Industrial Biotechnology,
2:194–208
Thompson GD, Dutton R, Sparks TC (2000) Spinosad – a case study: an example from a natural
products discovery programme. Pest Manage Sci 56:696–702
Ware GW (1994) The pesticide book, 4th edn. Thomson Publications, Fresno, CA, pp 386–390
Waterhouse DF, Norris KR (1987) Biological control: pacific prospects. Australian Centre Int Agr
Res. Inkata Press, Melbourne, p 454
Whalon ME, McGaughey WH (1998) Bacillus thuringiensis: use and resistance management. In:
Ishaaya I, Deheele D (eds) Insecticides with novel modes of action, mechanism and applica-
tion. Springer, New York, pp 106–137
Whiteley HR, Schnepf HE, Tomczak K, Lara JC (1987) Structure and regulation of the crystal
protein gene of Bacillus thuringiensis. In: Maramorosch K (ed) Biotechnology in invertebrate
pathology and cell culture. Academic Press, San Diego, pp 13–27
Zechendorf (1995) Novel approaches to integrated pest management. Lewis, Boca Raton, FL,
pp 231–257
Zehnder GW, Gelernter WD (1989) Activity of the M-ONE formulation of a new strain of Bacillus
thuringiensis against the Colorado potato beetle (Coleoptera: Chrysomelidae): relationship
between susceptibility and insect life stage. J Econ Entomol 82:756–761
Zimmermann G (2007) Review on safety of the entomopathogenic fungus Metarhizium aniso-
pliae. Biocontr Sci Technol 17:879–920
Chapter 10
Benefits of Arbuscular Mycorrhizal Fungi
to Sustainable Crop Production
M. Vosátka and J. Albrechtová
Abstract The majority of agricultural crops form relationships with arbuscular

mycorrhizal (AM) fungi, which affect physiology and, consequently, yields
and food qualities of plants. Mycorrhiza occur naturally in agroecosystems, but their
abundance decreases with soil degradation, pollution or excessive use of agrochemi-
cals. Additionally, mycorrhiza may be negatively affected by soil management
practices and disadvantageous crop rotation. Conversely, mycorrhizal fungi are usu-
ally more abundant in sustainable and medium-to-low-input production systems.
In order to exploit the beneficial effects of AM fungi, appropriate management
practices, such as the design of suitable crop rotations, appropriate tillage practices,
the introduction of multi-microbial inoculants, and the regulated use of agrochemicals,
have to be employed. An alternative strategy for improved use of AM in sustainable
crop production is to target crop breeding programs involving AM-favoring traits.
Understanding the multifarious activities of AM fungi is likely to provide a prospec-
tive tool for sustainable crop production in different agro-ecosystems.
10.1 Introduction
Mycorrhiza form a beneficial soil microbe–plant interaction that affects the physi-
ological traits of many crop plants, including yield and food-quality. Recently,
mycorrhiza have become a prospective tool for sustainable, low input crop production
J. Albrechtová (*)
Faculty of Science, Department of Plant Physiology, Charles University in Prague, Vinicna 5,
128 44, Prague, Czech Republic
Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pruhonice, Czech Republic
e-mail: albrecht@natur.cuni.cz
M. Vosátka
Institute of Botany, Academy of Sciences of the Czech Republic, 252 43 Pruhonice, Czech Republic
206 M. Vosátka and J. Albrechtová
systems. Mycorrhizas inhabit the ecosystems of terrestrial plants as a natural way to

acquire nutrients from soils (Pirozynski and Dalpe 1992). The majority of crop
plants form relationships with arbuscular mycorrhizal (AM) fungi, and their
responsiveness to mycorrhiza depends on numerous factors including genotype,
soil-related and climate properties, and agrochemical inputs into the cultivation
system. Extensive mycorrhizal research conducted during the last few decades
has focused on the mechanisms of the symbiosis and its role in the management of
a sustainable crop production. Systems high in sustainability include those which
aim to make the best use of environmental factors and services without damaging
these variables (Pretty 1995). The sustainability has, therefore, become one of the
most prominent issues in agricultural practices. Sustainability incorporates the
concepts of resilience (the capacity of systems to buffer shocks and stresses) and
persistence (the capacity of systems to sustain over long periods), and in turn
addresses many wider economic, social and environmental outcomes (Pretty
2008). Moreover, the soil quality is an integral indicator of sustainable ecosys-
tems as well (Herrick 2000).
Soil microorganisms including AM fungi form the basis of healthy and pro-
ductive soil environments. During the last century, soils have degraded consider-
ably on a global level, and as a result microbial diversity is disturbed. When the
world population was less than half what it is currently, its pressure on the
environment did not have such far-reaching consequences, and concerns about
the environment were not so vital (Lal 2008). The growing world population and
human needs such as food, energy and living environments have, however,
imposed increasing demands on agroecosystems. The twenty-first century faces
many global challenges involving soil functioning where mycorrhiza could play
an important role in combating these problems. Mycorrhiza’s problem-solving
potential applies to (1) increasing agronomic production, (2) enhancing carbon
sequestration in terrestrial ecosystems to stabilize the atmospheric CO2 concen-
tration, (3) converting degraded, polluted or desertified soils to restored land or
sustainable agroecosystems, and (4) developing sustainable farming/cropping
systems aimed at improving water use efficiency and soil properties to combat
increasing erosion and minimize risks of water pollution and eutrophication.
Thus, AM fungi are regarded as essential components of sustainable soil–plant
systems (Schreiner et al. 2003). As an ecological biofertilizer, a bioprotectant
against environmental stresses, an agent controlling root pathogens and a soil
improver acting as an anti-erosion agent, mycorrhiza possesses great potential in
sustainable or organic agriculture. Moreover, it appears that its contribution to
carbon sequestration is substantial; mycorrhizal fungi can be an important soil
carbon sink and often constitute 20–30% of total soil microbial biomass (Leake
et al. 2004). Under conditions of continuously increasing ambient CO2 concen-
trations, AM fungi are expected to increase their role. Based on meta-analysis,
global standing stocks of mycorrhizal fungi may increase substantially by up to
50% under elevated CO2 (Treseder 2004).
Phosphorus (P) availability in soil is often a limiting factor in plant production,
and the use of chemical fertilizers (e.g., manufactured water-soluble superphosphates)
10 Benefits of Arbuscular Mycorrhizal Fungi to Sustainable Crop Production 207
has played a significant role in meeting the growing population demands for foods.
However, the conventional agricultural practices based on the application of agro-
chemicals (chemical fertilizers and pesticides) cannot sustain the production base as
a healthy plant–soil system for very long (Khan et al. 2007). Also, consistently
increasing prices of chemical fertilizers warrants the implementation of sustainable
approaches. The new approaches to farming, referred to as sustainable agriculture,
require agricultural practices that maintain a long-term ecological balance in the
soil ecosystem, including the sustainable management of soil microbes. Under low
P or P-deficient conditions, interaction of plants with rhizospheric microorganisms
either improves the uptake of the available P or renders unavailable P sources that
would otherwise be accessible to the plant. Many lines of scientific evidence
highlight the importance of a targeted and sustainable microbial management of
soil resources. Management of sustainable agricultural systems needs to develop
technologies and practices that do not have adverse effects on environmental
variables. Additionally, these technologies and practices need to be accessible to
farmers in an effective manner that could lead to enhanced food productivity (Pretty
2008). Mycorrhizal biotechnology meets the demands of sustainable agriculture,
and in addition, it often improves food crop quality. However, efficient manage-
ment of biological soil resources in sustainable cropping systems remains the
largest challenge because of the complexity of its roles and interactions with
other parts of cropping systems. Special attention is paid to highlight the effects
of mycorrhiza on crop productivity and the principles underlying crop responsive-
ness to AM, including the occurrence and importance of AM in agro-ecosystems
depending on environmental factors and management practices. Also, the specifics
of AM fungi in horticultural production and the advantages, prospects, and draw-
backs of mycorrhiza-friendly management in sustainable plant–soil systems is
reviewed and discussed.
10.2 Principles of Crop Responsiveness to Mycorrhiza
Many important crops are mycotrophic, or responsive to mycorrhiza, except species

from the non-mycotrophic families Brassicaceae, Chenopodiaceae, and Amar-
anthaceae, which are never or rarely mycorrhized (Gerdemann 1968). Mycotrophic
agricultural and horticultural crops usually form symbioses with AM fungi, and
there are reports that approximately 160 zygomycetous fungal taxa of the order
Glomales (Glomeromycota) form mycorrhizal symbioses (Schüssler et al. 2001).
A number of AM fungi, including the most common ones Glomus intraradices/
Glomus fasciculatum group and Glomus mosseae, have a global distribution occur-
ing in both natural and anthropogenic (disturbed) habitats (Opik et al. 2006).
Morphological structures of mycorrhizal fungi and of entire symbiotic system
with plant roots determine the symbiotic effects and formulations of mycorrhizal
inocula.
In general, colonization with AM fungi result in improvements in plant fitness

and nutrition (Smith and Read 1997). A network of extraradical mycorrhizal
hyphae facilitate nutrient acquisition and transport many ions to roots, particularly
less mobile ions such as P, N, K, S, Ca, Zn, etc. In addition, AM fungi may enhance
the reabsorption of nutrients lost through root exudation (Hamel 2004). The contri-
bution of AM fungi to soil fertility has been partly attributed to production of
glomalin resulting in an accumulation of organic matter and protection against
erosion via forming water stable soil aggregates (Rillig 2004a). The application of
AM fungi facilitates the growth of plants by enhancing seedling growth and rooting
of cuttings, reducing phosphate and nitrate requirements, increasing survival rate
and development of plantlets, increasing resistance to abiotic stresses, increasing
flowering and fruiting, and by increasing crop uniformity (Azcon-Aguilar and
Barea 1997). In addition to the nutritional effects, AM fungi induce an array of
biochemical and molecular responses in host plants. For example, AM fungi have
shown an increased production of many allelochemicals (soluble and cell wall-
bound phenolics, terpenes, alkaloids, essential oils, and other secondary com-
pounds), and have also included up regulated expression of genes (e.g. phenylala-
nine ammonia-lyase, chalcone synthase, and chitinase coding genes) involved in
plant defences (Bi et al. 2007; Yao et al. 2007). Furthermore, AM fungi often
increase resistance or decrease susceptibility of plants to soil-borne pathogens
(Dassi et al. 1998; Brimner and Boland 2003; Whipps 2004; Dalpe 2005; Franco
et al. 2008). However, mycorrhizal-induced defenses and increased systemic resis-
tance in host plants are still not fully understood (Toussaint 2007).
Generally, the mycorrhizal fungi can be regarded as efficient biofertilizers for
sustainable agriculture (Piotrowski and Rillig 2008). In this regard, several aspects
of their interaction with target crops and the environment should, however, be taken
into account. Chief among them is the responsiveness of plants to mycorrhiza (i.e.,
the dependence of plant upon mycorrhiza), and the effectiveness of mycorrhizal
fungi species and their mixtures. These are terms commonly used in mycorrhiza
research with a variety of meanings, which have evolved during the last few
decades. Responsiveness to mycorrhiza refers to the dependency of plants upon
mycorrhiza, a measure of mycorrhizal fungus effectiveness (Janos 2007), and is
represented by the difference in growth between plants with and without mycorrhi-
za at any designated level of P availability. The difference in performance between
AM-colonized and non-colonized plants can be measured as a difference in bio-
mass accumulation, improved morphological parameters, yield, etc. Mycorrhiza
dependence denotes the inability of plants to grow or survive without mycorrhizas
and without leading to any increase in soil fertility. It can be assessed by examining
the effects of a range of soil nutrient contents (especially available phosphorus) on
the performance of plants lacking mycorrhizas (Janos 2007). Mycorrhiza depen-
dence can be calculated as the level of nutrient availability below which noncolo-
nized plants either cease to grow or fall below a given performance threshold. By
extension, variation in responsiveness can be partitioned into dependence and
nondependence components (Sawers et al. 2008). Dependence variation relates to
plant performance under a given set of abiotic conditions, and nondependence
variation describes differences in the interaction between plant and the fungus. As
such, nondependence differences include variation in the ability of lines to establish
colonization, variation in the efficiency of nutrient uptake, and variation in the
regulation of nutrient exchange between the fungus and host (Sawers et al. 2008).
The degree of mycotrophy depends to a greater extent on the plant genotype and
on environmental conditions (Hamel 1996). Many studies have investigated my-
corrhizal responsiveness variation in existing crop varieties, and it is apparent that
selection and breeding programs have often selected against AM associations, as
the programs are mainly aimed at maximum plant performance in high-input
production agro-ecosystems. For example, when comparing the performance of
wheat (Triticum aestivum) varieties developed before and after 1900, varieties
developed before 1900 were more responsive to AM colonization than those
developed later (Hetrick et al. 1992). The principle of this phenomenon lies in
selection for increased ability of modern lines to take up phosphate without AM
fungi under high phosphorus availability in soils (Sawers et al. 2008).
More insight into the complexity of plant responsiveness to AM brought recent
advances in the molecular basis of AM–plant interactions including P uptake
by mycorrhized plants (Paszkowski 2006). Plants use two distinct P uptake path-
ways: one acquiring P directly from the soil via P membrane transporters located in
the outer membrane of the rhizodermis and root hairs, and the second being active
in colonized plants when P is uptaken by the extraradical hyphal network and
delivered to arbuscules. In the arbuscules, it is absorbed by plant phosphate
transporters in the periarbuscular membrane (Sawers et al. 2008). In mycorrhized
plants, a switch from one pattern of gene expression to another corresponding to
changes in P uptake occurs. This process is not a simple superimposition of
additional gene expression on the noncolonized state (Sawers et al. 2008). More-
over, although cytological and physiological features of AM fungi seem to be
conserved, the molecular components may differ significantly between distantly
related plant species, as has been shown for P transporter genes in rice (Oryza
sativa) (Paszkowski et al. 2002). Sawers et al. (2008), however, imply that the
genetic determinants affecting the performance of AM-colonized and noncolonized
plants vary independently.
Mycorrhizal development increases the availability of nutrients other than
P. Nitrogen serves as a good example because of the ability of extraradical
mycelium of AM fungi to access small soil pores in the event of low nutrient status
or immobile nutrients such as NH4+ (Drew et al. 2004). Progress has been made
in exploring metabolic pathways of N uptake and transport by AM fungi
(Govindarajulu et al. 2005). For AM fungi, NH4+ is the most likely form of N
uptaken though the N uptake mechanisms remain largely unknown (Jackson et al.
2008). Nitrogen is synthesized and stored in the extraradical mycelium in the form
of arginine and is transported to the intraradical mycelium in host cells following its
generation from arginine breakdown (Jin et al. 2005). The presence of mycorrhiza
can affect the uptake of nitrogen depending on the type of fertilizer supplied and the
ratio of ammonium/nitrate in it. For example, corn (Zea mays) plants colonized by
Glomus aggregatum take up ten times more N from a 15NH4+ pathway than from a
15
NO3 pathway (Tanaka and Yano 2005).
10.3 Mycorrhiza in Agro-ecosystems
10.3.1 Occurrence
Mycorrhizas occur naturally in agro-ecosystems but their abundance is usually

lower compared to natural, nonmanaged ecosystems (Opik et al. 2006). The level
of AM fungi in soils and their efficacy decreases with soil degradation, pollution or
overfertilization. Also, high soil P fertility and/or fertilization, the use of systemic
fungicides, and the use of nonmycotrophic or low mycotrophic crops in rotation
have an adverse effect on mycorrhizal symbiosis (Plenchette et al. 2005). General-
ly, soil AM fungi are more abundant in low input cropping systems with low-to-
medium soil P content usually having less than 50 mg g 1 soil. In agro-ecosystems,
the inoculation effects of mycorrhizal fungi increases with lower availability of
essential nutrients and/or water, or where other environmental constraints are in
force. The occurrence of mycorrhizal fungi in a particular agro-ecosystem is greatly
dependent on the environmental conditions, management history, and current
management practices. Agricultural practices can directly or indirectly influence
mycorrhizal biodiversity. For example, the lowest AM species abundance has been
found in arable fields (Helgason et al. 1998; Daniell et al. 2001) and metal-
contaminated soils (Whitfield et al. 2004). Relatively low species richness (about
five fungal taxa per plant species) has been reported in habitats that remain under
anthropogenic influence (Opik et al. 2006). By contrast, taxon-rich fungal commu-
nities (over 20 taxa) were detected in different natural ecosystems, such as a boreal
herb-rich coniferous forest (Opik et al. 2008). Recent evidence suggests that the
richness of AM fungal taxa in soil is in a complex relationship with the management
history of a particular site and its production scheme (Hijri et al. 2006). For
example, conventional high-input agriculture with long-term maize monoculture
demonstrated low AM fungal diversity, while substantially higher diversity of AM
fungi was found in a field trial where a 7-year crop rotation was performed under
lower levels of inorganic fertilizer input and chemical pest control (Hijri et al.
2006). Sustainable agricultural practices including crop rotations, reduced or no-
tillage practices, and organic farming practices increase total microbial biomass
and caused a shift in the soil microbial community structure towards a more fungal-
dominated community (Six et al. 2006). It becomes apparent that low-input
agriculture involving an AM-friendly crop rotation may provide better conditions to
preserve AM fungi diversity by preventing the selection for the few AM fungi
species tolerating high nutrient levels.
Changes in the sequence of crops grown on agricultural land – crop rotation – are
known to enhance the yield of grain (e.g., wheat) crops by 20% or more (Kirkegaard
et al. 2008). Some of the principal mechanisms responsible for the benefits of crop
rotation include (1) effects on disease control, and (2) improved nitrogen nutrition
and water supply, besides affecting changes in rhizosphere biology and allelopathy
effects or soil structure (Kirkegaard et al. 2008). To successfully integrate mycor-
rhiza in cropping systems, different factors of production systems and management
practices have to be considered. However, the detailed knowledge of mycorrhizal
conditions in sustainable agro-ecosystems is scarce (Rillig 2004b). To improve it
further, efficient and fast quality indicators of AM fungi populations in the field,
including relative field mycorrhizal dependency, soil mycorrhizal infectivity, and
mycorrhizal fungi development on plants, need to be developed (Plenchette et al.
2005; Vosátka and Albrechtová 2008).
10.3.2 Mycorrhiza-Friendly Management in Sustainable

Plant–Soil Production Systems
In order to exploit beneficial effects of AM fungi in sustainable agricultural plant–

soil production systems, the appropriate management practices have to be applied.
These include the design of suitable crop rotation in cropping systems, appropriate
tillage practices, the introduction of multi-microbial inoculants, and the reduced use
of agrochemicals, including pesticides. The appropriate management of mycorrhizal
symbiosis could enable a reduction in the use of chemical fertilizers and water
supply (Verma and Arya 1998; Sharma and Adholeya 2004; Hart and Trevors 2005;
Subramanian et al. 2006; Wu and Xia 2006). In this context, many reports suggest a
substantial increase in soil fertility and, consequently, crop productivity following
AM inoculation in low-to medium input cropping systems (Harrier and Watson
2003; Johansson et al. 2004; Hart and Trevors 2005; Gosling et al. 2006; Liebig et al.
2006; Perner et al. 2006). Practical management of AM fungal populations and AM
symbiosis in agricultural soils and substrates can be of two types: (1) the manipula-
tion of indigenous AM fungi through selected agronomical practices, or (2) artificial
crop inoculation with AM inoculum (Sieverding 1991; Gianinazzi and Vosátka
2004; Vosátka and Albrechtová 2008). From an agronomic practice point of
view, the most important factors in the management of indigenous AM fungi popula-
tions are fertilization, its intensity and method of application, pesticide management,
tillage practices, and cropping system (Sieverding 1991). A better understanding of
the impacts of agronomic practices on soil microorganisms and the dissemination of
this information to end-users will ensure an opportunity for the utilization of the AM
fungi that could lead to enhanced sustainable agricultural production.
One of the major priority management issues in sustainable systems is how to
maintain nutrient levels (particularly P in the soluble form) compatible for AM
development. A significant positive effect of AM inoculation is reported in low or
P-deficient soils. This is particularly obvious when slow release biofertilizers are
used. In contrast, highly available soil P often limits AM colonization.
For example, Hao et al. (2008) showed a negative effect of AM application on

growth and P uptake by maize plants grown in soil rich in P compared to those
having low P supply. When P availability is relatively high with simultaneous
low N supply, AM fungi did not promote plant N acquisition suggesting that they
can act as a carbon drain to plants (Reynolds et al. 2005; Nogueira and Cardoso
2006). Even when P availability is low and AM colonization levels are high, as
may occur in organic and biodynamic agricultural systems, AM fungi may not
always facilitate plant growth for reasons not yet fully explained (Ryan and
Graham 2002).
Irrigation does not belong to the major factors affecting development and
efficacy of AM fungi. Mycorrhiza can function under irrigated-to-dry conditions
while frequent overlogging can be inhibitory to AM symbionts, since most AM
fungi seem to be rather resistant to drought stress (Auge 2001). The application of
agrochemicals to soils in order to attain optimum yields severely affects the
mycorrhizal symbiosis (Kjoller and Rosendahl 2000; Schweiger et al. 2001).
Such effects could be neutral, as reported for the impact of mefenoxam or fludiox-
onil on soybean (Glycine max) (Murillo-Williams and Pedersen 2008), or inhibitory,
as observed with certain fungicides, herbicides, or insecticides (Thingstrup et al.
2000; Pimienta-Barrios et al. 2003). Another factor affecting the colonization of
AM fungi are the tillage practices applied in agriculture systems. Tillage interferes
with AM fungi abundance and lowers the AM fungal diversity (Jansa et al. 2002,
2003). Therefore, such practices need to be considered while designing sustainable
farming systems (Gosling et al. 2006). To consolidate this hypothesis, different
tillage treatments including moldboard, shred-bedding, subsoil-bedding (Alguacil
et al. 2008), plowing, or chiseling (Jansa et al. 2003) have been investigated using
maize as a test crop. Although no differences in the effects of tillage treatments on
AM fungi diversity were observed, yet a moderate change in mycorrhizal diversity
was reported. Such variations in mycorrhizal diversity could be due to (1) the
differences in tolerance to the tillage-induced disruption of the hyphae among the
different AM fungal species, (2) changes in nutrient content of the soil, (3) changes
in microbial activity, and (4) changes in weed populations in response to soil tillage.
These findings therefore suggest that the frequent disturbances caused by tillage
practices should be avoided. Moreover, conservation tillage in combination with
high mycorrhization seems to be a strategy to reduce root pathogens, as was shown
for maize (Franco et al. 2008).
Crop rotation has proved to be an efficient tool for AM fungi management (Gosling
et al. 2006), and adopting an appropriate crop sequence is an important part
in developing sustainable production systems in many areas of the world (Kirkegaard
et al. 2008). For example, the use of nonmycotrophic or low mycotrophic crops in crop
sequences can lead to a substantial decrease or even the disappearance of indigenous
mycorrhizal populations (Harrier and Watson 2003; Plenchette et al. 2005).
The involvement of intercropping plants producing nonfavorable root exudates to
AM fungi development can also have negative effects on the maintenance of indige-
nous AM fungal populations. This phenomenon was observed for some cruciferous
plants that are inserted as cover crops between two cash crops to improve soil nitrate
fertility (Grodzinsky 1992). The consequences of practices using cruciferous nitrate

trapping plants, such as mustard (Brassica campestris), on mycorrhizal fungi survival
have not yet been fully considered (Plenchette et al. 2005). The allelopathic properties
of cereal crops can be related to low responsiveness to AM fungi or even carbon drain
effects in different crop rotation systems and have to be considered. The most
important grain crops like wheat, rice, maize, and sorghum (Sorghum bicolour) may
exhibit allelopathy and show variable response to mycorrhizal inoculation under
different soil environments (Javaid 2008).
An appropriate design of crop rotation involving the use of AM fungi may result
in improved mineral nutrition. The proper management of cover crops inducing
enhanced AM fungi colonization can increase the availability of soil P pool (both
organic and inorganic) by stimulating microbial activity and release of root exu-
dates (Nelson and Janke 2007). For example, legumes fixing biologically atmo-
spheric nitrogen are often supposed to be capable of bringing energy savings when
used as a cover crop. For maize production, it has been shown that under P-deficient
conditions, legumes supplied with phosphate rock increase P acquisition by a
subsequent maize crop compared to direct application of phosphate rock to maize
(Pypers et al. 2007). The results of this trial suggested that the legume in the rotation
system had other positive effects, possibly soil-microbial effects enhancing maize
growth and production. Nevertheless, caution should be exercised when simulta-
neous application of intercropping legumes and mycorrhizal inoculation is prac-
tised. Arbuscular mycorrhizas can alter competitive interactions between plants that
markedly differ in their dependence upon mycorrhizas, and also during intra- and
inter-specific competition between similarly dependent plant species (Schroeder-
Moreno and Janos 2008). Yao et al. (2008), while using AM fungi in a citrus–
leguminous Stylosanthes gracilis intercropping system, reported negative growth
effects of citrus due to the increased nutrient-competing capacity of S. gracilis and,
hence, suggested that AM fungi is not a potential biofertilizer candidate in a citrus–
S. gracilis intercropping system. However, the study of Brehmer et al. (2008)
concludes that legumes grown as bioenergy crops do not present energy savings,
and thus do not contribute to sustainability. The authors state that the benefits of
nitrogen fixation by legumes should be carefully assessed and best utilized within
the emerging sector of nonfood applications. The influence of AM fungi on soil
processes is complex and difficult to define, in part because it varies with plant and
fungal genotypes as well as with environmental conditions (Hamel 2004). Biocon-
trol can be another benefit of suitable crop rotation. Integrating appropriate break
crops into the farming system should contribute to disease control. Break crops
impede the life cycle of crop-specific pathogens by growing a nonhost crop, and
lead to better nutrient, water, and soil management (Kirkegaard et al. 2008). From a
practical point of view, the adoption of sustainable agricultural practices may not
yield desirable effects immediately, and hence numerous field trials in conventional
agriculture have turned to organic systems. Due to various reasons, the natural
populations of AM fungi in soils can be greatly reduced (Harrier and Watson 2003;
Plenchette et al. 2005; Gosling et al. 2006).
10.3.3 Specifics of Mycorrhiza Applications in Horticultural

Crop Production
High responsiveness to mycorrhiza is common for the majority of horticultural

crops (Plenchette et al. 1983; Azcon-Aguilar and Barea 1997). In this context, the
mycorrhizal biotechnology, i.e., introduction and/or maintenance and management
of mycorrhizal fungi populations in the cultivation system, should become an
important component of horticultural production (Vosátka and Albrechtová
2008). In floriculture, mycorrhizal application has shown a substantial increase in
the yield properties, such as aboveground biomass (Sramek et al. 2000), number of
flowers per plant (up to 24%) for marigold (Calendula officinalis) plants (Flores
et al. 2007), flower diameter, or shortening the period to flowering. The degree of
crop mycorrhizal responsiveness plays an important role, particularly when culti-
vation conditions involve either soil-less or fumigated substrates where indigenous
mycorrhizal fungal populations are either lacking or greatly reduced.
In high-value greenhouse crop production in horticulture, the introduction of
mycorrhiza can be important in soil-less substrates lacking microorganisms and
during the production of micropropagated plants (Vosátka and Albrechtová 2008).
For example, the AM fungi are not usually present in peat, while some ericoid and
ectomycorrhizal fungi are very common in peat substrates (Perner et al. 2006). Peat
based soil-less substrate is usually enriched with nutrients, and when excessive
P-enrichment of substrates is used, it can suppress the germination of endomycor-
rhizal spores. A mycorrhizal-friendly peat amendment can have a high N content
and low P content promoting spore growth of Gigaspora margarita Becker and Hall
(Ma et al. 2006). In horticultural crop systems, plants are usually transplanted after
the first propagation stage, and the conditions found in the transplanting substrate are
crucial for plant survival. Only a few reports are available on the use of mycorrhizal
inoculation when plants are grown in soil-less hydroponic culture systems as
described for the production of axenic arbuscular mycorrhizae (MacDonald 1981)
or nutrient film technique (NFT) in hydroponic culture systems (Mathew and
Johari 1988). George and Lee (2005) suggested that a modified NFT system might
be a useful way to mass-produce mycorrhizal crops and inoculum for commercial
horticultural purposes.
Mycorrhizal inoculation can be successfully used in horticulture for tissue
cultured plants. Micropropagation is a well-established and widely used technology
for propagation of many vegetables and spices (Vestberg and Estaun 1994). Plants
propagated under in vitro conditions can suffer from an initial lack of beneficial soil
microorganisms while they can greatly benefit from inoculation when transferred to
post vitro unsterile conditions. To alleviate transplantation shock and to increase
plant survival after transplantation, successful plant acclimatization has to be
achieved. The most crucial phase of acclimatization is the development of a func-
tional root system that mediates effective nutrient acquisition and water supply.
There are three stages during which mycorrhizal inoculation could be included in the
micropropagation procedures: (1) rooting phase, (2) at the beginning of acclimatization
(weaning) phase, and (3) after the acclimatization phase but before starting the post-
acclimatization period under greenhouse conditions (Azcon-Aguilar and Barea
1997; Taylor and Harrier 2003).
Studies on the inoculation of micropropagated plants again underlined the neces-
sity of selecting an efficient combination of mycorrhizal fungi and host plant
genotype for a particular substrate and cultivation conditions (Vestberg et al.
2002). As was documented in the case of red raspberry (Rubus idaeus) inoculated
with Gigaspora sp. (Taylor and Harrier 2000), the whole range of plant growth
responses from enhancement to growth depression can occur. Current possibilities of
using consortia of rhizotrophic microorganisms for the inoculation of micropropa-
gated plants are under investigation, and could lead to the development of composite
inoculants of mycorrhizal fungi and nitrogen-fixing and/or phosphate-solubilizing
bacteria (Vosátka et al. 2000; Gryndler et al. 2002; Vestberg et al. 2004).
10.4 Drawbacks and Potentials of Mycorrhizal

Applications in Sustainable Agriculture
During the last decade, mycorrhizal technology reached a more developed stage by
becoming an industry supported by extensive research and commercial applications
that emphasized ecological aspects of the use of mycorrhiza (Gianinazzi and
Vosátka 2004; Vosátka et al. 2008). Since large-scale inoculations with AM fungi
have become a reality, the drawbacks of these applications should be carefully
considered with regard to their profitability.
An important question for using AM fungi in sustainable crop production
systems is whether generic or tuned products should be used. One of the drawbacks
of using mycorrhizal inoculants in field is that there are already indigenous popula-
tions of AM fungi in soils. Such indigenous fungi can be highly competitive in
securing their colonization niche from the introduced fungi. Even though intro-
duced strains can be less efficient than indigenous ones (Schwartz et al. 2006), in
some cases the adaptation of the native fungi has shown a competitive advantage
over the introduced strains (Batkhuugyin et al. 2000; Oliveira et al. 2005). This is
often more pronounced in degraded ecosystems with extreme soil properties where
native populations of AM fungi can perform better in the environment of their
origin compared to other nonnative symbionts in degraded soils (Batkhuugyin et al.
2000). Furthermore, there can be significant differences in performance even
among different geographical isolates that belong to the same fungal species. It is
possible to isolate native mycorrhizal strains for a particular ecosystem, to produce
inoculum, and to re-introduce native strains in the field on a large scale. Some
commercial companies are applying inoculum tuning to target conditions where
native mycorrhizal populations have been reduced (Gianinazzi and Vosátka 2004;
Vosátka and Albrechtová 2008).
For the successful introduction of inocula into the field, numerous ecological
factors have to be considered. These include soil properties, level of fertilizer input,
and the degree and potential of existing fungal population (Vosátka and Dodd 2002).
To realize all the potentials of mycorrhizal management and applications, it is
absolutely vital to conduct reference field trials prior to large-scale applications,
which are of great importance for properly tuning the best combinations of mycorrhizal
fungal inoculum for target conditions. And, hence, a careful selection of symbionts
intended for use should be employed prior to large-scale production and application.
By tuning the appropriate inoculants to the target cultivation system, it is possible to
achieve economic feasibility of mycorrhizal technology (Vosátka and Dodd 2002).
Molecular identification of different isolates in a field to reliably distinguish
native and introduced populations is needed to evaluate ecological and economic
consequences of artificial inoculation into field conditions. Although, within spe-
cies, genetic differences among AM fungi have been shown to differentially affect
plant growth, very little is known about the degree of genetic diversity in AM
fungal populations (Croll et al. 2008). It is apparent that differences in crop
responsiveness to various mycorrhizal isolates can be based on genetic variability
in AM fungal populations that affects host–plant fitness. The AM fungi are asexu-
ally reproducing organisms that show coexistence of a population of many genomes
(Hijri and Sanders 2005). This implies a high genetic variation of within-AM fungi
populations, though the effects of within-AM fungi species or within-population
variation on plant growth have received less attention (Koch et al. 2006). Recently,
success has been achieved in molecular identification of different isolates of
G. margarita (Yokoyama et al. 2005) and G. intraradices using newly developed
simple sequence repeat, nuclear gene intron, and mitochondrial ribosomal gene
intron markers (Croll et al. 2008). Advances in molecular identification further
support the importance of adjusting the tuning of introduced commercial inocula to
a specific target combination of a crop production system.
Among different types of mycorrhiza, AM fungi exhibit relatively low specific-
ity in host–symbiont formation, which is advantageous for natural spreading of
arbuscular mycorrhiza in a plant community as well as for large-scale commercial
use of mycorrhizal inoculants in agriculture in different agro-ecosystems. However,
the last decade of AM fungi research has revealed far more selectivity of AM fungi–
plant associations, and a greater dependence of resulting functions on particular
host/fungus combinations than previously suggested (Piotrowski and Rillig 2008).
It has been proposed that genetic diversity, either within or among AM fungal
species, rather than the species richness of AM fungi, could play a pivotal role in the
diversity, functioning and overall performance of plants (Sanders 2004). Host
plants in a field could provide a heterogeneous environment favoring certain
genotypes. Such preferences, in turn, may partly explain the patterns of genetic
diversity within populations. The effects of specific host/AM fungi combinations
range from beneficial to parasitic; hence, the next step in AM fungi application will
entail an effort to employ advantageous combinations. The tuning of commercial
inoculum is regarded not as management practices but as necessity to achieve
optimum benefits from mycorrhizal association (Vosátka and Albrechtová 2008).
In crop breeding programs, progress has been made in the isolation and func-
tional analyses of genes controlling yield and tolerance to abiotic stresses (Takeda
and Matsuoka 2008), which have to be considered with respect to the type of
environment for which a crop cultivar is targeted. Crop breeding targeted to drought
tolerance focuses on physiological yield drivers such as water uptake, water use
efficiency, and harvest index (Reynolds and Tuberosa 2008). The AM fungi can
directly mediate improved drought tolerance via changes of plant physiological
traits, such as higher leaf water potential, transpiration rates, stomatal conductance,
and relative water content, and alleviate drought stress conditions, as reported for
citrus (Wu and Xia 2006), chillies (Capsicum annuum L.) (Mena-Violante et al.
2006), and tomato (Lycopersicon lycopersicum) plants (Subramanian et al. 2006).
Indirect effects of mycorrhiza on crop drought resistance can be mediated by the
introduction of glomalin into soil, quantified operationally in soils as glomalin-
related soil protein, which is able to aggregate soil particles and increase water
stable aggregates (Rillig 2004a) having a positive effect on soil moisture. In
conventional agriculture, the breeding programs have not considered mycorrhiza-
oriented breeding traits, and crops are selected to favor solely nutrient acquisition in
high input systems without considering the role of mycorrhiza in nutrient manage-
ment of soils (Sawers et al. 2008). This is a major drawback to mycorrhiza
application, though it can be circumvented by the proper selection of crop geno-
type. Thus, there is an urgent need to manipulate plant genetic factors that impact
the cost–benefit balance of mycorrhizal applications, or more specifically, the rate
at which resources are diverted to the fungus in return for a given level of enhanced
performance or reduction in production costs.
One of the main potentials of mycorrhizas for sustainable agriculture is that they
induce plant physiological changes that affect the quality and safety of food crops,
including a higher production of antioxidants or essential oils, or reduced uptake of
pollutants such as heavy metals to plant tissues (Toussaint 2007; Toussaint et al.
2007; Vosátka and Albrechtová 2008). Although no clear mechanism other than an
improvement in the nutritional status (mainly P) has been proposed (Toussaint
2007), yet the beneficial fungus–plant interactions has shown enhancement in
productivity of crops by synthesizing an increased level of active compounds (Rai
et al. 2001). For example, the suitable selection of host plant–fungus genotype led to
an altered accumulation of essential oil levels in AM-colonized plants of Mentha
arvensis (Freitas et al. 2004) and sweet basil Ocimum basilicum L. (Copetta et al.
2006, 2007; Toussaint et al. 2007). Thus, the use of mycorrhiza is being extended to
herbal plants in order to get maximum benefits by the steadily increasing herbal
medicine industry (Ernst 2000). However, to achieve good outcomes from mycor-
rhizal inoculations in crop production, it is necessary to incorporate inoculum in the
production systems in the most effective manner. The mycorrhiza should be intro-
duced in the plant propagation as soon as possible so that a reliable and effective
mycorrhization of crop plants is achieved. Unfortunately, there is no universal
method of inocula application, while some planting and plant production systems
require specific ways of application. Mycorrhizal inocula, however, could be applied
in several ways. For example, they can be applied in dry formulation form. For this, a
layer of inoculum is placed below the seeds or can be mixed with the growing
substrate for containerized plants. Either of these methods leads to its spreading into
the planting hole. When the mycorrhizal product is used as a gel formulation, it can
be easily applied either to the planting hole or the bare roots during transplanting.
Additionally, the root balls can be dipped into a gel product to cover their surface and
ensure mycorrhizal colonization of newly emerging roots. For large-scale applica-
tions under field environments, mechanical devices are needed (mixing tanks for
application in substrates or sowing machines for dispersing granulated inocula).
Currently, there is increasing demand for the incorporation of bioproducts from
organic agriculture and new alternatives that enhance sustainability in agriculture.
There are obviously certain problems that need to be seriously addressed. For exam-
ple, it is necessary to educate farmers about the benefits of mycorrhizal inoculation and
organic farming so that the reliance on agrochemicals is minimized. Also, the ques-
tionable capacity of organic agriculture to provide sufficient yield should be addressed
(Connor 2008). Some analyses have estimated the capability of organic agriculture
to feed three to four billion people, well below the present and projected world
population (Smil 2001). Recently, a new concept of eco-agriculture was proposed
for achieving productivity in perpetuity (Kesavan and Swaminathan 2008). That
includes developing farming/cropping systems that improve water use efficiency
and minimize risks of water pollution, contamination, and eutrophication thereby
converting the degraded/desertified soils to restored land use for enhancing biodiver-
sity and improving the environment (Lal 2008). The efficient management of mycor-
rhizas and soil biota, in general, forms an important component of this technology.
In developing countries, many farmers are using less agrochemicals and more
biocontrol agents like species of Trichoderma, Bacillus, and also mycorrhizal
products as an alternative to agrochemicals (Kesavan and Swaminathan 2008).
Apparently such bioagents are more often used due to economic reasons, since
agrochemicals are too expensive, rather than from mere interest in sustainable crop
production. Official marketing of mycorrhiza as a biocontrol agent is currently
unrealistic, because the registration of mycorrhizal products with biocontrol activity is
complicated and too costly for inoculant producers (Whipps 2004). Mycorrhizal
products are usually sold as growth promoters for two simple reasons: (1) a large-
scale screening process would be needed to specify strains acting as biocontrol
agents for specific plants and environmental conditions, and (2) biocontrol strains
would have to be formally registered (Whipps 2004). Despite all these facts, the
trend of using natural resources like AM fungi, seems to be increasing and could
become prevalent due to increasing demands placed on safety and quality of foods
across the globe. Another driving force for stronger focus on sustainable agriculture
in developing countries is indeed continuous depletion of soil and water resources.
10.5 Conclusion
In properly managed agriculture systems, mycorrhiza can act as a biofertilizer,

biocontrol agent and soil improver. When accurate inoculum tuning is accomplished
and used for formulation of mycorrhizal products, the large-scale application of
such fungi is not a threat either to the biodiversity or to soil fertility. Genotype-
related differences in host plant–AM fungus combinations can be discerned which
could be attributed to the differences in the balance between P uptake by the fungal
pathway and direct uptake via the roots of plants. Furthermore, maintaining sustain-
ability in agriculture requires attention on how new breeding programs could help to
attain maximum benefits following mycorrhizal symbiosis. Recent advances in
molecular breeding approaches are helping to optimize the use of AM–fungal
symbioses for improvement of crop productivity. However, there is also a need to
produce efficient varieties which are able to use AM fungi in more effective ways for
nutrient acquisition (Sawers et al. 2008).
Despite its potential and proven ability, microbe management currently plays
only a secondary role in agricultural practices. One of the reasons is that there is a
lack of legislative policy focusing explicitly on soil ecosystems and degradation
processes. And, currently, there is no international policy framework to guide
sustainable soil management, though there are some guidelines related to that
issue in the United Nations Convention to Combat Desertification (Stringer
2008). Another area of concern is the lack of awareness about the benefits of AM
fungi among farmers besides their inconsistent performance under different eco-
logical niches. The application of soil microbial inoculations is further compounded
by poor understanding of ecology, population dynamics, and functionality of AM
fungi over a range of environments (Khan et al. 2007). And, hence, if the microbes
are to be used in a predictable and useful way, focus has to be directed towards
understanding the basic biology of these microbes and their interaction with host
plants (Hart and Trevors 2005). Additionally, in order to expand the use of AM
fungi, inoculum tuning to monitor and predict the functioning of introduced
mycorrhiza in crop production systems able to improve soil fertility and pest
management should be given priority. Despite such problems, the mycorrhiza
industry has developed quickly. If farmers are provided with sufficient training as
to how these organisms are properly applied, the use of AM fungi is likely to
maximize not only the productivity of crops while reducing the use of agrochem-
icals but is also likely to reduce soil pollution.
Acknowledgement The authors acknowledge the funding of the Ministry of Education, Youth and
Sports of the Czech Republic from the projects No. 1P04OE187, the EU project E! 3375 EUROAGRI
+MYCOTAGRIF, the Centre for Bioindication and Revitalization 1M0571, and the Institutional
project AV0Z60050516 of the Institute of Botany, Academy of Sciences of the Czech Republic.
References
Alguacil MM, Lumini E, Roldan A, Salinas-Garcia JR, Bonfante P, Bianciotto V (2008) The
impact of tillage practices on arbuscular mycorrhizal fungal diversity in subtropical crops. Ecol
Appl 18:527–536
Auge RM (2001) Water relations, drought and vesicular-arbuscular mycorrhizal symbiosis.
Azcon-Aguilar C, Barea JM (1997) Applying mycorrhiza biotechnology to horticulture: signifi-

cance and potential. Sci Hortic-Amsterdam 68:1–24
Batkhuugyin E, Rydlova J, Vosátka M (2000) Effectiveness of indigenous and non-indigenous
isolates of arbuscular mycorrhizal fungi in soils from degraded ecosystems and man-made
habitats. Appl Soil Ecol 14:201–211
Bi HH, Song YY, Zeng RS (2007) Biochemical and molecular responses of host plants to
mycorrhizal infection and their roles in plant defence. Allelopathy J 20:15–27
Brehmer B, Struik PC, Sanders J (2008) Using an energetic and exergetic life cycle analysis to
assess the best applications of legumes within a biobased economy. Biomass Bioenerg
32:1175–1186
Brimner TA, Boland GJ (2003) A review of the non-target effects of fungi used to biologically
control plant diseases. Agr Ecosyst Environ 100:3–16
Connor DJ (2008) Organic agriculture cannot feed the world. Field Crop Res 106:187–190
Copetta A, Lingua G, Berta G (2006) Effects of three AM fungi on growth, distribution of
glandular hairs, and essential oil production in Ocimum basilicum L. var. Genovese. Mycor-
rhiza 16:485–494
Copetta A, Lingua G, Bardi L, Masoero G, Berta G (2007) Influence of arbuscular mycorrhizal
fungi on growth and essential oil composition in Ocimum basilicum var. Genovese. Caryologia
60:106–110
Croll D, Wille L, Gamper HA, Mathimaran N, Lammers PJ, Corradi N, Sanders IR (2008) Genetic
diversity and host plant preferences revealed by simple sequence repeat and mitochondrial
markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices. New Phytol
178:672–687
Dalpe Y (2005) Mycorrhizae: a potential tool for plant protection but not a panacea. Phytoprotec-
tion 86:53–59
Daniell TJ, Husband R, Fitter AH, Young JPW (2001) Molecular diversity of arbuscular mycor-
rhizal fungi colonising arable crops. FEMS Microbiol Ecol 36:203–209
Dassi B, Dumas-Gaudot E, Gianinazzi S (1998) Do pathogenesis-related (PR) proteins play a role
in bioprotection of mycorrhizal tomato roots towards Phytophthora parasitica? Physiol Mol
Plant P 52:167–183
Drew EA, Murray RS, Smith SE, Jakobsen I (2004) Beyond the rhizosphere: growth and function of
arbuscular mycorrhizal external hyphae in sands of varying pore sizes. Plant Soil 251:105–114
Ernst E (2000) Herbal medicines: where is the evidence? Growing evidence of effectiveness is
counterbalanced by inadequate regulation. Brit Med J 321:395–396
Flores AC, Luna AAE, Portugal VO (2007) Yield and quality enhancement of marigold flowers by
inoculation with Bacillus subtilis and Glomus fasciculatum. J Sustain Agr 31:21–31
Franco AD, Garcia JRS, Cano IG, Perez NM (2008) Impact of tillage and arbuscular mycorrhiza
inoculation on charcoal rot and yield of maize under semiarid conditions. Revista Fitotecnia
Mexicana 31:257–263
Freitas MSM, Martins MA, Vieira EIJC (2004) Yield and quality of essential oils of Mentha
arvensis in response to inoculation with arbuscular mycorrhizal fungi. Pesqui Agropecu Bras
39:887–894
George E, Lee YJ (2005) Development of a nutrient film technique culture system for arbuscular
mycorrhizal plants. HortScience 40:378–380
Gerdemann JW (1968) Vesicular-arbuscular mycorrhiza and plant growth. Annu Rev Phytopathol
6:397–418
Gianinazzi S, Vosátka M (2004) Inoculum of arbuscular mycorrhizal fungi for production sys-
tems: science meets business. Can J Bot 82:1264–1271
Gosling P, Hodge A, Goodlass G, Bending GD (2006) Arbuscular mycorrhizal fungi and organic
farming. Agr Ecosyst Environ 113:17–35
Govindarajulu M, Pfeffer PE, Jin HR, Abubaker J, Douds DD, Allen JW, Bucking H, Lammers PJ,
Shachar-Hill Y (2005) Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature
435:819–823
Grodzinsky AM (1992) Allelopathic effects of cruciferous plant in crop rotation. In: Rizvi SJH,
Rizvi V (eds) Allelopathy: basic and applied aspects. Chapman & Hall, London
Gryndler M, Vosátka M, Hrselova H, Catska V, Chvatalova I, Jansa J (2002) Effect of dual
inoculation with arbuscular mycorrhizal fungi and bacteria on growth and mineral nutrition of
strawberry. J Plant Nutr 25:1341–1358
Hamel C (1996) Prospects and problems pertaining to the management of arbuscular mycorrhizae
in agriculture. Agr Ecosyst Environ 60:197–210
Hamel C (2004) Impact of arbuscular mycorrhizal fungi on N and P cycling in the root zone. Can
J Soil Sci 84:383–395
Hao LF, Zhang JL, Chen FJ, Christie P, Li XL (2008) Response of two maize inbred lines with
contrasting phosphorus efficiency and root morphology to mycorrhizal colonization at differ-
ent soil phosphorus supply levels. J Plant Nutr 31:1059–1073
Harrier LA, Watson CA (2003) The role of arbuscular mycorrhizal fungi in sustainable cropping
systems. Adv Agron 79:185–225
Hart MM, Trevors JT (2005) Microbe management: application of mycorrhyzal fungi in sustain-
able agriculture. Front Ecol Environ 3:533–539
Helgason T, Daniell TJ, Husband R, Fitter AH, Young JPW (1998) Ploughing up the wood-wide
web? Nature 394:431
Herrick JE (2000) Soil quality an indicator of sustainable land management. Appl Soil Ecol 15:
75–83
Hetrick B, Wilson GWT, Cox TS (1992) Mycorrhizal dependence of modern wheat-varieties,
landraces, and ancestors. Can J Bot 70:2032–2040
Hijri M, Sanders IR (2005) Low gene copy number shows that arbuscular mycorrhizal fungi
inherit genetically different nuclei. Nature 433(7022):160–163
Hijri I, Sykorova Z, Oehl F, Ineichen K, Mäder P, Wiemken A, Redecker D (2006) Communities
of arbuscular mycorrhizal fungi in arable soils are not necessarily low in diversity. Mol Ecol
15:2277–2289
Jackson LE, Burger M, Cavagnaro TR (2008) Roots nitrogen transformations, and ecosystem
services. Annu Rev Plant Biol 59:341–363
Janos DP (2007) Plant responsiveness to mycorrhizas differs from dependence upon mycorrhizas.
Jansa J, Mozafar A, Anken T, Ruh R, Sanders IR, Frossard E (2002) Diversity and structure of
AMF communities as affected by tillage in a temperate soil. Mycorrhiza 12:225–234
Jansa J, Mozafar A, Kuhn G, Anken T, Ruh R, Sanders IR, Frossard E (2003) Soil tillage
affects the community structure of mycorrhizal fungi in maize roots. Ecol Appl 13:1164–
1176
Javaid A (2008) Allelopathy in mycorrhizal symbiosis in the Poaceae family. Allelopathy
J 21:207–217
Jin H, Pfeffer PE, Douds DD, Piotrowski E, Lammers PJ, Shachar-Hill Y (2005) The uptake,
metabolism, transport and transfer of nitrogen in an arbuscular mycorrhizal symbiosis. New
Phytol 168:687–696
Johansson JF, Paul LR, Finlay RD (2004) Microbial interactions in the mycorrhizosphere and their
significance for sustainable agriculture. Fems Microbiol Ecol 48:1–13
Kesavan PC, Swaminathan MS (2008) Strategies and models for agricultural sustainability in
developing Asian countries. Philos T R Soc B 363:877–891
Khan MS, Zaidi A, Wani PA (2007) Role of phosphate-solubilizing microorganisms in sustainable
agriculture – A review. Agron Sustain Dev 27:29–43
Kirkegaard J, Christen O, Krupinsky J, Layzell D (2008) Break crop benefits in temperate wheat
production. Field Crop Res 107:185–195
Kjoller R, Rosendahl S (2000) Effects of fungicides on arbuscular mycorrhizal fungi: differential
responses in alkaline phosphatase activity of external and internal hyphae. Biol Fert Soils
31:361–365
Koch AM, Croll D, Sanders IR (2006) Genetic variability in a population of arbuscular mycor-
rhizal fungi causes variation in plant growth. Ecol Lett 9:103–110
Lal R (2008) Soils and sustainable agriculture. A review. Agron Sustain Dev 28:57–64
Leake JR, Johnson D, Donnelly DP, Muckle GE, Boddy L, Read DJ (2004) Networks of power and
influence: the role of mycorrhizal mycelium in controlling plant communities and agroecosys-
tem functioning. Can J Bot 82:1016–1045
Liebig M, Carpenter-Boggs L, Johnson JMF, Johnson JMF, Wright S, Barbour N (2006) Cropping
system effects on soil biological characteristics in the Great Plains. Renew Agr Food Syst
21:36–48
Ma N, Yokoyama K, Marumoto T (2006) Stimulatory effect of peat on spore germination and
hyphal growth of arbuscular mycorrhizal fungus Gigaspora margarita. Soil Sci Plant Nutr
52:168–176
MacDonald RM (1981) Routine production of axenic vesicular-arbuscular mycorrhizas. New
Phytol 89:87–93
Mathew J, Johari BN (1988) Propagation of vesicular-arbuscular mycorrhizal fungi in moong
(Vigna radiata L) through nutrient film technique (NFT). Curr Sci India 57:156–158
Mena-Violante HG, Ocampo-Jimenez O, Dendooven L, Martinez-Soto G, Gonzalez-Castaneda J,
Davies FT, Olalde-Portugal V (2006) Arbuscular mycorrhizal fungi enhance fruit growth and
quality of chile ancho (Capsicum annuum L. cv San Luis) plants exposed to drought. Mycor-
rhiza 16:261–267
Murillo-Williams A, Pedersen P (2008) Arbuscular mycorrhizal colonization response to three
seed-applied fungicides. Agron J 100:795–800
Nelson NO, Janke RR (2007) Phosphorus sources and management in organic production systems.
Horttechnology 17:442–454
Nogueira MA, Cardoso EJBN (2006) Plant growth and phosphorus uptake in mycorrhizal rangpur
lime seedlings under different levels of phosphorus. Pesqui Agropecu Bras 41:93–99
Oliveira RS, Vosátka M, Dodd JC, Castro PML (2005) Studies on the diversity of arbuscular
mycorrhizal fungi and the efficacy of two native isolates in a highly alkaline anthropogenic
sediment. Mycorrhiza 16:23–31
Opik M, Moora M, Liira J, Zobel M (2006) Composition of root-colonizing arbuscular mycorrhi-
zal fungal communities in different ecosystems around the globe. J Ecol 94:778–790
Opik M, Moora M, Zobel M, Saks U, Wheatley R, Wright F, Daniell T (2008) High diversity of
arbuscular mycorrhizal fungi in a boreal herb-rich coniferous forest. New Phytol 179:
867–876
Paszkowski U (2006) A journey through signaling in arbuscular mycorrhizal symbioses. New
Phytol 172:35–46
Paszkowski U, Kroken S, Roux C, Briggs SP (2002) Rice phosphate transporters include an
evolutionarily divergent gene specifically activated in arbuscular mycorrhizal. P Natl Acad
Sci USA 99:13324–13329
Perner H, Schwarz P, George E (2006) Effect of mycorrhizal inoculation and compost supply on
growth and nutrient uptake of young leek plants grown on peat-based substrates. Hortscience
41:628–632
Pimienta-Barrios E, del Castillo-Aranda MEG, Muñoz-Urias A, Nobel PA (2003) Effects of
benomyl and drought on the mycorrhizal development and daily net CO2 uptake of a wild
platyopuntia in a rocky semi arid environment. Ann Bot 92:239–245
Piotrowski JS, Rillig MC (2008) Succession of arbuscular mycorrhizal fungi: patterns, causes, and
considerations for organic agriculture. Adv Agron 97:111–130
Pirozynski KA, Dalpe Y (1992) The geological history of the Glomaceae with particular reference
to mycorrhizal symbiosis. Symbiosis 7:1–36
Plenchette C, Fortin JA, Furlan V (1983) Growth responses of several plant species to mycorrhizae
in a soil of moderate P- fertility. I. Mycorrhizal dependency under field conditions. Plant Soil
70:199–209
Plenchette C, Clermont-Dauphin C, Meynard JM, Fortin JA (2005) Managing arbuscular mycor-

rhizal fungi in cropping systems. Can J Plant Sci 85:31–40
Pretty J (1995) Regenerating agriculture: policies and practice for sustainability and self-reliance.
Earthscan, London, UK; National Academy Press, Washington, DC, p 320
Pretty J (2008) Agricultural sustainability: concepts, principles and evidence. Philos T R Soc B
363:447–465
Pypers P, Huybrighs M, Diels J, Abaidoo R, Smolders E, Merckx R (2007) Does the enhanced P
acquisition by maize following legumes in a rotation result from improved soil P availability?
Rai M, Acharya D, Singh A, Varma A (2001) Positive growth responses of the medicinal plants
Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field
trial. Mycorrhiza 11:123–128
Reynolds M, Tuberosa R (2008) Translational research impacting on crop productivity in drought-
prone environments. Curr Opin Plant Biol 11:171–179
Reynolds HL, Hartley AE, Vogelsang KM, Bever JD, Schultz PA (2005) Arbuscular mycorrhizal
fungi do not enhance nitrogen acquisition and growth of old-field perennials under low
nitrogen supply in glasshouse culture. New Phytol 167:869–880
Rillig MC (2004a) Arbuscular mycorrhizae, glomalin, and soil aggregation. Can J Soil Sci
84:355–363
Rillig MC (2004b) Arbuscular mycorrhizae and terrestrial ecosystem processes. Ecol Lett 7:
740–754
Ryan MH, Graham JH (2002) Is there a role for arbuscular mycorrhizal fungi in production
agriculture? Plant Soil 244:263–271
Sanders IR (2004) Plant and arbuscular mycorrhizal fungal diversity – are we looking at the
relevant levels of diversity and are we using the right techniques? New Phytol 164:415–418
Sawers RJH, Gutjahr C, Paszkowski U (2008) Cereal mycorrhiza: an ancient symbiosis in modern
agriculture. Trends Plant Sci 13:93–97
Schreiner RP, Mishra RL, Mc Daniel KL, Benthlenfalvay GJ (2003) Mycorrhizal fungi influence
plant and soil functions and interactions. Plant Soil 188:199–209
Schroeder-Moreno MS, Janos DP (2008) Intra- and inter-specific density affects plant growth
responses to arbuscular mycorrhizas. Botany-Botanique 86:1180–1193
Schussler A, Schwarzott D, Walker C (2001) A new fungal phylum, the Glomeromycota:
phylogeny and evolution. Mycol Res 105:1413–1421
Schwartz MW, Hoeksema JD, Gehring CA, Johnson NC, Klironomos JN, Abbott LK, Pringle A
(2006) The promise and the potential consequences of the global transport of mycorrhizal
fungal inoculum. Ecol Lett 9:501–515
Schweiger PF, Spliid NH, Jakobsen I (2001) Fungicide application and phosphorus uptake by
hyphae of arbuscular mycorrhizal fungi into field-grown peas. Soil Biol Biochem 33:
1231–1237
Sharma MP, Adholeya A (2004) Effect of arbuscular mycorrhizal fungi and phosphorus fertiliza-
tion on the post vitro growth and yield of micropropagated strawberry grown in a sandy loam
soil. Can J Bot 82:322–328
Sieverding E (1991) Vesicular-arbuscular mycorrhiza management in tropical agrosystems.
Deutsche Gesellschaft fur Technische Zasammenarbeit (GTZ) GmBH. Eschborn, Germany,
p 371
Six J, Frey SD, Thiet RK, Batten KM (2006) Bacterial and fungal contributions to carbon
sequestration in agroecosystems. Soil Sci Soc Am J 70:555–569
Smil V (2001) Feeding the world: a challenge for the twenty-first century. MIT Press, Cambridge,
MA, p 360
Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic Press, San Diego, p 604
Sramek F, Dubsky M, Vosátka M (2000) Effect of arbuscular mycorrhizal fungi and Trichoderma
harzianum on three species of balcony plants. Plant Prod Sci 46:127–131
Stringer L (2008) Can the UN convention to combat desertification guide sustainable use of the
world’s soils? Front Ecol Environ 6:138–144
Subramanian KS, Santhanakrishnan P, Balasubramanian P (2006) Responses of field grown
tomato plants to arbuscular mycorrhizal fungal colonization under varying intensities of
drought stress. Sci Hortic-Amsterdam 107:245–253
Takeda S, Matsuoka M (2008) Genetic approaches to crop improvement: responding to environ-
mental and population changes. Nat Revs Gen 9:444–457
Tanaka Y, Yano K (2005) Nitrogen delivery to maize via mycorrhizal hyphae depends on the form
of N supplied. Plant Cell Environ. 28:1247–1254
Taylor J, Harrier LA (2000) A comparison of nine species of arbuscular mycorrhizal fungi on the
development and nutrition of micropropagated Rubus idaeus L. cv. Glen Prosen (red raspber-
ry). Plant Soil 225:53–61
Taylor J, Harrier LA (2003) Beneficial infuences of arbuscular mycorrhizal (AM) fungi on the
micropropagation of woody and fruit trees. In: Jain SM, Ishii K (eds) Micropropagation of
woody trees and fruits. Kluwer, Dordrecht, the Netherlands, pp 129–150
Thingstrup I, Kahiluoto H, Jakobsen I (2000) Phosphate transport by hyphae of field communities
of arbuscular mycorrhizal fungi at two levels of P fertilization. Plant Soil 221:181–187
Toussaint JP (2007) Investigating physiological changes in the aerial parts of AM plants: what do
we know and where should we be heading? Mycorrhiza 17:349–353
Toussaint JP, Smith FA, Smith SE (2007) Arbuscular mycorrhizal fungi can induce the production
of phytochemicals in sweet basil irrespective of phosphorus nutrition. Mycorrhiza 17:291–297
Treseder KK (2004) A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and
atmospheric CO2 in field studies. New Phytol 164:347–355
Verma RK, Arya ID (1998) Effect of arbuscular mycorrhizal fungal isolates and organic manure
on growth and mycorrhization of micropropagated Dendrocalamus asper plantlets and on
spore production in their rhizosphere. Mycorrhiza 8:113–116
Vestberg M, Estaun V (1994) Micropropagated plants, on opportunity to positively manage
mycorrhizal activities. In: Giananazzi S, Schuepp H (eds) Impact of arbuscular mycorrhizas
on sustainable agriculture and natural ecosystems. Birkhauser Verlag, Basel, Switzerland, pp
217–225
Vestberg M, Cassells AC, Schubert A, Cordier C, Gianinazzi S (2002) Arbuscular mycorrhizal
fungi and micropropagation of high value crops. In: Giananazzi S, Schuepp H, Barea JM,
Hasselwandter K (eds) Mycorrhizal technology in agriculture. Birkhauser Verlag, Basel,
Switzerland, pp 223–233
Vestberg A, Kukkonen S, Saari K, Parikka P, Huttunen J (2004) Microbial inoculation for
improving the growth and health of micropropagated strawberry. Appl Soil Ecol 27:
243–258
Vosátka M, Albrechtová J (2008) Theoretical aspects and practical uses of mycorrhizal technology
in floriculture and horticulture. In: Teixeira da Silva JA (ed) Floriculture, ornamental and plant
biotechnology: advances and topical issues, vol 5, 1st edn. Global Science Books, Isleworth,
UK, pp 466–479
Vosátka M, Dodd JC (2002) Ecological considerations for successful application of arbuscular
mycorrhizal fungi inoculum. In: Gianinazzi S, Schuepp H, Barea JM, Haselwandter K (eds)
Mycorrhizal technology in agriculture. Birkhauser Verlag, Basel, pp 235–248
Vosátka M, Jansa J, Vohnik M, Gryndler M (2000) Post-vitro mycorrhization and bacterization of
micropropagated strawberry, potato and azalea. Acta Hortic 530:313–324
Vosátka M, Albrechtová J, Patten R (2008) The international market development for mycorrhizal
technology In: Varma A (ed), Chapter 21. Springer, Berlin, pp 419–438
Whipps JM (2004) Prospects and limitations for mycorrhizas in biocontrol of root pathogens. Can
J Bot 82:1198–1227
Whitfield L, Richards AJ, Rimmer DL (2004) Relationships between soil heavy metal concentra-
tion and mycorrhizal colonisation in Thymus polytrichus in northern England. Mycorrhiza
14:55–62
Wu QS, Xia RX (2006) Arbuscular mycorrhizal fungi influence growth, osmotic adjustment and
photosynthesis of citrus under well-watered and water stress conditions. J Plant Physiol
163:417–442
Yao Q, Zhu HH, Zeng RS (2007) Role of phenolic compounds in plant defence: Induced by
arbuscular mycorrhizal fungi. Allelopathy J 20:1–13
Yao Q, Lin FX, Chen JZ, Lei XT, Zhu HH (2008) Responses of citrus seedlings and a leguminous
herb, Stylosanthes gracilis, to arbuscular mycorrhizal fungal inoculation. Proceedings of the
international symposium on citrus and other tropical and subtropical fruit crops 773:63–67
Yokoyama K, Tateishi T, Saito M, Marumoto T (2005) Application of a molecular method for the
identification of a Gigaspora margarita isolate released in a field. Soil Sci Plant Nutr 51:
125–128
Chapter 11
Enhancement of Rhizobia–Legumes Symbioses
and Nitrogen Fixation for Crops Productivity
Improvement
Hamdi Hussein Zahran
Abstract Rhizobia form a very interesting symbiotic relationship with leguminous

plants. This relationship has attracted the attention of biologists all over the world
due to the great impact of legumes for sustaining nutritional demands to humans
and animals. Great efforts have been made to improve the symbiotic N2-fixing
ability and productivity of legumes. The first step to improve legume productivity is
to select effective (N2-fixing) rhizobia to be used as inoculants for the respective
legumes. Recent studies reported about the wild-legume rhizobial ability to estab-
lish successful symbiosis with their original hosts, as well as with legume crops. In
addition to the traditional approaches, modern strategies like genetic and biotech-
nological tools are adopted to unravel several molecular and genetic mechanisms
controlling the rhizobia–legumes symbioses and to enhance N2 fixation and pro-
ductivity of the legumes. Extensive research work should be continued to improve
the inoculation technology using modern approaches, especially in N-poor lands.
Successful symbioses between the bacteria and the legumes are not sustained unless
the effects of environmental stresses such as salinity and drought are modulated.
The majority of arable lands of the globe experience one or more environmental
stresses, therefore stress-tolerant and effective N2-fixing legumes–rhizobia
symbioses will be the only productive systems on these lands.
11.1 Introduction
Cultivation in the dry land of arid climates is increasingly advocated as a strategy

to protect and reverse soil fertility decline, thus sustaining agricultural production
and prevent serious nitrogen (N) deficits in many tropical agro-ecosystems.
H.H. Zahran
Faculty of Science, Beni-Suef University, Beni-Suef, 62511, Egypt
e-mail: hamdi_zahran@hotmail.com
228 H.H. Zahran
Effective management of N in the environment is an essential element of agricultural

sustainability. An essential source of N input into the soil is the biologically-fixed
N2 (Graham 2008), which is used directly by the plant, and so is less susceptible to
volatilization, denitrification, and leaching. About 80% of this biologically-fixed N2
comes from symbioses involving leguminous plants and various rhizobial species.
Worldwide, legumes are grown on approximately 250 mha and fix about 90 million
metric tons (Tg) of N2 per year. N2 fixation-based systems (e.g., legumes and trees)
are the most promising and potentially profitable in extensive agricultural systems
for coping with the N export problems (Zahran 1999, 2001; Graham and Vance
2000). The significance of N2 fixation of rhizobium–legume symbiosis in global
agriculture, in the light of modern biotechnological developments, was recently
reviewed (Zahran 2005). The wide use of legumes as green manure or biofertilizer
is mainly associated with their ability to establish symbiotic associations with
N2-fixing bacteria (NFB), collectively called rhizobia (Zahran 2006a). These bacteria
are among the most studied group of microorganisms, mainly because of their
potential to replace N-fertilizers, with emphasis on their key role in achieving
sustainability of N-poor soils (Menna et al. 2006).
The significance of rhizobia–legume symbiosis for soil fertility improvement is
well known. The root-nodule bacteria (rhizobia and other newly discovered NFB)
establish substantial symbiotic associations with the leguminous plants to utilize
atmospheric N2. These diverse groups of bacteria form associations with crop
(forage and grain), as well as with wild legumes (Zahran 2006b). The leguminosae
is one of the most important and largest plant families and is composed of about 750
genera containing 16,000–19,000 species distributed worldwide and having major
impacts on agriculture, the environment, animal/human nutrition, and health. Plants
of the family leguminosae (Fabaceae) are able to form a symbiosis with bacteria
(termed rhizobia), which leads to the development of a new organ on the roots (and
sometimes stems) of the plant, the nodule, in which the bacteria fix N2. The
legume–Rhizobium symbiosis have tremendous ecological and agronomic impor-
tance and constitute a significant source of N, and consequently play an essential
role in structure ecosystems and sustainable agriculture (Dita et al. 2006).
Current bacterial taxonomy divides a group of Gram-negative bacteria, the
Proteobacteria, into a number of branches, a, b, g, d, and e. The a-branch has
seven orders, one of which is the rhizobiales, which has 11 families. Four of these
families (Bradyrhizobiaceae, Methylobacteriaceae, Phyllobacteriaceae, and Rhizo-
biaceae) contain genera that can nodulate and fix N2 in association with legumes
(Sprent 2008). The b-proteobacterial branch also contains seven orders. One of
these orders, Burkholderiales, is divided into five families, and one of these
families, the Burkholderiaceae, contains two genera, Burkholderia and Cupriavidus
having species known to nodulate legumes (Sprent 2008). Several species of
Burkholderia have been shown to nodulate mimosoid legumes (Barrett and Parker
2005; Chen et al. 2005), with varying levels of host specificity, some of which are
capable of fixing N2 ex planta. However, it remains to be seen how these b-rhizobia
become important in certain environments. The nodule-forming bacteria are cur-
rently divided into about 50–60 species within about 10–12 genera, with 8 genera
11 Enhancement of Rhizobia–Legumes Symbioses 229
among the alphaproteobacteria: Azorhizobium, Bradyrhizobium, Mesorhizobium,

Methylobacterium, Rhizobium, Sinorhizobium (Ensifer), Devosia, and Blastobacter,
and 2 genera (Burkholderia and Cupriavidus) within the b-proteobacteria, in
addition to some species in the g-proteobacteria (Barrett and Parker 2005, 2006;
Wang et al. 2006). However, the picture of rhizobia diversity is far from clear,
especially considering the wide geographical distribution and the large number
of leguminous species which might harbor novel root-nodule bacteria (Wei et al.
2007).
The desert areas are characterized by a water deficiency due to the atmospheric
dryness, and very low rainfall, resulting in seriously-degraded vegetation and a
progressive reduction of biodiversity in the ecosystem (Verdoy et al. 2006;
Essendoubi et al. 2007). Rhizobia encountered in desert areas play an important
role as root symbiotics involved in N2 fixation of some leguminous plants (e.g.,
Acacia), which are pioneering species that contribute to soil fertility (Zahran 2005).
Rhizobial bacteria occur naturally in most agricultural soils whose survival is affected
directly both by drought and salinity (Zahran 1999). To make optimal use of the
N2-fixation process, the physical and physiological conditions that affect the survival
of rhizobia during desiccation should be optimized (Vriezen et al. 2006).
The root-nodule bacteria from wild legumes are multipurpose bacteria with very
interesting characteristics; this concept was recently reviewed (Zahran 2006a).
They have a wide host range, a characteristic that offers these legumes an ecologi-
cal advantage as they can form nodules with other wild or crop legumes (Zahran
et al. 2003), and can be a source of genetic information to improve characteristics of
other rhizobia. The significance of rhizobia from wild legumes is their symbiotic
N2-fixing activity, which improves soil fertility and plant productivity. Woody
(trees) legumes like Faidherbia albida, Inga oerstediana, Prosopis lavegata, and
Pachycereus hollianus host NFB, and occasionally arbuscular mycorrhizal fungi
(AMF) may contribute to the soil organic carbon pool and soil fertility (Grossman
et al. 2006; Bernatchez et al. 2008; González Ruiz et al. 2008). These leguminous
trees usually create localized distributions of soil N, C, and P, through N2 fixation
and subsequent litter fall. The companion crops benefit from the N2 fixation of trees
by reabsorbing N mineralized from decomposing N-litter and prunings. Nitrogen
may transfer directly by belowground processes from tree to grass, e.g., via root
exudates and/or common mycorrhizal networks (Sierra and Nygren 2006). The
nodulation of four tree legumes (Calliandra calothyrsus, Gliricidia sepium,
Leucaena leucocephala, and Sesbania sesban) and the ecology of their native
rhizobial populations in tropical soils has been examined (Bala et al. 2003).
C. calothyrsus had the highest nodulation rate in the soils used; however, inocula-
tion tests showed L. leucocephala to be the most promiscuous species, G. sepium to
be the most effective symbiosis, and S. sesban to be the most specific for both
nodulation and symbiotic effectiveness.
Salinity of soil is one of the most serious forms of land degradation in the world,
especially in arid lands, where insufficient precipitation causes extensive reliance
on irrigation. Globally, about 7% of all land areas is affected by soil salinity (Unni
and Rao 2001). Of the total 1.5 billion ha of cultivated land, about 5% are affected
230 H.H. Zahran
by excess salt content. Among the most common effects of soil salinity is growth
inhibition by Na+ and Cl . Elevated Na+ in soil solution inhibits the uptake of other
nutrients (e.g., P, K, Fe, Cu, and Zn) directly by interfering with various transpor-
ters in the root plasma membrane (Giri et al. 2007). Salinity has profound effects on
crop production, therefore, reducing the spread of salinization and increasing the
salt-tolerance of high-yielding crops, are becoming important global issues. Mineral
nitrogen deficiency is another important limiting factor for plant growth in arid
zones, and rhizobia–legume symbioses are the primary sources of fixed N2 in these
habitats (Verdoy et al. 2006). Salinated soil contains very little N and thus is not
suitable for cultivation of most plants. An appropriate solution to this situation
would be the cultivation of plants that are able to fix N2 through symbiotic systems
(Chen et al. 2000). Most leguminous plants, however, are sensitive to even low
levels of salinity. In addition, most rhizobia are also sensitive to moderate and
higher levels of salinity during both the free-living stage and the symbiotic
process. Legumes used in the reclamation of degraded lands (e.g., salt-affected
lands) include P. juliflora, Acacia nilotica, A. auriculifonnis, Dalbergia sisso, and
Glyricidia maculate. However, both legume growth and the process of nodule
formation are more sensitive to salinity than are rhizobia (Zahran 1999). For
instance, Sinorhizobium meliloti tolerated up to 300 mM NaCl, while nodulation
and N2 fixation of its host (M. sativa) was inhibited at about 100 mM salt (Graham
and Vance 2000).
Major advances in our understanding of the relations between legumes and
rhizobia are being made using the legume models Medicago truncatula and Lotus
japonicus. Despite the taxonomic diversity, all rhizobia establish symbioses using
the same molecular mechanisms, involving signal molecule exchange between the
two partners. Host plants excrete inducing compounds (mainly flavonoids) that
activate regulatory NodD proteins and induce the expression of bacterial nodulation
(nod) genes. Nod genes are involved in the biosynthesis of the lipochitooligosac-
charides (nod factors) that are required for specific infection and nodulation of
legumes (Ba et al. 2002). This chapter reviews several reports to assess the signifi-
cance of symbiotic bacteria (rhizobia and other nodule bacteria) for the enhance-
ment of N2 fixation and the consequent effect on productivity improvement of the
economically important crop plants, either legumes or nonlegume-associated crops.
The recent developments in molecular genetics and their usefulness in improving
Rhizobium–legume symbiosis are also reviewed and discussed.
11.2 Molecular and Genetic Mechanisms Controlling the

Symbiosis
The soil bacteria (rhizobia) within the phylogenetic family Rhizobiaceae have the
unique ability to infect and establish a N2-fixing symbiosis on the roots or stems of
leguminous plants. The establishment of the symbiosis involves complex interac-
tions between the host and the rhizobia, resulting in the formation of a special
organ, the nodule, which the bacteria colonize as intracellular symbionts. Two
general developments have contributed to the recent explosion of research progress
in this area (Stacey et al. 2006): first, the adoption of two genetic model legumes,
M. truncatula and L. japonicus, and second, the application of modern methods in
functional genomics (transcriptomic, proteomic, and metabolomic analyses). The
first Rhizobium genes for nitrogen fixation (nif) and for nodulation (nod) were
cloned in the early 1980s (Long 2001), and soon many more nif, nod, and fix
(symbiotic fixation) genes were discovered. The rules for genome organization
are different for different rhizobia. Symbiotic genes are clustered or dispersed on
plasmids and can spread at high frequency by conjugation, while in others, the
genes are scattered among many chromosomes and plasmids. With all of this
genomic diversity it is no wonder that systematists have had a field day classifying
and reclassifying the root-nodule bacteria. Numerous genes coding for transporters
(sugar, peptide, nitrate, and H+-ATPase transporters) were shown to be upregulated
in L. japonicus and M. truncatula nodules, suggesting an important role of these
transporters in the exchange of carbon and nitrogenous compounds between
legumes and rhizobia.
The DNA microarray and transcript profiling studies of nodulation have been
considered only recently; however, the large numbers of expressed sequence tags
(ESTs) available for legume plants, in addition to cDNA and oligonucleotides, have
made large-scale transcriptomic studies on nodulation possible. Studying the gene
expression using DNA microarray became possible because of the availability of
the complete genomic sequence of one of the rhizobial strains (S. meliloti 1021).
The DNA microarray technology has revolutionized gene expression studies by
providing a powerful tool for parallel measurement of gene expression on a whole
genome scale. In contrast to the labor-intensive gene fusion technique, this tech-
nology achieves a higher coverage and provides the direct determination of mRNA
levels to monitor an expression profile (Rüberg et al. 2003). The microsatellites or
simple sequence repeats (SSRs), which are ubiquitous in genomes of various
organisms, can be used as genetic marker. The analysis of SSR in rhizobial genome
provides useful information for a variety of applications in population genetics of
rhizobia (Ya-mei et al. 2008). The occurrence, relative abundance, and relative
densities of SSRs of B. japonicum, M. loti, and S. meliloti were analyzed and are
available in the genomes sequenced database. The motif and distribution of SSRs
among the three rhizobia genomes were similar, and the tetranucleotide, pentanu-
cleotide and hexanucleotide repeats were predominant and indicated higher muta-
tion rates in these species. This marker can be used to find the gene position quickly
and exactly and then to scan, identify, and annotate the gene, and to study gene
function. Thus, the SSR marker had an important effect on functional genome
research of rhizobia.
Although rhizobia colonize roots in a way that is reminiscent of pathogenic
microorganisms, no host plant defense reactions are triggered during successful
symbiosis (Mithöfer 2002). The question is how defense responses in effective
symbiosis might be suppressed? A prerequisite for understanding the molecular
mechanisms underlying the control of defense in symbiosis is the identification of
232 H.H. Zahran
receptors, as well as the elucidation of subsequent signal transduction events

involved in the onset of plant defense for both rhizobial elicitors and suppressors
(Mithöfer 2002). Some of the defense-related genes, e.g., those coding for enzymes
of the phytoalexin biosynthesis pathway and genes coding for proteins that are
involved in cell wall modifications, were also regulated during the nodulation
process. Interestingly, the defense-related genes were upregulated during the early
stages of nodulation (recognition and infection) and then decreased in later stages
of nodule development, suggesting that the invading rhizobia suppress the host’s
defense to successfully colonize roots and so form nodules (Stacey et al. 2006).
Substantial progress has been made recently to understand how rhizobia enter
into symbiotic relationships with legumes. The establishment of the legume–
Rhizobium symbiosis requires recognition of the bacterial microsymbiont at the
root epidermis followed by initiation of plant infection and nodule organogenesis.
The symbiotic N2-fixing rhizobia harbor a set of nodulation (nod) genes that control
the synthesis of modified lipochitooligosaccharides, called nod factors; required for
nodulation (Moulin et al. 2004). Studies on nod factor activities, coupled with the
recent cloning of genes required for nodule initiation, are leading to an understand-
ing of the first steps in the signaling pathways. Moreover, studies have shown that
phytohormones (e.g., auxin and cytokinin) are involved in controlling or mediating
symbiotic responses. The challenge for the future will be to establish how nod
factor signaling integrates with phytohormone activities in the control of infection
and nodulation in the establishment of symbiosis (Mulder et al. 2005). The nodA
gene, which is essential for symbiosis, is responsible for the attachment of the fatty
acid group to the oligosaccharide backbone. PCR amplification, sequencing and
phylogenetic analysis of nodA gene sequences from a collection of diverse
Bradyrhizobium strains, isolated from various legume species and geographical
areas, revealed the monophyletic character of these strains. This study also revealed
a large nucleotide diversity of nodA sequences within this genus, ranging up to
21%, which seems consistent with the overall genetic variability of bradyrhizobial
strains and their ability to perform efficient symbiosis with many different plants
(Moulin et al. 2004).
Plants, like humans, contain hemoglobin. Three distinct types of hemoglobin,
with different functions exist in plants: symbiotic, nonsymbiotic, and truncated
(Hoy and Hargrove 2008). Symbiotic hemoglobins (sHbs) are proteins found in
millimolar quantities in legume root-nodules and sometimes in nonlegumes, for
example, Parasponia andersonii–rhizobium symbiosis (Ott et al. 2005). The main
function of sHbs in plants is well known: sHbS in the root-nodules are responsible
for facilitating diffusion of the oxygen necessary for N2 fixation, similar to the
activity of mammalian myoglobin (MB) in muscles. While using an RNA interfer-
ence approach to silence the expression of a three nodule-expressed leghemoglobin
genes in the legume L. japonicus, it was found that leghemoglobins indeed are
essential for N2 fixation in nodules (Downie 2005). Leghemoglobins, however, can
have a 20-fold higher affinity for oxygen than myoglobin. The oxygen-binding
characteristics of leghemoglobins are unusual in that they have an extremely fast O2
association rate and a relatively slow O2 dissociation rate, and so can buffer the free
oxygen concentration at around 7–11 nM (Downie 2005). Legumes can select for
greater mutualism, controlling nodule O2 supply and reducing reproduction of
rhizobia that fix less N2 (Denison and Kiers 2004).
11.3 Selection of Effective Symbiotic Rhizobia
Because of the value of rhizobia in the environmental and agricultural fields,

rhizobial analysis, based on symbiotic performance and cross inoculation range,
is essential for selection for field applications. Selection of effective strains of
rhizobia is mainly dependent on symbiotic properties of the bacteria. Symbiotic
properties are coded by genetic elements (e.g., plasmids) that can be transmitted
between different bacteria. Nevertheless, the symbiotic genes could be used as
markers for complete rhizobial characterization. Common nodulation genes (e.g.,
nodABC) or nitrogenase genes (e.g., nifHDK) have been useful in the study of
interactions with host plants and to clarify relationships between rhizobial system-
atic and symbiotic properties (Villegas et al. 2006).
The establishment of new legumes in agriculture often requires the introduction
of symbiotically competitive root-nodule bacteria as inoculants. However, prior to
the introduction of new species, the symbiotic interactions between the new legume
and the indigenous populations of root-nodule bacteria, and also the interaction of
any new inoculants with legumes already in the ecosystem, must be carefully
evaluated. The host recognizes the compatible and effective rhizobia from a soil
that harbors effective and ineffective rhizobia. Therefore, optimization of the
symbiosis between legume plants and their respective symbiotic rhizobia requires
the presence in the rhizosphere of competitive and infective strains of rhizobia
which are highly efficient at fixing N2. The success of inoculated strains of
Rhizobium in soil is largely dependent on their ability to colonize rapidly and
out-compete coexisting strains for nodule occupancy by specific strains of rhizobia.
Highly competitive strains for nodule formation need not always exhibit the highest
level of saprophytic fitness (Duodu et al. 2005). The basal rhizobial cell density,
rather than soil pH, is essential for colonization of the soil and selection by specific
host (Yates et al. 2008). A legume introduced into a new area will only form
nodules and fix N2 if compatible rhizobia are present in the soil. Rhizobial popula-
tion sizes estimated after trapping by the legume species, in soils from tropical areas
of Africa, Asia and Latin America (Bala et al. 2003), ranged from undetectable
numbers to 3.16104 cells g 1 of soil depending on the trap host species. Symbi-
otic effectiveness did not bear any close relationship with specific soil parameters,
but rhizobial numbers have been found highly correlated with soil acidity, particle
size, and exchangeable ions (Bala et al. 2003).
The traditional methods adopted for identifying the most effective rhizobial
strains to be used as inoculant is a time-consuming process, involving the produc-
tion of thousands of rhizobial cultures followed by greenhouse experiments and
field trials. Selection of effective strains of nodulating bacteria for legumes should
234 H.H. Zahran
consider the genetic characterization and taxonomic position (Menna et al. 2006).
Rhizobia are known to vary widely in their ability to nodulate many species of
legume host plants, ranging from species that appear to be highly specific (with a
very limited host range), such as Rhizobium galegae, compared with Rhizobium sp.
NGR 234 with a very wide host range and able to nodulate legumes from at least
112 genera, and some indigenous isolates of Bradyrhizobium, which nodulate
promiscuous varieties of soybean (Musiyiwa et al. 2005). Identification of effective
locally adapted strains with wide host ranges could be useful in the development of
rhizobial inoculants which can survive longer in agricultural soil and, hence, reduce
the need for regular inoculant applications.
It is essential to reclassify and evaluate the existing strains of rhizobia for their
symbiotic and N2-fixing abilities, using modern molecular genetic tools while
selecting bacteria for raising inoculants. A comprehensive study consistent with
this concept has been carried out in Brazil (Menna et al. 2006). The phylogeny of a
collection of rhizobial strains from a wide range of legumes was examined based on
the sequencing of the 16S rRNA genes. Host specificity of these strains was not
related to 16S rRNA genes, suggesting the diverse evolution of ribosomal and
symbiotic genes in such organisms (Menna et al. 2006). Similarly, strains of
R. leguminosarum bv. trifolii showed a different symbiotic pattern with Trifolium
spp., although their 16S rRNA gene sequences were identical (Yates et al. 2008).
Strains of R. leguminosarum bv viciae from pea (P. sativum L.) displayed a
relatively high degree of genetic diversity among both plasmid and chromosomal
components (Vessey and Chemining’wa 2006). Rhizobial strains from A. tortilis,
which are grouped in the genera Mesorhizobium and Sinorhizobium, exhibited very
similar symbiotic characters despite their chromosomal diversity (Ba et al. 2002).
11.4 Selection of Symbiotic Rhizobia from Crop

and Wild Legumes
11.4.1 Rhizobia from Crop Legumes
Soils in cultivated areas are rich in rhizobia from the so-called “cowpea group” that
effectively nodulate cowpea, peanut, and other tropical legumes. They are usually
slow-growing bacteria belonging to the genus Bradyrhizobium. Bacteria in the
genus Bradyrhizobium is a cosmopolitan and diverse group nodulating a variety
of legumes, as well as the nonlegume Parasponia. Polyphasic characterization of
these rhizobia from different hosts and from different geographic regions was done
by analyzing the variability of 16S rRNA gene RFLP, 16S–23S rRNA gene
intergeneric spacer (IGS) RFLP, G–C rich RAPD, and phenotype assays. The
evolution of “cowpea group” isolates of Bradyrhizobium nodulating cowpea and
peanut from different locations in South Africa were studied (Steenkamp et al.
2008). These bacteria are diverse, representing a number of distinct Bradyrhizobum
groups; horizontal gene transfer significantly influences the evolution of cowpea

and peanut root-nodule bacteria. The identification of the nodZ, noel and noeE
genes in the isolates tested indicated that African Bradyrhizobium species may
produce highly decorated nodulation factors, which represent an important adapta-
tion enabling nodulation of a great variety of legumes inhabiting the African
continent (Steenkamp et al. 2008). Based on these characteristics, mung bean
(Vigna radiata L.) rhizobia from China (Yang et al. 2008) were found to have
diverse slow-growing bacteria clustered into four groups, three related to Bradyr-
hizobium and the fourth group formed a miscellany of fast-growing isolates vari-
ously related to the genera Mesorhizobium, Rhizobium and Sinorhizobium. Studies
of the native rhizobial populations associated with peanut Arachis hypogaea
nodules in soils of Argentina, Morocco and Cameroon revealed that these popula-
tions are highly diverse and include slow-and fast-growing isolates (El-Akhal et al.
2008; Nkot et al. 2008). The bacteria isolated from Morocco were firstly character-
ized by restriction of the 16S rDNA region, and phylogeny was inferred from the
16S gene sequence. The isolates were grouped with species belonging to the
Bradyrhizobium and Rhizobium genera (El-Akhal et al. 2008); a high degree of
variability was detected among isolates in terms of their N2-fixing ability. Strains
from peanut (A. hypogaea L.) in Cameroon were examined after RFLP analysis of
16S–23S rDNA genes (Nkot et al. 2008) and a considerable level of genetic
diversity was determined among those peanut isolates. Fast-growing Rhizobium
isolates were obtained from root-nodules of the legumes belonging to the genera
Vicia, Lathyrus and Pisum from different agro-ecological areas in Italy (Moschetti
et al. 2005). Analysis of symbiotic properties showed a wide spectrum of nodula-
tion. The majority of the isolates were presumptively identified as R. legumino-
sarum bv. viciae both by their symbiotic properties and by the specific amplification
of the nodC gene, RFLP–PCR 16S rDNA analysis, and RAPD–PCR technique,
which showed a high level of genetic polymorphism.
11.4.2 Rhizobia from Wild Legumes
Wild (herb and tree) legumes harbor diverse and promiscuous root-nodule bacteria
(Zahran 2006a), which are currently classified into several rhizobial or nonrhizobial
genera. Further genetic and molecular studies are needed to elucidate the identifi-
cation and classification of many new isolates of rhizobia from wild legumes. To
develop new symbiotic legume species for agriculture, the genetic and symbiotic
diversity of rhizobial isolates from various wild legumes are being investigated
using numerical analysis, phenotypic characteristic, cross nodulation to selected
legumes, and modern genetic tools, such as amplified 16S ribosomal DNA restric-
tion analysis (ARDRA), RFLP analysis of 16S–23S rDNA intergeneric spacers
(IGS), 16S rRNA gene sequencing, and multi-locus sequence analysis of the PCR-
amplified nodC gene and DNA/DNA hybridization.
236 H.H. Zahran
The rhizobial strains isolated from several wild forage legumes collected from
various geographical regions were identified. For example, rhizobia from Lathyrus,
Vicia and Lotus grown in China (Han et al. 2008) clustered into nine genomic
species belonging to four genera: Bradyrhizobium, Mesorhizobium, Rhizobium, and
Sinorhizobium (Ensifer). The N2-fixing rhizobial isolates from root-nodules of
Medicago laciniata, and from Mediterranean soils in Tunisia and France (Villegas
et al. 2006), differed markedly from other S. meliloti or S. medicae isolates in their
symbiotic traits, such as nifDK RFLP diversity, nodA sequences, and N2-fixing
efficiency with annual Medicago species (M. truncatula, M. polymorpha and
M. sauvagei). The rhizobial isolates from root-nodules of Astragalus, Lespedeza
and Hedysarum in China (Wei et al. 2007) were divided into two groups: the first
was related to the Rhizobium–Agrobacterium group and the second was related to
R. galegae and R. huautlense. The bacteria isolated from nodules of Lotononis
angolensis were fast-growing, highly mucoid, and pink pigmented, and related to
genera other than rhizobia based on 16S rRNA phylogeny, while those isolated
from L. bainesii and L. listii were slow-growing, pink pigmented and attributed to
Methylobacterium (Yates et al. 2007). Sequencing of the 16S rRNA region of
Biserrula pelecinus (a new forage legume in Australia with high quality feed for
cattle and sheep and having many agronomic attributes) root-nodule bacterial
isolates revealed a close relationship between these bacteria and with both M. loti
and M. ciceri (Nandasena et al. 2004). As B. pelecinus can be nodulated by
Mesorhizobium spp. isolated from other legumes, particularly Lotus, there is an
opportunity to utilize this trait in cultivar development.
The leguminous trees have an important role in maintaining soil fertility. Many
legume trees were recently screened for their ability to form effective N2-fixing
nodules. Large numbers of the diverse root-nodule bacteria were isolated and
identified, and their symbiotic activities were assessed by using traditional meth-
ods, molecular methods, and genetic tools. Tree legumes such as Acacia, Prosopis,
Sesbania, Albizia, Leucaena, etc. have received increasing attention because of
their uses as fodder and wood, and are included in ambitious reforestation
programs in arid environments (Zakhia et al. 2004; Zahran 2006a). Some of the
legume trees have records of good N2 fixation capacities with their symbiotic
bacteria. The high diversity (rhizobial genotypes, host affinities and symbiotic
effectiveness) of the isolates require rigorous selection for host strain site
combinations in order to obtain effective and competitive inoculants for legume
trees and crops in rehabilitation and agro-forestry programs. A collection of
rhizobial isolates from A. tortilis subsp. raddiana, from various sites in the north
and south of Sahara (Africa), was very diverse (Ba et al. 2002). Based on whole
cell protein (SDS-PAGE) and 16S rDNA sequence analysis, most strains were
related to Mesorhizobium and Sinorhizobium genera, but with similar symbiotic
characters (Ba et al. 2002). The rhizobia isolated from root-nodules of Acacia
species native to Mexico constitute a diverse group of bacteria distinguished
from other Sinorhizobium species by their levels of DNA–DNA hybridization
and the sequence of 16S rRNA and nifH genes. Therefore, a new species,
S. americanus, was described (Toledo et al. 2003). The rhizobial strains isolated
from A. abyssinica, A. seyal, A. tortilis, F. albida, S. sesban, P. vulgaris and

V. unguiculata grown in southern Ethiopia was assessed using the BiologeTM
system and AFLP finger printing technique (Wolde-Meskel et al. 2004). The
rhizobia constituted metabolically and genomically diverse groups that are not
linked to reference species. The root-nodule bacteria from tree legumes (Acacia,
Prosopis, Sesbania and Faidherbia) and other legumes (Macroptilium, Phaseolus and
Vigna), growing in ecologically diverse sites in Kenya (Odee et al. 2002),
were analyzed by PCR–RFLP of the 16S rRNA gene and sequence analysis of a
230-bp fragment of this gene. The bacteria were assigned to four rhizobial genera
(Bradyrhizobium, Mesorhizobium, Rhizobium, and Sinorhizobium) and the
Agrobacterium. An analysis of the PCR-amplified 16S rDNA gene digestion pro-
files, using 5-endonucleases, indicated the presence of different lineages among the
taxa associated with P. juliflora nodules in arid soils of Morocco (Benata et al. 2008).
Nucleotide sequencing of the small subunit rRNA gene and blast analysis showed
that P. juliflora hosted bacteria belong to Rhizobium, Sinorhizobium and
Achromobacter xylosoxidans; the latter species is from b-proteobacteria.
Diversity of root-nodule bacteria (and sometime stem-nodule bacteria) from
the legume shrub Sesbania has received increasing interest. The symbiotic prop-
erties, LPS profiles, sym plasmid and rhizobiophage sensitivity of bacterial iso-
lates of three Sesbania species (S. sesban, S. aegyptica and S. rostrata) of the
semiarid Delhi region of India were analyzed (Sharma et al. 2005). The isolates
differed in their symbiotic efficiency, but 16S rDNA sequences revealed that root-
nodule isolates of Sesbania belonged to diverse rhizobial taxa (S. saheli,
S. meliloti, R. huautlense) whereas stem-nodule isolates were strictly Azorhizobium
caulinodans. Molecular systematics of rhizobia based on phylogenies inferred from
rrs, atpD, recA and nifH sequences were used for classification of Sesbania
isolates from Venezuelan wetlands (Vinuesa et al. 2005). The isolates were
consistently identified as M. plurifarium or R. huautlense. DNA–DNA hybridization
and 16S rRNA sequences support the inclusion of the rhizobial strains isolated from
root-nodules of Sesbania virgata in southeast Brazil in the genus Azorhizobium but not
in the species A. caulinodans, and hence the name A. doebereinerae was proposed
(Moreira et al. 2006). Rhizobial isolates from Albizia trees grown in China were
identified employing PCR–RFLP and sequencing of 16S rRNA genes, SDS-PAGE
of whole cell proteins, and clustering of phenotypic characters (Wang et al. 2006).
Albizia trees are nodulated by a wide range of rhizobial species within the genera
Rhizobium, Bradyrhizobium and Mesorhizobium. The bacterial isolates from root-
nodules of the woody legumes Wisteria sinensis, Cercis racemosa and Amorpha
fruticosa grown in China were characterized with phenotypic analysis, PCR-based
16S and 23S rRNA gene RFLP, Box PCR and 16S rRNA gene sequencing (Liu et al.
2005) and identified into three rhizobial genera (Rhizobium, Bradyrhizobium and
Mesorhizobium). The diversity of Retama raetam root-nodule bacteria, grown in arid
regions of Tunisia, was determined by 16S rRNA gene sequencing and phenotypic
analysis (Mahdhi et al. 2008). The bacteria were assigned to the genera Agrobacterium,
Rhizobium and Sinorhizobium, those of the latter genus showed a large diversity in
their symbiotic properties and N2-fixing ability.
238 H.H. Zahran
11.5 Selection of Symbiotic Rhizobia Tolerant

to Various Abiotic Stresses
11.5.1 The Problem of Soil Salinity
One of the most severe and widespread problems facing agriculture is the degrada-
tion of soil quality due to desiccation and salinity. Almost 40% of the world’s land
surface is affected by salinity-related problems (Zahran 1999; Vriezen et al. 2007).
Salinity may have a detrimental effect on soil microorganisms and, in general,
results in a decreased productivity of crop plants. Harsh environmental conditions
negatively affect the activity of most endogenous soil bacteria including rhizobia.
The crops grown in 90% of arable lands are subjected to one or more environmental
stresses. To face the threat of these stresses, several genetic improvement strategies
are available from classical plant breeding to more direct physiological and genetic
approaches. However, understanding the mechanisms underlying a specific stress is
essential (Dita et al. 2006). Various soil problems (such as extreme pH, salinity,
drought, and high temperature) hinder agriculture in arid areas. Some legumes are
relatively tolerant to such stress conditions, but nodule formation and N2 fixation
are greatly impaired by stress conditions. Therefore, for legume cultivation in arid
areas under environmentally friendly and economically attractive conditions (i.e.,
without applying fertilizer-N), the availability of stress-tolerant rhizobial strains is
an absolutely required precondition (Priefer et al. 2001).
Cultivation of agricultural crops in soil is limited by salt stress, which arises
from the excessive uptake of salt by plants and is an unavoidable consequence of
high ion concentrations. A higher salt amount in a soil, most commonly NaCl, has
detrimental effects on plant growth and productivity. In this regard, salinity affects
the activities of enzymes involved in nitrogen metabolism, but the mechanism
underlying this phenomenon still remains unclear. It is well known that the estab-
lishment of legume–Rhizobium symbiosis is susceptible to the effects of salinity.
The responses of plant genotypes to salinity differ widely, depending in the form
and dosage of salt and the stage of plant growth (Zilli et al. 2008).
11.5.2 Free-Living and Symbiotic Rhizobia Under Abiotic Stress
The symbiotic interactions between rhizobia and their host legumes have strongly
driven the investigation and application of biotechnology tools for legumes. Stress
parameters such as soil acidity, salinity and desiccation affect rhizobial persistence
and nodulation efficiency. The responses of various rhizobia to desiccation were
recently reviewed (Vriezen et al. 2007). An adaptation to increased salt or osmotic
stress elevates the ability of rhizobia to survive desiccation by accumulation of
osmoprotectants, desiccation protectants, and heat-shock proteins (Vriezen et al. 2006).
Four strains of rhizobia nodulating Acacia were isolated from the Moroccan
desert soil by trapping seedlings of A. gummifera and A. raddiana, and were studied
for their ability to tolerate high salinity and dry conditions (Essendoubi et al. 2007).
Three strains were halotolerant, and grew at 1 M NaCl and accumulated glutamate
and mannosucrose. Such strains showed high resistance to desiccation, whose
tolerance to dryness was stimulated by osmotic pretreatment. The accumulation of
solutes and sugars in these rhizobia represents both an osmoadaptive response and a
part of a desiccation tolerance mechanism (Essendoubi et al. 2007). High salt
concentrations may have detrimental effects on rhizobial populations as a result of
direct toxicity, as well as through osmotic stress. Howieson and Ballard (2004)
suggested that stressful environments limit rhizobial communities to less than 100
cells g 1 soil, at some time during the season. One of the salt-induced responses in
rhizobia is changes in cell morphology and size, and modifications in the pattern of
polysaccharides (Zahran 1999). The latter responses may have an impact on the
symbiotic interaction between rhizobia and their legumes because extracellular
polysaccharides (EPS) and lipo-polysaccharides (LPS) are essential factors for
the development of root nodules. A Rhizobium mutant (EPS deficient, exo ),
showed decreased levels of LPS in the presence of 350 mM NaCl (Unni and
Rao 2001). The response and adaptation of rhizobia to environmental stresses are
probably complex phenomena involving many physiological and biochemical
processes that likely reflect changes in gene expression and consequently the
activity of enzymes and transport proteins (Wei et al. 2004; Domı́nguez-Ferreras
et al. 2006). Some species of rhizobia adapt to saline conditions by intracellular
accumulation of low-molecular-weight solutes called osmolytes, such as glutamate,
glycine betaine and polyamines, or by accumulation of K+. As salt tolerance of
rhizobia is a phenotype, in which many regulatory mechanisms are involved, it is
necessary to study salt-tolerant rhizobial genes. Different genes involved in salt
tolerance of S. meliloti were identified using random Tn5-1063 insertional muta-
genesis (Wei et al. 2004). The adaptation of S. meliloti to salt is a complex
multilevel regulatory process in which many different genes can be involved.
The rhizobia–legume symbioses are more sensitive to salt or osmotic stress than
free-living rhizobia. Salt stress may inhibit the initial steps of the symbiosis (root
colonization and infection), but it also has a depressive effect on N2 fixation.
Nevertheless, salt-tolerant rhizobia are important to establish a salt-tolerant symbi-
otic system. Selection of adapted strains of rhizobia under stress conditions is
possible and they can be used as inoculants for successful lupin growth (Raza
et al. 2001). Objectives of genomic research on rhizobia are the identification of
genes and regulatory networks relevant for symbiosis and the consideration of the
free-living status, especially adaptations to fluctuated soil environmental conditions
(e.g., temperature, osmolarity, pH, and nutrient supply). DNA microarrays have
been used to examine gene expression in response to various abiotic stresses in
rhizobia (Domı́nguez-Ferreras et al. 2006). Based on the complete S. meliloti
genome sequence, Rüberg et al. (2003) established DNA microarrays as a compre-
hensive tool for systematic genome-wide gene expression analysis in S. meliloti
1021. Gene expression in S. meliloti in response to an osmotic upshift, imposed by
240 H.H. Zahran
the addition of 0.38 M NaCl, was monitored using the DNA microarray tool. About
137 genes showed significant changes in gene expression resulting from the osmotic
upshift. From these genes, 52 were induced and 85 were repressed (Rüberg et al.
2003). DNA microarrays were used to investigate genome-wide transcriptional
responses of S. meliloti to a sudden increase in external osmolarity elicited by
addition of either NaCl or sucrose to exponentially-growing cultures (Domı́nguez-
Ferreras et al. 2006). The genetic data correlated well with the microarray results
and suggested that pSymB contains a large number of genes upregulated after an
osmotic upshift, which may play an active role in the osmoadaptation of S. meliloti.
A well-characterized promoter is now available to drive expression of rhizobial
stress-tolerance genes of interest under acidic conditions. Selection of stress-
adapted symbioses, or elite rhizobial strains or legume genotypes, can overcome
these stress factors. Rhizobium mutants whose adaptation to high salinity is affected
have deficiencies in their symbiotic capacity (Nogales et al. 2002). Salt-tolerance
genes of S. fredii (a halotolerant rhizobium strain grow at 0.6 M NaCl) were
identified by the construction and screening of a transposon Tn5-1063 library
containing over 30,000 clones (Jiang et al. 2004). This strain could have developed
sophisticated mechanisms to maintain its intracellular steady osmotic and ionic
state. Several genes involved in osmotic tolerance of this strain were identified,
which are important to improve salt tolerance of salt-sensitive rhizobia.
Bacterial isolates from Canavalia rosea, an indigenous leguminous halophyte
grown in the seaside area of southern Taiwan, were effective symbionts for the
original host and able to grow at NaCl concentrations up to 3–3.5% (w/v). Based on
SDS-PAGE of proteins, pulsed-field gel electrophoresis (PFGE) analysis and
ARDRA results, the 12 isolates were highly diverse. The 16S rRNA and nifH
gene sequences were determined for isolates with distinct ARDRA patterns and
compared with other members of the rhizobial species. The isolates were proposed
to be classified into the genus Sinorhizobium and distinguished from the current
species of the genus (Chen et al. 2000). Rhizobia were isolated from the root-
nodules of Glycyrrhiza glabra and G. uralensis, growing in the arid and semiarid
regions of northwestern China (Ge-Hong et al. 2008). The taxonomic position and
stress tolerance were determined to select the promising putative inoculant strain.
Based on physiological and biochemical characteristics, the isolates were clustered
into three groups. One isolate was found to have high tolerance to NaCl, pH and
temperature. Based on the sequence analysis of 16S rDNA and nodA gene, this
isolate was placed in the genus Mesorhizobium.
11.5.3 Symbiotic N2 Fixation of Legumes Under Abiotic Stresses
Optimum performance of the N2-fixing symbiosis depends upon preselection of

both symbiotic partners for adaptation to the target environment, which may in
some cases present a challenge to rhizobial survival or nodulation. Symbiotic N2
fixation in legumes is limited, especially in semiarid conditions. Legumes are
classified as salt-sensitive crops, and their limitation in productivity is associated

with poor development of symbiotic root-nodule bacteria, reduction in the N2
fixation capacity and lower growth of the host plant. N2 fixation of legumes is
usually affected by abiotic stress; the response of root-nodules of legumes to
various kinds of stress was examined extensively at the physiological and biochemi-
cal levels. However, improvement of N2 fixation of these legumes under abiotic
stressed environments requires a better knowledge of the key elements acting at
the molecular level.
The response of Rhizobium–Phaseolus symbiosis to salt stress was investigated,
and each of the symbiotic partners was examined separately for their salt tolerance
(Bouhmouch et al. 2005). Plant cultivars of Phaseolus showed genotypic variations
with respect to salt tolerance. Rhizobium strains (e.g., R. tropici and R. giardinii),
were tolerant up to 250 mM NaCl but failed to nodulate Phaseolus under saline
conditions (50 mM NaCl). The unsuccessful nodulation could be related to the
inhibition of one or more steps of the early events of the infection process under salt
stress (Zahran and Sprent 1986). In contrast, common bean plants (P. vulgaris)
inoculated with a salt-tolerant strain (wild-type R. tropici) formed more active
symbiosis than did its decreased salt-tolerant mutant derivatives (Tejera et al.
2004). The growth, nodule weight and nitrogenase activity of salinized common
bean (P. vulgaris) plants were improved by abscisic acid (ABA) pretreatment
(Khadri et al. 2007). ABA treatment limited Na+ translocation to shoots, which is
a strategy of bean to limit Na+ toxicity. When water-stressed P. vulgaris was
inoculated with salt-tolerant rhizobia, the nodulation, shoot dry weight and grain
yield were significantly increased under nonirrigated conditions (Mnasri et al.
2007). The improvement of the cultivation of P. vulgaris under arid conditions
can take place by enhancing stress tolerance of N2-fixing rhizobia (Priefer et al.
2001).
Alterations of plant growth, nitrogenase activity and nutrient concentration as a
consequence of salt treatment was studied in five chickpea (Cicer arietinum)
cultivars from Spain and Syria, in symbiosis with Mesorhizobium ciceri (Tejera
et al. 2006). One cultivar (ILC1919) was moderately salt tolerant at 100 mM NaCl,
and exhibited less N2 fixation inhibition, a higher root-to-shoot ratio, normalized
nodule weight and shoot K/Na ratio, and a reduced foliar accumulation of Na+. The
results revealed the effectiveness of these nutritional and physiological indicators
in the selection of salinity-tolerant and symbiotic N2-fixing chickpea plants.
Since nitrogenase, the enzyme responsible for N2 fixation, is O2 labile, nodules
have evolved mechanisms to regulate their permeability to O2 and maintain the
infected-cell O2 concentration at the lowest level (about 5–50 nM) compared with
approximately 250 mM for cells in equilibrium with air (Aydi et al. 2004). Nodule
conductance to O2 diffusion is a major factor of the inhibition of N2 fixation by soil
salinity. However, salinity did not significantly change the nodule conductance and
nodule permeability of the plant, and the salt tolerance of this variety appears to be
associated with stability in nodule conductance and the capacity to form nodules
under salt constraint (L’taief et al. 2007). Salinity stress (75 mM NaCl) signifi-
cantly increased the nodule conductance of M. truncatula plants inoculated with
242 H.H. Zahran
S. meliloti (Aydi et al. 2004). The sensitivity to salinity appears to be associated

with an increase in nodule conductance that supports the increased respiration of
N2-fixing nodules under salinity (Aydi et al. 2004). Nodule conductance (oxygen
uptake) in chickpea inoculated with M. ciceri was measured under salt (25 mM
NaCl) treatment (L’taief et al. 2007). The symbiotic performance of three strains of
rhizobia (M. ciceri, M. mediterraneum and S. medicae) with chickpea was inves-
tigated under salt stress (25 mM NaCl) conditions (Mhadhbi et al. 2004). M. ciceri,
the most efficient strain, seemed to allow the best tolerance to chickpea plants under
salt stress. The activity of enzymes involved in nitrogen metabolism, as well as
oxidative stress generation and heme oxygenase (HO) gene and protein expression
and activity, were analyzed in soybean (Glycine max L.) nodules exposed to 50, 100
and 200 mM NaCl (Zilli et al. 2008). Nitrogenase activity and leghemoglobin
content were diminished while ammonium content increased only at 200 mM
NaCl. The synthesis of several enzymes involved in nitrogen metabolism was
only changed at higher levels of salt (200 mM NaCl). HO activity, protein synthesis
and gene expression were significantly increased under 100 mM NaCl treatment.
The data demonstrated that the upregulation of HO, as part of an antioxidant
defense system, could protect N2 fixation and assimilation under saline stress
conditions. Under osmotic stress conditions, plants accumulate some compatible
osmolytes (e.g., proline, glycine betaine, etc.) to protect major processes such as
cell respiration, photosynthetic activity, nutrient transport, and nitrogen and carbon
metabolism. L. japonicus and M. truncatula, as legume models, provided a conve-
nient system to study plant–Rhizobium interactions (López et al. 2008). Plant
growth parameters, nitrogenase activity, and activities of trehalose-6-phosphate
synthase and trehalase of L. japonicus and M. truncatula decreased with
NaCl treatments (25 and 50 mM NaCl). Legume root-nodule N2-fixing activity
was severely affected by osmotic stress (100 mM NaCl) in M. truncatula
(Verdoy et al. 2006). High proline accumulation level was found in the trans-
genic M. truncatula plants under osmotic stress. N2 fixation in the transgenic
M. truncatula plants was significantly less affected by salt treatment compared to
the wild-type plants. This is the first time that a produced transgenic legume
displayed N2-fixing activity with enhanced tolerance to osmotic stress, and that
the essential role of proline in the maintenance of N2-fixing activity under osmotic
stress was ascertained (Verdoy et al 2006).
Under stress treatments, some genes of nodules are downregulated while others
are upregulated as an adaptive response to abiotic stress. To identify molecular
markers of drought stress in nodules of soybean, the suppression subtractive
hybridization (SSH) was chosen as a method to isolate rare but differentially
expressed genes (Clement et al. 2008). In this work, 56 cDNA fragments were
validated as drought stress-induced genes by reverse northern hybridization, and
analysis was focused on two genes, encoding respectively a ferritin and a metal-
lothionein, which are involved in homeostasis (cellular metal balance) and detoxi-
fication of metals. These two genes showed high accumulation of transcripts
restricted to infected cells of nodules in response to drought. The growth of Lotus
creticus, a major forage legume in the arid climate of Tunisia, was studied under
salt stress (Rejili et al. 2007). The presence of salt (50–400 mM) negatively affected
plant growth, such negative effects being more obvious on aerial organs than on
roots. The content of Na+ in both parts (above and below ground) increased;
however, plants probably expressed a certain mechanism of compartmentation.
Under salt stress, salt-tolerant plants maintain a high concentration of K+ and a
low concentration of Na+ in the cytosol (Zhu 2003; Zahran et al. 2007). They do this
by regulating the expression and activity of K+ and Na+ transporters and of H+
pumps that generate the driving force for transport. The effect of salinity (EC up to
3.8 dS m 1) on yield and nitrogen uptake of four grain legumes (broad bean,
chickpea, lentil and soybean) was examined (Van Hoorn et al. 2001). Salinity
affected crop yield, crop total nitrogen uptake, and the nitrogen contribution of
the soil. A salinity effect on N2 fixation explains, at least partly, the salt sensitivity
of grain legumes. Exposure of mungbean to water deficit results in variability in
grain yield, nitrogen accumulation, and grain quality (Thomas et al. 2004). Nodule
activities are dramatically affected in most legumes under several environmental
constrains, an effect which results in crop yield loss. A negative response to drought
stress effects on N2 fixation by root-nodules of P. sativum is mainly controlled in
nodules rather than by a systemic nitrogen signal (Marino et al. 2007).
Wild forage legumes are the most potential forage legumes grown in salt-
affected soils (Ashraf and Bashir 2003; Zahran et al. 2007) which play a vital
role in long-term maintenance of soil productivity because of their high N2-fixing
ability. The effects of salt stress on growth and metabolic changes in nodules and
other plant parts of two leguminous species, P. vulgaris (salt sensitive) and
S. aculeata (salt tolerant), were investigated (Ashraf and Bashir 2003). Plants of
P. vulgaris were subjected to 3.5 dS m 1 of NaCl and those of S. aculeata to
13 dS m 1 of NaCl. S. aculeate showed a small reduction in the number of root-
nodules, high proline content in leaves, high glycine betaine content in all plant
parts, a high photosynthetic rate, and low uptake of Cl in the leaves. The nodules
of S. aculeata seemed to have played an active role in the high salt tolerance of the
species because the nodules had lower concentrations of both Na+ and Cl and a
higher level of glycine betaine as compared to those found in P. vulgaris. Many
legumes form symbioses with both rhizobia and AM- fungi. Dual inoculation with
both microorganisms results in a tripartite mutualistic symbiosis and generally
increases plant growth to a greater extent than inoculation with only one organism.
Enhanced acquisition of P by the host and effects on molecular signaling between
the three symbionts may explain the synergism of AMF and rhizobia. De Varennes
and Goss (2007) investigated how the rate of colonization by indigenous AM fungi
(AMF) affects the interaction between S. meliloti and M. truncatula and AMF. The
effects of dual inoculation of V. faba plants with NFB such as Azospirillum
brasilense and AMF were investigated at five levels (0–6.0 dS m 1) of NaCl
using irrigating water (Rabie and Al Madini 2005). The AMF infection significantly
increased tolerance to salinity, mycorrhizal dependency, P level, phosphatases,
nodule number, nitrogen level, protein content, and nitrogenases in all salinized
V. faba plants, in comparison with control and non-AMF-colonized plants, either in
the absence or presence of NFB. The study provides evidence for benefits of NFB to
244 H.H. Zahran
AMF in the protection of host plants against the detrimental effects of salt. The
bacterial–AMF–legume tripartite symbioses could be a new and interesting ap-
proach for increasing tolerance of legumes to salinity conditions (Rabie and Al
Madini 2005). The effects of a mycorrhizal fungus (Glomus fasciculatum) on
growth and mineral acquisition by A. nilotica seedlings over a wide range of soil
salinity was investigated (Giri et al. 2007). Mycorrhizal plants maintained greater
root and shoot biomass and higher P, Zn, and Cu concentrations than uninoculated
plants at all salinity levels. Mycorrhizal fungus alleviated deleterious effects of
salinity on plant growth that could be primarily related to improved P nutrition.
The improved K/Na ratios in root and shoot tissues of mycorrhizal plants may
help in protecting disruption of K-mediated enzymatic processes under salt stress
conditions.
11.6 Improvement of Inoculation Technology
Many of the microbial inoculants applied worldwide are based on solid peat
formulations. This has been mostly true for well-developed legume inoculants
due to peat bacterial protection properties. Commercial legume inoculant formula-
tions include powder or granular carriers, and broth cultures or liquid formulations,
for agricultural applications. Due to unavailability of peat, more readily available
alternative carriers for inoculant production should be investigated. An appropriate
material for carrying microorganisms must offer special properties, such as water-
holding capacity, chemical and physical uniformity, and lack of compounds toxic to
microbial strains, and be environmentally safe. However, osmotic stress, nutrient
deficiencies (especially phosphorous), and the lack of efficient strains of rhizobia
are major factors limiting symbiotic N2 fixation and yield of legumes. Ideally,
inoculation is required in the absence of compatible resident rhizobia, where the
native rhizobial population density is very low, or where the resident rhizobia are
less infective than alternative (inoculant) strains. Soils lacking in compatible
rhizobia are found in areas where indigenous legumes are absent or where levels
of pH, osmotic stress, high temperature, and heavy metals are detrimental to
rhizobial populations. Chemining’wa and Vessey (2006) found that effective
strains of R. leguminosarum bv viciae occur broadly in agricultural soils; neverthe-
less, there is a tendency for increased symbiotic efficiency with the use of commer-
cial inoculation.
Effective rhizobial inoculants, inoculation carriers able to support viable rhizo-
bia, and appropriate methods of storage, are required for successful symbiosis.
Rhizobial density and the period for which these are maintained are highly variable.
Even where strains are carefully evaluated, the lack of competitiveness with
indigenous rhizobia and inconsistency in their performance can lead to a decline
in nitrogen-fixing efficiency under field environments. And, hence, before applying
rhizobia, the establishment and persistence of such strains must be considered in
inoculation studies. Perlite and compost from the cork industry were superior to
peat in maintaining survival of different rhizospheric bacteria. Similarly, some

culture media maintained more than 109 cfu mL 1 of S. fredii or B. japonicum
after 3 months of storage. Soybean plants inoculated with two highly effective
rhizobia, using the carriers cork compost, perlite and liquid formulations, produced
seed yields that were not significantly different to those produced by peat-based
inoculants (Albareda et al. 2008). Responses to bean rhizobia inoculation and P
fertilization were investigated (Zaman-Allah et al. 2007), and a significant effect of
inoculation and P supply on nodulation, N content and growth was found. It is
concluded that inoculation with suitable rhizobia with supply of additional P is a
technology that may improve symbiotic N2 fixation and yield in the common bean.
Rhizobial inoculation of legume seeds or soils has been well studied. However,
much less work has been done regarding the association and growth-promoting
activities of rhizobia with nonlegumes. Efforts at extending N2-fixing ability to
important nonleguminous crops such as cereals has long been a major goal of
workers in the field of biological N2 fixation and would be a useful technology
for increasing crop yields (Matiru and Dakora 2004). Although some inoculation
attempts have resulted in nodule formation in cereal plants, there was no evidence
of N2 fixation. Nevertheless, associated rhizobia produce molecules such as auxins,
cytokinins, abscisic acids, IAA, lumichrome, riboflavin, lipochitooligosaccharides
and vitamins that promote plant growth. Therefore, rhizobial colonization and
infection of cereal roots would be expected to increase plant development
and grain yield (Matiru and Dakora 2004). Roots of sorghum (Sorghum bicolour)
and millet (Pennisetum americanum) were easily infected by rhizobia from five
unrelated legume genera. Plant growth and P uptake, in sorghum in particular, were
significantly increased by rhizobial inoculation, suggesting that field selection of
suitable rhizobia/cereal combinations could increase yields and produce fodder for
livestock production (Matiru and Dakora 2004). Inoculation of rice (Oryza sativa)
seedlings with R. leguminosarum bv. trifolii and Rhizobium sp. stimulated rice
growth and increased grain and straw yields (Biswas et al. 2000). The tested
rhizobial strains produced IAA in vitro, and some of them reduced acetylene to
ethylene in association with rice under laboratory growth conditions. Enhancement
of legume N2 fixation by coinoculation of rhizobia with plant growth-promoting
bacteria is thus important for improving nitrogen availability in sustainable agri-
culture.
Endophytes recovered from the surface-sterilized root-nodules of pigeonpea
(Cajanus cajan), designated as nonrhizobial isolates (Rajendran et al. 2008),
facilitated plant growth and increased the fresh weight and symbiotic traits when
coinoculated with the rhizobial bioinoculant strain, isolated from nodules of the
same plant. The nonrhizobial isolates were not able to nodulate pigeonpea and nor
did they show significant plant growth promotion when inoculated alone without
Rhizobium spp. The nonrhizobial isolates were identified as Bacillus megaterium
following amplified ribosomal DNA restriction analysis and partial sequences of
16S rRNA genes studies. Several papers have reported on dual inoculation with
rhizobia and NFB (e.g., Azospirillum) and P-solubilizing bacteria (e.g., Pseudomonas).
The legume growth, yield, and P and N uptake, could be increased by coinoculation
246 H.H. Zahran
(Graham and Vance 2000). A general positive effect of Azospirillium–Rhizobium

coinoculation on the expression of nod genes by R. tropici and R. etli, and on
nodulation, was observed in the presence of root exudates (Dardanelli et al. 2008).
The negative effects obtained under salt stress on nod gene expression and on nod
factor appearance were relieved in coinoculated plants. The effect of dual inoculation
of P-solubilizing bacteria (Pseudomonas putida) on the symbiosis of S. meliloti and
B. japonicum with alfalfa and soybean, respectively, was evaluated (Rosas et al.
2006). Modification of shoot and root dry weights occurred in soybean but not in
alfalfa in the presence of P. putida.
11.7 Breeding and Selection for Enhanced N2 Fixation

in Crop Legumes
Biotechnology is a powerful tool that has potential to contribute to sustainable

agriculture. Over the years, biotechnology has emerged as a promising tool to
overcome stresses in plants, but to date, progress has been limited in legumes.
However, molecular approaches, such as marker-assisted breeding, genetic trans-
formation, tissues cultures, gene expression, transcriptomics, transcription factors,
proteomics, etc., can contribute to speed-up classical breeding. Dita et al. (2006)
reviewed many of the modern biotechnological techniques and evaluated their
applications to improve tolerance of legumes to abiotic stress. The use of genetic
and genomic analysis to help identifying DNA regions tightly linked to agronomic
traits in crops, the so-called molecular markers, can facilitate breeding strategies for
crop improvement. Numerous molecular marker-related techniques have been used
in legumes in relation to abiotic stress. As a result, genetic maps for many species
have been established and quantitative trait loci (QTLs) have been located in some
legumes, as reported for soybean, under salt stress (Lee et al. 2004). This improved
the knowledge of genetic control of specific resistance and/or tolerance in many
legumes by providing information on the chromosomal number and location, and
individual or interactive effects of the QTLs involved. In plants, there is a poor
understanding of most abiotic stress responses. Thus, the successful use of genetic
transformation requires a better physiological and molecular understanding of these
stresses. Enhancement of resistance against both hyperosmotic stress and Na+
toxicity is necessary for successful molecular breeding of salt-tolerant plants.
Introduction of genes for osmolyte biosynthesis is useful to increase hyperosmotic
tolerance of plant cells (Yoshida 2002). Overexpression of a single-gene controlling
vacuolar or plasma membrane Na+/H+ antiport protein in plants (e.g., M. intertexta;
Zahran et al. 2007) provided them with a high level of salt tolerance. Identification
of differentially expressed genes is particularly important to understand stress
responses in plants. With respect to abiotic stress, gene expression analyses have
been mainly based on studies with cloned genes. Transcriptomic tools are a good
option for legume breeding to environmental stresses. Using a modified cDNA-AFLP
technique in soybean, 140 differentially expressed cDNA fragments were obtained

by comparing control and osmotic-treated plants. Some of the responsive genes
encoded ion transporters, transcription factors and redox enzymes (Umezawa et al.
2002). Further, recent technological developments have allowed the establishment
of valuable methods for quantitative and qualitative protein profiling. This ap-
proach is very important in evaluating stress responses because mRNA levels do
not always correlate with protein accumulation (Dita et al. 2006).
Graham and Vance (2000) reviewed the developments and research areas which
are critical for improvement of N2 fixation. Genetic improvement in nodulation and
nitrogen fixation can be done by indirect selection for growth and seed yield in
plants grown under N limitation conditions, and by direct selection for nodule mass,
acetylene reduction activity and xylem ureide content. For all legumes, there is
great potential to increase the percent of legume N derived from N2 fixation as well
to enhance the total N2 fixed through improved management and genetic modifica-
tion of the plant. Herridge and Rose (2000) reviewed strategies and operational
frameworks for conducting selection and breeding programs to enhance N2 fixation,
and methods for measuring N2 fixation that have been used in such programs. Three
general strategies for increasing legume N2 fixation through breeding. Firstly, there
is the maximizing of legume (biomass) and seed yield under constraints imposed
by agronomic management and the environment. This approach assumes a capacity
for N2 fixation sufficient to satisfy increased N demand of larger plants, but
has particular application to lower-yielding plants such as common bean, lentil
(L. esculentum), mungbean, and chickpea. Secondly, legume ability to nodulate and
fix N2 in the presence of soil nitrate is enhanced. Variation for symbiotic nitrate
tolerance exists in natural plant populations and has also been created through
mutagenesis. Thirdly, legume nodulation is optimized through specific nodulation
traits (e.g., mass and duration). Crop improvement through genetic engineering has
become a reality, and it is now possible to transform many grain legumes using
A. tumefaciens as a vector for legume transformation; the inserted DNA can be
either a specific gene with a specific biochemical function, a regulatory gene that
controls a network of other genes, or multiple genes to generate long-term durable
resistance. Rhizobia were examined for their ability to be used as potential vehicles
for plant transformation. Several species of rhizobia were successfully transformed
with broad host-range plasmids of different replicons. A genetic binary vector
(pPZP211) was maintained in M. loti and stably inherited during nodulation
(Vincze and Bowra 2006).
11.8 Conclusion
The information available in the literature highlights the significance of N2-fixing

rhizobia–legumes symbioses for improving soil fertility and, consequently, plant
productivity. The symbiotic bacteria that colonize nodules of these plants are very
diverse, with prominent ecological characteristics and specific genetic traits.
248 H.H. Zahran
These bacteria are classified into more than 70 genomic species belonging to several
genera. With all of this genomic diversity, it is no wonder that the systematics of
root-nodule bacteria are changing every day. Recent developments in the field
of Rhizobium biology has taken place due to the advances in the molecular and
genetic techniques. Such techniques provide an effective tool to characterize
and identify the bacterial isolates vis-a-vis examining the physiological responses of
the host plant. Molecular identification of rhizobia depends on the symbiotic and
ribosomal genes. However, in some cases, host specificity of rhizobial strains was
not related to ribosomal genes (16S rRNA). The evolution of ribosome and symbi-
otic genes may have been diverse. The symbiotic and N2-fixing genes appear to be
spread in various bacterial taxa and not restricted to what is known as rhizobium
bacteria of the alphaproteobacteria. In recent years, plenty of new strains of
symbiotic bacteria were classified in nonrhizobial genera, namely Burkholderia
and Cupriavidus, in the b-proteobacteria.
Symbiotic characteristics are some of the basic criteria for selecting compatible
rhizobia. Numerous species of the newly isolated and identified nodule bacteria
have shown substantial symbiotic activities and proved that legumes exhibit biodi-
versity with regards to symbiotic bacteria and also the host plants. Enhancement of
N2 fixation of the existing symbiotic systems, and those newly discovered symbi-
otic systems, is possible through the inoculation of legumes with compatible and
effective strains of bacteria. However, inoculation technology and delivery system
is needed to be improved. Successful symbiosis can be obtained by coinoculation or
mixed inoculation with other plant-growth promoting rhizobacteria including N2
fixers. Legumes’ N2 fixation enhancement is likely to improve the productivity of
associated nonlegume plants by providing them with N and growth-promoting
substances. Suitable rhizobia–cereal combinations could increase yields and pro-
duce fodder for livestock. Bacterial–AMF–legume tripartite symbioses could also
be viable alternatives to enhance N2 fixation of legumes especially under salt stress
conditions.
The N2-fixing activity of root-nodules is severely affected by osmotic stress.
However, elucidation of genes involved in the regulation of ion transport and their
expression has increased our understanding about how legumes circumvent the
stress conditions. One of the strategies adopted to combat the problem of soil
salinity is the usage of transgenic leguminous plants. The transgenic plants in
certain cases are more salt tolerant than wild-type plants. For example, transgenic
plants (e.g., M. truncatula) accumulated higher levels of proline and fixed N2
actively more than wild-type under osmotic stress, and is the first transgenic legume
that displayed N2-fixing activity with enhanced tolerance to osmotic stress. How-
ever, selection of a salt-tolerant effective Rhizobium–legume symbiosis is of para-
mount importance. The symbiotic bacteria have shown a variable response to
abiotic stresses such as salinity, acidity, and aridity. Some strains of symbiotic
bacteria exhibit substantial stress-tolerant abilities. Understanding the mechanisms
of osmoadaptation of rhizobia at the molecular level would help to rationally design
and engineer better strains for field applications.
References
Albareda M, Rodrı́guez-Navarro DN, Camacho M, Temprano FJ (2008) Alternatives to peat as a

carrier for rhizobia inoculants: solid and liquid formulations. Soil Biol Biochem 40:2771–2779
Ashraf M, Bashir A (2003) Salt stress induced changes in some organic metabolites and ionic
relations in nodules and other plant parts of two crop legumes differing in salt tolerance. Flora
198:486–498
Aydi S, Drevon J-J, Abdelly C (2004) Effect of salinity on root-nodule conductance to the oxygen
diffusion in the Medicago truncatula-Sinorhizobium meliloti symbiosis. Plant Physiol Bio-
chem 42:833–840
Ba S, Willems A, De Lajudie P, Roche P, Jeder H, Quatrini P, Neyra M, Ferro M, Promé J-C,
Gillis M, Boivin-Masson C, Lorquin J (2002) Symbiotic and taxonomic diversity of rhizobia
isolated from Acacia tortilis subsp: raddiana in Africa. Syst Appl Microbiol 25:130–145
Bala A, Murphy PJ, Osunde AO, Giller KE (2003) Nodulation of tree legumes and the ecology of
their native rhizobial populations in tropical soils. Appl Soil Ecol 22:211–223
Barrett CF, Parker MA (2005) Prevalence of Burkholderia sp. nodule symbionts on four mimosoid
legumes from Barro Colorado Island, Panama. Syst Appl Microbiol 28:57–65
Barrett CF, Parker MA (2006) Coexistence of Burkholderia, Cupriavidus, and Rhizobium sp.
nodule bacteria on two Mimosa spp. in Costa Rica. Appl Environ Microbiol 72:1198–1206
Benata H, Mohammed O, Noureddine B, Abdelbasset B, Abdelmoumen H, Muresu R,
Squartini A, El Idrissi MM (2008) Diversity of bacteria that nodulate Prosopis juliflora in
the eastern area of Morocco. Syst Appl Microbiol 31:378–386
Bernatchez F, Jeannotte R, Begg CBM, Hamel C, Whalen JK (2008) Soil fertility and arbuscular
mycorrhizal fungi related to trees growing on smallholder farms in Senegal. J Arid Environ
72:1247–1256
Biswas JC, Ladha JK, Dazzo FB, Yanni YG, Rolfe BG (2000) Rhizobial inoculation influences
seedling vigor and yield of rice. Agron J 92:880–886
Bouhmouch I, Souad-Mouhsine B, Brhada F, Aurag J (2005) Influence of host cultivars and
Rhizobium species on the growth and symbiotic performance of Phaseolus vulgaris under salt
stress. J Plant Physiol 162:1103–1113
Chemining’wa GN, Vessey JK (2006) The abundance and efficacy of Rhizobium leguminosarum
bv. viciae in cultivated soils of the eastern Canadian prairie. Soil Biol Biochem 38:294–302
Chen W-M, Lee T-M, Lan C-C, Cheng C-P (2000) Characterization of halotolerant rhizobia
isolated from root nodules of Canavalia rosea from seaside areas. FEMS Microbiol Ecol
34:9–16
Chen W-M, James EK, Chou J-H, Sheu S-Y, Yang S-Z, Sprent JI (2005) b-rhizobia from Mimosa
pigra, a newly discovered invasive plant in Taiwan. New Phytol 168:661–675
Clement M, Lambert A, Herouart D, Boncompagni E (2008) Identification of new up-regulated
genes under drought stress in soybean nodules. Gene 426:15–22
Dardanelli MS, Fernández De Córdoba FJ, Espuny MR, Rodrı́guez Carvajal MA, Soria Dı́az ME,
Gil Serrano AM, Okon Y, Megı́as M (2008) Effect of Azospirillum brasilense coinoculated
with Rhizobium on Phaseolus vulgaris flavonoids and nod factor production under salt stress.
De Varennes A, Goss MJ (2007) The tripartite symbiosis between legumes, rhizobia and indige-
nous mycorrhizal fungi is more efficient in undisturbed soil. Soil Biol Biochem 39:2603–2607
Denison RF, Kiers ET (2004) Why are most rhizobia beneficial to their plant hosts, rather than
parasitic? Microb Infect 6:1235–1239
Dita MA, Rispail N, Prats E, Rubiales D, Singh KB (2006) Biotechnology approaches to overcome
biotic and abiotic stress constraints in legumes. Euphytica 147:1–24
Domı́nguez-Ferreras A, Pérez-Arnedo R, Becker A, Olivares J, Soto MJ, Sanjuan J (2006)
Transcriptome profiling reveals the importance of plasmid pSymB for osmoadaptation of
Sinorhizobium meliloti. J Bacteriol 188:7617–7625
250 H.H. Zahran
Downie JA (2005) Legume haemoglobins: symbiotic nitrogen fixation needs bloody nodules. Curr
Biol 15:196–198
Duodu S, Bhuvaneswari TV, Gudmundsson J, Svenning MM (2005) Symbiotic and saprophytic
survival of three unmarked Rhizobium leguminosarum biovar trifolii strains introduced into the
field. Environ Microbiol 7:1049–1058
El-Akhal MR, Rincon A, Arenal F, Lucas MM, El Mourabit N, Barrijal S, Pueyo JJ (2008) Genetic
diversity and symbiotic efficiency of rhizobial isolates obtained from nodules of Arachis
hypogaea in northwestern Morocco. Soil Biol Biochem 40:2911–2914
Essendoubi M, Brhada F, Eljamali JE, Filali-Maltouf A, Bonnassie S, Georgeault S, Blanco C,
Jebbar M (2007) Osmoadaptative responses in the rhizobia nodulating Acacia isolated from
south-eastern Moroccan Sahara. Environ Microbiol 9:603–611
Ge-Hong W, Xue-Ying Y, Zhi-Xin Z, Ya-Zhen Y, Lindström K (2008) Strain Mesorhizobium sp.
CCNWGX035: a stress-tolerant isolate from Glycyrrhiza glabra displaying a wide host range
of nodulation. Pedosphere 18:102–112
Giri B, Kapoor R, Mukerji KG (2007) Improved tolerance of Acacia nilotica to salt stress by
arbuscular mycorrhiza, Glomus fasciculatum may be partly related to elevated K/Na ratios in
root and shoot tissues. Microb Ecol 54:753–760
González Ruiz T, Rodrı́guez Zaragoza S, Ferrera Cerrato R (2008) Fertility islands around
Prosopis laevigata and Pachycereus hollianus in the drylands of Zapotitlán Salinas, México.
J Arid Environ 72:1202–1212
Graham PH (2008) Ecology of the root-nodule bacteria of legumes. In: Dilworth MJ, James EK,
Sprent JI, Newton WE (eds) Nitrogen-fixing leguminous symbiosis. Springer, Dordrecht, The
Netherland, pp 23–58
Graham PH, Vance CP (2000) Nitrogen fixation in perspective: an overview of research and
extension needs. Field Crops Res 65:93–106
Grossman JM, Sheaffer C, Wyse D, Bucciarelli B, Vance C, Graham PH (2006) An assessment of
nodulation and nitrogen fixation in inoculated Inga oerstediana, a nitrogen-fixing tree shading
organically grown coffee in Chiapas, Mexico. Soil Biol Biochem 38:769–784
Han TX, Wang ET, Han LL, Chen WF, Sui XH, Chen WX (2008) Molecular diversity and
phylogeny of rhizobia associated with wild legumes native to Xinjiang, China. Syst Appl
Herridge D, Rose I (2000) Breeding for enhanced nitrogen fixation in crop legumes. Field Crops
Res 65:229–248
Howieson J, Ballard R (2004) Optimizing the legume symbiosis in stressful and competitive
environments within southern Australia – some contemporary thoughts. Soil Biol Biochem
36:1261–1273
Hoy JA, Hargrove MS (2008) The structure and function of plant hemoglobins. Plant Physiol
Biochem 46:371–379
Jiang JQ, Wei W, Du BH, Li XH, Wang L, Yang SS (2004) Salt-tolerance genes involved in cation
efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characteri-
zation of TN5 mutants. FEMS Microbiol Lett 239:139–146
Khadri M, Tejera NA, Lluh C (2007) Sodium chloride-ABA interaction in two common bean
(Phaseolus vulgaris) cultivars differing in salinity tolerance. Environ Exp Bot 60:211–218
Lee GJ, Boerma HR, Villagarcia MR, Zhou X, Carter TE Jr, Li Z, Gibbs MO (2004) A major QTL
conditioning salt tolerance in S-100 soybean and descendent cultivars. Theor Appl Genet
109:1610–1619
Liu J, Wang ET, Chen WX (2005) Diverse rhizobia associated with woody legumes Wisteria
sinensis, Cercis racemosa and Ammorpha fruticosa grown in the temperate zone of China. Syst
Appl Microbiol 28:465–477
Long SR (2001) Genes and signals in the Rhizobium-legume symbiosis. Plant Physiol 125:69–72
López M, Herrera-Cervera JA, Iribarne C, Tejera NA, LLuch C (2008) Growth and nitrogen
fixation in Lotus japonicus and Medicago truncatula under NaCl stress: nodule carbon
metabolism. J Plant Physiol 165:641–650
L’taief B, Sifi B, Zaman-Allah M, Drevon J-J, Lachaâl M (2007) Effect of salinity on root-nodule
conductance to the oxygen diffusion in the Cicer arietinum-Mesorhizobium ciceri symbiosis.
J Plant Physiol 164:1028–1036
Mahdhi M, Nzoué A, DeLajudie P, Mars M (2008) Characterization of root-nodulating bacteria on
Retama raetam in arid Tunisian soils. Prog Nat Sci 18:43–49
Marino D, Frendo P, Ladrera R, Zabalza A, Puppo A, Arrese-Igor C, González EM (2007)
Nitrogen fixation control under drought stress. Localized or systemic? Plant Physiol
143:1968–1974
Matiru VN, Dakora FD (2004) Potential use of rhizobial bacteria as promoters of plant growth for
increased yield in landraces of African cereal crops. Afr J Biotechnol 3:1–7
Menna P, Hungria M, Barcellos FG, Bangel EV, Hess PN, Martı́nez-Romero E (2006) Molecular
phylogeny based on the 16S rRNA gene of elite rhizobial strains used in Brazilian commercial
inoculants. Syst Appl Microbiol 29:315–332
Mhadhbi H, Jebara M, Limam F, Aouani ME (2004) Rhizobial strain involvement in plant growth,
nodule protein composition and antioxidant enzyme activities of chickpea-rhizobia symbioses:
modulation by salt stress. Plant Physiol Biochem 42:717–722
Mithöfer A (2002) Suppression of plant defence in rhizobia-legume symbiosis. Trends Plant Sci
7:440–444
Mnasri B, Aouani ME, Mhamdi R (2007) Nodulation and growth of common bean (Phaseolus
vulgaris) under water deficiency. Soil Biol Biochem 39:1744–1750
Moreira FMD, Cruz L, De Faria SM, Marsh T, Martı́nez-Romero E, Pedrosa FD, Pitard RM,
Young JPW (2006) Azorhizobium doebereinerae sp. nov. microsymbiont of Sesbania virgata
(Caz.) Pers. Syst Appl Microbiol 29:197–206
Moschetti G, Peluso A, Protopapa A, Anastasio M, Pepe O, Defez R (2005) Use of nodulation
pattern, stress tolerance, nodC gene amplification, RAPD-PCR and RFLP-16S rDNA analysis
to discriminate genotypes of Rhizobium leguminosarum biovar viciae. Syst Appl Microbiol
28:619–631
Moulin L, Béna G, Boivin-Masson C, Ste˛pkowski T (2004) Phylogenetic analysis of symbiotic
nodulation genes support vertical and lateral gene co-transfer within the Bradyrhizobium
genus. Mol Phylogenet Evol 30:720–732
Mulder L, Hogg B, Bersoult A, Cullimore JV (2005) Integration of signalling pathways in the
establishment of the legume-rhizobia symbiosis. Physiol Plant 123:207–218
Musiyiwa K, Mpepereki S, Giller KE (2005) Symbiotic effectiveness and host ranges of indige-
nous rhizobia nodulating promiscuous varieties in Zimbabwean soils. Soil Biol Biochem
37:1169–1176
Nandasena KG, O’Hara GW, Tiwari RP, Yates RJ, Kishinevsky BD, Howieson JG (2004)
Symbiotic relationships and root nodule ultrastructure of the pasture legume Biserrula
pelecinus L. – a new legume in agriculture. Soil Biol Biochem 36:1309–1317
Nkot LN, Krasova-Wade T, Etoa FX, Sylla SN, Nwaga D (2008) Genetic diversity of rhizobia
nodulating Arachiss hypogaea L. in diverse land use systems of humid forest zone in Camer-
oon. Appl Soil Ecol 40:411–416
Nogales J, Campos R, Ben Abdelkhalek H, Olivares J, LLuch C, Sanjuan J (2002) Rhizobium
tropici genes involved in free-living salt tolerance are required for the establishment of
efficient-nitrogen-fixing symbiosis with Phaseolus vulgaris. Mol Plant Microb Interact
15:225–232
Odee DW, Haukka K, McInroy SG, Sprent JI, Sutherland JM, Young JPW (2002) Genetic and
symbiotic characterization of rhizobia isolated from tree and herbaceous legumes grown in
soils from ecologically diverse sites in Kenya. Soil Biol Biochem 34:801–811
Ott T, Van Dongen JT, Günther C, Krusell L, Desbrosses G, Vigeolas H, Bock V, Czechowski T,
Geigenberger P, Udavardi MK (2005) Symbiotic leghemoglobins are crucial for nitrogen
fixation in legume root nodules but not for general plant growth and development. Curr Biol
15:531–535
252 H.H. Zahran
Priefer UB, Aurag J, Boesten B, Bouhmouch I, Defez R, Filali-Maltouf A, Miklis M, Moawad H,

Mouhsine B, Prell J, Schlüter A, Senatore B (2001) Characterization of Phaseolus symbionts
isolated from Mediterranean soils and analysis of genetic factors related to pH tolerance.
J Biotechnol 91:223–236
Rabie GH, Al Madini AM (2005) Role of bioinoculants in development of salt-tolerance of Vicia
faba plants under salinity stress. Afr J Biotechnol 4:210–222
Rajendran G, Sing F, Desai AJ, Archana G (2008) Enhanced growth and nodulation of pigeon pea
by co-inoculation of Bacillus strains with Rhizobium spp. Bioresource Technol 99:4544–4550
Raza S, Jørnsgård B, Abou-Taleb H, Christiansen JL (2001) Tolerance of Bradyrhizobium sp.
(Lupini) strains to salinity, pH, CaCO3 and antibiotics. Lett Appl Microbiol 32:379–383
Rejili M, Vadel AM, Guetet A, Neffatti M (2007) Effect of NaCl on the growth and the ionic
balance K+/Na+ of two populations of Lotus creticus (L.) (Papilionaceae). South Afr J Bot
73:623–631
Rosas SB, Andrés JA, Rovera M, Correa NS (2006) Phosphate-solubilizing Pseudomonas putida
can influence the rhizobia-legume symbiosis. Soil Biol Biochem 38:3502–3505
Rüberg S, Tian Z-X, Krol E, Linke B, Meyer F, Wang Y, Pühler A, Weidner S, Becker A (2003)
Construction and validation of a Sinorhizobium meliloti whole genome DNA microarray:
genome-wide profiling of osmoadaptive gene expression. J Biotechnol 106:255–268
Sharma RS, Mohammed A, Mishra V, Babu CR (2005) Diversity in a promiscuous group of
rhizobia from three Sesbania spp. colonizing ecologically distinct habitats of the semi-arid
Delhi region. Res Microbiol 156:57–67
Sierra J, Nygren P (2006) Transfer of N fixed by a legume tree to the associated grass in a tropical
silvopastrol system. Soil Biol Biochem 38:1893–1903
Sprent JI (2008) 60Ma of legume nodulation: what’s new? what’s changing? J Exp Bot 59:1081–
1084
Stacey G, Libault M, Brechenmacher L, Wan J, May GD (2006) Genetics and functional genomics
of legume nodulation. Curr Opin Plant Biol 9:110–121
Steenkamp ET, Ste˛pkowski T, Przymusiak A, Botha WJ, Law IJ (2008) Cowpea and peanut in
Southern Africa are nodulated by diverse Bradyrhizobium strains harboring nodulation
genes that belong to the large pantropical clade common in Africa. Mol Phylogenet Evol
48:1131–1144
Tejera NA, Campos R, Sanjuan J, Lluch C (2004) Nitrogenase and antioxidant enzyme activities in
Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying toler-
ance to salt stress. J Plant Physiol 161:329–338
Tejera NA, Soussi M, LLuch C (2006) Physiological and nutritional indicators of tolerance to
salinity in chickpea plants growing under symbiotic conditions. Environ Exp Bot 58:17–24
Thomas RMJ, Fukai S, Peoples MB (2004) The effect of timing and severity of water deficit on
growth, development, yield accumulation and nitrogen fixation of mungbean. Field Crops Res
86:67–80
Toledo I, Lloret L, Martı́nez-Romero E (2003) Sinorhizobium americanus sp. Nov., a new
Sinorhizobium species nodulating native Acacia spp. in Mexico. Syst Appl Microbiol 26:54–64
Umezawa T, Mizuno K, Fujimura T (2002) Discrimination of genes expressed in response to the
ionic or osmotic effect of salt stress in soybean with c-DNA-AFLP. Plant Cell Environ
25:1617–1625
Unni S, Rao KK (2001) Protein and lipopolysaccharide profiles of salt-sensitive Rhizobium sp. and
exopolysaccharide deficient mutant. Soil Biol Biochem 33:111–115
Van Hoorn JW, Katerji N, Hamdy A, Mastrorilli M (2001) Effect of salinity on yield and nitrogen
uptake of four grain legumes and on biological nitrogen contribution from the soil. Agric Water
Manage 51:87–98
Verdoy D, De La Peña TC, Redondo FJ, Lucas MM, Pueyo JJ (2006) Transgenic Medicago
truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced
tolerance to osmotic stress. Plant Cell Environ 29:1913–1923
Vessey JK, Chemining’wa GN (2006) The genetic diversity of Rhizobium leguminosarum bv.
viciae in cultivated soils of the eastern Canadian prairie. Soil Biol Biochem 38:153–163
Villegas MDC, Rome S, Mauré L, Domergue O, Gardan L, Bailly X, Cleyet-Marel J-C, Brunel B
(2006) Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv.
medigaginis) of S. meliloti. Syst Appl Microbiol 29:526–538
Vincze E, Bowra S (2006) Transformation of rhizobia with broad-host range plasmids by using a
freeze-thaw method. Appl Environ Microbiol 72(2290):2293
Vinuesa P, Silva C, Lorite MJ, Izaguirre-Mayoral ML, Bedmar EJ, Martı́nez-Romero E (2005)
Molecular systematics of rhizobia based on maximum likelihood and Bayesian phylogenies
inferred from rrs, atpD, recA and nifH sequences, and their use in the classification of Sesbania
microsymbionts from Venezuelan wetlands. Syst Appl Microbiol 28:702–716
Vriezen JAC, De Bruijn FJ, Nüsslein K (2006) Desiccation responses and survival of Sinorhizo-
bium meliloti USDA 1021 in relation to growth phase, temperature, chloride and sulfate
availability. Lett Appl Microbiol 42:172–178
Vriezen JAC, De Bruijn FJ, Nüsslein K (2007) Response of rhizobia to desiccation in relation to
osmotic stress, oxygen and temperature. Appl Environ Microbiol 73:3451–3459
Wang FQ, Wang ET, Zhang YF, Chen WX (2006) Characterization of rhizobia isolated from
Albizia spp. in comparison with microsymbionts of Acacia spp. and Leucaena leucocephala
grown in China. Syst Appl Microbiol 29:502–517
Wei W, Jiang J, Li X, Wang L, Yang SS (2004) Isolation of salt-sensitive mutants from
Sinorhizobium meliloti and characterization of gene involved in salt tolerance. Lett Appl
Wei GH, Zhang ZX, Chen C, Chen WM, Ju WT (2007) Phenotypic and genetic diversity of
rhizobia isolated from nodules of the legume genera Astragalus, Lespedeza and Hedysarum in
northwestern China. Microbiol Res 163:651–662
Wolde-Meskel E, Terefework Z, Lindström K, Frostegård A (2004) Rhizobia nodulating African
Acacia spp. and Sesbania sesban trees in Southern Ethiopian soils are metabolically and
genomically diverse. Soil Biol Biochem 36:2013–2025
Ya-mei G, Yi-qiang H, Hui T, Dong-mei S, Yan-jie W, Wei-dong W (2008) Analysis of simple
sequence repeats in genomes of rhizobia. Agric Sci China 7:1189–1195
Yang JK, Yuan TY, Zhang WT, Zhou JC, Li YG (2008) Polyphasic characterization of mung bean
(Vigna radiata L.) rhizobia from different geographical regions of China. Soil Biol Biochem
40:1681–1688
Yates RJ, Howieson JG, Reeve WG, Nandasena KG, Law IJ, Bräu L, Ardley JK, Nistelberger HM,
Real D, O’Hara GW (2007) Lotononis angolensis forms nitrogen-fixing, lupinoid nodules with
phylogenetically unique, fast-growing, pink-pigmented bacteria, which do not nodulate
L. bainesii or L. listii. Soil Biol Biochem 39:1680–1688
Yates RJ, Howieson JG, Reeve WG, Bräu L, Speijers J, Nandasena KG, Real D, Sezmis E,
O’Hara GW (2008) Host-strain mediated selection for an effective nitrogen-fixing symbi-
osis between Trifolium spp. and Rhizobium leguminosarum biovar trifolii. Soil Biol
Biochem 40:822–833
Yoshida K (2002) Plant biotechnology–genetic engineering to enhance plant salt tolerance.
J Biosci Bioeng 94:585–590
Zahran HH (1999) Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and
in an arid climate. Microbiol Mol Biol Rev 63:968–989
Zahran HH (2001) Rhizobia from wild legumes: diversity, taxonomy, ecology, nitrogen fixation
and biotechnology. J Biotechnol 91:143–153
Zahran HH (2005) Rhizobial nitrogen fixation in agriculture: biotechnological perspectives.
In: Ray RC (ed) Microbial biotechnology in agriculture and aquaculture, vol 1. Science,
Enfield, USA, pp 71–100
Zahran HH (2006a) Wild-legume rhizobia: biodiversity and potential as biofertilizers. In:
Rai MK (ed) Handbook of microbial biofertilizers. Haworth, New York, pp 203–222
254 H.H. Zahran
Zahran HH (2006b) Nitrogen (N2) fixation in vegetable legumes: biotechnological perspectives.

In: Ray RC, Ward OP (eds) Microbial biotechnology in horticulture, vol 1. Science, Enfield,
USA, pp 49–82
Zahran HH, Sprent JI (1986) Effects of sodium chloride and polyethylene glycol on root-hair
infection and nodulation of Vicia faba L. plants by Rhizobium leguminosarum. Planta
167:303–309
Zahran HH, Abdel-Fattah M, Ahmad MS, Zaki AY (2003) Polyphasic taxonomy of symbiotic
rhizobia from wild leguminous plants gowing in Egypt. Folia Microbiol 48:510–520
Zahran HH, Marin-Mansano MC, Sánchez-Raya AJ, Bedmar EJ, Venema K, Rodrı́guez-Rosales
MP (2007) Effect of salt stress on the expression of NHX-type ion transporters in Medicago
intertexta and Melilotus indicus. Physiol Plant 131:122–130
Zakhia F, Jeder H, Domergue O, Willems A, Cleyet-Marel J-C, Gillis M, Dreyfus B, De Lajudie P
(2004) Characterization of wild legume nodulating bacteria (LNB) in the infra-arid zone of
Tunisia. Syst Appl Microbiol 27:380–395
Zaman-Allah M, Sifi B, L’taief B, El Aouni MH, Drevon J-J (2007) Rhizobial inoculation and P
fertilization response in common bean (Phaseolus vulgaris) under glasshouse and field condi-
tions. Exp Agric 43:67–77
Zhu J-K (2003) Regulation of ion homeostasis under salt stress. Curr Opin Plant Biol 6:441–445
Zilli CG, Balestrasse KB, Yannarrelli GG, Polizio AH, Santa-Cruz DM, Tomaro ML (2008) Heme
oxygenase up-regulation under salt stress protects nitrogen metabolism in nodules of soybean
plants. Environ Exp Bot 64:83–89
Chapter 12
Monitoring the Development of Nurse Plant
Species to Improve the Performances of
Reforestation Programs in Mediterranean Areas
R. Duponnois, M. Hafidi, J. Thioulouse, A. Galiana, L. Ouahmane,

B. Dreyfus, and Y. Prin
Abstract In the Mediterranean basin, a millenarian history of overexploitation has

lead to the loss of most primeval forests and an increase of the surface area covered
by shrublands that represent stages of degradation of mature forests. In this situa-
tion, and since environmental characteristics act as barriers to succession, human
intervention is usually necessary to improve recovery of woodlands. Reafforesta-
tion is a common practice in Mediterranean areas to achieve this aim but its
performances are very low with high rates of early mortality making this practice
unprofitable in ecological as well as in economic terms.
In degraded semiarid ecosystems, shrub and tall-grass species grow following a
patchy distribution. Traditionally, shrubs growing near to newly planted trees are
considered heavy competitors, and are consequently removed before planting.
However, the vegetation patches usually constitute “fertility islands” or “resource
islands” which could promote the tree species development. It has previously been
assessed that some native plant species could act as “nurse plants” through their
positive impacts on soil abiotic characteristics (i.e., soil nutrient contents), but they
also exhibit a positive influence on soil microbiota, especially on symbiotic
R. Duponnois(*), B. Dreyfus
IRD. Laboratoire Commun de Microbiologie IRD/ISRA/UCAD, Centre de Recherche de Bel Air,
BP1386, Dakar, Sénégal
e-mail: robin.duponnois@ird.sn
M. Hafidi and L. Ouahmane
Equipe Ecologie et Environnement associée au CNRST, Faculté des sciences Semlalia, Université
Cadi Ayyad, Marrakech, Maroc
J. Thioulouse
Université Lyon 1; CNRS; UMR 5558 Laboratoire de Biométrie et Biologie Evolutive, 43
boulevard du 11 novembre 1918, VilleurbanneF-69622, France
A. Galiana and Y. Prin
CIRAD. UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2, Laboratoire des Symbioses Tropicales et
Méditerranéennes (LSTM), Montpellier, France
L. Ouahmane
Centre Régional de Recherche Foresti‘re, Marrakech, Maroc
256 R. Duponnois et al.
microorganisms including rhizobia and mycorrhizal fungi. In this chapter, an

attempt is made to assess the beneficial effects of plant nurses on the growth of
Mediterranean tree species like Cupressus species, and on the bio-functioning of
soils. Furthermore, the potential benefits of native plant species in the rehabilitation
of degraded areas especially in stressful conditions is reviewed and discussed.
12.1 Introduction
Shrub and tall-grass species growth following a patchy distribution are characteri-
stic of the plant communities in semiarid ecosystems and more particularly in
Mediterranean areas. The vegetation patches usually form “fertility islands”
(Garner and Steinberger 1989) or “resource islands” (Schlesinger et al. 1996) that
could be involved in the development of native plant species (Callaway 1995,
1997). It has been previously reported that some native plants species improve
their own environment by self-promoting changes in water infiltration, organic
matter, etc. (Bochet et al. 1999; Valladares and Pugnaire 1999), and could act as
“nurse plants” through their positive impacts on the survival of other native plant
species (Carrillo-Garcia et al. 2000). It is now well accepted that the spatial proxi-
mity among plants could be beneficial in environments such as Mediterranean-
type ecosystems that are characterized by abiotic stress (Boucher et al. 1982;
Callaway and Walker 1997; Gomez-Aparicio et al. 2004).
In semiarid Mediterranean ecosystems, desertification processes result from
scarce and irregular rainfall, long dry and hot summers, and man-mediated degrada-
tive activities like deforestation, overgrazing, non-regulated cultivation techniques,
etc. (Francis and Thornes 1990). Desertification generally alters natural plant com-
munities including population structure, succession pattern, and species diversity
(Barea and Jeffries 1995). In addition, these disturbances are often accompanied by
degradation of physico-chemical and biological soil properties, such as nutrient
availability, microbial activity, soil structure, etc., that largely determine soil quality
and fertility (Garcia et al. 1997a, b; Albaladejo et al. 1998; Requena et al. 2001). For
instance, it is well known that land degradation is usually linked with reductions in
the belowground microbial diversity and/or activity (Kennedy and Smith 1995).
Among components of soil microbiota, arbuscular mycorrhizal (AM) fungi are
known to be key components of natural systems in semiarid ecosystems and
particularly important in counteracting desertification of Mediterranean ecosystems
(Carpenter and Allen 1998; Brundrett 1991). The mycorrhizal symbiosis mobilizes
and transports nutrients to roots (Smith and Read 1997), reduces water stress (Augé
2001), and improves soil aggregation in eroded soils (Caravaca et al. 2002). AM
fungal symbiosis also changes root functions (e.g., root exudation) (Graham et al.
1981; Marshner et al. 1997), modifies carbohydrate metabolism of the host plant
(Shachar-Hill et al. 1995), and synergistically interacts with rhizosphere popula-
tions (Andrade et al. 1997; 1998). The structure and functionalities of these
microbial communities surrounding AM roots differ from those of the rhizosphere
12 Monitoring the Development of Nurse Plant Species to Improve the Performances 257
(Duponnois et al. 2005), and this microbial compartment has been named “mycor-
rhizosphere” (Linderman 1988). It has also been reported that AM fungi affect the
diversity of plant communities (van der Hejden et al. 1998; Klironomos et al. 2000;
O’Connor et al. 2002) and influence competitive relationships between plants (West
1996; Marler et al. 1999; van der Heijden et al. 2003).
It is well known that the AM fungal activity is generally low in degraded
semiarid Mediterranean ecosystems (Maremammani et al. 2003). However, an
increased activity of fungal inoculum is needed in both natural and artificial
processes of re-vegetation. It has already been shown that AM inoculation of plants
is very effective in establishing plants on disturbed soils (Estaun et al. 1997). In
order to increase and maintain high populations of infective AM propagules in soil,
two main cultural practices can be considered: (1) screening of AM fungal isolates
(native or exotic isolates) for their effect on the plant growth under controlled con-
ditions and a cultural substrate inoculation with the most efficient AM strains, and (2)
adoption of field practices to manage and improve the inoculum potential of indi-
genous AM fungi. The inoculation practice is generally used in tree nurseries to help
tree establishment in field conditions (Plenchette 2000; Franson and Bethlenfalvay
1989). However, previous studies have reported that, although AM inoculation
improved plant growth, it could also strongly modify soil microbial activities
(Dabire et al. 2007). In some conditions, to overcome the negative influences of AM
inoculation on soil microbiota, it is possible to increase AM soil potential through
the management of highly mycotrophic plants (Azcon and Barea 1997). In the
following section, focus is placed on highlighting the impacts of some Mediterranean
shrubs on microbial functionalities and AM potential and evaluating their signi-
ficant use in forestry practices to enhance the performance of afforestation program.
In this context, most of the reported results will be directed to Cupressus spp. and
Lavendula species. In Morocco, the area of natural and introduced cypress stands,
one natural species, Cupressus atlantica, and two introduced species, Cupressus
sempervirens and C. arizonica, has declined, and numerous reports indicate a
complete absence of natural regeneration. Although attempts have been made to
replant these species, the rate of success has been very low. Lavandula species are
representative plant species in Mediterranean shrublands and belong to the natural
succession in semiarid Mediterranean ecosystems (Barea et al. 1992). They have
been classified as “obligatory mycorrhizal” (Brundrett 1991) or as “highly dependent
on mycorrhiza” (Habte and Manjunath 1991).
12.2 Impacts of “Nurse Plant Species” on Soil Microbial

Functionalities and AM Fungus Communities
A study has been conducted in the N’Fis valley (Haut Atlas mountains, Morocco) at
the Idni station (8 170 0200 W, 31 540 3400 N, 1,700 m above sea level) in a natural
stand of C. atlantica (Ouahmane et al. 2006). This area was covered by a sparse
and degraded vegetation mainly composed of grasses (i.e., Stipa nitens Ball.)
and various shrub species, such as Cistus salviifolus L., Lavandula dentata L.,
L. stoechas L., Thymus pallidus Coss., Polygala balansae Coss., Globularia
alypum L. and Thymus satureioides Coss. Soil samples were collected from the
rhizosphere of L. dentata, L. stoechas, and T. satureioides (abundant shrub species
and always recorded in the vicinity of C. atlantica adult trees) and of C. atlantica.
Control samples were collected from bare soil sites, away from plant influence.
The soil carbon and nitrogen contents were higher under C. arizonica than under the
other sampling areas due to the leaf litter formation and the fact that, in forest eco-
systems, most of the soil nitrogen is in organic form (Kaye and Hart 1997)
(Table 12.1), while soluble phosphorus contents were significantly lower under
the targeted plant species than in the bare soil (Table 12.1). Therefore, the shrub
species did not demonstrate greater effects on soil chemical characteristics and, in
contrast, decreased soil P content. The main impacts of these shrub species were
recorded on soil functionalities and AM soil potential.
Microbial functional diversity in rhizosphere and in bare soil was assessed by
measuring the patterns of in situ catabolic potential (ISCP) of microbial commu-
nities (Degens and Harris 1997). Organic compounds comprising of a range of
amino acids, carbohydrates, organic acids, and amides were screened for differ-
ences in substrate-induced respiration (SIR) between soil treatments. The results
show that microbial communities are very different according to the soil origin
(Fig. 12.1). In particular, SIR responses to carboxylic acids were significantly
higher in the C. atlantica and T. satureioides rhizosphere soils than in the soils of
other origins (Fig. 12.1). Soluble organic acids are involved in plant nutrient
acquisition and they particularly act as biological weathering agents of minerals
in soils. They could be of high molecular weight (HMW) (i.e., humic substances) or
low molecular weight (LMW) produced by plant roots and soil microorganisms
(Ochs 1996). In the process of P acquisition from minerals, among the identified
carboxylic acids, dicarboxylic (oxalic, tartaric, malic, fumaric, malonic acids) and
tricarboxylic acids (citric acid) have been found very effective in P mobilization
(Ryan et al. 2001). Since the highest SIR responses with most of these organic acids
have been measured under C. atlantica and one of the targeted shrub species
(T. satureioides), it suggests that these plant species and their associated microflora
Table 12.1 Chemical characteristics of the rhizosphere soils collected from Lavandula dentata,
L. stoechas, Thymus satureioides, Cupressus atlantica and the bare soil (control) in the C. atlantica
stand located in the N’Fis valley (Haut Atlas mountains, Morocco)
Control Plant species
L. dentata L. stoechas T. satureioides C. atlantica
pH 7.5 a 7.0 a 7.5 a 7.4 a 7.7 a
Total carbon (%) 1.58 a 1.60 a 1.73 a 1.80 a 3.15 b
Total nitrogen (%) 0.09 a 0.10 ab 0.12 ab 0.10 a 0.14 b
C/N 17.2 a 15.7 a 14.7 a 18.8 a 22.9 a
Soluble P (mg kg1) 19.7 d 7.9 a 9.8 ab 11.8 bc 13.1 c
Data in the same line followed by the same letter are not significantly different (p<0.05) according
to one-way analysis of variance. Adapted from Ouahmane et al. (2006)
Gallic
2
–2 2
–2 Formic Oxalic
TS
Ascorbic
Ketoglutaric
BS
Ketobutyric
Hydroxybutyric
Quinic
LS Succinic
LD
Malonic
Tartric
Fumaric
CA
Fig. 12.1 Between-group analysis (BGA) of the substrate-induced respiration (SIR) responses
with respect to the rhizosphere and the bare soils. LS, LD, TS, CA and BS represent Lavandula
stoechas, L. dentata, Thymus satureioides, Cupressus atlantica and bare soils, respectively
(Ouahmane et al. 2006)
(AM fungi and mycorrhizosphere microbiota) excreted higher amounts of such

organic acids which could exert a selective influence on soil microbial communities
through a multiplication of microorganisms that catabolize organic acids.
Beside the positive effects of “nurse” plant species on soil microbial function-
alities, they also increase the mycorrhizal soil infectivity. Three Lavandula species
(L. multifida, L. dentata, L. stoechas) have been studied for their AM dependencies
(Ouahmane et al. 2007). After inoculating with the AM fungus, Glomus intraradices,
the mycorrhizal dependency of L. stoechas was 33% whereas those recorded for
L. dentata and L. multifida were 63 and 58%, respectively (Ouahmane et al.
2007). These results confirmed the high mycorrhizal dependency of these plant
species (Azcon and Barea 1997). Lavender plants are very mycotrophic and enrich
their cultural soil in AM fungal propagules. The L. multifida have been found to
increase the mycorrhizal soil infectivity (Table 12.2) in six different soils as
determined by MPN (most probable number) method (Ouahmane et al. 2007). In
addition, this positive contribution is linked with the total soil P contents since the
highest increases were recorded in soils with the lowest soil total P contents. Hence,
Table 12.2 Responses of soil mycorrhizal infectivity to Lavandula multifida plantation in six
sandy soils after 5 months culturing
Soils pH Total C (g C Organic matter Total N Total P MSIa
1 1 1
(H2O) kg ) (%) (g kg ) (g kg )
1 5.8 3.99 0.7 0.26 68.5 533.3
2 4.9 2.85 0.5 0.19 78.7 1121.9
3 5.4 6.62 1.1 0.46 119.2 144.9
4 5.7 3.17 0.5 0.12 48.4 443.0
5 6.1 7.29 1.3 0.60 200.7 87.2
6 5.7 3.09 0.5 0.47 90.0 131.3
a
(MSI of soil planted with L. multifida) – (MSI of soil unplanted with L. multifida) after 5 months
culture in each soil. Adapted from Ouahmane et al. (2007)
as lavender plants have a patchy distribution in Cupressus stands in Haut Atlas

Mountains in Morocco, this Lavandula species could act as a “nurse plant” for
natural regeneration of Cupressus young seedlings by (1) enhancing soil microbial
activities (in particular those involved in P mobilization), and (2) enhancing the
mycorrhizal soil infectivity.
12.3 Response of Cupressus sp. Growth to the “Nurse”

Plant Effects
Recognizing the benefits recorded for lavender plants on soil diversity and func-
tioning, several studies have been undertaken to determine their potential impacts
on the early growth of Cupressus sp. To test the potential benefits from the
association between Cupressus sp. and “nurse plant” on the growth of each plant
partner, Ouahmane et al. (2007) conducted an experiment in controlled conditions
combining the following treatments: C. arizonicaþL. multifida, L. multifida alone
and C. arizonica alone in 20-l pots filled with non-disinfected sandy soil. The
results show that the height of plant species was increased in the dual cultivation
treatment (Fig. 12.2). In addition, after 4 months culturing, the growth of L. multi-
fida and C. arizonica was generally higher when they were cultured together than
those recorded for singly cultured treatment (Fig. 12.2). A similar effect was
observed for the mycorrhizal colonization of the plants (Table 12.3). Other studies
have also been conducted to evaluate the capacity of the “fertility islands” created
by the nurse plants to facilitate the early growth of Cupressus spp.. Soil samples
were collected under L. dentata, L. stoechas and T. satureioides rhizosphere and
from an area free of cover plant (control). Soils were then packed in 1-dm3 pots and
one C. arizonica seedling was planted per pot. After 6 months of culturing, the
height, stem diameter, N and P foliar contents, AM colonization, and shoot and root
biomass were significantly higher in the soils originating from the nurse plants than
in the bare soil (Table 12.4). These results suggest that C. atlantica has to be
mycorrhizal in order to reach its optimal growth and that this Mediterranean tree
species is “highly dependent on mycorrhizas” (Habte and Manjunath 1991). These
80
70
60
Height (cm) 50
40
30
20
10
0
0 3 5 7 9 11 13 14 16
Time (week)
Fig. 12.2 Time course changes in plant height (in cm) of Cupressus arizonica and Lavandula
multifida seedlings in the mono and dual cultivation treatments. Height growth of C. arizonica
seedlings: empty square C. arizonica alone; filled square C. arizonicaþL. multifida – height
growth of L. multifida seedlings; empty circle L. multifida alone; filled circle L. multifidaþ
C. arizonica (adapted from Ouahmane et al. 2007)
Table 12.3 Growth and mycorrhizal colonization of Cupressus arizonica and Lavandula multifida
after 4 months culturing in a non-disinfected sandy soil
Plant species Treatment Shoot biomass (mg Root biomass (mg Mycorrhizal
dry weight/plant) dry weight/plant) colonization (%)
C. arizonica Alone 3,393 a 735 a 4a
þ L. Multifida 3,456 b 1,499 b 92 b
L. multifida Alone 1,020 a 1,102 a 10 a

þ C. arizonica 1,032 a 2,120 b 50 b
For each plant species, data in the same column followed by the same letter are not significantly
different (p<0.05) according to one-way analysis of variance. Adapted from Ouahmane et al.
(2007)
Table 12.4 Growth and mycorrhizal colonization of Cupressus atlantica seedlings planted in the
rhizosphere soils collected from Lavandula dentata, L. stoechas, Thymus satureioides, C. atlantica
and the bare soil (control) after 6 months culturing in greenhouse conditions
Control Soil origin
L. dentata L. stoechas T. satureioides C. atlantica
Height (cm) 14.2 a 18.6 b 21.0 cd 23.0 d 19.4 bc
Stem diameter (mm) 2.02 a 2.72 bc 2.72 bc 2.94 c 2.54 b
Shoot biomass (mg dry 330 a 634 bc 738 c 666 bc 486 ab
weight/plant)
Root biomass (mg dry 76 a 176 c 157 bc 115 abc 104 ab
weight/plant)
N (mg/plant) 0.79 a 1.56 b 1.82 c 2.03 d 1.48 b
P (mg/ plant) 0.033 a 0.107 c 0.115 c 0.147 d 0.090 b
AM colonization (%) 35 b 48 b 50 b 75 c 54 b
Data in the same line followed by the same letter are not significantly different (p<0.05)
according to one-way analysis of variance. Adapted from Ouahmane et al. (2007)
studies confirmed that plant nurses mainly act on Cupressus seedling growth
through their impact on AM fungus communities. AM fungi are important agents
in promoting plant co-existence (Allen and Allen 1990). Moreover, the abundance
and diversity of AM fungi are known to have a strong effect on the direction of
succession (Medve 1984). This AM fungal effect is mainly important in early
successional ecosystems where plant and soil have been severely disturbed and
where AM fungi are absent or are in low abundance and patchily distributed
(Hart et al. 2003). Hence, the use of plant nurses as promoting agent of mycorrhizal
soil infectivity could be of great interest in restoring a self-sustaining vegetation
cover in order to act against desertification.
12.4 Conclusion
The use of nurse shrubs facilitates seedling establishment in many different ecolog-
ical settings in Mediterranean mountains (Gomez-Aparicio et al. 2004) suggesting
that the removal of shrubs is not an appropriate practice for reforestation in
Mediterranean mountains. Therefore, a new paradigm for the science of restoration
of Mediterranean forests emerges from all these ecological studies (Maestre et al.
2001, 2002; Gomez-Aparicio et al. 2004). It is based on the natural spatial patterns
of regeneration of woody vegetation with shrubs as micro-sites for recruitment.
Furthermore, since the benefits of positive interactions between plant species is
widely recognized (Callaway et al. 2002), this innovative forestry practice might be
more relevant under the predicted rise in temperatures, dryness, and rainfall varia-
bility for the Mediterranean region under global warming (IPCC 2001). Since the
primary limitation of plant fitness is generally represented by the severity of the
physical environment, the enhancement of environmental conditions by nurse
shrubs can be of crucial importance in many stressful environments.
References
Albaladejo J, Martinez-Mena M, Roldan A, Castillo V (1998) Soil degradation and desertification

induced by vegetation removal in a semiarid environment. Soil Use Manage 14:1–5
Allen EB, Allen MF (1990) The mediation of competition by mycorrhizae in successional and
patchy environments. In: Grace JB, Tilman D (eds) Perspectives in plant competition. Aca-
demic Press, New York, pp 367–389
Andrade G, Mihara KL, Linderman RG, Bethlenfalvay GJ (1997) Bacteria from rhizosphere and
hyphosphere soils of different arbuscular mycorrhizal fungi. Plant Soil 192:71–79
Andrade G, Mihara KL, Linderman RG, Bethlenfalvay GJ (1998) Soil aggregation status and
rhizobacteria in the mycorrhizosphere. Plant Soil 202:89–96
Augé RM (2001) Water relations, drought and vesicular arbuscular mycorrhizal symbiosis.
Azcon R, Barea JM (1997) Mycorrhizal dependency of a representative plant species in Mediter-
ranean shrublands (Lavandula spica L.) as a key factor to its use for revegetation strategies in
desertification-threatened areas. Appl Soil Ecol 7:83–92
Barea JM, Jeffries P (1995) Arbuscular mycorrhizas in sustainable soil plant systems. In: Hock B,
Varma A (eds) Mycorrhiza structure, function, molecular biology and biotechnology. Springer,
Heidelberg, pp 521–559
Barea JM, Azcon R, Azcon-Aguilar C (1992) The use of 15N to assess the role of VA mycorrhiza
in plant N nutrition and its application to evaluate the role of mycorrhiza in restoring
Mediterranean ecosystems. In: Read DJ, Lewis DH, Fitter AH, Alexander IJ (eds)
Mycorrhizas in Ecosystems. Structure and Function. CAB International, Wallingford, UK,
pp 190–197
Bochet E, Rubio JL, Poesen J (1999) Modified topsoil islands within patchy Mediterranean
vegetation in SE Spain. Catena 38:23–44
Boucher DH, James S, Keeler KH (1982) The ecology of mutualism. Ann Rev Ecol Syst
13:315–347
Brundrett MC (1991) Mycorrhizas in natural ecosystems. In: Macfayden A, Begon M, Fitter AH
(eds) Advances in ecological research, vol 21. Academic Press, London, pp 171–313
Callaway RM (1995) Positive interactions among plants. Bot Rev 61:306–349
Callaway RM (1997) Positive interactions in plant communities and the individualistic-continuum
concept. Oecologia 112:143–149
Callaway RM, Walker LR (1997) Competition and facilitation: a synthetic approach to interac-
tions in plant communities. Ecology 78:1958–1965
Callaway RM, Brooker RW, Choler P, Kikvidze Z, Lortie CJ, Michalet R, Paolini L, Pugnaire FI,
Newingham B, Aschehoug ET, Armas C, Kikidze D, Cook BJ (2002) Positive interactions
among alpine plants increase with stress. Nature 417:844–848
Caravaca F, Barea JM, Figueroa D, Roldan A (2002) Assessing the effectiveness of mycorrhizal
inoculation and soil compost addition for reafforestation with Olea europaea subsp. sylvestris
through changes in soil biological and physical parameters. Appl Soil Ecol 20:107–118
Carpenter AT, Allen MF (1998) Responses of Hedysarum boreale Nutt. to mycorrhizas and
Rhizobium: plant and soil nutrient changes in a disturbed shrub-steppe. New Phytol
109:125–132
Carrillo-Garcia A, Bashan Y, Bethlenfalvay GJ (2000) Resource island soils and the survival of the
giant cactus, cardon, of Baja California Sur. Plant Soil 218:207–214
Dabire AP, Hien V, Kisa M, Bilgo A, Sangare KS, Plenchette C, Galiana A, Prin Y, Duponnois R
(2007) Responses of soil microbial catabolic diversity to arbuscular mycorrhizal inoculation
and soil disinfection. Mycorrhiza 17:537–545
Degens BP, Harris JA (1997) Development of a physiological approach to measuring the catabolic
diversity of soil microbial communities. Soil Biol Biochem 29:1309–1320
Duponnois R, Colombet A, Hien V, Thioulouse J (2005) The mycorrhizal fungus Glomus
intraradices and rock phosphate amendment influence plant growth and microbial activity in
the rhizosphere of Acacia holosericea. Soil Biol Biochem 37:1460–1468
Estaun V, Save R, Biel C (1997) AM inoculation as a biological tool to improve plant re-vegetation
of a disturbed soil with Rosmarinus officinalis under semi-arid conditions. Appl Soil Ecol
6:223–229
Francis DF, Thornes JB (1990) Matorral: erosion and reclamation. In: Albaladejo J, Stocking MA,
Diaz E (eds) Soil degradation and rehabilitation in Mediterranean environmental conditions.
CSIC, Murcia, Spain, pp 87–115
Franson RI, Bethlenfalvay GJ (1989) Infection unit method of vesicular arbuscular mycorrhizal
propagule determination. Soil Sci Soc Am J 53:754–756
Garcia C, Hernandez T, Costa F (1997a) Potential use of dehydrogenase activity as an index of
microbial activity in degraded soils. Commun Soil Sci Plan Anal 28:123–124
Garcia C, Hernandez T, Roldan A, Albaladejo L (1997b) Biological and biochemical quality of a
semiarid soil after induced revegetation. J Environ Qual 26:1116–1122
Garner W, Steinberger Y (1989) A proposed mechanism for the formation of fertile islands in the
desert ecosystem. J Arid Environ 16:257–262
Gomez-Aparicio L, Zamora R, Gomez JM, Hodar JA, Castro J, Baraza E (2004) Applying plant
facilitation to forest restoration: a meta-analysis of the use of shrubs as nurse plants. Ecol Appl
14:1128–1138
Graham JH, Leonard RT, Menge JA (1981) Membrane-mediated decrease in root exudation
responsible for phosphorus inhibition of vesicular-arbuscular mycorrhiza formation. Plant
Physiol 68:548–552
Habte M, Manjunath A (1991) Categories of vesicular–arbuscular mycorrhizal dependency of host
species. Mycorrhiza 1:3–12
Hart MM, Reader RJ, Klironomos JN (2003) Plant coexistence mediated by arbuscular mycorrhi-
zal fungi. Trends Ecol Evol 18:418–423
IPCC [Inter-Government Panel on Climate Change] (2001) In: Watson RT, The Core Writing
Team (eds) Climate change 2001: third assessment report of the Intergovernmental Panel on
Climate Change(WG I & II). Cambridge University Press, Cambridge, UK
Kaye JP, Hart SC (1997) Competition for nitrogen between plants and soil microorganisms. Tree
12:139–143
Kennedy AC, Smith KL (1995) Soil microbial diversity and the sustainability of agriculture soils.
Plant Soil 170:75–86
Klironomos JN, McCune J, Hart M, Neville J (2000) The influence of arbuscular mycorrhizae on
the relationship between plant diversity and productivity. Ecol Lett 3:137–141
Linderman RG (1988) Mycorrhizal interactions with the rhizosphere microflora: the mycorrhizo-
sphere effect. Phytopathology 78:366–371
Maestre FT, Bautista S, Cortina J, Bellot J (2001) Potential for using facilitation by grasses to
establish shrubs on a semiarid degraded steppe. Ecol Appl 11:1641–1655
Maestre FT, Bautista S, Cortina J, Diaz G, Honrubia M, Vallejo R (2002) Microsite and mycor-
rhizal inoculums effects on the establishment of Quercus coccifera in a semiarid degraded
steppe. Ecol Eng 19:289–295
Maremammani A, Bedini S, Matosevic I, Tomei PE, Giovannetti M (2003) Type of mycorrhizal
associations in two coastal nature reserves of Mediterranean basin. Mycorrhiza 13:33–40
Marler MJ, Zabinski CA, Callaway RM (1999) Mycorrhizae indirectly enhance competitive
effects of an invasive forb on a native bunchgrass. Ecology 80:1180–1186
Marshner P, Crowley DE, Higashi M (1997) Root exudation and physiological status of a root-
colonizing fluorescent pseudomonad in mycorrhizal and non-mycorrhizal pepper (Capsicum
annuum L.). Plant Soil 189:11–20
Medve RJ (1984) The mycorrhizae of pioneer species in disturbed ecosystems in western
Pennsylvania. Am J Bot 71:787–794
O’Connor PJ, Smith SE, Smith FA (2002) Arbuscular mycorrhizas influence plant diversity and
community structure in a semiarid herbland. New Phytol 154:209–218
Ochs M (1996) Influence of humidified and non-humidified natural organic compounds on mineral
dissolution. Chem Geol 132:119–124
Ouahmane L, Hafidi M, Kisa M, Boumezouch A, Thioulouse J, Plenchette C, Duponnois R (2006)
Some Mediterranean plant species (Lavandula spp. and Thymus satureioides) act as “plant
nurses” for the early growth of Cupressus atlantica. Plant Ecol 185:123–134
Ouahmane L, Hafidi M, Kisa M, Boumezouch A, Thioulouse J, Duponnois R (2007) Lavandula
species as a source of arbuscular mycorrhizal propagules facilitating the early development of
Cupressus arizonica. Appl Soil Ecol 34:190–199
Plenchette C (2000) Receptiveness of some tropical soils from banana fields in Martinique to the
arbuscular fungus Glomus intraradices. Appl Soil Ecol 15:253–260
Requena N, Perez-Solis E, Azcon-Aguilar C, Jeffries P, Barea JM (2001) Management of indi-
genous plant–microbe symbioses aids restoration of desertified ecosystems. Appl Environ
Ryan PR, Delhaise E, Jones DL (2001) Function and mechanism of organic anion exudation from
plant roots. Annu Rev Plant Physiol 52:527–560
Schlesinger WH, Raikes JA, Hartley AE, Cross AF (1996) On the spatial pattern of soil nutrients in
desert ecosystems. Ecology 7:364–374
Shachar-Hill Y, Pfeffer PE, Douds D, Osman SF, Doner LW, Ratcliffe RG (1995) Partitioning of in-
termediary carbon metabolism in vesicular arbuscular mycorrhizal leeks. Plant Physiol 108:7–15
Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn. Academic Press, London
Valladares F, Pugnaire FI (1999) Tradeoffs between irradiance capture and avoidance in semiarid
environments assessed with a crown architecture model. Ann Bot 83:459–469
van der Heijden MGA, Wiemken A, Sanders IR (2003) Different arbuscular mycorrhizal fungi
alter coexistence and resource distribution between co-occuring plant. New Phytol
157:569–578
van der Hejden MGA, Klironomos JN, Ursic M, Moutoglis P, Streitwolf-Engel R, Boller T,
Wiemken A, Sanders IR (1998) Mycorrhizal fungal diversity determines plant biodiversity
ecosystem variability and productivity. Nature 396:69–72
West HM (1996) Influence of arbuscular mycorrhizal infection on competition between Holcus
lanatus and Dactylis glomerata. J Ecol 84:429–438
Chapter 13
Pea Cultivation in Saline Soils: Influence
of Nitrogen Nutrition
Etelvina Figueira
Abstract Salinity is one of the most important abiotic factors limiting plant growth
and productivity. This is especially acute in arid and semiarid regions of the world.
In this chapter, attention is paid to evaluate the influence of different forms of
nitrogen on the tolerance and the yield of pea (Pisum sativum L.) plants cultivated
under different saline conditions. Results show that the nitrogen form influenced
pea growth, yield and ionic content, both under the presence and absence of salinity.
Under nonsaline conditions, the highest growth and yields were obtained by
NO3NH4, whereas under salinity, NO3 displayed the highest values. The inocula-
tion with a salt-tolerant strain allowed the nodule formation process to remain
unaffected by salt. Nevertheless, these plants had lower yields than NO3-fed plants,
although similar values to NH4þ-supplemented plants, indicating that, by relying
solely on biological N2 fixation or NH4þ fertilization, pea yields under saline
conditions will be compromised.
13.1 Introduction
Salinity stress is of great importance in arid and semiarid areas of the world, since
most crops are sensitive to salinity, evidencing sharp yield decreases in the presence
of moderate salt concentrations (Subbarao and Johansen 2004). More than 6% of
the total land area of the world is salt affected. Most of these salt-affected areas
have arisen from natural causes, by the accumulation of salts over long periods of
time in arid and semiarid zones (Rengasamy 2002). Apart from natural salinity, a
significant proportion of recently cultivated agricultural land has become saline
owing to land clearing or irrigation, both of which cause water tables to rise and
E. Figueira
Centre for Cell Biology, Departamento de Biologia, Universidade e Aveiro, Campus de Santiago,
3810-193, Aveiro, Portugal
e-mail: efigueira@ua.pt
268 E. Figueira
concentrate the salts in the root zone. Of the 1,500 million ha of land farmed by dry
land agriculture, 32 million ha (2%) are affected by secondary salinity of varying
degrees. Of the current 230 million ha of irrigated land, 45 million ha (20%) are salt
affected (Munns and Tester 2008).
Tolerance to salinity may have different meanings according to the objectives by
which it is considered. If tolerance is viewed from an ecological perspective, the
most relevant is that species colonizing a habitat can complete their life cycle and
create conditions for their permanence. From an agronomic point of view, the most
important factor is productivity. If salinity causes toxic effects and osmotic and
nutritional imbalances, a large part of photosynthates will be used as osmotic
solutes or in ion exclusion, reducing growth and productivity and putting their
cultivation in salt-affected agricultural soils at risk.
The focus of this chapter is to evaluate the influence that different nitrogen
sources have on salt tolerance of pea (Pisum sativum), during its growth and
productivity. To achieve this goal, growth, productivity, seed protein content,
and ion contents were determined and compared between Pisum sativum plants
grown for 75 days under the presence (90 mM NaCl) or absence (0 mM NaCl) of
salinity and four types of nitrogen nutrition (NO3, NH4+, NH4NO3, or symbioti-
cally fixed N2 by an halotolerant isolate of Rhizobium leguminosarum).
13.2 Nitrogen Nutrition Under Salt Stress
13.2.1 Cell Ionic Status
As the boundary between the outside and inside, the plasma membrane is one of the
most important structures that cells have to adjust their composition. Plasmalemma
proteins control which solutes will be accumulated and which will be excluded,
creating gradients through the membrane. This process is important in many aspects
of plant growth and development, including mineral nutrition (Ward 1997). Some
ions and solutes, such as H+, Na+, and Ca2+, are excluded from the cytosol of cells,
while others, such as K+, NH4+, NO3, Cl, SO42, HPO4, sugars, organic acids,
and amino acids are accumulated. One of the fundamental characteristics of
membrane transport in plants is that protons are actively excluded from the cytosol
to the cell outside. This process creates a pH gradient, usually between 1.5 and
2 units (Marschner 1995; Ward 1997), and a voltage gradient, usually between 100
and 200 mV (Marschner 1995; Ward 1997) in the plasmalemma. Thus, in electro-
physiological terms the absorption of cations can occur passively, while anion
absorption uses up energy.
As the selectivity of ion channels and transporters is based on the ionic charge it
leads to competition between ions of the same electrical charge. The competition
between Cl and NO3, between K+ and Na+, or between Ca2+ and Na+ (Marschner
1995; Tyerman 1992; Fox and Guerinot 1998) results in severe nutritional imbalances
13 Pea Cultivation in Saline Soils: Influence of Nitrogen Nutrition 269
to plants when the intracellular ratios of these ions are very different from those in
the soil solution. In the presence of saline conditions, Na+ and Cl are often the
most abundant ions. Therefore, it is not surprising that these soils are characterized
by low activities of nutrient ions and high ratios of Na+/Ca2+, Na+/K+, and Cl/
NO3. In these circumstances, nutritional disorders may develop and plant growth
be reduced.
13.2.2 Nitrogen as a Plant Macronutrient
Nitrogen is the fourth most abundant element in organisms, constituting about 1.5–5%
of the plant dry weight (Below 1995). Due to the high requirements of this element,
about 70% of the ions absorbed by plants are nitrogen-containing compounds
(Marschner 1995). Among the mineral nutrients, nitrogen often limits the growth
and productivity of many crops (Marschner 1995). Nitrogen is incorporated in
numerous organic compounds that include proteins, nucleic acids, chlorophylls,
and growth regulators, all playing crucial roles in plant growth and development.
13.2.2.1 Inorganic Nitrogen Nutrition
Nitrogen is available to plants in the soil in a variety of forms including ammonium

(NH3 and NH4+), nitrate, amino acids, soluble proteins, and other nitrogen-containing
compounds, but plants absorb nitrogen primarily in an inorganic form as NO3 or
ammonium (Williams and Miller 2001). Under natural conditions, the nitrogen
present in the soil originates from biological fixation, or animal and plant decom-
position. The vast majority (over 90%) of nitrogen present in the soil is contained in
organic matter, which is relatively stable and is not directly available to plants
(Below 1995). However, part of the organic nitrogen may become available
through mineralization by microorganisms in the soil (Marschner 1995). The
form of nitrogen provided, cationic or anionic, affects their availability to plants
due to differences in the mobility of the two ions in the soil solution. The cation
NH4+ binds to the negatively charged particles in the soil and becomes relatively
immobile. In contrast, the NO3 anion is repelled, which increases its bioavailabil-
ity for root uptake. However, this form of nitrogen is easily lost by leaching and
denitrification (Bray 1983; Below 1995).
Plants acquire the vast majority of their nitrogen through the root system. This
process involves the movement of inorganic nitrogen, NO3 or NH4+, through the
plasma membrane. The high-affinity transport systems in roots are able to scavenge
NH4+ and NO3 from the soil at concentrations between 1mM and 1 mM, whereas
the activity of low-affinity transport systems becomes evident when these ions are
above 5 mM (Jackson et al. 2008). All NO3 transport systems need energy to
overcome an unfavorable electrochemical gradient between the outside solution
and the symplast of root cells. Ammonium transport can be passive if uptake occurs
270 E. Figueira
through low-affinity systems or active through high-affinity systems (Williams and

Miller 2001; Grossman and Takahasi 2001). Whereas NH4+ can be directly used in
the synthesis of amino acids, NO3 must first be reduced to NH4+. This reduction is
a process that uses up energy and may be the reason why NH4+ inhibits NO3
uptake (Gazzarrini et al. 1999; Gessler et al. 1998; Siddiqi et al. 2002).
13.2.2.2 N2 Fixation and Plant Nutrition
Eukaryotic cells do not possess the biochemical "machinery" to react with diatomic
nitrogen. Only a few prokaryotes, including species of the genera Azotobacter,
Azotococcus, Clostridium, Klebesiella, Bacillus, Azopirillum, Nostoc, Anabaena,
Rhizobium, Bradyrhizobium, and Frankia, have enzymes that reduce N2 (Bray
1983; Marschner 1995). Some of them establish endosymbiosis, such as the associ-
ation between plants of the family Fabaceae and bacteria of the genera Rhizobium,
Bradyrhizobium, Azorhizobium, Mesorhizobium, and Ensifer. Free-living rhizobia
do not fix N2, this function is accomplished only in association with the host plants.
Bacteria colonize legume roots at the beginning of the vegetative growth and leads
to the formation of nodules. In nodules, bacteria undergo structural (such as loss of
cell wall) and metabolic (as the expression of nitrogenases) changes that enable
them to fix the N2, turning into bacteroids (Brewing et al. 1993). Individually or in
small groups, each bacteroid is surrounded by a membrane, forming a structure
called the symbiosome which is the site of N2 fixation. The NH4+ resulting from the
reduction of NO3, from the symbiotic fixation of N2, or absorbed directly, is
assimilated into glutamate and glutamine. Ammonium assimilation usually occurs
at root level, whereas nitrate assimilation can occur both in roots and leaves.
Glutamate and glutamine are the precursors of other amino acids, generating all
the amino acids required for protein synthesis. They are also the raw material
responsible for generating a series of other compounds, such as chlorophylls,
plant growth regulators, and nucleic acids (Below 1995).
13.2.2.3 N2 Fixation in Plants Under Salinity Stress
Many studies have reported the inhibitory effects of salinity in the fixation of N2
(Lauter and Munns 1986; Cordovilla et al. 1994, 1996, 1999a, 1999b; Tu 1981;
Salehi et al. 2008; Tawfik 2008). In saline environments, the nodulation reduction
may be due to the decrease in rhizobial populations, because of its sensitivity to salt,
affecting the survival and distribution of rhizobia in the soil and rhizosphere of
plants (Singleton et al. 1982; Craig et al. 1991). For example, Pereira et al. (2008)
found that 50 mM NaCl strongly inhibited the growth of three of the five Rhizobium
populations compatible with pea plants, when grown in YEM medium supplemen-
ted with salt. Elsheikh and Wood (1995) selected strains of Rhizobium and
Bradyrhizobium for their salt tolerance and reported that one of them only grew
in the absence of NaCl, and the others showed different tolerances but were not able
to grow above 340 mM. However, Zharan et al. (1994) isolated strains of Rhizobium
with different salt tolerances; some grew at 1.7 M NaCl. Pereira et al. (2008) also
recorded isolates that grew at the same level of halotolerance (1.6 M NaCl).
In the nodulation process, salt inhibition is regarded more as an effect on the
establishment of nodules than on the reduction of Rhizobium population. There are
several studies showing that the most sensitive process to salinity is the establish-
ment of the nodules (Lauter and Munns 1986; Cordovilla et al. 1999a). For
example, Singleton and Bholool (1983) found a strong reduction (50%) in the
number of nodules in the roots of soybean [Glycine max (L.) Merr] growing
under 26.6 mM NaCl. In a similar study, Elsheikh and Wood (1990) observed
that nodulation in chickpea (Cicer arietinum L.) was completely inhibited by
61.6 mM NaCl, even when inoculated with a strain tolerant to salt, while Abd-
Alla et al. (1998) observed that salinity (30 and 60 mM NaCl) inhibited the number
of nodules and the biomass of four cultivars of G. max inoculated with a salt-
tolerant strain of Bradyrhizobium. The reduction in the number of nodules has been
associated with the effect of salinity on cell expansion inhibition, and to root hairs
number and curling (Sprent and Zahran 1988; Tu 1981). In Pisum sativum plants
growing in the presence (90 mM) and absence of NaCl, it was observed that salinity
delayed nodule formation. In contrast to control plants, 15-day-old salt-stressed
plants had no nodules. However, salinity had no effect on nodule number at later
stages, although nodule senescence started earlier (Table 13.1). These results show
that the susceptibility of the nodulation process to salinity is not always verified and
that the selection of a halotolerant Rhizobium strain with good N2-fixing ability is
important to legume nodulation in saline conditions.
The effects of salinity on the symbiosis between rhizobia and host may also be
on the N2-fixation process directly. Several hypotheses aim to explain the negative
Table 13.1 Number and color of root nodules produced on halotolerant

Rhizobium-inoculated Pisum sativum plants grown in soils treated with
(90 mM NaCl) or without (0 mM NaCl) salts
Circle diameter reflects the number of nodules present in roots: less than 30
(small circles); and higher than 30 (big circles); nodule absence is also
registered. The relative abundance of different nodule colors is also presented:
white white nodules, gray pink nodules, stripes green nodules, black brown
nodules
272 E. Figueira
effects of salt on N2 fixation: a decline in the supply of photosynthates to the

nodules (Bekki et al. 1987; Georgiev and Atkins 1993); lower availability of
reduced compounds to bacteroids (Delgado et al. 1993, 1994); changes in the
oxygen diffusion barrier (Delgado et al. 2006; Serraj et al. 1994; Wahab and Zahran
1981); or inhibition of several enzymes important to N2 assimilation. Serraj et al.
(1994) showed that exposure of G. max plants to 0.1 M NaCl resulted in a rapid
decrease in nitrogenase activity. Abd-Alla et al. (1998) found that 30 mM was
enough to cause a sharp decline in the activity of nitrogenase in three of the four
cultivars of G. max used in the study. Bourgeais-Chaillou et al. (1992) described
reductions in the activity of glutamine synthetase (GS) and glutamate synthase
(GOGAT) in the root nodules of plants under salt stress. Cordovilla et al. (1996,
1999a, 1999c) reached the same conclusion and showed that GOGAT was more
inhibited than GS; thus concluding that GOGAT limited the assimilation of ammo-
nium in the nodules of Vicia faba L. plants under salt stress. In other works, the
same authors (Cordovilla et al. 1994, 1999c) concluded that N2 fixation was more
sensitive to salinity than the assimilation of ammonium.
The influence of salt stress in N2 fixation is higher than on the absorption of
inorganic nitrogen due to the higher energy costs to fix N2, leaving less energy
available for growth and for salinity coping. The accumulation of intracellular
organic solutes in the nodules, such as proline and glycine betaine, has been
correlated with salt tolerance (Robson and Bottomley 1991; Botsford and Lewis
1990). A more efficient way to acquire salt tolerance will be to compartmentalize
subcellularly inorganic compounds in the vacuoles, thus avoiding ion toxicity
(Zahran 1991). The improvement of ion partitioning in the nodule cells could
promote sustainable legume growth by means of N2 fixation in the presence of
salinity. According to Zahran (1991), this seems to be a good criterion for selecting
legumes for cultivation under salt conditions. Thus, although the importance that
salt-tolerant plant species and cultivars may have on the establishment of an
effective symbiosis under salt stress, the need for salt-tolerant rhizobia cannot be
ignored. Over time, only the rhizobia tolerant to salt can survive and persist in a
saline soil. It is imperative that soils contain salt-tolerant populations of rhizobia
compatible with the legume crops to be cultivated, in order to establish an effective
symbiosis and to promote good growth and yield in saline soils.
13.3 Plant Growth Under Salt Stress
13.3.1 Growth in Saline Conditions
Plants absorb most of the mineral nutrients from the soil. That is why they are
greatly affected by soil characteristics. Most crops are glycophytes and evolved
under conditions of low salinity in the soil. Therefore, they possess mechanisms for
nutrient absorption in nonsaline soils and are often unable to grow in this type of
environment without evidencing damage. Plant growth and productivity involve the
integrated effect of diverse environmental factors and metabolic processes that act
with different intensities throughout the plant life cycle. The first problem that
plants have to overcome when growing in an adverse environment is to establish.
Establishment is only possible if plants build up a good photosynthetic capacity to
produce enough biomass to support their vegetative growth and reproduction.
Maintenance of the photosynthetic capacity can become a problem when plants
grow under stressful conditions, such as those propitiated by salinity. Salinity is
characterized by an excess of ions, frequently Na+ and Cl, in whose presence most
plants grow poorly (Figueira and Caldeira 2005; Dudley1994; Marschner 1995).
13.3.2 Nitrogen Nutrition and Growth Under Salinity
Under saline conditions, the three main constraints imposed by salinity on growth
are (1) osmotic stress that originates with the increase of the soil osmotic potential,
(2) ion toxicity which is associated to excessive uptake of Cl and Na+; and (3)
nutritional imbalances due to the decrease of absorption and/or transport to shoots
as well as to an altered distribution of mineral nutrients (Marschner 1995; Munns
and Tester 2008). The decrease in the turgor pressure caused by the defective
osmotic regulation of the shoot is regarded as the main factor causing the inhibition
of leaf elongation, leading to a reduction in leaf area and to the partial closure of
stomata. The prolonged stomata closure causes considerable reduction in the CO2
fixation per leaf area and per plant (Marschner 1995). The higher respiration rates
and the shorter leaf duration also contribute to reduction in photosynthetic rates
(Yeo 1998; Munns and Tester 2008). In fact, many studies report reductions of the
photosynthetic capacity in plants subjected to salt stress (Below 1995; Marschner
1995; Yeo 1998). The decrease of photosynthates and the additional energy costs
with the exclusion, partitioning, excretion, osmotic adjustment, and repair of
cellular damage necessary to cope with salinity, contribute to the decline of plant
growth under saline conditions (Marschner 1995; Munns and Tester 2008). In pea
plants grown under different salt concentrations (0 and 90 mM NaCl) and nitrogen
nutrition (NO3, NH4+, NO3NH4, Rhizobium inoculation), it was observed that both
salinity and N form affected plant growth (Fig. 13.1). Root growth was reduced by
salinity and irrespective to the duration of salt exposure the N form also affected
root growth. Under saline conditions, N2-fixing plants had a higher root biomass
than plants fed with mineral N (Fig. 13.1a, b). In the absence of salinity, shoot
biomass was lower in N2-fixing than in mineral N-fed plants, and NO3NH4 pro-
duced the highest shoot biomass (Fig. 13.1c). The enhanced biomass of shoots may
certainly be related to the effect that a combined nitrogen nutrition of anions and
cations has on the maintenance of cell pH, through the rates of H+ production
(assimilation of NH4+) and consumption (assimilation of NO3), with lower energy
costs in the maintenance of the citosolic pH homeostasis, usually between 7.3 and
7.6 (Marschner 1995). Under salt stress, however, the response pattern changed.
Although biomass was reduced in all N treatments, N2-fixing plants were less and
274 E. Figueira
Fig. 13.1 Root and shoot weight of Pisum sativum grown during 75 days at two levels of salinity:
(a roots without NaCl supply, b roots with 90 mM NaCl supply, c shoots without NaCl supply,
d shoots with 90 mM NaCl supply) and with different N forms ( filled diamond NO3, filled
triangle NH4+, filled square NO3NH4, open circle Rhizobium inoculation). Root and shoot fresh
weight (g plant1) values are meansstandard errors of 16 replicates
NH4+-nourished plants more affected by salt. Other authors also observed that
NH4+ was the N form causing the highest decreases in plant growth: Leidi et al.
(1992) in cotton (Gossypium hirsutum L.) and peanut (Arachis hypogaea L.), Lewis
et al. (1989) in maize (Zea mays L.), and Feigin (1990) in muskmelon (Cucumis
melo L.). Among the different N forms, NO3 induced the highest growth of pea
plants under moderate salt conditions (Fig. 13.1d), as also reported by Leidi et al.
(1992) and Silberbush and Lips (1991a, 1991b).
The high concentrations of inorganic solutes, especially Na+ and Cl absorbed
by roots from the salinized media, directly affect plant growth. Above certain
levels, the accumulation of ions are toxic to cells and will have a negative impact
on plant metabolism and growth (Munns and Tester 2008). In severe cases of
toxicity, plants show not only growth decreases but also marginal chlorosis and
necrosis in mature leaves. Later on or with increasing salinity, the damaged areas
will increase and organ or plant death may occur.
13.4 Salinity and Nitrogen Nutrition Influence on Productivity
13.4.1 The Importance of Seeds
Seeds perpetuate species over time and space, and are especially important in those
species that do not propagate vegetatively. Perhaps for this reason, plants spend
abundant resources to ensure that enough energy and nutrients exist for germination
and establishment. In Pisum sativum, cotyledons contribute actively to the growth

of plantlets in the first 2 weeks following germination, losing 88% of their weight
during this time (Lovell 1977). The richness of the seeds in minerals and organic
compounds such as sugars, starch, fats, and proteins explain their excellent nutri-
tional value and, perhaps for that reason, quickly resulted in their use as a food.
Thus, besides the ecological importance that seeds have in the perpetuation of the
species, the economic significance related to productivity maximization also
became an important issue.
13.4.2 Seed Production
Legumes are generally considered either sensitive or moderately tolerant to salinity

(Läuchli 1984; Zahran 1991). For crops, salinity tolerance is normally expressed in
terms of the yield decrease associated with a given level of salinity. A yield
decrease of 50% is usually considered a critical level for evaluating the relative
salt tolerance of crops (Maas and Hoffman 1977). Pea is one of the most tolerant
crop legumes, with 50% yield reduction in the presence of 100 mM NaCl (Subbarao
and Johansen 1994). When pea plants were grown under different forms of N
nutrition and salt conditions, it was observed that, under nonsaline conditions,
productivity was two to four times higher when the source of nitrogen was
NO3NH4. The highest seed weight in plants grown under this form of N is mainly
due to the production of a higher number of seeds per plant, as a result of the
increase in pod number (results not shown). Salinity decreased productivity, mark-
edly in NO3NH4 and NH4+ and only slightly in NO3-nourished plants, where
salinity did not change productivity significantly (Fig. 13.2). Doré et al. (1998) also
Fig. 13.2 Seed production of Pisum sativum grown at two levels of salinity (0 and 90 mM NaCl)
and with different N forms (NO3, NH4+, NO3NH4, Rhizobium inoculation). Seed fresh weight
(g plant1) values are meansstandard errors of 12 replicates. Significantly (P<0.001) different
values are marked (*)
276 E. Figueira
observed variability in the productivity of pea plants cultivated under different

growth conditions, and these authors also found that changes in productivity were
related to the number and not the size of the seeds.
The productivity of plants exclusively dependent on N2 fixation are generally
smaller than those fed with inorganic nitrogen, due to the higher energy costs
associated initially to anabolic processes leading to the formation of nodules and
to the metabolic "machinery" necessary for N2 fixation, and later to energy costs
with the N2 fixation process itself and with the maintenance of conditions that
enable N2 fixation. The high energy cost of N2 fixation explains why, in Pisum
sativum plants grown under different forms of nitrogen nutrition (NO3, NH4+,
NO3NH4, Rhizobium inoculation), those dependent on symbiotic fixation showed
lower productivity (Fig. 13.2). Since inorganic nitrogen uptake is less costly to the
plant than maintaining the capacity to reduce atmospheric N2 (Lee et al. 2003),
under conditions of increased soil nitrogen (e.g., fertilization), N2 fixation is
inhibited. NO3 generally has a greater inhibitory effect on N2 fixation than
NH4+ (Hartwig 1998; Lee et al. 2003). However, fixation has the advantage of
freeing plants from the dependence of available nitrogen for their growth and
development in N-limited soils, allowing sustainable legume production.
13.4.3 Seed Protein Content
A deficient nitrogen nutrition not only impacts significantly on productivity but also
on the seed protein levels, especially in pulses, due to their high protein content
(25% of P. sativum seed dry weight), indicating that seeds could be an appropriate
organ to assess the nutritional status of plants (Atta et al. 2004). Protein synthesis
has long been identified as one of the processes strongly affected by salinity
(Gibson et al. 1984; Yeo 1998), due to the requirement of ion homeostasis that
favors K+ over Na+ (Leigh and Wyn-Jones 1984), even in plants with as high salt
tolerance as halophytes (Yeo 1998; Greenway and Munns 1980). In most cases,
salinity decreases the protein level as a result of reduced protein synthesis. However,
in some cases, an increase in the levels of proteins is reported (Dubey and Runi
1987; Joshi 1987; Dubey 1994). Pisum sativum plants grown under different
nitrogen sources (NO3, NH4+, NO3NH4, Rhizobium inoculation) and salinity
conditions ( 0 and 90 mM NaCl) showed that both factors influenced protein levels
in peas. Under nonsaline conditions, nutrition with NH4+ propitiated the accumula-
tion of 64% more proteins than the other nitrogen treatments (Fig. 13.3a). However,
the interaction of salinity with the nitrogen source changed this scenario. The
amount of protein increased significantly in NO3- and NO3NH4-treated plants to
levels similar to those previously provided by NH4+, but the seed protein content of
plants dependent on N2 remained low (Fig. 13.3a). However, when calculating the
total seed protein per plant, the value was lower under salt than under control
conditions for all treatments except for NO3, since in the other treatments (NH4+,
NO3NH4 and Rhizobium inoculation) salinity caused a massive reduction in
Fig. 13.3 Seed proteins of Pisum sativum grown for 75 days under different conditions of salinity
(0 and 90 mM NaCl) and nitrogen sources (NO3, NH4+, NO3NH4, Rhizobium inoculation):
a expressed in mg proteins per gram of seed fresh weight, b expressed in mg proteins per plant.
Values are means of 12 replicates. Significantly (P<0.001) different values are marked (*)
productivity (seed weight per plant) that protein increment did not compensate
for (Fig. 13.3b). From a food production perspective, the amount of total protein
produced by plants is more important than the protein concentration in the seed.
Results clearly show that salinity has a strong impact in pea productivity, except for
the NO3 nutrition, suggesting that this form of N should be used as a source of
N nutrition for pea cultivation in salt-affected areas.
13.5 Salinity Influence and Nitrogen Nutrition on Ion

Accumulation
13.5.1 Vegetative Organs
Among the various factors contributing to plant growth, the availability of nutrients
plays a vital role. Inorganic elements regulate plant metabolism by integrating
organic molecules or acting as cofactors or activators of a large number of enzymes.
Their availability for plant growth is influenced by biotic and abiotic factors,
by synergistic and antagonistic interactions, and by uptake rates. Many of the
278 E. Figueira
elements are absorbed or excreted actively, using energy supplied mostly by root
aerobic respiration at the expense of carbohydrates and O2 (Velagaleti and
Schweitzer 1994).
The form of nitrogen supplied to plants (NO3, NH4+ or fixed N2) has a vast
impact on the relationships between anions and cations. Plants nourished with NH4+
are characterized by a high ratio of absorption of cations over anions. The opposite
situation occurs in plants absorbing NO3, due to the higher consumption of
protons used in the cotransport through plasma membrane and to the accumulation
of anions within the cell. Thus, in plants supplied with NH4+, absorption of anions is
favored over cations. In Pisum sativum plants grown in the presence of salt (90 mM
NaCl) and different N sources (NO3, NH4+, NO3NH4, fixed N2), the lower salt
tolerance of plants under NH4+ nutrition may be related to the lower absorption of
K+ and to the higher accumulation of Na+ and Cl in the shoot (Figs. 13.4, 13.5,
13.6). Other authors also reported a similar influence of NH4+ nutrition in plants
grown under saline and nonsaline conditions. For instance, Smart and Bloom
(1998) observed that exposure of tomato (Lycopersicon esculentum L.) plants to
ammonium led to a lower absorption of K+ and an increase in the efflux of protons to
the apoplast. Martı́nez and Cerdá (1989) observed that the addition of NH4+ to the
growing medium decreased the accumulation of K+ in cucumber (Cucumis sativus L.)
plants growing in saline conditions. These authors also found that the increase
of the ratio NH4+/NO3 induced plants to accumulate more Cl and Na+ and less
K+ in the leaves. Plants dependent on symbiotically fixed N2 accumulated lower
concentrations of any of the ions analyzed (Figs. 13.4, 13.5, 13.6) than plants
grown in inorganic nitrogen nutrition (NO3, NH4+, NO3NH4). Thus, the organs
Fig. 13.4 Potassium concentration in roots and shoots of Pisum sativum grown for 75 days at two
salinity levels (0 and 90 mM NaCl) and with four nitrogen nutrition sources: (a NO3, b NH4+,
c NO3NH4, d Rhizobium inoculation): filled circle shoots with 90 mM NaCl, open circle shoots
with 0 mM NaCl, filled triangle roots with 90 mM NaCl, open triangle roots with 0 mM NaCl.
Values are meansstandard errors of six replicates
Fig. 13.5 Sodium concentration in roots and shoots of Pisum sativum grown for 75 days at two
salinity levels (0 and 90 mM NaCl) and with four nitrogen nutrition (a NO3, b NH4+, c NO3NH4,
d Rhizobium inoculation): filled circle shoots with 90 mM NaCl, open circle shoots with 0 mM
NaCl, filled triangle roots with 90 mM NaCl, open triangle roots with 0 mM NaCl. Values are
meansstandard errors of six replicates
Fig. 13.6 Chloride concentration in roots and shoots of Pisum sativum grown for 75 days at two
salinity levels (0 and 90 mM NaCl) and with four nitrogen nutrition (a NO3, b NH4+, c NO3NH4,
d Rhizobium inoculation): filled circle shoots with 90 mM NaCl, open circle shoots with 0 mM
NaCl, filled triangle roots with 90 mM NaCl, open triangle roots with 0 mM NaCl. Values are
meansstandard errors of six replicates
280 E. Figueira
of these plants must have experienced lower toxicity, and in turn a smaller inhibi-
tion would be caused by salt on vegetative and reproductive growth compared to
plants nourished with NO3NH4 and NH4. However, plants tolerated better salt
stress when NO3 was the N form provided. In plants nourished with NO3,
pH increases, enhancing cations absorption and inhibiting the absorption of other
anions, and thus these plants frequently have higher K+ contents than plants
nourished with NH4+ (Marschner 1995). In Pisum sativum plants grown under
different nitrogen sources, plants nourished with NO3 showed a higher alloca-
tion of Na+ in the root (Fig. 13.5), and higher K+/ Na+ ratios and higher Ca2+
concentrations (results not shown) in the shoot compared to plants nourished
with other forms of N. The first recorded response to an increase in Na+ around
roots is an increase in cytosolic-free Ca2+. (Munns and Tester 2008). The best
characterized signaling pathway specific to salinity stress involves increases in
cytosolic-free Ca2+ (Zhu 2002). Calcium ions play a role in maintaining the
integrity of the plasma membrane and the activation effect that Ca2+ has on
plasmalemma P-ATPases can contribute to facilitate the absorption of K+ at low
pH values (Marschner 1995). The influence that Ca2+ has on the maintenance of
high K+/Na+ rates highlights the important role that Ca2+ plays in the mainte-
nance of K+ concentrations, simultaneously restricting the Na+ uptake in salt-
stressed plants. Martı́nez and Cerdá (1989) also observed thatm when NO3 was
the only source of nitrogen available, the accumulation of K+ in plants increased
under saline conditions compared to plants supplied with mixed nitrogen nutri-
tion. A study conducted by Subbarao et al. (1990) showed that the increase in
saline tolerance of three different species of Atylosa was linked to a higher
efficiency in the regulation of Na+ and Cl acropetal transport and to the
maintenance of K+ selectivity. The shoot exclusion mechanism observed in
plants of Pisum sativum under NO3 nutrition seems to have lost much of its
effectiveness in older plants over a 15-day period (between 60 and 75 days), the
concentration of Na+ increased about five times in the shoot and decreased three
times in the root (Fig. 13.5). The Cl ratio between shoots and roots is kept
close to the unit, however, at the end of the growth increases six times
(Fig. 13.6). Thus, for most of the growth period, plants dependent on NO3
accumulated 20–50% less Cl, 30–70% less Na+, and 0.4–3.6 times more K+ in
the shoots than plants nourished with other forms of N, which certainly con-
tributed to the higher salt tolerance observed in plants nourished with nitrate.
In most situations, Cl influx requires energy and is probably catalyzed by a Cl/
2H+ cotransporter (Felle 1994; Sanders 1980). The cytosolic Cl concentration is
likely in the range of 10–20 mM, but may be higher in saline conditions (Munns and
Tester 2008). Cl tissue concentrations as high as 400 mM are tolerated by most
species, and even sensitive species can tolerate 250 mM (Munns and Tester 2008).
The absorption of Cl is often inhibited by NO3 (Marschner 1995; Grattan and
Grieve 1994), not only because of the influence of pH, but also due to the
competition between the two anions (Caldwell et al. 1986; Glikey and Staehelin
1989; Marschner 1995; Tyerman 1992). This competition is of great importance in
the tolerance to salinity. Rogers et al. (1997) observed that the difference in salinity
tolerance shown by two populations of white clover (Trifolium repens L.) was due
to the lower absorption of Cl and to an increase of its restriction efficiency from
the shoot. Läuchli and Wieneke (1979) recorded that some varieties of G. max
exhibited different salt tolerances and that these differences could be related to the
restriction of Cl transport in the xylem. Rogers et al. (1997) and Läuchli and
Wieneke (1979) concluded that within these species salt tolerance seems to be
related to Cl exclusion, in particular from the shoot. Chloride concentration
differences in Pisum sativum plants grown under different forms of nitrogen
nutrition and NaCl concentrations (Fig. 13.6) showed that Cl accumulation is
not only genotypically fixed but is also dependent on the N source.
13.5.2 Seeds
In pea plants grown in the presence of NaCl, Na+ concentrations in seeds are about
ten times lower than those found in vegetative organs, with the N form supplied
Fig. 13.7 Potassium (a) sodium (b) and chloride (c) concentrations in seeds of Pisum sativum
plants grown for 75 days under different conditions of salinity (0 and 90 mM NaCl) and nitrogen
sources (NO3, NH4+, NO3NH4, Rhizobium inoculation). Values are meansstandard errors of 16
replicates. Significantly (P<0.001) different values are marked (*)
282 E. Figueira
influencing Na+ levels (Fig. 13.7b), as also reported by Greenway and Munns
(1980), Hajibagheri et al. (1987), and Marschner (1995), who suggested that Na+
is excluded from reproductive and young vegetative organs. Chloride, that was
preferentially accumulated in the shoot, was also excluded from seeds, although
less effectively than Na+, and concentrations varied with the N form supplied to
plants (Fig. 13.7c). The large acropetal translocation of ions that occurred at the
later stages of Pisum sativum plants growth was also described by Pate and Flinn
(1977), and was explained by these authors as a physiological process that mobi-
lizes organic nutrients and minerals to the growing seeds. Although this process is
advantageous for seeds, it also changes the mechanisms of salt tolerance, with
decreases in the efficiency of Na+ and Cl exclusion from the shoot, leaving this
organ more vulnerable to salinity, but maintaining low Na+ and Cl concentrations
in the reproductive organs and thus not compromising seeds viability.
13.6 Conclusion and Future Prospects
In this chapter, a better knowledge of Pisum sativum responses to salinity as well as

some of the factors that influence these responses is provided. Plants nourished with
NO3 experienced lower toxicity in the shoots than those of other N treatments, due
to lower accumulations of Na+ and Cl and to higher K+ concentrations. Thus, it is
possible to grow Pisum sativum in moderate salinity levels without significant
decreases in productivity if plants are provided with adequate levels of nitrogen
in the form of nitrate. Plants dependent on symbiotically-fixed nitrogen had lower
productivity, but were not as affected by salinity as plants dependent on ammonium
or ammonium nitrate.
This chapter also raised some issues that require urgent attention. Can the study
of salt tolerance of different Pisum sativum cultivars extend the known range of this
species tolerance? Would the screening of new Rhizobium strains increase the
nodulation efficiency in Pisum sativum growing in saline and nonsaline conditions?
Which are the organic solutes that cells of Pisum sativum accumulate in order to
adjust the cytoplasm osmotically? What are the concentration gradients of sodium
and chloride through tonoplast in each of the plant organs? How are xylem loading
and acropetal transport of ions regulated? What will be the growth and yield
responses of plants under salt stress to a mixed nutrition of symbiotic nitrogen
and low levels of nitrate?
The answer to these questions could be approached in future in order to reach a
better understanding of the factors determining salt tolerance in legumes, and allow
more efficient pulses cropping in salinized areas without massive reductions in
productivity or high inputs of inorganic N fertilization.
Acknowledgments I thank Georgina Hodge for her help and suggestions. This work was
supported by the Centre for Cell Biology.
References
Abd-Alla MH, Vuong TD, Harper JE (1998) Genotypic differences in dinitrogen fixation response
to NaCl stress in intact and grafted soybean. Crop Sci 38:72–77
Atta S, Maltese S, Cousin R (2004) Protein content and dry weight of seeds from various pea
genotypes. Agronomie 24:257–266
Bekki A, Trinchant JC, Rigaut J (1987) Nitrogen fixation (C2H2 reduction) by Medicago nodules
and bacteroides under sodium chloride stress. Physiol Plantarum 71:61–67
Below FE (1995) Nitrogen metabolism and crop productivity. In: Pessarakli M (ed) Handbook of
plant and crop physiology. Marcel Dekker, New York, pp 275–301
Binzel ML, Hess FD, Bressan RA, Hasegawa PM (1988) Intracellular compartmentation of ions in
salt adapted tobacco cells. Plant Physiol 86:607–614
Bohnert HJ, Su H, Shen B (1999) Molecular mechanisms of salinity tolerance. In: Shinozaki K,
Yamaguchi-Shinozaki K (eds) Molecular responses to cold, Drought, heat and salt stress in
higher plants. University of Arizona, Arizona, pp 29–60
Botsford JL, Lewis TA (1990) Osmoregulation in Rhizobium meliloti: production of glutamic acid
in response to osmotic stress. Appl Environ Microbiol 56:488–494
Bourgeais-Chaillou P, Alfocea FP, Guerrier G (1992) Comparative effects of N-sources on growth
and physiological responses of soybean exposed to NaCl-stress. J Exp Bot 43:1225–1233
Bray CM (1983) Nitrogen metabolism in plants. Longman, New York
Brewing NJ, Ambrose MJ, Downie JA (1993) Root nodules, Rhizobium and nitrogen fixation.
In: Casey R, Davies DR (eds) Peas: genetics, molecular biology and biothechnology. Cab
International, Wallingford, pp 237–290
Caldwell JH, Van Brunt J, Harold FM (1986) Calcium-dependent anion channel in the water mold,
Blastocladiella emersonii. J Membrane Biol 39:85–97
Cordovilla MP, Ligero F, Lluch C (1994) The effect of salinity, N fixation and assimilation in
Vicia faba. J Exp Bot 279:1483–1488
Cordovilla MD, Ligero F, Lluch C (1996) Growth and nitrogen assimilation in nodules in response
to nitrate levels in Vicia faba under salt stress. J Exp Bot 47:203–210
Cordovilla MD, Ligero F, Lluch C (1999a) Effect of salinity on growth, nodulation and nitrogen
assimilation in nodules of faba bean (Vicia faba L.). Appl Soil Ecol 11:1–7
Cordovilla MD, Berrido SI, Ligero F, Lluch C (1999b) Rhizobium strain effects on the growth and
nitrogen assimilation in Pisum sativum and Vicia faba plant growth under salt stress. Plant Sci
140:127–136
Cordovilla MP, Ligero F, Lluch C (1999c) Effects of NaCl on nitrogen fixation and assimilation of
inoculated and KNO3 fertilized of Vicia faba L. and Pisum sativum L. Plant Sci 140:127–136
Craig GF, Atkins CA, Bell DT (1991) Effect of salinity on growth of four strains of Rhizobium and
their infectivity and effectiveness on two species of Acacia. Plant Soil 133:253–262
Delgado MJ, Ligero F, Lluch C (1993) Nitrogen fixation and carbon metabolism by nodules and
bacteroids of pea plants under sodium chloride stress. Physiol Plantarum 89:824–829
Delgado MJ, Ligero F, Lluch C (1994) Effect of salt stress on growth and nitrogen fixation by pea,
faba bean, common bean and soybean plants. Soil Biol Biochem 26:371–376
Delgado MJ, Garrido JM, Ligero F, LIuch C (2006) Nitrogen fixation and carbon metabolism by
nodules and bacteroids of pea plants under sodium chloride stress. Physiologica plantarum
89:824–829
Doré T, Meynard JM, Sebillotte M (1998) The role of grain number, nitrogen nutrition and stem
number in limiting pea crop (Pisum sativum) yields under agricultural conditions. Eur J Agron
8:29–37
Dubey RS (1994) Protein synthesis by plants under stressful conditions. In: Pessarakli M (ed)
Handbook of plant and crop stress. Marcel Dekker, New York, pp 277–299
Dubey RS, Runi M (1987) Proteases and proteins in germinating rice seeds in relation to salt
tolerance. Plant Physiol Biochem 12:9–15
284 E. Figueira
Elsheikh EAE, Wood M (1990) Effect of salinity on growth, nodulation and nitrogen yield of
chickpea (Cicer arietinum L.). J Exp Bot 231:1263–1269
Elsheikh EAE, Wood M (1995) Nodulation and N2 fixation by soybean inoculated with salt-
tolerant rhizobia or salt-tolerant bradyrhizobia in saline soil. Soil Biol Biochem 27:657–661
Feigin A (1990) Interactive effects of salinity and ammonium/nitrate ratio on growth and chemical
composition of melon plants. J Plant Nutr 13:1257–1269
Felle H (1994) The H+/Cl symporter in root-hair cells of Sinapis alba. An electrophysiological
study using ion-selective microelectrodes. Plant Physiol 106:1131–1136
Figueira EMDAP, Caldeira GCN (2005) Effect of nitrogen nutrition on salt tolerance of Pisum
sativum during vegetative growth. J Plant Nutr Soil Sci 168:359–363
Fox TC, Guerinot ML (1998) Molecular biology of cation transport in plants. Annu Rev Plant
Physiol Plant Mol Biol 49:669–696
Gazzarrini S, Lejay T, Gojon A, Ninnemann O, Frommer WB, Von Wiren N (1999) Three
functional transporters for constitutive, diurnally regulated, and starvation induced uptake of
ammonium into Arabidopsis roots. Plant Cell 11:937–947
Georgiev GI, Atkins CA (1993) Effects of salinity on N2 fixation, nitrogen metabolism, export and
diffusive conductance of cowpea root nodules. Symbiosis 15:239–255
Gessler A, Schneider S, Von Sengbusch D, Weber P, Hanemann U (1998) Field and laboratory
experiments on net uptake of nitrate and ammonium by the roots of spruce (Picea abies) and
beech (Fagus sylvatica) trees. New Phytol 138:275–285
Gibson TS, Speirs J, Brady CJ (1984) Salt tolerance in the plants. II. In vivo translaction of m-RNAs
from salt-tolerant and salt-sensitive plants on wheat germ ribosomes: responses to ions and
compatible solutes. Plant Cell Environ 7:579–587
Glikey JC, Staehelin LA (1989) A new organelle related to osmoregulation in ultrarapidly frozen
Pelvetia embryos. Planta 178:425–435
Grattan SR, Grieve CM (1994) Mineral nutrient acquisition and response by plants grown in saline
environments. In: Pessarakli M (ed) Handbook of plant and crop physiology. Marcel Dekker,
New York, pp 203–226
Greenway H, Munns R (1980) Mechanisms of salt tolerance in nonhalophyes. Annu Rev Plant
Physiol 31:149–190
Grossman A, Takahasi H (2001) Macronutrient utilization by photosynthetic eukaryotes and the
fabric of interactions. Annu Rev Plant Physiol Plant Mol Biol 52:163–210
Hajibagheri MA, Harvey DMR, Flowers TJ (1987) Quantitative ion distribution within root cells
salt-sensitive and salt-tolerant maize varieties. New Phytol 105:367–379
Hartwig UA (1998) The regulation of symbiotic N2 fixation: a conceptual model of N feedback
from the ecosystem to the gene expression level. Perspect Plant Ecol Evol Syst 1:92–120
Jackson LE, Martin Burger M, Cavagnaro TR (2008) Roots, nitrogen transformations, and
ecosystem services. Annu Rev Plant Biol 59:341–363
Joshi S (1987) Effect of soil salinity on nitrogen metabolism in Cajanus cajan L. Indian J Plant
Physiol 30:223–231
Läuchli A (1984) Salt exclusion: an adaptation of legumes for crops and pastures under saline
conditions. In: Staples R, Toenniessen GH (eds) Salinity tolerance in plants: strategies for crop
improvement. Wiley, New York, pp 171–187
Läuchli A, Wieneke J (1979) Studies on growth and distribution of Na+, K+ e Cl in soybean
varieties differing in salt tolerance. Z Pflanzenk Pflanzens 142:3–13
Lauter DJ, Munns DN (1986) Salt resistance of chickpea genotypes in solutions salinised with
NaCl and Na2SO4. Plant Soil 95:271–279
Lee TD, Tjoelker MG, Reich PB, Russelle MP (2003) Contrasting growth response of an N2-fixing
and nonfixing forb to elevated CO2: dependence on soil N supply. Plant Soil 255:475–486
Leidi EO, Sliberbush M, Soares MIM, Lips SH (1992) Salinity and nitrogen nutrition studies on
peanut and cotton plants. J Plant Nutr 15:591–604
Leigh RA, Wyn-Jones RG (1984) A hypothesis relating critical potassium concentration for
growth to the distribution and functions of this ion in the plant cell. New Phytol 97:1–13
Lewis OAM, Leidi EO, Lips SH (1989) Effect of nitrogen source on growth response to salinity
stress in maize and wheat. New Phytol 111:155–160
Lovell PH (1977) Correlative influences in seedling growth. In: Sutcliffe JF, Pate JS (eds) The
physiology of the garden pea. Academic, London, pp 265–290
Maas EV, Hoffman GJ (1977) Crop salt tolerance – current assessment. Am Soc Civil Eng
103:115–134
Marschner H (1995) Mineral nutrition of higher plants. Academic, London
Martı́nez V, Cerdá A (1989) Influence of N source on rate of Cl, N, Na+, and K+ uptake by
cucumber seedlings grown in saline conditions. J Plant Nutr 12:971–983
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681
Pate JS, Flinn AM (1977) Fruit and seed development. In: Sutcliffe JF, Pate JS (eds) The
physiology of the garden pea. Academic, London, pp 431–468
Pereira SIA, Lima AIG, Figueira EMAP (2008) Rhizobium leguminosarum isolated from agricul-
tural ecosystems subjected to different climatic influences: the relation between genetic
diversity, salt tolerance and nodulation efficiency. In: Liu TX (ed) Soil ecology research
development. Nova Science, New York, pp 247–263
Rengasamy P (2002) Transient salinity and subsoil constraints to dryland farming in Australian
sodic soils: an overview. Aust J Exp Agric 42:351–361
Robson AD, Bottomley PJ (1991) Limitations in the use of legumes in agriculture and forestry. In:
Dilworth MJ, Glenn AR (eds) Biology and biochemistry of nitrogen fixation. Elsevier,
Amesterdam, pp 320–349
Rogers ME, Noble CL, Halloran GM, Nicolas ME (1997) Selecting for salt tolerance in white
clover (Tripolium repens): chloride ion exclusion and its heritability. New Phytol 135:645–654
Salehi M, Salehi F, Poustini K, Heidari-Sharifabad H (2008) The effect of salinity on the nitrogen
fixation in 4 cultivars of Medicago sativa L. in the seedling emergence stage. Res J Agric Biol
Sci 4:413–415
Sanders D (1980) The mechanism of Cl transport at the plasma-membrane of Chara corallina.
I Cotransport with H+. J Membr Biol 53:129–141
Serraj R, Roy G, Drevon JJ (1994) Salt stress induces a decrease in the oxygen uptake of soybean
nodules and in their permeability to oxygen diffusion. Physiol Plantarum 91:161–168
Siddiqi MY, Malhotra B, Min XJ, Glass ADM (2002) Effects of ammonium and inorganic carbon
enrichment on growth and yield of a hydroponic tomato crop. J Plant Nutr Soil Sci 165:191–197
Silberbush M, Lips SH (1991a) Potassium, nitrogen, ammonium/nitrate ratio, and sodium chloride
effects on wheat growth. I. Shoot and root growth and mineral composition. J Plant Nutr
14:751–764
Silberbush M, Lips SH (1991b) Potassium, nitrogen, ammonium/nitrate ratio, and sodium chloride
effects on wheat growth. II. Tillering and grain yield. J Plant Nutr 14:765–773
Singleton PW, Bholool BB (1983) Effect of salinity on functional components of the soybean-
Rhizobium japonicum symbiosis. Crop Sci 23:451–460
Singleton PW, El Swaify SA, Bohlool BB (1982) Effect of salinity on Rhizobium growth and
survival. Appl Environ Microbiol 44:884–890
Smart DR, Bloom AJ (1998) Investigations of ion absorption during NH4+ exposure. I. Relation-
ship between H+ efflux and NO3 absorption. J Exp Bot 49:95–100
Sprent JI, Zahran HH (1988) Infection, development and functioning of nodules under drought and
salinity. In: Beck DP, Materon LA (eds) Nitrogen fixation by legumes in Mediterranean
agriculture. Martinus Nijhoff, Dordrecht, pp 145–151
Subbarao GV, Johansen C (1994) Strategies and scope for improving salinity tolerance in crop
plants. In: Pessarakli M (ed) Handbook of plant and crop stress. Marcel Dekker, New York, pp
559–579
Subbarao GV, Johansen C, Jana MK, Kumar Rao JFDK (1990) Effects of sodium/ calcium ratio in
modifying salinity response of pigeonpea (Cajanus cajan). J Plant Physiol 136:439–443
Tawfik KM (2008) Evaluating the use of rhizobacterin on cowpea plants grown under salt stress.
Res J Agric Biol Sci 4:26–33
286 E. Figueira
Tu JC (1981) Effect of salinity on Rhizobium-root-hair interaction, nodulation and growth of

soybean. Can J Plant Sci 61:231–239
Tyerman SD (1992) Anion channels in plants. Annu Rev Plant Physiol Plant Mol Biol 43:351–373
Velagaleti RR, Schweitzer SM (1994) General effects of salt stress on growth and symbiotic
nitrogen fixation in soybean. In: Pessarakli M (ed) Handbook of plant and crop stress. Marcel
Dekker, New York, pp 461–471
Wahab AMA, Zahran HH (1981) Effects of salt stress in the nitrogenase activity and growth of
four legumes. Biol Plant 23:16–23
Ward JM (1997) Patch-clamping and other molecular approaches for the study of plasma mem-
brane transporters demystified. Plant Physiol 114:1151–1159
Williams LE, Miller AJ (2001) Transporters responsible for the uptake and partitioning of
nitrogenous solutes. Annu Rev Plant Physiol Plant Mol Biol 52:659–688
Yeo AR (1998) Molecular biology of salt tolerance in the context of whole-plant physiology. J Exp
Bot 49:915–926
Zahran HH (1991) Conditions for successful Rhizobium-legume symbiosis in saline environments.
Biol Fertil Soils 12:73–80
Zahran HH, Rsanen LA, Karsisto M, Lindstrom K (1994) Alteration of lipopolysaccharide and
protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress. World J. Microbiol.
Biotechnol. 10:100–105
Zhu JK (2002) Salt and drought signal transduction in plants. Annu Rev Plant Biol 53:247–273
Chapter 14
Plant Growth-Promoting Diazotrophs and
Productivity of Wheat on the Canadian Prairies
Anthony O. Anyia, Daniel J. Archambault, Carols J. Bécquer,

and Jan J. Slaski
Abstract Nitrogen is an essential plant nutrient, which is limiting for wheat

production in Canada. To achieve higher yields, farmers often apply large amounts
of nitrogen fertilizers at a considerable cost. The prospects of extending biological
nitrogen fixation (BNF) to non legume crops, as a way to overcome the high cost
and ecological issues of nitrogenous fertilizers, has remained a dream to date.
Several studies have reported colonization and N2 fixation by rhizobia strains in
chemically-induced para-nodules (nodule-like structures). The extent to which
nitrogen fixation in para-nodules and intercellular spaces benefits plants, especially
under field conditions remains unclear. Exploiting the benefits of diazotrophs as
plant growth-promoting rhizobacteria (PGPR) appears to be a more promising
approach, assuming that issues of lack of consistency of growth stimulation can
be resolved. This chapter describes experiences and progress made in Canada
towards the application of Azorhizobium caulinodans and native rhizobia strains
as inoculants for improving wheat production.
A.O. Anyia (*)

Alberta Research Council, Bioresource Technologies, P.O. Bag 4000, Vegreville, Alberta,
Canada, T9C 1T4
e-mail: Anthony.Anyia@arc.ab.ca
D.J. Archambault
Laurentian University, 935 Ramsey Lake Road, Sudbury, Ontario, Canada, P3E 2C6
e-mail: darchambault@laurentian.ca
C.J. Bécquer
Ministerio de la Agricultura, Instituto de Ganaderı́a Tropical, Estación Experimental
Sancti-Spiritus, Cuba
e-mail: becquer@pastos.yayabo.inf.cu
J.J. Slaski
Alberta Research Council, Bioresource Technologies, P.O. Bag 4000, Vegreville, Alberta,
Canada, T9C 1T4
e-mail: Jan.Slaski@arc.ab.ca
288 A.O. Anyia et al.
14.1 Introduction
14.1.1 Wheat in Canada
Wheat (common, Triticum aestivum and durum, T. turgidum) is the most important
cereal crop in the world, which, together with rice and maize, account for about
73% of world cereal production. Canada is one of the world’s largest producers and
exporters of wheat. In 2007, Canada ranked as the eighth largest wheat-producing
country in the world (Fig.14.1). Over the last decade, the annual production of
wheat in Canada has consistently exceeded 20 million tons, except in 2002 when
production dropped to about 16 million tons due to severe drought in the Prairie
Provinces. Wheat is Canada’s largest crop both in terms of area seeded and
production. All Canadian provinces, except Newfoundland and Labrador, grow
wheat; Saskatchewan, Manitoba, Alberta, and Ontario are the leading wheat pro-
ducers in Canada. The annual revenue derived from wheat export in Canada
exceeds $5 billion, making wheat the highest earner of all exported agricultural
products. Only one class of durum is grown in Canada, spring amber durum;
however, there are several classes of common wheat grown in Canada, based on
seed hardness and color, and on sowing time (autumn or spring).
14.1.2 Nitrogen Use in Wheat Production
Canadian wheat yields are relatively low averaging 2.4 tha1 between 1998 and
2007 compared to the world average of 2.7 tha1 (FAOStart, Statistics Canada).
120.0
109.9
100.0
Million metric tonnes
80.0 74.9
60.0 53.6
49.4
40.0 33.2
23.5 21.4 20.6 17.7 16.5
20.0
0.0
a
na
y
ce
a
ey
SA
n
an
ad
di
si
ta
an
hi
rk
st
In
us
U
s
m
an
C
Tu
ki
kh
Fr
R
er
C
Pa
za
G
Ka
Fig. 14.1 Wheat-producing top 10 countries of the world (FAOSTAT data, 2007)
14 Plant Growth-Promoting Diazotrophs and Productivity of Wheat 289
Nitrogen is a very important plant nutrient whose limitation can affect yield
considerably. The emergence of nitrogen-containing synthetic fertilizers is one of
the factors that contributed to the “green revolution” in the middle of the twentieth
century. The Haber-Bosch process allows for the production of ammonia from
nitrogen and hydrogen, which are the main inputs used in the production of all
nitrogen fertilizers. The hydrogen used in the Haber-Bosch process is derived from
natural gas. Farmers in Canada as in many other countries are highly dependent on
nitrogen fertilizers to achieve high yields. In terms of tonnage, nitrogen accounted
for about 60% of the total sales of chemical fertilizers in Canada in 2004 (Canadian
Fertilizer Institute). Production of chemical nitrogen fertilizers, besides being
very cost intensive, also depletes nonrenewable resources and poses human and
environmental hazards. To complement and eventually substitute mineral fertilizers
with biologically fixed nitrogen would represent an economically beneficial and
ecologically sound alternative (Glick et al. 1999).
Analysis of nitrogen use on the Canadian prairies over the last three decades
shows a steady increase from approximately 125,000 metric tons per year in 1969 to
more than 1.25 million tons per year since 1996. The Canadian Fertilizer Institute
reported total sales of nitrogen-containing fertilizers of 2.57 million metric tons in
western Canada in the year ending June 30, 2000. Data show that nitrogen applica-
tions represent 22.1 and 33%, respectively, of the operating costs of corn and wheat
production. In the black soils zone of Alberta, a wheat farmer would spend
approximately three times more on nitrogen fertilizer than his counterpart who
grows feed peas (Pisum sativum) inoculated with rhizobia. This suggests that the
introduction of nitrogen-fixing bacteria to wheat could significantly reduce the cost
of nitrogen fertilizer input. Norman Borlaug, in his Nobel Peace Prize lecture in
1970, highlighted the need to extend the symbiotic nitrogen fixation of legumes
with rhizobia to the world’s major cereals, maize (Zea mays), wheat and rice (Oryza
sativa) to sustain the green revolution (Borlaug 1970). Haber, who invented the
famous “Haber-Bosch process” of ammonia production, in his 1920 Nobel Prize
acceptance speech noted that “it may be that this solution is not the final one.”
Nitrogen bacteria teach us that nature, with her sophisticated forms of the chemistry
of living matter, still understands and utilizes methods, which we do not as yet
know how to imitate (Cocking 2005).
14.2 Nitrogen Fixation and Non legume Plants
Many researchers have long dreamt of the prospects of extension of biological

nitrogen fixation (BNF) to non legume crops. The extension was thought to be able
to overcome the high cost and ecological issues of nitrogenous fertilizers. When in
1969 Van Overbeek urged botanists to tackle the problem of nitrogen deficiencies
with boldness (Quispel 1991), molecular biology was still in its infancy. Van
Overbeek stated then that “we need to device more efficient ecosystems, put root
nodules on cereal crops, and put nitrogen-fixing chloroplasts in their leaves.” At
that time, many thought that, with increased knowledge and advances in molecular
biology, nitrogen fixation would be easily achieved in non legume crops. Several
decades later, nitrogen fixation by non legumes is still only a dream. The complexi-
ty of the genes of the nitrogenase enzyme complex involved in nitrogen fixation has
so far prevented their successful transfer with function in plant cells. Quispel (1991)
concluded that “in the light of our present knowledge the prospect of active
nitrogenase in plant cells seems farther removed than it appeared in 1969.” As
sad as it may be, the same conclusion still holds today.
A 1987 patent (Publication No.: WO/1987/004182) by NIELSEN, Sven-
Erik SO/RENSEN, Grete, Mo/rch, published at the World Intellectual Property
Organization claims to have invented “Rhizobia transformants that nodulate and
fix nitrogen in non legumes. The nodulated non legume plants can be grown without
nitrogenous fertilizer and have at least the same or higher protein content, dry matter
content and nitrogen content than their nonnodulated counterparts which are fertilized
by the addition of nitrogenous fertilizer. The straw remaining after harvesting the
nodulated non legumes is also high in protein content”. There is no indication if
these transformants were ever tested in field experiments and how well they per-
formed. As was noted by Quispel (1991) “we are able to select and modify bacterial
strains, host plants and bacteria–plant combinations, which are highly efficient
under laboratory conditions. Successful field inoculation methods have been devel-
oped. Yet results in the field are mostly disappointing since the introduced selected
bacteria have to compete with the indigenous Rhizobium populations in the soil.”
14.3 Plant Growth-Promoting Effects of Diazotrophs
Bacteria are abundantly present in the rhizosphere and in close vicinity of the root.
It has long been recognized that several genera of these rhizobacteria have the
ability to promote plant growth, and these have been termed plant growth-promoting
rhizobacteria or PGPR. Some of these rhizobacteria interact with the plant in
different mutually beneficial ways and may thereby promote plant growth or
yield by direct or indirect mechanisms. Direct growth promotion may be through
biofertilization by synthesis of elements or compounds utilizable by the plant, or by
aiding the plant in uptake of nutrients and/or water. Indirect growth promotion on
the other hand may entail biocontrol of infection by phyto-pathogenic organisms.
The bacteria that provide some benefit to plants are of two general types: those that
form a symbiotic relationship, which involves formation of specialized structures or
nodules on host plant roots, and those that are free-living in the soil; the latter are
often found near, on, or even within the roots of plants (Kloepper et al. 1988).
Examples of symbiotic diazotrophs include: (1) rhizobia (includes soil bacteria of
the genera Rhizobium, Azorhizobium and Bradyrhizobium), which interact with
leguminous plants (belonging to the Fabaceae family) to form N2-fixing nodules;
(2) Frankia – “actinorhizal” nitrogen fixers that form similar symbiosis to rhizobia,
with a number of woody plant species; and (3) symbiotic cyanobacteria which,
unlike rhizobia and Frankia, do not form nodules. The host range of cyanobacteria
varies widely from fungi, mosses, and liverworts to waterferns (Azolla), some
Gymnosperms (e.g., Cycas and Macrozamia) and Angiosperms (Gunnera) (Quispel
1991). In these symbioses, the plant supplies energy materials to the diazotrophs,
which in turn reduce atmospheric nitrogen to ammonia. This ammonia is trans-
ferred from the bacteria to the plant to meet the plant’s nutritional nitrogen needs
for the synthesis of proteins, enzymes, nucleic acids, and chlorophyll. Some
examples of free-living growth-promoting rhizobacteria are Azobacter species,
Azospirillum species, pseudomonads, bacilli, and Burkholderia species (Brown
1974; Elmerich 1984; Kloepper et al. 1989; Quispel 1991).
Associative nitrogen fixation in rice was reported to have supplied 20–25% N of
the total need in a study at International Rice Research Institute, Philippines (Ladha
et al. 1987). Rice seedlings inoculated with Burkholderia spp. previously isolated
from rice plants contained a relatively higher level of N due to BNF (Baldani et al.
2000). Sevilla and Kennedy (2000) suggested that Acetobacter diazotrophicus
could promote rice growth, which might be related to the transfer of biologically
fixed N, although other factors such as auxin production could be involved.
Inoculation of wheat with Azospirillum brasilense and A. lipoferum significantly
increased the biomass and grain yield as well as branching of root hairs (Kennedy
and Tchan 1992).
14.4 Beneficial Effects of Rhizobia in Non legume Crops
According to Saikia and Jain (2007) advances in agricultural sustainability will

require an increase in the utilization of BNF as a major source of nitrogen for plants.
The beneficial effects of legumes in crop rotation have long been known. Albrecht
Thaer – the famous German pioneer of agricultural research – said this in 1856:
“Latterly the practice of sowing white clover (Trifolium repens) with the last crop
has become very general. Only a very few apathetic and indolent agriculturalists or
men who are firmly wedded to their opinions and customs neglect this practice.”
Legumes have also been used for soil improvement for centuries because of their N
and non-N rotational benefits to non legume crops (Lupwayi et al. 2005). Seeding a
non legume crop after legumes in a rotation can reduce the amount of nitrogen
required to maintain yield. Noel et al. (1996) suggested that Rhizobium not only
interacts with the roots of legumes but may also interact beneficially with nonlegu-
minous plants. These authors concluded that “as Rhizobium may be found as a free-
living rhizobacterium and appears to promote growth of non legumes as well as
host legume species, the term PGPR seems to apply.”
Yanni et al. (2001) investigated the benefits of inoculating rice with Rhizobium
leguminorarum bv trifolii. The authors exploited the long-term association between
this Rhizobium and rice in the Egyptian Nile delta where rice has been rotated
successfully with berseem clover (Trifolium alexandrinum L) for ages. Greenhouse
and field inoculation results of these experiments show an increase in plant
performance and grain yield, which was associated more with rhizobial effects on
rice root architecture for enhanced uptake of soil nutrients. The authors suggested
that some rhizobia may have evolved an additional ecological niche enabling them
to form a three-component life cycle including a free-living heterotrophic phase in
soil, a N2-fixing endosymbiont phase within legume root nodules, and beneficial
growth promoting endocolonizer phase within cereal roots in the same crop rotation.
Azorhizobium caulinodans is a stem and root nodulating nitrogen-fixing bacte-
ria, which was isolated from the stem nodules of Sesbania rostrata (Dreyfus et al.
1988). The nitrogenase enzyme of A. caulinodans is reported to be more tolerant of
oxygen than other rhizobia studied so far (Dreyfus et al. 1983). Several studies have
shown that this bacterium is able to colonize intercellular spaces of cortex, xylem
and root meristems of several non legumes through crack entry of emerging lateral
roots (Sabry et al. 1997; Reddy et al. 1997; Webster et al. 1997; Cocking 2001). In
the studies of Sabry et al. (1997), inoculation of aseptically grown wheat with
A. caulinodans resulted in stimulation of root development accompanied by
increased yield and N content. Since inoculated plants showed some level of
acetylene reduction activity (a measure of N2 fixation), the increase in plant growth
and N content were attributed to nitrogen fixation by the bacterium. In follow-up
studies with A. caulinodans by Mathews et al. (2001) in a “temperate” controlled
environment, inoculation of wheat again resulted in increased yield and plant
N content. However, application of a non-N2-fixing strain of A. caulinodans or a
filter-sterilized supernatant of the bacterium produced similar growth stimulation
effects as the N2-fixing strain. Mathews et al. (2001) concluded that response of
wheat to A. caulinodans was not due to N2 fixation but probably related to plant
growth substances produced by the bacteria in culture.
14.5 Trials with A. caulinodans in Alberta, Canada
In a preliminary greenhouse study with a genetically improved strain of A. cauli-

nodans ORS 571 (Geelen et al. 1995) using soil beds to simulate field conditions,
Archambault and Li (2002) reported that inoculation of wheat (variety CDC Teal )
with A. caulinodans increased biomass and grain yield by 49 and 34%, respectively.
In subsequent growth chamber and greenhouse experiments, growth of wheat cv.
CDC Teal was consistently enhanced by inoculation with A. caulinodans ORS 571
(Anyia et al. 2004). In one greenhouse experiment, leaf area, leaf area index, and
biomass of inoculated plants were significantly higher in inoculated plants than in
the uninoculated controls at 7 weeks after seeding (Table 14.1 and Fig. 14.2).
Following successful and consistent positive results with A. caulinodans ORS
571 inoculation with wheat, it was thought that similar effects could be achieved
under field conditions. During the summer of 2004, field trials were conducted at
one location in Vegreville, Alberta, Canada, and in another location at Whitewater,
Wisconsin, USA, using seeds of spring wheat pre-inoculated with A. caulinodans
ORS 571. The trials were repeated in 2005 at four locations in Alberta, Canada,
Table 14.1 Means and standard errors of growth parameters at 7 weeks of inoculated and
uninoculated plants of wheat cv. CDC Teal grown in soil beds in a greenhouse
Treatment Height (cm) Leaf area (cm2) Leaf area index Tillers Biomass
(g5 plants)
Control 37.720.72 52.125.01 0.350.03 0.830.19 4.890.50
Inoculated 38.090.50 62.115.32 0.500.04 1.170.10 5.630.36
Difference (%) 1 19 41 40 15
Fig. 14.2 Wheat cv. CDC Teal grown in the presence (right) and absence (left) of A. caulinodans
representing different agro-ecological zones. One location in the southern part of

the province was irrigated due to low precipitation. A. caulinodans inoculant was
also tested in eight field locations including five winter wheat and three spring
wheat plots in the USA. Results of the 2 years of field testing are presented in
Table 14.2. In 2004, inoculated plots at the Vegreville location consistently showed
greater biomass production and grain yields than control plots. Overall, across all
four nitrogen levels used in the trial, grain yield of inoculated plots increased by
10% over controls (Table 14.1). The yield advantage of inoculated treatments over
controls, although consistent at all nitrogen levels used, was however not statisti-
cally significant at p¼0.05. In Whitewater, Wisconsin, inoculated plots showed
significant positive effects. Grain yield increased by 6.4% (Table 14.2) while plant
vigor increased by 10.6% (data not shown).
In the 2005 trial, grain yield of spring wheat at all locations in Alberta, Canada,
except one showed positive effects of inoculation, which was again not statistically
significant. Grain yield of inoculated plots were 4% (Vegreville), 8% (St. Vincent)
and 14% (Taber) higher than the corresponding uninoculated control plots. In the
USA, across all eight trials, inoculation showed an average increase of 1.3%.
Considering the Wisconsin trials alone, inoculation with A. caulinodans caused
an average increase of 6.5%. Half the trial locations (winter and spring wheat)
did not respond significantly to inoculation. Although A. caulinodans inoculation
showed very positive effects at some of the locations tested, the effects were
not consistent. Compared with the greenhouse results, which were highly consis-
tent and significant, those of the field trials were disappointing. Results of the
Table 14.2 Field testing of wheat inoculated with A. caulinodans in Canada and USA
Location Year Control Inoculated % increase
Alberta, Canada, trial spring wheat (extrapolated grain yield in kg ha1)
Vegreville 2004 2,664 2,932 10.1
Vegreville 2005 2,975 3,095 4.0
St Vincent 2005 4,199 4,533 8.0
Innisfail 2005 2,712 2,637 2.8
Taber 2005 4,142 4,705 13.6
Wisconsin and Idaho, USA, trial spring wheat (grain yield in kg/ha)
Whitewater, WI 2004 1,197 1,273 6.4
Genesee, ID 2005 1,496 1,578 5.5
Moscow, ID 2005 1,768 1,768 0.0
Nezperce, ID 2005 1,605 1,632 1.7
Wisconsin and Idaho, USA, trial winter wheat (grain yield in kg/ha)
Whitewater, WI 2005 2,068 2,231 7.9
Whitewater, WI 2005 2,422 2,585 6.7
Genesee, ID 2005 2,286 2,258 1.2
Lewiston, ID 2005 2,912 2,857 1.9
Nezperce, ID 2005 2,830 2,830 0.0
A. caulinodans trial did not meet the minimum requirement of 95% significance
required for registration of new inoculants or soil amendments in Canada.
Several reasons could be responsible for the difference observed between effects
of inoculation in greenhouse and field studies. Two obvious ones in the experiments
with A. caulinodans in Canada would be the methods of inoculation and pretreat-
ment of plant growth medium. Firstly, in the greenhouse and growth chamber
experiments, plants were grown in presteamed soil, which eliminated or minimized
competition from indigenous soil microbes. Secondly, pre-inoculated seeds were
used in field trials while in the greenhouse trials, seeds were pre-inoculated and
multiple soil inoculations were performed subsequently. It is common knowledge
that inoculation methods usually affect N2 fixation in traditional rhizobium–legume
symbioses. Soil inoculation (e.g., granular inoculants) is usually more effective
than seed inoculation for initiating nodulation and N2 fixation (Lupwayi et al.
2005). Multiple soil inoculations ensured that high number of cells of A. caulino-
dans was introduced to the soil, which perhaps aided this strain in its competition
with indigenous soil microbes. In addition to the two obvious differences between
the greenhouse and field experiments, it should be mentioned that A. caulinodans is
adapted to conditions in tropical and subtropical environments. Early spring soil
temperatures are usually very cold in the Canadian prairies. Lupwayi et al. (2005)
suggested that the most adaptable rhizobia or legume genotypes are usually the ones
isolated from similar environments. This might explain why attempts to re-isolate
the microbes in inoculated plots during the growing season were not successful.
Inconsistency of response is a perennial problem limiting the wide acceptance of
PGPR as substitutes for chemical fertilizers in nonleguminous crops. Andrews et al.
(2003) noted that the effects of Azotobacter and Azospirillum inoculants on growth
and yield of graminaceous crops have been tested in many experiments in several
countries with inconsistent results obtained. Several reasons were suggested to

account for the inconsistent results including unfavorable soil environment, com-
petition with better adapted indigenous soil bacteria, or predation by protozoans
(Dowling and Broughton 1986; Quispel 1991; Jjemba and Alexander 1999).
Although most of the effects of A. caulinodans inoculation on field grown wheat
were positive, the trials were discontinued because the effects were not deemed to
be large enough. Irrespective of the inconsistency of response observed in the field,
it was obvious that the positive inoculation trends could not be attributed to chance
alone. More fundamental research is needed to optimize field inoculation methods
and to enhance survival and adaptation of A. caulinodans to temperate soil and
temperature conditions.
14.5.1 Monitoring of Introduced Microbes in Field Soils
All field locations used for testing of A. caulinodans were placed under strict
monitoring requirements by the Canadian regulatory authorities. On each trial
site, the experimental plots were spatially isolated from other field operations
with a 12-m fallowed buffer zone. The buffer zone acted as inoculum trap providing
effective trial confinement isolating treated crop and minimizing treatment drift
to nontarget plants. The entire perimeter was fenced to restrict access to wildlife
or unauthorized personnel. Wheat seeds were inoculated with liquid culture of
A. caulinodans at a rate of 2.8 mL kg1. The inoculum was estimated to contain
approximately 9.4108 CFU per mL. Rhizosphere soil and root samples collected
from the experimental sites at different time intervals during the growing season
were analyzed for presence of A. caulinodans using BIOLOG1 system and PCR
technology. Soil and plant samples for PCR and BIOLOG1 analysis were extracted
and analyzed using methods described by Yeates et al. (1998) and Slaski et al.
(2002), respectively. The substrate utilization pattern of the isolate was detected
using an Emax BIOLOG1 plate reader. The detected pattern was compared to a
pattern produced from the original stock culture of A. caulinodans. The minimum
detection limits of the PCR and BIOLOG1 were estimated to be about 10 cells and
2,000 cells per gram of soil, respectively. None of the samples tested was positive
for A. caulinodans suggesting that the microbe did not persist or spread in any of the
locations tested. A. caulinodans does not produce spores, and thus low soil tem-
peratures (below 0 C) may cause cell rupture and death of the organism. Low soil
temperature was suggested as an important environmental factor drastically affect-
ing survival of A. caulinodans in temperate climatic conditions of UK soils (Bullard
1999). The author reported that moderate temperatures 18/8 C reduced introduced
populations of A. caulinodans by an order of magnitude in 35 days. Therefore, it
appears that hostile fall and winter conditions of Alberta may have totally extermi-
nated this strain from the environment. The depth of frost in prairie soils is typically
greater than that in any other agricultural land in Canada, mostly due to the length
of the winter season and the typically shallow snow depths. Frozen soils at depths
below 40 cm have been observed in April on some of the field sites tested, and
therefore, for the organism isolated from tropical soils, environmental conditions
were not favorable for it to thrive in the test locations.
14.6 Native Canadian Rhizobia and Wheat Production
Empirical data suggest that native rhizobia strains are usually more adapted than
introduced strains. In a search for rhizosphere bacteria capable of promoting growth
of wheat on the Canadian Prairies, several strains of rhizobia were isolated,
identified, and characterized from rangeland ecosystems in the Castor region of
central Alberta. This region is prone to drought due to low precipitation and high
temperatures in the summer months. Sixteen native rhizobia strains identified as
belonging to Sinorhizobium meliloti (Bécquer et al. 2008) were isolated from roots
of Melilotus officinalis and Medicago sativa. The host legumes are adapted to the
local pedological and climatic conditions prevalent in the region. Plant growth-
promoting effects of the isolated rhizobia strains were evaluated in a greenhouse
experiment using a Canadian variety of wheat, CDC Teal. The seeds disinfected
with alcohol and 8% sodium hypochloride (Webster et al. 1997) were planted in
pots containing standard soil mixture with a low N level, while fertilized control
received 150 ppm N kg1 in a form of NH4NO3. Three milliliters per plant of
inoculum containing approximately 108 cells mL1 was applied 5 days after
seeding. Two subsequent inoculations were performed 20 and 30 days after seed-
ing. Wheat plants were grown to maturity under 29/25ºC (day/night) and 14 h of
light. In general, inoculation with native rhizobia led to increase in root weight and
plant height with no obvious effects on aerial dry matter (Bécquer et al. 2007). All
but one of the isolated strains enhanced plant height while 12 strains stimulated root
growth. Although changes in root morphology of wheat plants inoculated with
rhizobia were not studied, literature data suggest that rhizobial inoculants could
induce a higher volume of root hairs and lateral roots, which favors more efficient
nutrient extraction (Biswas et al. 2000). Stimulation of root growth accompanied by
increased yield and N content by A. caulinodans have also been reported (Sabry
et al. 1997). Similarly, inoculation of barley (Hordeum vulgare) with Mesorhizo-
bium mediterraneum (strain PECA21) considerably increased the dry matter yield
and the content of macroelements in the plant (Peix et al. 2001). A well-developed
root system is advantageous for the wheat plants cultivated in semiarid zones or for
crops experiencing periodic soil moisture deficit. Thus, stimulation of root growth
by inoculation with rhizobia may increase production of wheat in drought condi-
tions through enhanced efficiencies of water and nutrient uptake/utilization.
The nine best performing native rhizobia strains identified in the greenhouse
studies described above were evaluated for wheat growth promotion under field
conditions at the Sancti-Spı́ritus Experimental Station in Cuba. In addition to the
nine strains isolated from rangeland ecosystem of central Alberta, another strain
(CAS2) isolated from legume grown in soil contaminated with petrochemical
sludge containing high concentration of salts and heavy metals, was tested. The
well-described rhizobia strains (USDA 191 and ATCC 10004) were included in the
field trial as reference strains. All strains were used to inoculate seeds of wheat
(T. aestivum, L., var. Cuba-204) provided by the National Institute of Fundamental
Research of Tropical Agriculture (INIFAT), La Habana, Cuba. Inoculation was
performed by immersion of seeds in a bacterial inoculum containing approximately
108 cells mL1 at room temperature for 24 h. Liquid inoculum was reapplied at the
base of young seedlings 18 days after sowing to ensure adequate number of rhizobia
cells in the rhizosphere. A fertilized and unfertilized control was included for
comparison with Rhizobium inoculation effects. The fertilized treatment received
150 kg N ha1 NH4NO3. The experimental design was a randomized complete
block with four replicates. Since the experiment was conducted on sandy loam soil
with low mineral content (P2O5: 2.63 mg/100 g; K2O: 10.00 mg/100 g; OM: 1.61%;
pH: 5.4), a second application of fertilizer (NPK: 9–13–17) was made 21 days after
sowing on the basis of 88 kg N ha1.
Plant growth-promoting and grain yield-enhancing effects of inoculation with
nine Canadian native Rhizobium strains were observed (Fig. 14.3). Two strains,
CAC2 and CAC 5, significantly improved plant agronomic parameters including
grain yield, weight of 1,000 seeds, yield of total aboveground biomass, and stem
height of the Cuban wheat cv. Cuba-204. Several of the native Canadian strains
250
200 a a
abc abc ab
abc abcd
bcd
bcde
Grain yield (g/m 2 )
cde
150 efg
defg
efg
fg
100
50
0
4
l
91
2
14
16
17
2
ro
ro
00
AC
AC
AC
AC
AC
AC
AS
nt
nt
A1
AC
AC
AC
10
co
co
SD
C
C
te
ed
U
lu
liz
AT
so
rti
Ab
Fe
Fig. 14.3 Effects of inoculation with Rhizobium strains isolated from forage legumes of Alberta,
Canada on grain yield of wheat cv. Cuba 204 (Bécquer et al. 2007, modified)
tested clearly demonstrated plant growth-promoting effects on wheat grown in the

field in Cuba. Positive growth-promoting effects of these strains were also observed
on sorghum grown at the Sancti-Spiritus experimental station in Cuba (Bécquer,
personal communication). The next steps would be to further characterize these
strains and test their growth-promoting effects on wheat under field conditions in
the Canadian prairies.
14.7 Conclusion
Although the process of BNF offers an economically attractive and ecologically

sound means of reducing chemical nitrogen use in agriculture, N2 fixation by non
legumes remains a dream. Quispel (1991) suggested that only in endophytic
systems are the prerequisites for effective nitrogen fixation likely to be fulfilled in
non legume rhizobial interactions. Colonization and N2 fixation by A. caulinodans
in para-nodules (nodule-like) structures induced by plant growth hormones (2,4-D)
treatment has been reported (Chen et al. 1993). Intercellular colonization of wheat
by A. caulinodans has also been reported by several authors. While some levels of
nitrogenase activity have been detected in wheat plants inoculated with A. cauli-
nodans (Sabry et al. 1997), the extent to which the fixed nitrogen benefits the plant
remains to be verified. In the absence of BNF by non legumes, most researchers
have focused on the growth-promoting effects of diazotrophs in non legume
systems. The theory proposed by Yanni et al. (2001), suggesting that some rhizobia
can form a three-component life cycle including a free-living heterotrophic phase in
soil, a N2-fixing endosymbiont phase within legume root nodules, and beneficial
growth-promoting endocolonizer phase within cereal roots in the same crop rotation,
would need to be further verified. In addition to the known rotational benefits of
legumes, optimization of conditions that promote endophytic colonization of cereal
roots in rotation with legumes may improve crop performance while decreasing the
use of chemical fertilizers required to maintain yield. Despite perennial inconsis-
tencies in the positive effects of PGPR in Canada, the potential for the use of PGPR
in non legume crops remains interesting, especially given the potential environ-
mental benefits over the use of conventional agricultural methods. More fundamental
research is needed to optimize field inoculation methods that will enhance survival
and adaptation of PGPR, especially of A. caulinodans, in temperate conditions.
References
Andrews M, James EK, Cummings SP, Zavalin AA, Vinogradova LV, McKenzie BA (2003) Use of
nitrogen fixing bacteria inoculants as a substitute for nitrogen fertiliser for dryland graminaceous
crops: Progress made, mechanisms of action and future potential. Symbiosis 35:209–229
Anyia AO, Archambault DJ, Slaski JJ (2004) Growth promoting effects of the diazotroph
Azorhizobium caulinodans on Canadian wheat cultivars. Proceedings of the 4th International
Crop Science Congress, Brisbane, Australia, pp 201–202. www.cropscience.org.au/icsc2004/

poster/2/5/6/863_anyiaa.htm#TopOfPage
Archambault DJ, Li X (2002) Potential benefit of endophytic nitrogen fixing bacteria to wheat
(Triticum aestivum, L.) and barley (Hordeum vulgare). Stage 1 Investment Project Final Report
(unpublished internal report). Alberta Research Council, Vegreville, Alberta, Canada
Baldani VLD, Baldani JI, Dobereiner J (2000) Inoculation of rice plants with the endophytic
diazotrophs Herbasprillum seropedicae and Burkholderia spp. Biol Fertil Soil 30:485–491
Bécquer CJ, Salas B, Archambault D, Slaski JJ, Anyia A (2007) Inoculation of wheat (Triticum
aestivum, L.) with rhizobia adapted to livestock ecosystems of Alberta, Canada. Pastos
Forrajes 30:133–140
Bécquer CJ, Salas B, Archambault D, Slaski J, Anyia A (2008) Selection of rhizobia adapted to
livestock production ecosystems from Alberta, Canada, inoculated in corn (Zea mays, L.).
Stage I: greenhouse. Pastos Forrajes 31:341–354
Biswas JC, Ladha JK, Dazzo FB (2000) Rhizobia inoculation improves nutrient uptake and growth
of lowland rice. Soil Sci Soc Am J 64:1644–1650
Borlaug NE (1970) The green revolution: peace and humanity. Nobel peace price. CIMMYT,
Mexico DF
Brown ME (1974) Seed and root bacterization. Annu Rev Phytopathol 12:181–197
Bullard M (1999) Further studies with Azorhizobium caulinodans as a novel diazotrophic
symbiont in temperate cereals. Final Project Report (MAFF Project; Code CE0153), Ministry
of Agriculture, Fisheries and Food, ADAS Arthur Rickwood, Mepal Ely, Cambridgeshire
Chen TW, Scherer S, Boger P (1993) Nitrogen fixation of Azorhizobium in artificially induced root
para-nodules in wheat. In: Nester EW, Verma DPS (eds) Advances in molecular genetics of
plant-microbe interactions. Kluwer, Netherlands, pp 593–606
Cocking EC (2001) Xylem colonization of tomato by Azorhizobium caulinodans ORS571. Acta
Biol Hungarica 52:189–194
Cocking E (2005) OBPC Symposium: Maize 2004 and beyond – Intracellular colonization of
cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic
nitrogen fertilizers. In Vitro Cell Dev Biol Plant 41:369–373
Dowling DN, Broughton WJ (1986) Competition for nodulation of legumes. Annu Rev Microbiol
40:131–157
Dreyfus B, Elmerich C, Dommergues YR (1983) Free-living Rhizobium strains able to grow on N2
as sole nitrogen source. Appl Environ Microbiol 45:711–713
Dreyfus B, Garcia JL, Gillis M (1988) Characterization of Azorhizobium caulinodans gen. nov.,
sp. nov., a stem nodulating nitrogen-fixing bacterium isolated from Sesbania rostrata. Int
J Syst Bacteriol 38:89–98
Elmerich C (1984) Molecular biology and ecology of diazotrophs associated with nonleguminous
plants. Biotechnology 2:967–978
Geelen D, Montagu MV, Holsters M (1995) Cloning of an Azorhizobium caulinodans endogluca-
nase gene and analysis of its role in symbiosis. Appl Environ Microbiol 61:3304–3310
by plant growth promoting bacteria. Imperial College Press, London, p 267
Jjemba PK, Alexander M (1999) Possible determinants of rhizosphere competence of bacteria.
Kennedy IR, Tchan YT (1992) Biological nitrogen fixation in non-leguminous field crops: Recent
advances. Plant Soil 141:93–118
Kloepper JW, Lifshitz R, Schroth MN (1988) Pseudomonas inoculants to benefit plant production.
ISI Atlas Sci Anim Plant Sci 8:60–64
Kloepper JW, Lifshitz R, Zablotowicz RM (1989) Free-living bacterial inocula for enhancing crop
productivity. Trends Biotechnol 7:39–43
Ladha JK, Tirol-Padre PGC, Watanabe I (1987) Nitrogen fixing (C2H2-reducing) activity and plant
growth characters of 16 wetland rice varieties. Soil Sci Plant Nutr 33:187–200
Lupwayi NZ, Rice WA, Clayton GW (2005) Rhizobial inoculants for legume crops. J Crop Improv
15:289–321
Mathews SS, Sparkes DL, Bullard MJ (2001) The response of wheat to inoculation with the
diazotroph Azorhizobium caulinodans. Aspects Appl Biol 63:35–42
Noel TC, Sheng C, Yost CK, Pharis RP, Hynes MF (1996) Rhizobium leguminosarum as a plant
growth-promoting rhizobacterium: Direct growth promotion of canola and lettuce. Can
Peix A, Rivas-Boyero AA, Mateos PF, Rodriguez-Barrueco C, Martı́nez-Molina E, Velazquez E
(2001) Growth promotion of chickpea and barley by a phosphate solubilizing strain of Mesorhi-
zobium mediterraneum under growth chamber condition. Soil Biol Biochem 33:103–110
Quispel A (1991) A critical evaluation of the prospects for nitrogen fixation with non legumes.
Plant Soil 137:1–11
Reddy PM, Ladha JK, So RB, Hernandez RJ, Ramos MC, Angeles OR, Dazzo FB, de Bruijn K
(1997) Rhizobial communication with rice roots: induction of phenotypic changes, mode of
invasion and extent of colonization. Plant Soil 194:81–98
Sabry SRS, Saleh SA, Batchelor CA, Jones J, Jotham J, Webster G, Kothari SL, Davey MR,
Cocking EC (1997) Endophytic establishment of Azorhizobium caulinodans in wheat. Proc R
Soc Lond B 264:341–346
Saikia SP, Jain V (2007) Biological nitrogen fixation with non legumes: An achievable target or a
dogma? Curr Sci 92:317–322
Sevilla M, Kennedy C (2000) Colonization of rice and other cereals by Acetobacter diazotrophi-
cus, an endophyte of sugarcane. In: Ladha JK, Reddy PM (eds) The quest for nitrogen fixation
in rice. International Rice Research Institute, Manila, Philippines, pp 151–165
Slaski JJ, Archambault DJ, Li X, Macyk T (2002) BIOLOG1 system as a tool for the evaluation of
functional diversity of microbial communities in soils. 39th Alberta Soil Science Workshop,
Nisku, AB, pp 204–209
Webster G, Gough C, Vasse J, Batchelor CA, O’Callaghan KJ, Kothari SL, Davey MR, Denarie J,
Cocking EC (1997) Interactions of rhizobia with rice and wheat. Plant Soil 194:115–122
Yanni YG, Rizk RY, Abd El-Fattah FK, Squartini A, Corich V, Giacomini A, de Bruijn F,
Rademaker J, Maya-Flores J, Ostrom P, Vega-Hernandez M, Hollingsworth RI, Martinez-
Molina E, Mateos P, Velazquez E, Wopereis J, Triplett E, Umali-Garcia M, Anarna JA, Rolfe
BG, Ladha JK, Hill J, Mujoo R, Ng PK, Dazzo FB (2001) The beneficial plant growth
promoting association of Rhizobium leguminosarum bv. trifolii with rice roots. Aust J Plant
Physiol 28:845–870
Yeates C, Gillings MR, Davison AD, Altavilla N, Veal DA (1998) Methods for microbial DNA
extraction from soil for PCR amplification. Biol Proc Online 1:40–47
Chapter 15
Factors Affecting the Variation of Microbial
Communities in Different Agro-Ecosystems
Munees Ahemad, Almas Zaidi, Md Saghir Khan, and Mohammad Oves
Abstract Soil microbial communities play an important role in supplying essential

nutrients to plants by decomposing various organic matters. Composition, structure
and functions of microbial communities in soil are, however, under the constant
control of the environment including various agricultural management practices.
Due to scarcity of convenient methods for exploration, our understanding of the
different degrees and dynamics of microbial community variations are limited. An
attempt will be made to understand such structural and functional variations
employing molecular tools. Earlier it was believed that it is the plant community
that exerts control over the microbial community, but recently, some findings have
suggested that it is actually the microbial community that acts as a driver of plant
community structure and dynamics. Attention will therefore be paid to highlight
some of these issues, and the effect of various farm management practices on the
composition and functions of microbial communities This is likely to lead to the
development of best management practices for improving soil fertility and, conse-
quently, agricultural productivity to improve the sustainability of agro-ecosystems.
15.1 Introduction
Microorganisms are a fundamentally important component of the soil habitat where

they play key roles in ecosystem functioning through controlling nutrient cycling
reactions essential for maintaining soil fertility, and also contribute to the genesis
and maintenance of soil structure (Kirk et al. 2004). Despite their importance to the
functioning of ecosystems, they are rarely explicitly considered in individual
ecosystem or global process models. In addition to methodological limitations, a
Md. S. Khan(*), M. Ahemad, A. Zaidi, and M. Oves

Faculty of Agricultural sciences, Department of Agricultural Microbiology, Aligarh Muslim
University, Aligarh, 202002, Uttar Pradesh, India
302 M. Ahemad et al.
primary reason for this gap is their overwhelming diversity. Estimates of soil
microbial diversity range from thousands to a million microbial species in a few
grams of soil (Torsvik and Øvreås 2002; Gans et al. 2005), and how this diversity
affects ecosystem processes is largely unknown (Torsvik et al. 2002; Crawford
et al. 2005; Azam and Malfatti 2007). Furthermore, it is extremely difficult to
assess, identify and track each individual microorganism in an ecosystem. Due in
part to the lack of convenient and appropriate methods for exploration, our under-
standing of the different degrees and dynamics of microbial community variation as
induced by soil type, plant type, or plant development is so far limited (Mahaffee
and Klöpper 1997; Duineveld et al. 1998).
Despite all these problems, in recent times, attention has been focused on the
impacts that microbial communities have on soil fertility and crop productivity in
different agro-ecosystems. In soils, biologically mediated processes are known to
exert profound influences on the status of soil health by: (1) degrading organic
residues, (2) transformation of organic matter, (3) mineralization of nutrients, and
(4) formation of soil aggregates (Haynes 1999). However, a great variety of abiotic
and biotic factors shape soil- and plant-associated habitats and modify the compo-
sitions and activities of inhabiting microbial communities, which in turn bear upon
the quality of their environment, the growth of plants, and the production of root
exudates (Bever et al. 1997). For example, changes in physico-chemical variables
such as pH and nutrient levels (Kennedy et al. 2004, 2005), floristic community
change (Grayston et al. 1998; Grayston et al. 2004), changes in soil physical
structure (Ibekwe et al. 2002; Sessitich et al. 2006), and the impacts of grazing
animals have all been proposed as principal causes of shifts in microbial commu-
nity structure, although it is still not fully understood which environmental factors
influence microbial community change. Microbial communities in root-asso-
ciated habitats respond with respect to density, composition, and activity to the
abundance and great diversity of organic root exudates, eventually yielding plant
species-specific microflora which may also vary during plant development stages
(Lynch and Whipps 1990; Bowen and Rovira 1991; Mahaffee and Klöpper 1997;
Weisskopf et al. 2006). Thus, interactions between plants and soil microbes
are highly dynamic in nature and based on coevolutionary pressures (Reinhart
and Callaway 2006). Such microbial communities are greatly influenced by plant
species (Innes et al. 2004; Batten et al. 2006), genotypes within species (Kowalchuk
et al. 2006), and diversity of carbon substrates and signaling compounds provided
by the plants (Zak et al. 2003; Broeckling et al. 2008).
Agricultural improvement by applying chemical fertilizers has become a com-
mon practice in different parts of the world. However, the excessive and injudicious
application of these fertilizers cause change in the soil chemical and physical
properties by disturbing the soil microbial biomass and activities, an early indicator
of soil fertility (Brookes 1995; Trasar-Cepeda et al. 1998). For instance, N fertili-
zation has been shown to significantly affect soil microbial biomass and activities
(Saliana-Garcia et al. 1997; Li et al. 2002), and long-term application of NH4+ or
NH4+ forming fertilizers may lead to changes in the soil microbial communities
in terms of promoting nitrifying populations, enhancing nitrification rates, and
15 Factors Affecting the Variation of Microbial Communities 303
increasing potential risks of groundwater contamination with NO3–N (Tabatabai

et al. 1992). Likewise, phosphate application affects soil microbial community
structures in grassland ecosystems (Rooney and Clipson 2009). The understanding
of interactions between fertilizers, plants and microbial communities in soils, in
turn will lead to better understanding of the health of soils and the design of
strategies for crop improvement in different agro-ecosystems.
15.2 Microbial Community Structure and Functional Analysis
It has long been recognized that the activity of soil microorganisms plays an
intrinsic role in residue decomposition, nutrient cycling, and crop production.
And, hence, the microbial diversity at the functional level plays a crucial role in
the long-term stability of an ecosystem. Variations in the structure and function of
microbial communities in soils have, however, been linked primarily to the quan-
tities and qualities of soil organic materials (Saetre and Bååth 2000), which in turn
are greatly affected by the availability and biochemical composition of the litter
(Johansson 1995), vegetation (Priha et al. 2001), and root exudates (Grayston and
Campbell 1996). A patchy distribution of organic litter may result in a high degree
of biochemical compartmentalization (Bauhus et al. 1998; Priha and Smolander
1999; Côté et al. 2000; Priha et al. 2001) and may ultimately cause a spatial
aggregation of the forest soil microbiota (Saetre and Bååth 2000). Thus a post-
fire successional sequence from deciduous to coniferous species will have a
significant impact on the morphological and physiological profiles of soil micro-
bial communities (Bormann and Sidle 1990; Bending et al. 2002; Merilä et al.
2002). This may be due to differences in the structure and composition of leaf and
needle litter, which alter nutrient availability in the soil (Priha and Smolander
1997) and cause shifts in microbial populations as the community adapts to
new environmental conditions. Understanding microbial community structure
shifts following implementation of various land use and management systems,
therefore may lead to the development of best management practices for different
agro-ecosystems.
Microbial diversity describes complexity and variability at different levels of
biological organization. It includes genetic variations within taxons (species), and
the number (richness) and relative abundance (evenness) of taxons and functional
groups (guilds) in communities. Important aspects of diversity at the ecosystem
level are the range of processes, complexity of interactions, and number of trophic
levels. Although animal and plant ecologists can quantify and identify different
species through (relatively) easily identifiable traits, it is extremely difficult to do
this with microbial communities, because microbes vary greatly in their activities.
Thus, measures of microbial diversity should include multiple methods integrating
holistic measures at the total community-level and partial approaches targeting
structural or functional subsets. However, the assessment of the composition and/or
function of soil microbial communities still presents a number of challenges, due
to which less than 1% of bacterial species and an unknown percentage of fungi

have been so far recovered (Rozak and Colwell 1978; Van Elsas et al. 1997).
Unfortunately, changes in microbial community structure and diversity due to
seasonal and temporal variations in nutrient or physical conditions are slow and
gradual, making it difficult to further interpret the data and obtain conclusive results.
Disturbances in microbial community equilibrium influenced by changes in envi-
ronmental conditions and soil management practices have been reported (Sun
et al. 2004). The challenge is how to quantify the changes and link them to the
corresponding ecosystems.
In the premolecular biology era, microbial communities were determined by
isolating and screening them onto commercially available standard laboratory
media, and the total numbers and/or species of microbial communities in a particu-
lar soil were assessed using conventional methods, such as the most probable
number (MPN) and plate-counting techniques. Such methods, however, have
certain limitations (Torsvik et al. 1990), such as they address only the culturable
populations present in the rhizosphere and provide only limited information about
the functional diversity of the microbial communities. Similarly, microscopic
techniques can be used to obtain information about numbers and, potentially,
spatial distribution, but these approaches lack the discriminating ability to assess
diversity and distinguish between multiple microbial populations. Recently, several
modern methods including BIOLOGTM analysis, phospholipid fatty acid (PLFA)
analysis and nucleic acid-based analysis have been developed to identify and
characterize soil microbial diversity. Despite various limitations (Konopka et al.
1998; Garland 1999; Preston-Mafham et al. 2002), the Biolog system has been used
to characterize microbial communities associated with boreal forest soils (Staddon
et al. 1998b; Adkins et al. 2001), various crop types (Garland and Mills 1991;
Garland 1996), grasslands (Zak et al. 1994), tree species (Grayston and Campbell
1996), plant rhizospheres (Grayston et al. 1998; Kent and Triplett 2002), and
environmental samples. In addition, differences between soil microbial commu-
nities along a climatic gradient (Staddon et al. 1998a) and bacterial communities
affected by soil types have also been analyzed (Winding 1994).
Garland and Mills (1991) compared the patterns of carbon source utilization at
community-level for microbial communities of different habitats, such as samples
from freshwater, saltwater, estuarine, and hydroponic solutions, from the rhizo-
sphere of hydroponically grown wheat, and from soils, using commercially avail-
able microtiter plates that contain 95 carbon substrate. This and other studies
suggest that, in addition to establishing ecologically relevant classifications of
microbial communities, substrate utilization profiles might offer information with
regard to community function, metabolic potential (Winding 1993), or functional
diversity (Zak et al. 1994; Haack et al. 1995). When inoculum density was con-
trolled, patterns of positive and negative responses exhibited by microbial commu-
nities to each of the carbon sources were reproducible. Even so, the rates and
extents of substrate oxidation by the communities were reproducible but were not
simply the sum of those exhibited by community members when tested separately.
Replicates of the same model community clustered when analyzed by principal
components analysis (PCA), and model communities with different compositions

were clearly separated on the first PCA axis, accounting for >60% of the dataset
variation. However, the substrates were interpreted by PCA to be most significant in
distinguishing the communities that changed with reading time, reflecting the
nonlinearity of substrate oxidation rates. Although whole-community substrate
utilization profiles were reproducible signatures for a given community, the extent
of oxidation of specific substrates and the numbers or activities of microorganisms
using those substrates in a given community were not correlated.
Compared to substrate utilization profiles, PLFA profiles have been shown to be
more sensitive and can be used as biomarkers to identify and quantify microbial
biomass as they are essential components of microorganisms (Priha and Smolander
1999; Waldrop et al., 2000; Grayston et al. 2003). The two community-based
microbiological measurements, namely potential C source utilization patterns in
Biolog microtiter plates and PLFA profiles, have been employed to examine
metabolic fingerprints of soil microbial communities and changes in species com-
position of Palouse and Ritzville silt loams collected from the rhizosphere of wheat
(Triticum aestivum L.), barley (Hordeum vulgare L.), pea (Pisum sativa L.), jointed
goatgrass (Aegilops cylindrica L.), and downy brome (Bromus tectorum L.) grown
under field and greenhouse environments. PCA of PLFA profiles and C source
utilization patterns were used to describe changes in microbial biomass and meta-
bolic fingerprints from the two soil types. Biomass measurements from extractable
PLFA profiles per g dry weight ranged from 28.8 nmol in wheat soil in the
greenhouse to 71.4 nmol in pea soil in the field. In general, biomass was higher
in all the field samples than in the greenhouse samples. PCA of the two soils with
different plants in the field and greenhouse showed clear separation. PCA of C
utilization patterns on the effects of environment on soil microbial community
yielded similar results with PLFA measurements. However, higher variability
observed among different plants with the Biolog data resulted in the low amount
of variance for Biolog data explained by the first two dimensions of the PCA
suggesting that PLFA may be more sensitive for community analysis than the
Biolog technique (Ibekwe and Kennedy 1998).
Furthermore, the introduction of bio-molecular approaches like nucleotide
sequence analysis of ribosomal RNA genes (the 16S rRNA genes in bacteria and
18S rRNA genes in fungi) into microbial ecology has allowed characterization of
microbial communities without pure cultures. In such approaches, the total micro-
bial community DNA can be directly extracted from soils; hence, the repeated
subculturing of microbial communities could be avoided. Such DNA is used as a
template for amplification of ribosomal genes from all the members of that
community in the polymerase chain reaction (PCR), a technique that allows for
high resolution analysis of the microbial community structure. The resulting
individual gene products can then be cloned and sequenced to affirm the identity
of the microbial species from which the original target sequence was amplified
(Giovannoni et al. 1990; Hugenholtz et al. 1998). Furthermore, the heterogeneous
mix of PCR products recovered from the environment by specific amplification of
bacterial 16S rDNA sequences can be separated by a culture-independent method,
denaturing gradient gel electrophoresis (DGGE), which separates the DNA frag-
ments based on their nucleotide content rather than size alone (Muyzer et al. 1993).
The benefit of this approach is that a molecular fingerprint of the community
structure is generated for each soil, such that each band in each lane of the gel
theoretically represents a different bacterial species. Molecular approaches, in
general, are rapid, specific and more reliable, compared to conventional culturing
techniques, and have provided a further insight into the identification of the
remaining 99% of microbial species.
Both conventional and molecular approaches have been employed to assess the
impact of management practices, changes in seasons, and environment stress on
microbial community structure and functions. For instance, de Lima et al. (1999)
isolated 82 bacterial strains from the sugarcane (Saccharum officinarum) agro-
ecosystem and grouped them in 16 different genera and 35 species of bacteria.
The microbial diversity was found greater in wastewater collected from the stabili-
zation ponds and in the soil sample collected from the sugarcane plantation burned
before harvesting. Such variations in microbial communities supported the very
concept of Odum’s (1971) classical observation that communities with low energy
cost for maintaining the entropy (high respiration: biomass ratio) divert their energy
supply into diversity, which may be happening to the microbiota of the environ-
ments. A high respiration:biomass ratio observed in unproductive soil irrigated with
vinasse in the tableland soil of the Usina Japungu (de Luna and Grisi 1996) also
supported this theory. However, Atlas (1984) further pointed out that diversity
changes in response to environmental stress may lead to an increase in diversity by
selective toxicity causing elimination of dominant organism, or diversity may
decrease by elimination of many species. To substantiate this further, Torsvik
et al. (1997) observed a substantial reduction in diversity in perturbed soil due to
agriculture, as compared to undisturbed environments. Also, molecular analysis
using PCR amplification of small-subunit rRNA of microbial diversity in Amazonia
soils, demonstrated microbial population shifts related to deforestation in the
Amazonian forest, with predominance of Bacillus and high G+C Gram-positive-
like sequences in pasture and predominance of Clostridium and unclassified bacte-
ria in the forest (Borneman and Triplett 1997). Of these organisms, Bacillus
seems to be a natural indicator of inhospitable environmental conditions whose
endospore-forming ability certainly explains their occurrence in these situations.
15.3 Factors Causing Change in Microbial Community

Structure and Function
Soil microbial communities are subjected to a range of factors that can be broadly
classified as: (1) stress factors that constantly limits microbial growth and do not
change markedly over time, and (2) disturbance factors that involves rapid
changes and often causes destruction of organism biomass. Although both stress
fumigants Organic/inorganic
amendments
Pesticides Soil treatments Heavy metals
DIF
TY F ER
SI
Type/physico-chemical Fertilization
manuring
properties
R (N/P)
BIAL DIVE
EN
T
Abioticfactors Soil management
FACTOR
of soils practices
RO
Moisture/ Particle Tillage

rainfall size distribution
S
IC AF
M F EC
TING
Plant type Bioticfactors Rhizosphere/

rhizoplane
Introduction of GMO
Fig. 15.1 Environmental factors influencing the microbial structure and functions
and disturbance are strong drivers of the microbial community, they exert their
effects independently from one another (Williamson and Wardle 2007). The factors
causing changes in microbial community structure and functions (Fig. 15.1) are
reviewed and discussed in the following section.
15.3.1 Rhizosphere, Root Exudates and Soil pH
The plant rhizosphere, a term introduced by Hiltner to denote the region of the soil
that is subjected to the influence of plant roots (Hiltner 1904), is a dynamic
environment in which many factors affect the structure and composition of the
microbial communities that colonize the roots. The rhizosphere communities vary
spatially in a radial direction from the root surface, including the endorhizosphere,
rhizoplane, and rhizosphere zones (Assmus et al. 1995; Bosse and Frenzel 1997;
Gilbert and Frenzel 1998), as well as in specific root locations along the root axis
(Gilbert and Frenzel 1998; De Leij et al. 1994). Microbial communities associated
with the rhizosphere also vary depending on the plant species (Grayston et al.
1998), the soil type (Campbell et al. 1997), and cultural practices (Table 15.1) such
as crop rotation or tillage (Lupwayi et al. 1998). Understanding the structure and
Table 15.1 Effect of plant and soil types on microbial community structure
System and factors studied Methods used Results and conclusion References
Plant cultivar (maize) and soil- PCR-TGGE Soil type showed higher effect Da Silva
effect on a specific bacterial than plant cultivar on et al.
group Paenibacillus Paenibacillus communities (2003)
Plant species (chickpea, rape, PCR-DGGE Bacterial community structure Marschner
and Sudan grass); soil type in the rhizosphere was et al.
(sandy, sandy loam, and affected by a complex (2001)
clay); root zone location interaction between soil
type, plant species, and root
zone location
Soil type, plant type (clover, PCR-TGGE The plant species type had the Wieland
bean, alfalfa), plant age greatest effect on microbial et al.
community structure (2001)
Microbial community in the Biolog-CLPP; Soil type affected microbial Buyer et al.
spermosphere as affected FAME community structure more (1999)
by soil type and seed type than seed type
Soil type, plant type (wheat, Biolog-CLPP Plant effect with significant Grayston
ryegrass, bentgrass, and difference in microbial et al.
clover) communities from the (1998)
different plant species
Plant (maize) development, Cultivation– Between the factors studied, Chiarini
cultivar, and soil-effect plating soil had the dominant effect et al.
enumeration on microbial diversity (1998)
Plant type (canola, wheat); FAME Effect of plant type stronger Germida
soil type than that of soil type soil- et al.
effect stronger than plant (1998)
effect
Plant (flax, tomato) and soil Cultivation; Soil-effect stronger than plant Latour
type; effect on fluorescent REP-PCR, effect et al.
pseudomonads RFLP (1996)
Adapted from Garbeva et al. (2004)
species composition of these communities is fundamental to understanding how

soil biological processes are influenced by such factors.
Generally, the microbes that inhabit the rhizosphere serve as an intermediary
link between the plant, which requires soluble inorganic nutrients, and the soil,
which contains the necessary nutrients but mostly in complex and inaccessible
forms. However, the magnitude of the rhizosphere effect depends mainly on the
nature and amount of root exudates, a complex mixture of chemicals and organic
compounds secreted into the soil by the roots that affects microbial inter-
actions (Bais et al. 2004). Root exudate concentrations are determined by many
factors including species and nutritional status of the plant, plant age, and edaphic
and climatic factors (Marschner 1995). About 5% to 60% (Marschner 1995) of the
photosynthetic carbon fixed by the plant is released into rhizosphere by exudation
through plant roots. Root exudates may consist of water soluble compounds such as
amino acids, sugars, hormones, and vitamins that leak from the root surfaces, or
actively secreted polymeric carbohydrates and enzymes. The products of the roots
may also include gases such as CO2 and ethylene, lysates released when cells
autolyse, solid materials including cell walls, sloughed cells and root border cells,
and eventually parts as large as root hairs or roots themselves (Kang and Mills
2004) (Table 15.2). The effect of root exudates on the rhizosphere microbes is
likely to be more intense, stimulatory or inhibitory. Depending on the composition
of the exudates, plant may be able to: (1) enhance the possibilities and success of
symbiotic relationships, (2) affect the soil microbial community, (3) alter the
physical and chemical properties of the soil, and (4) inhibit the propagation or
growth of another plant species. In fact, most rhizosphere bacteria and fungi are
highly dependent on associations with plants that are clearly regulated by root
exudates (Yang and Crowley 2000; Bais et al. 2004), and in the rhizosphere, density
of microorganisms can reach 1010–1012 organisms g1 soil. The influence of
individual plants is reflected in the rhizosphere as the R:S (rhizosphere to nonrhizo-
sphere ratio). For bacteria and fungi, values commonly range from 5 to 20. Actino-
mycetes, a somewhat less affected group of microorganisms of the rhizosphere,
may reveal R:S ratios between 2 and 12.
Based on differences in root exudation and rhizodeposition in different root
zones, rhizosphere microbial communities can vary in structure and composition in
different root locations or in relation to soil type, plant species, nutritional status,
age, stress, disease, and other environmental factors (Griffiths et al. 1999; Mahaffee
and Klöpper 1997). As an example, Wieland et al. (2001)) observed while studying
32 microcosms in three habitats, namely soil, rhizosphere, and rhizoplane, that the
type of plant species (clover, bean, or alfalfa) had the greatest effect in plant-
associated habitats and also affected soil patterns through the variation of microbial
communities as represented by the patterns of a sequence-specific separation of
rRNA target sequences. Plant development demonstrated a minor habitat-dependent
effect that was partly obscured by replicate variation. Ruiyu et al. (2007), while
investigating the dynamics of microbial populations and their functional diversities
in the seedling rhizospheres of rice (Oryza sativa) cultivars with varied allelopathic
activities by employing agar plate bioassay, fumigation, and Biolog analysis,
concluded that the rice cultivars significantly affected the microbial carbon content
in their associated rhizospheric soil. Moreover, microbial carbon contents and the
respiration rate of the soils varied with the type of cultivar. The microbial flora in
the rhizospheric soil of different rice cultivars was dominated by bacteria (58.4–
65.6%), followed by actinomycete (32.2–39.4%) and fungi (2.2–2.8%). Moreover,
Biolog analysis showed that the value of average well color development (AWCD)
differed significantly among rice cultivars. It was always the highest in the rhizo-
spheric soil of the strongly allelopathic rice cv. PI312777 and the lowest in the
rhizospheric soil of the poorly allelopathic rice cv. Lemont. The AWCD value
reached the maximum in all the sampled soils after 144 h of incubation. The AWCD
values from the rhizospheric soils of PI312777, IAC47, Iguape Cateto, and Lemont
were 1.89, 1.79, 1.60, and 1.43 times higher than that of the control soil, respectively.
PCA identified three principal component factors (PCF) in relation to carbon
sources, accounting for 70, 11, and 7% of the variation, respectively, and 19
categories of carbon sources were positively correlated to the three principal
Table 15.2 Components of root exudates, functions in the rhizosphere, and examples identified in
root exudates of different plant species
Exudate Rhizosphere functions Specific compounds identified in
component root exudates
Organic acids Nutrient source Citric, glutaric
Chemoattractant signals to microbes Oxalic, malonic
Chelators of poorly soluble mineral Malic, aldonic
nutrients
Acidifiers of soil Fumaric, erythronic
Detoxifiers of Al Succinic, ferulic
nod gene inducers Acetic, butanoic, butyric syringic,
valeric, rosmarinic, Glycolic,
trans-cinnamic, piscidic,
vanillic, formic tetronic,
aconitic, lactic, pyruvic
Amino acids Nutrient source a- and (b-alanine, proline
Chelators of poorly soluble mineral Asparagine, valine
nutrients
Chemoattractant signals to microbes Aspartate, tryptophan, cystein,
ornithine, cystine histidine,
glutamate, arginine,glycine,
homoserine, isoleucine,
phenylalanine, leucine,
aminobutyric acid, lysine,
aminoadipic acid, methionine,
serine, threonine
Sugars and Promoters of plant and microbial growth Glucose, deoxyribose
vitamins Nutrient source Fructose, oligosaccharides,
galactose, biotin, maltose
thiamine, ribose, niacin, xylose,
pantothenate, rhamnose,
rhiboflavin, arabinose, raffinose
Enzymes Catalysts for P release from organic acid/alkaline phosphatase
molecules
Biocatalysts for organic matter Invertase, amylase, protease
transformations
Purines Nutrient source Adenine, guanine, cytidine, uridine
Inorganic ions Chemoattractant signals to microbes HCO3, OH, H+, CO2, H2
and gases
Phenolics Nutrient source Liquiritigenin, luteolin
Chemoattractant signals to microbes Daidzein, 40 ,7-dihydroxyflavanone
Microbial growth promoters Genistein, 40 ,7-dihydroxyflavone
nod gene inducers in rhizobia Coumetrol, 4,40 -dihydroxy -20 -
methoxychalcone
nod gene inhibitors in rhizobia Eriodictyol, 40 -7-dihydroxyflavone
Root border Produce signals that control mitosis,
cells produce signals controlling gene
expression, stimulate microbial
growth, release chemoattractant,
synthesize defense molecules for the
rhizosphere, act as decoys that keep
root cap infection-free, release
mucilage and proteins
Compiled from Dakora (2003), Dakora and Phillips (2002), and Bais et al. (2004)
components. In addition, the total microbial population in the rhizospheric soil was
significantly positively correlated with AWCD, microbial biomass carbon, micro-
bial respiration, and Shannon index. There was a significantly positive correlation
between the total microbial population and the inhibition rate (IR) on the root length
of lettuce owing to the different allelopathic activities of the rice cultivars. The
results suggested that changes in microbial population, activity, and functional
diversity in the rhizospheres are highly cultivar-dependent.
During the growth of new roots, exudates secreted in the zone of elongation
behind the root tips support the growth of primary root colonizers that utilize easily
degradable sugars and organic acids. In the older root zones, carbon is deposited
primarily as sloughed cells and consists of more recalcitrant materials, including
lignified cellulose and hemicellulose, so that fungi and bacteria in these zones are
presumably adapted to crowded, oligotrophic conditions. Other nutritionally dis-
tinct sites include the sites of lateral root emergence and the secondary, nongrowing
root tips, which are relatively nutrient-rich environments colonized by mature
communities. Soil chemical changes related to the release of organic and inorganic
compounds, and the respective products of their microbial metabolism, are impor-
tant factors affecting microbial populations, availability of nutrients, solubility of
toxic elements in the rhizosphere, and thereby the ability of plants to cope with
adverse soil chemical conditions. Organic compounds in root exudates are continu-
ously metabolized by root-associated microorganisms and in the rhizosphere.
Mobilization of micronutrients or heavy metals in the rhizosphere has also been
related to rhizosphere acidification and to complexities with organic acids in root
exudates (Marschner 1995; Pinton et al. 2001; Waisel et al., 1991).
Soil pH is an important factor affecting the functioning of soil microbial com-
munities in soils, and may influence rates of substrate utilization; the values of
substrate utilization were significantly higher in soils of higher pH (Anderson and
Joergensen 1997). And, hence, soil pH has been found as the most influential
environmental factor responsible for discrimination among microbes (Grayston
et al. 2003). For example, soil acidity has shown a considerable decrease in the
availability of carbon to microbial communities (Anderson and Domsch 1993;
Baath et al. 1995), and to slower bacterial growth rates (Baath 1998). However,
studies have also shown optimum pH for growth of bacterial communities to be
correlated with soil pH from which the communities are extracted, indicating that
different bacterial communities are adapted to different pH values (Baath 1996;
Andersson and Ingvar Nilsson 2001). For instance, bacterial communities asso-
ciated with coniferous forest soils may contain larger proportions of Gram-positive
bacteria adapted to the acidifying environment; in contrast, increases in soil pH may
result in larger proportions of Gram-negative bacteria (Frostegard et al. 1993;
Pennanen 2001). Additionally, numbers of bacteria have been shown to decrease
in acidified soils (Baath et al. 1980), possibly because bacteria are less adapted to
acidic conditions in soil compared to fungi (Matthies et al. 1997). However,
microbial biomass C and inorganic N do not respond to changed soil moisture
while N2O and CO2 efflux and respiration increase after increasing moisture in the
agricultural soils. Moreover, in the agricultural systems, reductions in both
the measures of microbial diversity and the resistance of the microbial community
to change after a perturbation have been found to be associated with lower micro-
bial responses to increased moisture availability (Steenwerth et al. 2005).
15.3.2 Soil Management Practices
Agricultural management practices have strong impacts on soil microbes including

both the indices related to biomass and activity as well as those related to commu-
nity composition (Follett and Schimel 1989; Aon et al. 2001; Steenwerth et al.
2003; Potthoff et al. 2006). Therefore, in recent times, attention has been focused on
the influence that agricultural management practices have on biological and bio-
geochemical properties of soils (Gregorich et al. 1994; Franzluebbers et al. 1995).
Soil microbial biomass and activities are frequently used as an indicator of changes
in soil chemical and physical properties resulting from soil management and
environmental stress in agricultural ecosystems (Brookes 1995; Trasar-Cepeda
et al. 1998). Understanding how management practices influence soil fertility and
agricultural productivity is essential to improve the sustainability of agro-ecosystems
(Wardle et al. 1999). The effect of different management regimes and perturbations
on the soil microbial community (e.g., crop rotations, manure applications, and
tillage) has been studied in a wide range of soil and management systems.
15.3.2.1 Tillage
Cultivation and tillage practices profoundly disrupt the soil by: (1) breaking up soil
aggregates, (2) increasing soil compaction, (3) exposing previously protected
organic matter, and (4) mixing soil horizons (Beare 1997; Allison et al. 2005). In
turn, such alterations lead to a significant decline in microbial biomass, reduce the
abundance of fungi, aerobic microorganisms, and facultative anaerobes, while
increasing the relative abundance of Gram-negative bacteria (Doran 1980; Beare
1997). Tillage practice in agricultural soils not only affects the microbial diversity
but also influences species composition, N transformation processes mediated by
microbes, and interactions between organisms and the soil pore network, and
modulates the role of soil structure in mediating oxygen movement to sites of
microbial activity in soil (Young and Ritz 2000). The agricultural soils which are
tilled frequently and subjected to crop rotations have shown lower microbial
diversity compared to those observed in soils which are either not tilled or have
been infrequently tilled (Øvreås and Torsvik 1998). For example, by extracting
microbial RNA, Buckley and Schmidt (2001) found that the abundance of groups
comprising of Gram-positive bacteria (aerobes) and fungi were significantly lower
in fields abandoned from agriculture 7 years earlier. In another study, Fraterrigo
et al. (2006) reported that old farms had similar compositional patterns, despite
having experienced relatively little tillage and having been abandoned since 1930.
The high bulk density of former farms (Fraterrigo et al. 2005) and the elevated
abundance of Gram-negative bacterial markers suggest that these enduring micro-
bial patterns may be partly due to a continued state of soil hypoxia; a common
condition in highly managed soils. Moreover, tillage events contribute to decreased
soil quality by increasing emissions of greenhouse gases (i.e., CO2, NO, or N2O),
and increasing the potential for nitrate leaching to groundwater. Such negative
aspects require proper attention before considering the benefits of tillage for
increasing the health and productivity of crops (Jackson et al. 2003). In addition,
research is also needed to assess potential effects of long-term agricultural man-
agement practices that may mask microbial responses to recent management
change, as well as to identify conditions that lead to high microbial community
resiliency in response to management so that communities remain similar under a
given crop despite different preceding crops (Stromberger et al. 2007).
15.3.2.2 Nutrient Management
Agricultural improvement through fertilizer application by the field practitioners is

being practised around the world to achieve maximum plant productivity; but this
practice is not only expensive, but the repeated and injudicious application of
chemical fertilizers also leads to the loss of soil fertility (Gyaneshwar et al. 2002)
by disturbing microbial diversity and catabolic activity (Zhang et al. 2007;
Guanghua et al. 2008), and consequently reduces yield of crops. For instance, N
fertilization has been shown to significantly affect soil microbial biomass and
activities (Ladd et al. 1994; Saliana-Garcia et al. 1997). Long-term application of
NH4+ or NH4+-forming fertilizers may lead to changes in the structure of soil
microbial communities in terms of promoting nitrifying populations, enhancing
nitrification rates, and increasing potential risks of groundwater contamination with
NO3–N (Tabatabai et al. 1992). However, such deleterious effects of N fertilizers
could be reduced by controlled release and deep application (0–10 cm) of mineral
fertilizers (Chu et al. 2005). Similarly, the long-term effect of different sources of
phosphate fertilizers, like single superphosphate, North Carolina phosphate rock,
partially acidulated North Carolina phosphate rock, and diammonium phosphate,
on microbial activities, like basal respiration, substrate-induced respiration, inhibi-
tion of substrate-induced respiration by streptomycin sulfate (fungal activity) and
actidione (bacterial activity), and microbial biomass C of pasture soils, has been
reported (Bolan et al. 1996). The fertilizer addition caused an initial decrease in
basal and substrate-induced respiration but had no effect on total microbial bio-
mass. The initial decline in basal and substrate-induced respiration with the ferti-
lizer addition was restored within 8 weeks after incubation. In the field experiment,
the fertilizer addition had no significant effect on basal respiration but increased
substrate-induced respiration and microbial biomass C (Bolan et al. 1996). Similarly,
the application of triple superphosphate (at 94 kg ha1) has produced a substantial
reduction in microbial respiration and metabolic quotient (qCO2) (Chandini and

Dennis 2002). Recently, multidimensional scaling plots and canonical corres-
pondence analysis revealed that phosphate (K2PO4 at 25 kg P ha1) addition and
its interaction with upland grassland plant species (Agrostis capillaries, Festuca
ovina, Lolium perrene) resulted in considerable changes in the fungal and bacterial
communities of upland soil. Both fungal and bacterial community structures were
significantly affected by phosphate suggesting that phosphate application may be an
important contributor to microbial community structural change during agricultural
management (Rooney and Clipson 2009). Similarly, Vineela et al. (2008) studied the
long-term effect of fertilization on soil microbial communities and found that the
bacterial counts were higher in treatments where combinations of organic and
inorganic fertilizers were applied compared to control. Fungal population was higher
in treatments under continuous inorganic fertilization whereas a high number of
bacteria were found in integrated use of organic and inorganic fertilizers. At most
of the locations, soil organic C and microbial biomass C showed a significant (p
0.05) positive correlation with microbial populations. In contrast, Sun et al. (2004))
while examining 16S rRNA gene fingerprints of bacterial communities in six agro-
ecosystems soils treated with manure for over a century or different fertilizers for
over 70 years reported that the bacterial community structure and diversity in the
manure-treated soil was more closely related to the structure in the untreated soil than
that in soils treated with inorganic fertilizers. In addition, soils treated with P and N–P
had bacterial community structures more closely related to each other than to those of
soils given other treatments, suggesting that bacterial community structure was
closely related to agro-ecosystem management practices conducted for over 70
years. In contrast, application of mineral fertilizers (e.g., N, P, K) in combination
with farmyard manure (FYM) had greater numbers of soil microbes and more
complex structure of the ammonium-oxidizing bacteria (AOB) community
than those receiving mineral fertilizers alone, suggesting that mineral fertilizers
along with FYM could be more effective for increasing the quantity of soil microbes,
enriching the AOB community, and improving the soil bio-fertility (Guanghua et al.
2008).
15.3.3 Impact of Seasonal Variations
Another important issue to elucidate is how seasonal variations influence qualitative

variation in community composition. In this regard, the impact of seasonal varia-
tions on soil microbial communities of wheat grown under a field of an experimental
farm in The Netherlands was assessed in different seasons over a 1-year period,
using both cultivation-based and molecule-based methods (Smit et al. 2001). Fatty
acid-based typing of bacterial cultures obtained via plating revealed a diverse
community including predominantly Gram-positive bacteria, and only a few isolates
represented Proteobacteria and green sulfur bacteria. Interestingly, Micrococcus,
Arthrobacter, and Corynebacterium were detected throughout the year, while
Bacillus was found only during the month of July. The isolate diversity was lowest in
July, and the most abundant bacteria, Arthrobacter oxydans, and members of the
genus Pseudomonas, were found in reduced numbers in July. Moreover, molecular
analysis suggested that diversity of cloned 16S ribosomal DNA (rDNA) sequences
was greater than the diversity among cultured isolates. In addition, based on analysis
of 16S rDNA sequences, there was a more even distribution among five main
divisions, Acidobacterium, Proteobacteria, Nitrospira, cyanobacteria, and green
sulfur bacteria, but no clones were found belonging to the Gram-positive bacteria,
which dominated the cultured isolates. Cluster analysis of the patterns revealed that
the bacterial community observed in July was clearly different from those observed
in the other months. Thus, both molecular- and cultivation-based methods indicated
that the community present in July had the largest difference from the communities
of the other months. Based on the distribution of 16S rDNA sequences among the
bacterial divisions found in this study and in literature, it was suggested that the ratio
between the number of Proteobacteria and Acidobacterium organisms might be
indicative of the trophic level of the soil. Effects of seasonal shifts on rhizosphere
microbial populations of pea, wheat, and sugar beet (Beta vulgaris var. amythyst)
have also been determined by culturing, rRNA gene density gradient gel electro-
phoresis, and Biolog. Culturable bacterial and fungal rhizosphere community den-
sities were stable in pea and wheat rhizospheres, with dynamic shifts observed in the
sugar beet rhizosphere. Successional shifts in bacterial and fungal diversity as plants
mature demonstrated that different plants select and define their own functional
rhizosphere communities. Assessment of metabolic activity and resource utilization
by bacterial community-level physiological profiling demonstrated greater simila-
rities between different plant species rhizosphere communities at the same rather
than at different developmental stages. Marked temporal shifts in diversity and
relative activity were observed in rhizosphere bacterial communities with the
developmental stage for all plant species studied. Shifts in the diversity of fungal
and bacterial communities were more pronounced in maturing pea and sugar beet
plants. This extensive study demonstrates that plant species select for specialized
microbial communities that change in response to plant growth and plant inputs
(Houlden et al. 2008).
15.3.4 Pesticides
A wide variety of pesticides including herbicides, fungicides and herbicides are

used in agricultural practices in order to improve the yield of various crops. The use
of such pesticides beyond recommended concentrations or their accumulation into
soil following continuous application leads to changes in microbial populations
agro-ecosystem. For instance, the application of endosulfan, profenophos with
alphamethrin, and methamidophos has shown considerable inhibition in bacterial
population in cotton (Gossypium hirsutum) agro-ecosystem while monocrotophos
and bifenthrin with acetamiprid enhanced the bacterial population of soil. Fungal
population was depressed with endosulfan while monocrotophos, methamidophos,

endosulfan with dimethoate, fenpropathrin, bifenthrin with acetamiprid, or with
ethion or with a mixture of carbosulfan and chloropyrifos and profenophos alone or
with ethion or cypermethrin or alphamethrin stimulated fungal counts (Iqbal et al.
2001). In another study, Ekundayo (2003) investigated the effect of 11 pesticides on
the populations of bacteria, actinomycetes, fungi, and protozoa by treating a garden
soil with their recommended rates. Phenylmercuric acetate (agrosan) at 50 mg g1
inhibited bacterial density severely. Pentachloronitrobenzene (PCNB) at 240,000
mg g1 reduced bacterial population from 4.6106 to 2.1102 cells g1, whereas
tetramethylthiuram disulphide (thiram) at 100 mg g1 suppressed it by twofold. Soil
application of 1-naphthylmethylcarbamate (Vetox 85) at 100 mg g1 and
1,2,3,4,5,6,-hexachlorocyclohexane (Gamalin 20) at 1,300 mg g1 repressed the
bacterial numbers by two orders each. PCNB reduced the actinomycetes density
from 3.4105 to 3.2102 cells g1 and completely eliminated all fungal and
protozoan propagules from the soil. The Gammalin 20 completely wiped out all
the fungi, whereas phenylmercuric acetate totally eliminated all the protozoa and
reduced the fungal population from 3.4104 to 60 cells g1. In general, protozoa
and fungi were more susceptible to fungicides than bacteria and actinomycetes.
PCNB, 1,2,3,4,5,6,-hexachlorocyclohexane and phenylmercuric acetate were toxic
particularly to soil microorganisms, whereas the herbicides dacthal, preforan and
dual were quite harmless in soil at application rates of 0.1, 0.06 and 0.02 mg g1,
respectively. Wang et al. (2006) showed that both a low and a higher dose of
methamidophos (0, S-dimethyl phosphoroamidothioate) in soil significantly de-
creased microbial biomass C (Cmic) by 41–83% compared to control. In contrast,
the respiration activity of the applied soils was significantly higher than the control.
Pesticide application also significantly increased the soil total of N and P. In
addition, substrate richness, Shannon and Simpson indices of microbial commu-
nities under chemical stresses increased significantly. Moreover, the populations of
methamidophos-metabolized bacteria also increased significantly. It was concluded
that methamidophos reduces microbial biomass and enhances functional diversities
of soil microbial communities; meanwhile, some species of bacteria may be
enriched in soils under methamidophos stress. Similarly, Ratcliff et al. (2006)
reported that commercial formulation herbicide (glyphosate) applied at the recom-
mended field rate to a clay loam and a sandy loam forest soil has a benign affect on
community structure and produced a nonspecific, short-term stimulation of bacteria
at a high concentration.
15.4 Resilience of Microbial Communities
Although microbial communities are highly sensitive to both natural and artificial
disturbance, the community might still be resilient and quickly return to its predis-
turbed composition. Microorganisms in general possess a number of features that
help them to acquire resilience against several adverse environmental variables.
Such factors that protect them from disturbances include, firstly, the fast growth
rates of microorganisms; if their abundance is suppressed by a disturbance, they
have the potential to recover quickly. Secondly, their high degree of physiological
flexibility; for example, purple nonsulfur bacteria, which act as a phototroph under
anoxic conditions are a heterotroph under aerobic conditions suggesting that if the
relative abundance of some microbes decreased initially, such organisms possess
the ability to acclimatize rapidly to the new abiotic conditions over time and could
return to their original abundance. And thirdly, their rapid evolution potential; if
physiological adaptation is not possible, then the rapid evolution (through muta-
tions or horizontal gene exchange) could allow microbes to adapt to new environ-
mental conditions and recover from disturbance (Allison and Martiny 2008). Thus,
there are three ways in which microbial composition might not matter substantially
to ecosystem functioning even if there are any disturbances in the agro-ecosystems:
(1) ability of microbial communities to resist changes; however, in the majority of
cases microbial communities have been found sensitive to elevated CO2, mineral
fertilization, temperature changes, and C amendments (Allison and Martiny 2008),
(2) microbial composition might be resilient and quickly return to its original state,
and (3) even if microbial composition changes, the new community might be
functionally similar to the original one. Although this hypothesis is currently
difficult to test, recent studies suggest that many microbial communities are proba-
bly not functionally redundant and different communities are not functionally
similar (Allison and Martiny 2008).
15.5 Conclusion
Soil microbial communities are integrally involved in biogeochemical cycles and

their activities are crucial to maintaining soil fertility and productivity and improv-
ing the functioning of the soil ecosystem. A reasonable selection of sensitive and
robust soil indicators is, however, required to distinguish the trends in improvement
and deterioration of soil quality in various agro-ecosystems. Novel methods and
approaches enable us to explore the variation in compositions and functional
diversity of microbial communities. Studies of sequence information from organ-
isms in soil microhabitats and their gene expression under different conditions will
provide guidelines for designing new and improved culturing methods that resem-
ble their natural niches. However, despite the importance of soil microorganisms,
little is known about the distribution of microorganisms in the soil or the manner in
which microbial community structure responds to changes in environmental con-
ditions and farm management practices that have shown a considerable impact on
soil biota, affecting nutrient cycling processes and ecosystem functioning. Under-
standing the mechanisms that regulate microbial community structure and activity
following implementation of various management practices may lead to the
development of best management practices for improving soil fertility and, conse-
quently, agricultural productivity to improve the sustainability of agro-ecosystems.
References
Adkins A, Wright RB, Scott GAJ, Adkins A, Wright RB, Scott GAJ (2001) Microbial diversity
and activity in a stressed boreal forest ecosystem (Manitoba, Canada), vol Abstract I-37.
American Society for MicroBiology, Orlando, FL
Allison SD, Martiny JBH (2008) Resistance, resilience, and redundancy in microbial commu-
nities. Proc Natl Acad Sci 105:11512–11519
Allison VJ, Miller RM, Jastrow JD, Matamala R, Zak DR (2005) Changes in soil microbial
community structure in a tallgrass prairie chronosequence. Soil Sci Soci Am J 69:1412–1421
Anderson TH, Domsch KH (1993) The metabolic quotient for CO2 (qCO2) as a specific activity
parameter to assess the effects of environmental conditions, such as pH, on the microbial
biomass of forest soils. Soil Biol Biochem 25:393–395
Anderson TH, Joergensen RG (1997) Relationship between SIR and FE estimates of microbial
biomass C in deciduous forest soils at different pH. Soil Biol Biochem 29:1033–1042
Andersson S, Ingvar Nilsson S (2001) Influence of pH and temperature on microbial activity,
substrate availability of soil-solution bacteria and leaching of dissolved organic carbon in a
mor humus. Soil Biol Biochem 33:1181–1191
Aon MA, Cabello MN, Sarena DE, Colaneri AC, Franco MG, Burgos JL, Cortassa S (2001) I.
Spatio-temporal patterns of soil microbial and enzymatic activities in an agricultural soil. Appl
Soil Ecol 18:239–254
Assmus B, Hutzler P, Kirchhof G, Amann R, Lawrence JR, Hartmann A (1995) In situ localization
of Azospirillum brasilense in the rhizosphere of wheat with fluorescently labeled, rRNA-
targeted oligonucleotide probes and scanning confocal laser microscopy. Appl Environ Micro-
biol 61:1013–1019
Atlas RM (1984) Use of microbial diversity measurements to assess environmental stress. In:
Klug MJ, Reddy CA (eds) Current perspectives in microbial ecology. ASM, Washington,
pp 540–545
Azam F, Malfatti F (2007) Microbial structuring of marine ecosystems. Nat Rev Microbiol 5:782–791
Baath E (1996) Adaptation of soil bacterial communities to prevailing pH in different soils. FEMS
Baath E (1998) Growth rates of bacterial communities in soils at varying pH: a comparison of the
thymidine and leucine incorporation techniques. Microb Ecol 36:316–327
Baath E, Berg B, Lohm U, Lundgren B, Lundkvist H, Rosswall T, Soderstrom B, Wiren A (1980)
Effects of experimental acidification and liming on soil organisms and decomposition in a
Scots pine forest. Pedobiologia 20:85–100
Baath E, Frostegard A, Pennanen T, Fritze H (1995) Microbial community structure and pH
response in relation to soil organic matter quality in wood–ash fertilized, clear-cut or burned
coniferous forest soils. Soil Biol Biochem 27:229–240
Bais HP, Park S, Weir TL, Callaway RM, Vivanco JM (2004) How plants communicate using the
underground information superhighway. Trends Plant Sci 9:26–32
Batten KM, Scow KM, Davies KF, Harrison SP (2006) Two invasive plants alter soil microbial
community composition in serpentine grasslands. Biol Invasions 8:217–230
Bauhus J, Paré D, Côté L (1998) Effects of tree species, stand age and soil type on soil microbial
biomass and its activity in a southern boreal forest. Soil Biol Biochem 30:1077–1089
Beare MH (1997) Fungal and bacterial pathways of organic matter decomposition and nitrogen
mineralization in arable soils. In: Brussaard L, Ferrera-Cerrato R (eds) Soil ecology in
sustainable agricultural systems. Lewis, Boca Raton, Florida, USA, pp 37–70
Bending GD, Turner MK, Jones JE (2002) Interactions between crop residue and soil organic
matter quality and the functional diversity of soil microbial communities. Soil Biol Biochem
34:1073–1082
Bever JD, Westover KM, Antonovics J (1997) Incorporating the soil community into plant
population dynamics: the utility of the feedback approach. J Ecol 85:561–573
Bolan NS, Currie LD, Baskaran S (1996) Assessment of the influence of phosphate fertilizers on
the microbial activity of pasture soils. Biol Fertil Soils 21:284–292
Bormann BT, Sidle RC (1990) Changes in productivity and distribution of nutrients in a chrono-
sequence at Glacier-Bay national park, Alaska. J Ecol 78:561–578
Borneman J, Triplett EW (1997) Molecular microbial diversity of soils from eastern Amazonia:
evidence for unusual microorganisms and microbial population shifts associated with defores-
tation. Appl Environ Microbiol 63:2647–2653
Bosse U, Frenzel P (1997) Activity and distribution of methane-oxidizing bacteria in flooded rice
soil microcosms and in rice plants (Oryza sativa). Appl Environ Microbiol 63:1199–1207
Bowen GD, Rovira AD (1991) The rhizosphere, the hidden half. In: Waisel Y, Eshel A, Kafkafi U
(eds) Plant roots – the hidden half. Marcel Dekker, New York, pp 641–649
Broeckling CD, Broz AK, Bergelson J, Manter DK, Vivanco JM (2008) Root exudates regulate
soil fungal community composition and diversity. Appl Environ Microbiol 74:738–744
Brookes PC (1995) The use of microbial parameters in monitoring soil pollution by heavy metals.
Biol Fertil Soils 19:269–289
Buckley DH, Schmidt TM (2001) The structure of microbial communities in soil and the lasting
impact of cultivation. Microb Ecol 42:11–21
Buyer JS, Roberts DP, Russek-Cohen E (1999) Microbial community structure and function in the
spermosphere as affected by soil and seed type. Can J Microbiol 45:138–144
Campbell CD, Grayston SJ, Hirst DJ (1997) Use of rhizosphere carbon sources in soil carbon
source tests to discriminate soil microbial communities. J Microbiol Methods 30:33–41
Chandini TM, Dennis P (2002) Microbial activity, nutrient dynamics and litter decomposition in a
Canadian Rocky Mountain pine forest as affected by N and P fertilizers. For Ecol Manage
159:187–201
Chiarini L, Bevivino A, Dalmastri C, Nacamulli C, Tabacchioni S (1998) Influence of plant deve-
lopment, cultivar and soil type on microbial colonization of maize root. Appl Soil Ecol 8:11–18
Chu HY, Hosen Y, Yagi K, Okada K, Ito O (2005) Soil microbial biomass and activities in
Japanese Andisol as affected by controlled release and application depth of urea. Biol Fertil
Soils 42:89–96
CÔté L, Brown S, Paré D, Fyles J, Bauhus J (2000) Dynamics of carbon and nitrogen mineraliza-
tion in relation to stand type, stand age and soil texture in the boreal mixed wood. Soil Biol
Biochem 32:1079–1090
Crawford JW, Harris JA, Ritz K, Young IM (2005) Towards an evolutionary ecology of life in soil.
Trends Ecol Evol 20:81–87
Da Silva KRA, Salles JF, Seldin L, van Elsas JD (2003) Application of a novel Paenibacillus-
specific PCR-DGGE method and sequence analysis to assess the diversity of Paenibacillus
spp. in the maize rhizosphere. J Microbiol Methods 54:213–231
Dakora FD (2003) Defining new roles for plant and rhizobial molecules in sole and mixed plant
cultures involving symbiotic legumes. New Phytol 158:39–49
Dakora FD, Phillips DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient
environments. Plant Soil 245:35–47
De Leij FAAM, Whipps JM, Lynch JM (1994) The use of colony development for the characteri-
zation of bacterial communities in soil and on roots. Microb Ecol 27:81–97
de Lima TCS, Grisi BM, Bonato MCM (1999) Bacteria isolated from a sugarcane agroecosystem:
their potential production of polyhydroxyalcanoates and resistance to antibiotics. Revista de
Microbiologia 30:214–224
de Luna RG, Grisi BM (1996) Biomassa e atividade microbianas de solos cultivados com cana-
de-açúcar, sob efeito da vinhaça. Rev Nordest Biol 11:15–29
Doran JW (1980) Soil microbial and biochemical changes associated with reduced tillage. Soil Sci
Soc Am J 44:765–771
Duineveld BM, Rosado AS, van Elsas JD, van Veen JA (1998) Analysis of the dynamics of
bacterial communities in the rhizosphere of the chrysanthemum via denaturing gradient gel
electrophoresis and substrate utilization patterns. Appl Environ Microbiol 64:4950–4957
Ekundayo EO (2003) Effect of common pesticides used in the Niger Delta basin of southern
Nigeria on soil microbial populations. Environ Monit Assess 89:35–41
Follett RF, Schimel DS (1989) Effect of tillage practices on microbial biomass dynamics. Soil Sci
Soc Am J 53:1091–1096
Franzluebbers AJ, Zuberer DA, Hons FM (1995) Comparison of microbiological methods for
evaluating quality and fertility of soil. Biol Fertil Soils 19:135–140
Fraterrigo JM, Turner MG, Pearson SM, Dixon P (2005) Effects of past land use on spatial
heterogeneity of soil nutrients in southern Appalachian forests. Ecol Monogr 75:215–230
Fraterrigo JM, Balser TC, Turner MG (2006) Microbial community variation and its relationship
with nitrogen mineralization in historically altered forests. Ecology 87:570–579
Frostegard A, Tunlid A, Baath E (1993) Phospholipid fatty acid composition, biomass, and activity
of microbial communities from two soil types experimentally exposed to different heavy
metals. Appl Environ Microbiol 59:3605–3617
Gans J, Wolinsky M, Dunbar J (2005) Computational improvements reveal great bacterial
diversity and high metal toxicity in soil. Science 309:1387–1390
Garbeva P, van Veen JA, van Elsas JD (2004) Microbial diversity in soil: selection of microbial
populations by plant and soil type and implications for disease suppressiveness. Annu Rev
Phytopathol 42:243–270
Garland JL (1996) Patterns of potential C source utilization by rhizosphere communities. Soil Biol
Biochem 28:223–230
Garland JL (1999) Potential and limitations of BIOLOG for microbial community analysis.
In: Bell CR, Brylinsky M, Johnson-Green P (eds) Microbial biosystems: new frontiers.
Proceedings of the 8th International Symposium on Microbial Ecology. Atlantic Canada
Society for Microbial Ecology, Halifax, Canada, pp 1–7
Garland JL, Mills AL (1991) Classification and characterization of heterotrophic microbial com-
munities on the basis of patterns of community level sole-carbon-source utilization. Appl
Germida JJ, Siciliano SD, Freitas JR, Seib AM (1998) Diversity of rootassociated bacteria
associated with fieldgrown canola (Brassica napus L.) and wheat (Triticum aestivum L.).
FEMS Microbiol Ecol 26:43–50
Gilbert B, Frenzel P (1998) Rice roots and CH4 oxidation: the activity of bacteria, their distribution
and the microenvironment. Soil Biol Biochem 30:1903–1916
Giovannoni SJ, Britschgi TB, Moyer C, Field KG (1990) Genetic diversity in Sargasso Sea
bacterioplankton. Nature 345:60–63
Grayston SJ, Campbell CD (1996) Functional biodiversity of microbial communities in the
rhizosphere of hybrid larch (Larix eurolepis) and Sitka spruce (Picea sitchensis). Tree Physiol
16:1031–1038
Grayston SJ, Wang S, Campbell C, Edwards A (1998) Selective influence of plant species on
microbial diversity in the rhizosphere. Soil Biol Biochem 30:369–378
Grayston SJ, Campbell CD, Bardgett RD, Mawdsley JL, Mawdsley Clegg CD, Ritz K,
Griffiths BS, Rodwell JS, Edwards SJ, Davies WJ, Elston DJ, Millard P (2003) Assessing
shifts in microbial community structure across a range of grasslands of differing manage-
ment intensity using CLPP, PLFA and community DNA techniques. Appl Soil Ecol
25:63–84
Grayston SJ, Campbell CD, Bardgett RD, Mawdsley JL, Clegg CD, Ritz K, Griffith BS, Rodwell
JS, Edwards SJ, Davies WJ (2004) Assessing shifts in microbial community structure across a
range of grasslands of differing management intensity using CLPP, PLFA and community
DNA techniques. Appl Soil Ecol 25:63–84
Gregorich EG, Carter MR, Angers DA, Monreal CM, Ellert BH (1994) Towards a minimum set to
assess soil organic matter quality in agricultural soils. Can J Soil Sci 76:367–385
Griffiths BS, Ritz K, Ebblewhite N, Dobson G (1999) Soil microbial community structure: effects
of substrate loading rates. Soil Biol Biochem 31:145–153
Guanghua W, Junjie L, Qi Xiaoning Q, Jian J, Yang W, Xiaobing L (2008) Effects of fertilization
on bacterial community structure and function in a black soil of Dehui region estimated by
Biolog and PCR-DGGE methods. Acta Ecol Sin 28:220–226
Gyaneshwar P, Kumar GN, Parekh LJ, Poole PS (2002) Role of soil microorganisms in improving
P nutrition of plants. Plant Soil 245:83–93
Haack SK, Garchow H, Klug MJ, Forney LJ (1995) Analysis of factors affecting the accuracy,
reproducibility, and interpretation of microbial community carbon source utilization patterns.
Haynes RJ (1999) Size and activity of the soil microbial biomass under grass and arable manage-
ment. Biol Fertil Soils 30:210–216
Hiltner L (1904) Uber neuere Erfahrungen und Probleme auf dem Gebiet der Bodenbakteriologie
und unter besonderer Berucksichtigung der Grundungung und Brache. [German]. Arb Dtsch
Landwirtsch Ges 98:59–78
Houlden A, Timms-Wilson TM, Day MJ, Bailey MJ (2008) Influence of plant developmental stage
on microbial community structure and activity in the rhizosphere of three field crops. FEMS
Hugenholtz P, Goebel BM, Pace NR (1998) Impact of culture-independent studies on the
emerging phylogenetic view of bacterial diversity. J Bacteriol 180:6793–6794
Ibekwe AM, Kennedy AC (1998) Phospholipid fatty acid profiles and carbon utilization patterns
for analysis of microbial community structure under field and greenhouse conditions. FEMS
Ibekwe AM, Kennedy AM, Frohne PS, Papiernik SK, Yang CH, Crowley DE (2002) Microbial
diversity along a transect of agronomic zones. FEMS Microbiol Ecol 26:151–163
Innes L, Hobbs PJ, Bardgett RD (2004) The impacts of individual plant species on rhizosphere
microbial communities in soils of different fertility. Biol Fertil Soils 40:7–13
Jackson LE, Calderon FJ, Steenwerth KL, Scow KM, Rolston DE (2003) Responses of soil
microbial processes and community structure to tillage events and implications for soilquality.
Geoderma 114:305–317
Johansson M (1995) The chemical composition of needle and leaf litter from Scots pine, Norway
spruce and white birch in Scandinavian forests. Forestry 68:49–62
Kang S, Mills AL (2004) Soil bacterial community structure changes following disturbance of the
overlying plant community. Soil Sci 169:55–65
Kennedy N, Connolly J, Clipson N (2004) Impact of lime and nitrogen and plant species on fungal
community structure in grassland microcosms. Environ Microbiol 7:780–788
Kennedy NM, Gleeson DE, Connolly J, Clipson NJW (2005) Seasonal and management influences
on bacterial community structure in an upland soil. FEMS Microbiol Ecol 53:329–337
Kent AD, Triplett EW (2002) Microbial communities and their interactions in soil and rhizosphere
ecosystems. Annu Rev Microbiol 56:211–236
Kirk JL, Beaudette LA, Hart M, Moutoglis P, Klironomos JN, Lee H, Trevors JT (2004) Methods
of studying soil microbial diversity. J Microbiol Methods 58:169–188
Konopka A, Oliver L, Turco JRF (1998) The use of carbon substrate utilization patterns in
environmental and ecological microbiology. Microb Ecol 35:103–115
Kowalchuk GA, Hol WHG, van Veen JA (2006) Rhizosphere fungal communities are influenced
by Senecio jacobaea pyrrolizidine alkaloid content and composition. Soil Biol Biochem
38:2852–2859
Ladd JN, Amato M, Li-Kui Z, Schultz JE (1994) Differential effects of rotation, plant residue and
nitrogen fertilizer on microbial biomass and organic matter in the Australian alfisol. Biol Fertil
Soils 26:821–831
Latour X, Corberand T, Laguerre G, Allard F, Lemanceau P (1996) The composition of fluorescent

Pseudomonas population associated with roots is influenced by plant and soil type. Appl
Li X, Inubushi K, Sakamoto K (2002) Nitrous oxide concentrations in an Andisol profile and
emissions to the atmosphere as influenced by the application of nitrogen fertilizres and manure.
Biol Fertil Soil 35:108–113
Lupwayi NZ, Rice WA, Clayton GW (1998) Soil microbial diversity and community structure
under wheat as influenced by tillage and crop rotation. Soil Biol Biochem 30:1733–1741
Lynch JM, Whipps JM (1990) Substrate flow in the rhizosphere. Plant Soil 129:1–10
Mahaffee WF, Klöpper JW (1997) Temporal changes in the bacterial communities of soil,
rhizosphere, and endorhiza associated with field-grown cucumber (Cucumis sativus L.).
Microb Ecol 34:210–223
Marschner P, Yang CH, Lieberei R, Crowley DE (2001) Soil and plant specific effects on bacterial
community composition in the rhizosphere. Soil Biol Biochem 33:1437–1445
Matthies C, Erhard HP, Drake HL (1997) Effects of pH on the comparative culturability of fungi
and bacteria from acidic and less acidic forest soils. J Basic Microbiol 37:335–343
Merilä P, Strömmer R, Fritze H (2002) Soil microbial activity and community structure along a
primary succession transect on the landuplift coast of western Finland. Soil Biol Biochem
34:1647–1654
Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by
denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes
for 16S rRNA. Appl Environ Microbiol 59:695–700
Odum EP (1971) Fundamentals of ecology. W.B. Saunders, Philadelphia
Øvreås L, Torsvik V (1998) Microbial diversity and community structure in two different
agricultural soil communities. Microb Ecol 36:303–315
Pennanen T (2001) Microbial communities in boreal coniferous forest humus exposed to heavy
metals and changes in soil pH-a summary of the use of phospholipid fatty acids, Biologw and
3H thymidine incorporation methods in field studies. Geoderma 100:91–126
Pinton R, Varanini Z, Nannipieri P (2001) The rhizosphere. Biochemistry and organic substances
at the soil-plant interface. Marcel Dekker, New York
Potthoff M, Steenwerth KL, Jackson LE, Drenovsky RE, Scow KM, Joergensen RG (2006) Soil
microbial community composition as affected by restoration practices in California grassland.
Preston-Mafham J, Boddy L, Randerson PF (2002) Analysis of microbial community functional
diversity using sole-carbon-source utilization profiles-a critique. FEMS Microbiol Ecol 42:1–14
Priha O, Smolander A (1997) Microbial biomass and activity in soil and litter under Pinus
sylvestris, Picea abies and Betula pendula at originally similar field afforestation sites. Biol
Priha O, Smolander A (1999) Nitrogen transformations in soil under Pinus sylvestris and Betula
pendula at originally similar forest sites. Soil Biol Biochem 31:965–977
Priha O, Grayston SJ, Hiukka R, Pennanen T, Smolander A (2001) Microbial community structure
and characteristics of the organic matter in soils under Pinus sylvestris, Picea abies, and Betula
pendula at two forest sites. Biol Fertil Soils 33:17–24
Ratcliff AW, Busse MD, Shestak CJ (2006) Changes in microbial community structure following
herbicide (glyphosate) additions to forest soils. Appl Soil Ecol 34:114–124
Reinhart KO, Callaway RM (2006) Soil biota and invasive plants. New Phytol 170:445–457
Rooney DC, Clipson NJW (2009) Phosphate addition and plant species alters microbial community
structure in acidic upland grassland soil. Microb Ecol 57:4–13
Rozak DB, Colwell RR (1978) Survival strategies of bacteria in the natural environment. Microbiol
Rev 51:365–379
Ruiyu L, Hong R, Junjian Z, Cuiping Y, Chenying Y, Liangsheng C, Wenxiong L (2007) Impact of

allelopathic rice seedlings on rhizospheric microbial populations and their functional diversity.
Acta Ecol Sin 27:3644–3654
Saetre P, Bååth E (2000) Spatial variation and patterns of soil microbial community structure in a
mixed spruce-birch stand. Soil Biol Biochem 32:909–917
Saliana-Garcia JR, Hons FM, Matocha JE (1997) Long-term effects of tillage and fertilization on
soil organic matter dynamics. Soil Sci Soc Am J. 61:152–159
Sessitich A, Weilharter A, Gerzabek MH, Kirchmann H, Kandeler E (2006) Microbial population
structure in soil particle size fractions of a long-term fertilizer field experiment. Appl Environ
Smit E, Leeflang P, Gommans S, Broek JVD, Mil SV, Wernars K (2001) Diversity and seasonal
fluctuations of the dominant members of the bacterial soil community in a wheat field as
determined by cultivation and molecular methods. Appl Environ Microbiol 67:2284–2291
Staddon WJ, Duchesne LC, Trevors JT (1998a) Impact of clearcutting and prescribed burning on
microbial diversity and community structure in Jack pine (Pinus banksiana Lamb.) clear-cut
using Biologe gram-negative microplates. World J Microbiol Biotechnol 14:119–123
Staddon WJ, Trevors JT, Duchesne LE, Colombo CA (1998b) Soil microbial diversity and
community structure across a climatic gradient in western Canada. Biodivers Conserv
7:1081–1092
Steenwerth KL, Jackson LE, Calderón FJ, Stromberg MR, Scow KM (2003) Soil microbial
community composition and land use history in cultivated and grassland ecosystems of coastal
California. Soil Biol Biochem 35:489–500
Steenwerth KL, Jackson LE, Calderon FJ, Scow KM, Rolston DE (2005) Response of microbial
community composition and activity in agricultural and grassland soils after a simulated
rainfall. Soil Biol Biochem 37:2249–2262
Stromberger M, Shah Z, Westfall D (2007) Soil microbial communities of no-till dryland agroe-
cosystems across an evapotranspiration gradient. Appl Soil Ecol 35:94–106
Sun HY, Deng SP, Raun WR (2004) Bacterial community structure and diversity in a century-old
manure-treated agroecosystem. Appl Environ Microbiol 70:5868–5874
Tabatabai MA, Fu MH, Basta NT (1992) Effects of cropping systems on nitrification in soils.
Commun Soil Sci Plant Anal 23:1885–1891
Torsvik V, Øvreås L (2002) Microbial diversity and function in soil: from genes to ecosystems.
Curr Opin Microbiol 5:240–245
Torsvik V, Salte K, Sorheim R, Goksoyr J (1990) Comparison of phenotypic diversity and DNA
heterogenity in a population of soil bacteria. Appl Environ Microbiol 56:776–781
Torsvik VL, Daae FL, Goksfyr J, Sfrheim R, Vreas L (1997) Diversity of bacteria in soil and
marine environments. In: Martins MT, Sato MIZ, Tiedje JM, Hagler LCN, Döbereiner
J, Sanchez PS (eds) Progress in microbial ecology. SBM–ICOME, São Paulo, pp 115–120
Torsvik V, Øvreås L, Thingstad TF (2002) Prokaryotic diversity-magnitude, dynamics, and
controlling factors. Science 296:1064–1066
Trasar-Cepeda C, Leiros C, Gil-Sotres F, Seoane S (1998) Towards a biochemical quality index
for soils: an expression relating several biological and biochemical properties. Biol Fertil Soils
26:100–106
Van Elsas JD, Trevors JT, Wellington EHM (1997) Modern soil microbiology. Marcel Dekker,
New York, NY
Vineela C, Wani SP, Ch S, Padmaja B, Vittal KPR (2008) Microbial properties of soils as affected
by cropping and nutrient management practices in several long-term manurial experiments in
the semi-arid tropics of India. Appl Soil Ecol 40:165–173
Waisel Y, Eshel A, Kafkafi U (1991) Plant roots- the hidden half. Marcel Dekkar, New York
Waldrop MP, Balser TC, Firestone MK (2000) Linking microbial community composition to
function in a tropical soil. Soil Biol Biochem 32:1837–1846
Wang M, Ming G, Zang H, Hua X, Yao J, Pang Y, Yang Y (2006) Effect of methamidophos and
urea application on microbial communities in soils as determined by microbial biomass and
community level physiological profiles. J Environ Sci Health B 41:399–413
Wardle DA, Yeates GW, Nicholson KS, Bonner KI, Watson RN (1999) Response of soil microbial
dynamics, activity and plant litter decomposition to agricultural intensification over a seven-
year period. Soil Biol Biochem 31:1707–1720
Weisskopf L, Tomasi N, Santelia D, Martinoia E, Langlade NB, Tabacchi R, Abou-Mansour E
(2006) Isoflavonoid exudation from white lupin roots is influenced by phosphate supply, root
type and cluster-root stage. New Phytol 171:657–668
Wieland G, Neumann R, Backhaus H (2001) Variation of microbial communities in soil, rhizo-
sphere and rhizoplane in response to crop species, soil type, and crop development. Appl
Williamson WM, Wardle DA (2007) The soil microbial community response when plants are
subjected to water stress and defoliation disturbance. Appl Soil Ecol 37:139–149
Winding A (1993) Fingerprinting bacterial soil communities using Biologw microtiter plates.
In: Ritz K, Dighten J, Giller KE (eds) Beyond the biomass: compositional and functional
analysis of soil microbial communities. Wiley, Chichester, United Kingdom, pp 85–94
Winding A (1994) Fingerprinting bacterial soil communities with BIOLOGE microtitre plates. In:
Ritz K, Dighton J, Giller KE (eds) Beyond the biomass: compositional and functional analysis
of soil microbial communities. Wiley, Chichester, pp 85–94
Yang C, Crowley DE (2000) Rhizosphere microbial community structure in relation to root
location and plant iron nutritional status. Appl Environ Microbiol 66:345–351
Young IM, Ritz K (2000) Tillage, habitat space and function of soil microbes. Soil Tillage Res
53:201–213
Zak JC, Willig MR, Moorhead DL, Wildman HG (1994) Functional diversity of microbial
communities: a quantitative approach. Soil Biol Biochem 26:1101–1108
Zak DR, Holmes WE, White DC, Peacock AD, Tilman D (2003) Plant diversity, soil microbial
communities, and ecosystem function: are there any links? Ecology 84:2042–2050
Zhang P, Zheng J, Pan G, Zhang X, Li L, Tippkotter R (2007) Changes in microbial community
structure and function within particle size fractions of a paddy soil under different long-term
fertilization treatments from the Tai Lake region, China. Colloids Surf B Biointerf 58:264–270
Chapter 16
Strategies for Utilizing Arbuscular
Mycorrhizal Fungi and Phosphate-Solubilizing
Microorganisms for Enhanced Phosphate
Uptake and Growth of Plants in the Soils
of the Tropics
Nelson Walter Osorio and Mitiku Habte
Abstract One of the major constraints for plant productivity in tropical regions is
low soil phosphate (Pi) availability. Phosphate ions are rendered unavailable for
plant uptake due to adsorption onto the surface of soil minerals and precipitation by
free aluminum and iron ions. In highly weathered soils, this is so intense that plant
crops commonly exhibit Pi-deficiency. High rates of soluble Pi-fertilizers are
employed to meet plant P demands. However, the large quantity of Pi required in
order to offset the high Pi-retention capacity of the soils and the high cost associated
with it makes it inaccessible to the vast majority of growers in the region.
An alternative means of improving plant Pi-uptake from insoluble native and
applied rock phosphate is the use of arbuscular mycorrhizal (AM) fungi. These
fungi form a symbiotic association with most plants and improve the efficiency of
associated plants to take up Pi from the soil solution. Other soil microorganisms
commonly known as phosphate-solubilizing microorganisms (PSM) can replenish
soil solution Pi by solubilizing complex phosphorus compounds found in soil or
added to it, mostly through the release of organic acids. In this chapter, an attempt is
made to highlight the interactions of these two distinct groups of soil microorgan-
isms and the mechanisms by which they facilitate plant available Pi and enhance
plant growth in the soils of the tropics.
N.W. Osorio(*)
Soil Microbiology Research Group, Universidad Nacional de Colombia at Medellin, College of
Sciences, Calle 59A No. 63-20, Medellin, Colombia, USA
e-mail: nwosorio@unal.edu.co
M. Habte
University of Hawai’i at Manoa, College of Tropical Agriculture and Human Resources, St. John
Room 102, 3190 Maile Way, Honolulu, HI, USA, 96822
326 N.W. Osorio and M. Habte
16.1 Introduction
Phosphate (Pi)-fixation is a serious problem in agricultural soils, particularly in

highly weathered soils and those formed from volcanic ash (Trolove et al. 2003;
Sanchez and Uehara 1980). It is estimated that the soils that exhibit high Pi-fixation
capacity occupy 1,018 million ha in the tropics (Sanchez and Logan 1992). In
tropical America, there are 659 million ha affected by high Pi-fixation, 210 in
Africa, and 199 in Asia. The term Pi-fixation is used to describe reactions that
remove bioavailable Pi from the soil solution into the soil solid phase (Barber
1995). There are two types of reactions: (1) Pi-sorption onto the surface of soil
minerals, and (2) Pi-precipitation by cations such as Al3+ and Fe3+ in the soil
solution (Havlin et al. 1999). Of these, phosphate sorption is particularly strong
on iron and aluminum hydrous-oxides (crystalline or noncrystalline) that predomi-
nate in the highly weathered soils of humid regions and acid savannas (Mattingly
1975). Jones (1981) characterized the Pi-sorption by 11 Puerto Rican soils and
found that the surface area of Goethite was a primary factor accounting for Pi-
sorption, with Gibbsite and Hematite contributing little to Pi-sorption. A similar
result for Hawaiian soils has been reported (Jackman et al. 1997). Thus, soil Pi-
sorption was satisfactorily predicted by soil mineralogical composition. In soils
derived from volcanic parent materials, humus-Al/Fe complexes, Allophanes,
Ferrihydrite, and Goethite are the soil minerals responsible for the strong Pi-
sorption (Jackman et al. 1997; Shoji et al. 1993; Schwertmann and Herbillon
1992; Parfitt 1989). On the other hand, in calcareous soils, Pi is sorbed on the
surface of calcium carbonate (Mattingly 1975).
In acidic soils, Pi-precipitation occurs with active forms of aluminum [Al3+, Al
(OH)2+, Al(OH)2+) and iron (Fe3+)], while in neutral and alkaline soils, it occurs
mostly with calcium (Ca2+) (Bohn et al. 1985). The extent of dominance of these
cations depends mainly on the degree of soil weathering and soil pH. Phosphate
ions precipitate to form initially amorphous (noncrystalline) compounds, becoming
much more stable as crystalline forms are formed over time (Brady and Weil 1999).
Amorphous minerals are slightly more soluble than their crystalline forms because
they have smaller particle size, and consequently greater surface area. For instance,
the crystalline mineral variscite (AlPO4.2H2O) has a surface area of 1.54 m2 g1
(Taylor and Gurney 1964) whose solubility product (Ksp) is 1030.5 (Bache 1963).
In contrast, its amorphous aluminum-phosphate counterpart has a surface area of
10.5 m2 g1 (Juo and Ellis 1968) and its Ksp is 1028.1 (Veith and Sposito 1977). In
alkaline soils, Pi-compounds are similarly transformed to more insoluble forms.
Initially Pi-ions precipitate to calcium-monohydrogen-phosphate (Ksp ¼106.6)
(Stumm and Morgan 1995), which is then converted to calcium-orthophosphate
(Ksp ¼1024), and finally to apatite (Ksp ¼1055.9) (Snoeyink and Jenkins 1980).
Fox and Kamprath (1970) showed that the degree of P fixation varies among
soils, with Andisols, Oxisols, and Ultisols (USDA soil taxonomy) having a high
Pi-fixation capacity (Buol et al. 1997). These soils are usually acidic and generally
have higher amounts of exchangeable Al and clay minerals that can sorb high
16 Strategies for Utilizing Arbuscular Mycorrhizal Fungi 327
Table 16.1 Categories of soil Pi-sorption as measured by Pi-sorption isotherms following the
method of Fox and Kamprath (1970) and usual mineralogy in each category
Category P0.2 (P: mg kg1)a Usual mineralogy
Very low (VL) <10 Quartz, organic materials
Low (L) 10–100 2:1 clays, quartz, and 1:1 clays
Medium (M) 100–500 1:1 clays with oxides
High (H) 500–1,000 Oxides, moderately weathered ash
Very high (VH) >1,000 Desilicated amorphous materials
a
Amount of P required to achieve a soil solution P of 0.2 mg L1
amounts of Pi (Sanchez and Uehara 1980). Juo and Fox (1977) proposed several
categories for soil Pi-sorption capacity in tropical soils as measured by Pi-sorption
isotherms and the usual mineralogy of each category (Table 16.1).
The immediate Pi-source for plants is the soil solution, which usually contains a
low Pi-concentration (P: 0.001–0.01 mg L1) (Barber 1995; Fox 1979). When Pi is
removed from the soil solution by roots and/or by Pi-fixation reactions, a gradient of
Pi-concentration is created between the solid phase and the soil solution around the
roots. Sorbed Pi on the soil solid surfaces must desorb in order to replenish Pi in the
soil solution (Do Carmo Harta and Torrent 2007). Hence, Pi diffuses from the solid
phase, where it is more concentrated, to the soil solution around the root surface
where its concentration is continuously being depleted. However, the rate of
Pi-diffusion is quite slow (1012–1015 m2 s1) (Schachtman et al. 1998) which
limits the Pi-supply and creates a depletion zone of Pi around the roots of approxi-
mately of 1–2 mm (Barber 1995). Phosphate ions beyond the zone of depletion
cannot be accessed by the root surface (Barber 1995). Phosphate ions that slowly
diffuse into the soil solution originate mainly from Pi weakly sorbed on soil colloids
and from those freshly precipitated (Lindsay 2001; Stevenson 1986). Soil Pi-supply
depends on the Pi-buffering capacity of soils (Pypers et al. 2006), which can be
estimated from the relationship between the concentration of Pi in soil solution
(intensity factor, I) and the quantity of labile Pi in the solid phase (quantity factor,
Q) (Holdford 1997; Barber 1995).
16.2 Phosphate Fertilizer Management
Sanchez and Uehara (1980) discussed different strategies to increase soil Pi-availability
of acidic tropical soils with high Pi-fixation capacity. One strategy consists of
applying a high dose of soluble Pi-fertilizers (500–1,000 mg kg1), followed by
small amounts of annual application. Although a great part of the added Pi is
fixed, it may be released over several years, thus generating a residual effect.
These Pi-fertilization rates are, however, not added by most farmers in develop-
ing countries due to its high cost. The proportion of the added Pi taken up by
the first crop is quite low, ranging from 5 to 10%, suggesting that 90–95% of
the added phosphatic fertilizer could be fixed in the soils in chemical forms that
slowly release Pi for uptake by plants (Engelstad and Terman 1980).
Rock phosphates (RP) are highly recommended for acid soils with a high
Pi-fixation capacity because other more soluble Pi forms are quickly fixed and are
more expensive (Yusdar et al. 2007; Randhawa et al. 2006; Msolla et al. 2005).
However, the greater the reactivity of a RP, the greater is its desirability, because
less reactive minerals are very insoluble (Shrivastava et al. 2007; Ojo et al. 2007;
Hammond and Leon 1992). There is an increasing interest in tropical countries to
use organic amendments and RP concurrently. In this context, several studies
conducted across the globe have shown that the effectiveness of RP to increase
plant growth and crop yields could be enhanced by mixing it with farmyard
manures, compost, and green manures (Msolla et al. 2007; Yusdar et al. 2007;
Shrivastava et al. 2007). Moreover, added manures can also facilitate desorption of
sorbed Pi from soil particles (Redding et al. 2006). Some treatments on RP such as
“fine grinding,” thermal alteration, and fusion with silica, sodium or magnesium
carbonate have been satisfactorily used (Sanchez and Uehara 1980). These treat-
ments are oriented to increase solubility (due to a lower particle size) and/or reduce
Pi-sorption by including silicates that compete for the Pis soprtion sites. On the
other hand, since RP are more soluble in acidic conditions, their acidulation with
strong acids has been employed to produce more soluble fertilizers, such as super-
phosphates (Young and Davies 1980). Partial acidulation has also been applied;
however, it increases the cost of production (Havlin et al. 1999). The direct
application of nonacidulated RP is recommended for acid soils but not for neutral
and alkaline soils. However, several authors have used RP successfully in alkaline
soils with simultaneous inoculation of P-solubilizing microorganisms, which can
release Pi rapidly and, in turn, increases plant Pi-uptake (Khan et al. 2007; White-
law 2000; Kucey et al. 1989). For instance, Bar-Yosef et al. (1999) tested Pseudo-
monas cepacia for P-solubilizing activity on a RP and recorded that it produced
superphosphate. These researchers found that this bacterium produced gluconic
acid and 2-ketogluconic acid using glucose as the sole carbonaceous substrate.
Once these acids were dissociated in solution, the protons reacted with the RP and
released Pi into the solution that was then reprecipitated with Ca2+ to form super-
phosphate fertilizers.
The use of mycorrhizal fungi to increase the efficiency of Pi-uptake by plant is
also reported (Habte and Osorio 2001). For instance, Manjunath et al. (1989)
evaluated the effectiveness of AM-fungus (Glomus aggregatum) to enhance plant
Pi-uptake of Leucaena leucocephala grown in an Oxisol fertilized with RP
(P: 340–5,440 mg kg1). Although plant dry weight and shoot P concentration
did not increase significantly in uninoculated soils, when soil was inoculated with
G. aggregatum, a significant increase in plant dry weight and tissue P concentra-
tion was observed. However, in order to obtain adequate growth of mycorrhizal
plants, it was necessary to apply a high (at least 2,720 mg kg1) P level. Despite
the benefits of mycorrhizal inoculation, it is clear that it is necessary to apply
a high rate of RP. This imposes economic limitations on use of the mycorrhizal
association as a strategy to manage Pi-deficient soils. It is clear that AM fungi
absorb only Pi from the soil solution, as plant roots do, and there is no evidence
of their ability to solubilize insoluble soil P minerals (Bolan 1991). The use of
Pi-solubilizing microorganisms may increase the amount of available Pi in soil

solution and, consequently, enhance the effectiveness of AM fungi to increase
plant Pi-uptake.
16.3 Arbuscular Mycorrhizal Pi-Uptake
Some plants adopt different strategies to grow in P deficient soils: (1) changes
in root morphology such as the production of an elongated root system with fine
roots and abundance of root hairs, (2) release of phosphatase enzymes that release
Pi from organic compounds, and (3) production and release of organic acids that
solubilize Pi-compounds (Radersma and Grierson 2004; McCully 1999; Hetrick
1991). Plants with less root plasticity need to form symbiotic associations with
mycorrhizal fungi that colonize the cortical tissue of roots (Smith et al. 2003; Smith
2002; Sylvia 1999) if they are to grow normally in Pi-deficient soils. During
plant–AM fungal interactions, the plant supplies carbonaceous compounds to the
fungus, while the fungus provides nutrients, particularly diffusion-limited ones
such as Pi, Cu2+, and Zn2+ (Lynch and Ho 2005; Hamel 2004; Marschner 1995;
Barber 1995; Habte and Manjunath 1991). It is clear that AM fungi can only take up
soluble Pi from the same Pi-pool that is available for uptake by roots (Cardoso et al.
2006; Bolan 1991). In turn, roots can absorb available Pi from distances not
exceeding a few millimeters from their surface while mycorrhizal hyphae can
extend to several centimeters from the root surface, exploring a greater volume of
soil (Jeffries et al. 2003; Habte and Osorio 2001; Miyasaka and Habte 2001).
For example, 47 days after mycorrhizal inoculation of Trifolium subterraneum,
Jacobsen et al. (1992) found mycorrihzal hyphae spreading from the root surface
to 11 cm with the proportion between mycorrhizal hyphae and root length of
1–10 mcm1 of infected root while Barber (1995), however, reported a lower
value of 0.8 mcm1 of root.
Mycorrhizal hyphae have a higher affinity for absorbing Pi than roots. Schachtman
et al. (1998) reported that the hyphae of Gigaspora margarita had an affinity
constant for Pi (Km) of 2.5mM (P: 0.077 mg L1), while many plants usually
exhibited a Km of 6–44mM (P: 0.19–1.36 mg L1), particularly those highly
dependent on the mycorrhizal association (Barber 1995; Nye and Tinker 1977).
In addition, Barber (1995) affirmed that due to the small radius of the mycorrhizal
hypha (1–3mm) there is no Pi-depletion zone around the hypha which allows the
mycorrhizal hypha to take up Pi more effectively due to a higher and more constant
Pi concentration. In comparison, Li et al. (1991) found a very narrow Pi-depletion
zone around the mycorrhizal hypha. Roots with a greater radius (150mm), however,
generate a zone of depletion of at least 1 mm, resulting in low Pi concentration
around the root surface. Smith and Read (1997) reported P influx in mycorrhizal
roots of 3- to 5-fold higher than nonmycorrhizal roots (1011 mol m1 s1).
Plant species exhibit different degrees of mycorrhizal dependency (MD) to
produce maximum growth at a given level of soil fertility (Plenchette et al.
1983). Habte and Manjunath (1991) found that the MD of several plant species
was determined by root characteristics, such as root length, root density, root
surface area, and incidence and length of hair roots of the host species, which
help them to explore and absorb Pi from the soil solution. Since MD was signifi-
cantly affected by soil solution Pi-concentration, they proposed that MD should
be estimated at different levels of available Pi, particularly at the soil solution P
of 0.02 mg L1.
16.4 Phosphate-Solubilizing Microorganisms
Many soil microorganisms can solubilize inorganic soil P compounds, reversing the
process of Pi-fixation (Khan et al. 2007; Gyaneshwar et al. 2002; Rao 1992). Soil
bacteria of the genus Pseudomonas, Enterobacter, and Bacillus are particularly
active as Pi-solubilizers (Canbolat et al. 2006; Pandey et al. 2006; Xavier and
Germida 2003; Kim et al. 1998a, 1998b). Soil fungi especially those of the genus
Penicillium and Aspergillus have also been demonstrated to be effective phosphate-
solubilizing microorganisms (PSM) (Reddy et al. 2002; Whitelaw 2000). Kucey
et al. (1989) found in Mollisols of Canada that 0.5 and 0.1% of the total population
of bacteria and fungi, respectively, exhibited the ability to solubilize insoluble
Pi-compounds. Although Pi-solubilizing bacteria have received greater attention,
Whitelaw (2000) and Kucey (1983) indicated that Pi-solubilizing fungi are more
effective in solubilizing P compounds. Moreover, bacteria on repeated subculturing
can lose their ability to solubilize P, while subcultures of Pi-solubilizing fungi
maintain this ability (Rashid et al. 2004; Whitelaw 2000). Apparently, there are
some bacterial genes that could be repressed by high levels of Pi that control
the production of some organic acids (e.g., gluconic acid); however, in spite of
the progress made in the genetic control of Pi-solubilizing bacteria (Rodriguez et al.
2006), this is not completely understood and requires more study.
During the 1950s and 1960s, inoculation with Bacillus megaterium var.
phosphaticum (phosphobacterin) in Russian soils (mainly Mollisols) was the best-
known use of PSM (Kucey et al. 1989; Stevenson 1986). The mechanisms of
Pi-solubilization were not fully understood, but the mineralization of organic
P was proposed as the major mechanism. Trials carried out in many locations
demonstrated little consistency in plant response; apparently other factors such as
liming and/or organic material addition affected the effectiveness of phosphobac-
terin. The lack of response to phosphobacterin in many locations pointed to a
possible intensified organic matter decomposition, and the poor understanding of
the mechanisms of P solubilization carried out by this microorganism discouraged
its use. Since then, the focus on microbial solubilization of P has been directed
towards understanding the mechanisms of the dissolution of inorganic P compounds
(Kucey et al. 1989).
Inoculation with PSM has produced positive results on growth, yield, and
Pi-uptake in several plant species (Wani et al. 2007; Khan and Zaidi 2007; El-Azouni
Table 16.2 Effect of PSM inoculation on plant Pi-uptake of mycorrhiza-free and mycorrhized
plants grown in temperate soils
Soil type/plant P added PSM Increase of plant P Reference
uptake due to PSM
inoculation (%)
AMF + AMF
Mollisol pH 7.7 None Penicillium bilaji 73 Kucey
Plant: wheat RP 47 (1988)
Mollisol pH 8.0 None Penicillium bilaji 25 Asea et al.
(1988)
Mollisol pH >7.0 None Penicillum bilaji 62 Kucey et al.
Plant: wheat RP 36 (1989)
MAP 19
Calcareous RP Penicillium sp. 17 Salih et al.
TypicTorrifluvent RP Aspergillus foetidus 19 (1989)
pH 8.2 TSP Penicillium sp. 8
TSP Aspergillus foetidus 4
Mollisol pH 7.6 None Penicillium bilaji 39 Gleddie
TSP 18 (1993)
TSP 18
TSP 7
Hapludoll pH 6.2 None Aspergillus 58 (grain) Singh and
RP awamori 13 (grain) Singh
(1993)
Calcareous mixed RP Azospirillum 33 0–33 Toro et al.
with sand, Penicillium 33 0 (1996)
pH 6.7 Unidentified 33 22
Plant: kudzu Pseudomonas 33 33
Calcareous, None Aspergillus 30 39 Omar
pH 7.5 Penicillum 24 26 (1998)
Plant: wheat Aspergillus + 78 49
Penicillium
RP Aspergillus 116 24
Penicillum 118 11
Aspergillus + 151 57
Penicillium
Vertic Epiaqualf RP Enterobacter 54 (35 days)a 124 Kim et al.
mixed with sand agglomerans 27 (55 days) 27 (1998a)
and vermiculite, 8 (75 days) 11
pH 5.9
Plant: Tomato
Sandy soil pH 7.6 None Bacillus 26 52 Singh and
Plant: wheat Cladosporium 47 69 Kapoor
Bacillus + 73 98 (1999)
Cladosporium
RP Bacillus 248
Cladosporium 301
Bacillus + 51 344
Cladosporium
Rhodic Haplustox None Mortierella sp. 9 13 Osorio and
Plant: leucaena RP 14 73 Habte
(2001)
(continued)
Table 16.2 (continued)

Soil type/plant P added PSM Increase of plant P Reference
uptake due to PSM
inoculation (%)
AMF + AMF
Alluvial sandy Azotobacter 39 99.6 Khan and
loam pH 7.2 chroococcum Zaidi
Plant: wheat Bacillus sp. 77 120 (2007)
Penicillium variable 94
Typic Haplustox None Mortierella sp. 0 0 Osorio
Plant: leucaena RP b (150 mg P kg1) 0 40 (2008)
RP c (300 mg P kg1) 0 66
Typic Haplustoll None Mortierella sp. 155 26 Osorio
Plant: leucaena (2008)
a
Days after planting; b, c150 and 300 mg of P kg1 of soil, respectively
2008) (Table 16.2). Some effective Pi-solubilizing fungi that have shown a substantial
increase in plant growth and P uptake by different plants including Aspergillus
niger (Omar 1998), Aspergillus flavus (Rashid et al. 2004), Penicillium bilaji (Kucey
et al. 1989), Penicillium italicum (El-Azouni 2008), Penicillium radicum (Whitelaw
2000), and Mortierella sp. (Osorio 2008). Salih et al. (1989) inoculated a calcareous
soil (Typic Torrifluvent, pH 8.2) with two PSM, Penicillium sp. and Aspergillus sp.
They observed that, when the soil was fertilized with RP, the PSM inoculation
increased sorghum (Sorghum bicolor) Pi-uptake by 17 and 19%, respectively,
compared to the sole application of RP. Also, Kucey (1988) inoculated a Mollisol
(pH 7.7) of Canada with P. bilaji, the soil was either fertilized or unfertilized with RP
(Table 16.2). Wheat (Triticum aestivum) Pi-uptake increased only by 4% with RP
alone, 14% with the PSM inoculation alone, and 12% with a combination of RP and
P. bilaji. In a similar experiment on a Mollisol (pH 8), Asea et al. (1988) found that
the addition of RP increased wheat plant Pi-uptake by only 2%; P. bilaji inoculation
alone significantly increased it by 26%, and the combination of both (RP and
P. bilaji) by 28% (Table 16.2).
Several authors have reported that soil microorganisms can increase soil
P availability (Table 16.3). Although in some soils this increase does not have-
practical implications, it has been important in other soils (Marschner et al.
2006). For instance, the increases in soil P availability reported by Goenadi
(1995) and Goenadi et al. (1995) in two acidic Ultisols fertilized with RP
were significant. The effectiveness of PSM to enhance plant Pi-uptake has been
questioned by some authors (Bolan 1991, Tinker 1980) due to several reasons:
(1) organic substances required for these microorganisms are scarce in nonrhizo-
spheric sites, (2) antagonism and competition by other microorganisms in the
rhizosphere can reduce the effectiveness of PSM, and (3) low translocation of
solubilized Pi through the soil because it can be refixed by soil components. Of
these, the latter point is more important in soils with a high Pi-fixation capacity as
discussed earlier.
Table 16.3 Enhancement of the soil Pi-available by phosphate-solubilizing microorganisms

PSM Soil P source SAP increase Reference
(mg kg1)
Aspergillus Fluvaquent, pH 5.4 Nil P fertilizer 2 Banik and Dey
niger SAP: 9 mg kg1 (1981b)
A. fumigates Fluvaquent, pH 7.4 RP and farmyard 3 Banik and Dey
SAP: 7 mg kg1 manure (1982)
Penicillium Mollisol, pH 7.7 RP 2 Kucey (1988)
bilaji SAP: 4 mg kg1
Penicillium Torrifluvent, pH 8.2 RP 2 Salih et al. (1989)
sp. SAP: 4 mg kg1 TSP 9
A. foetidus RP 1
TSP 5
A. awamori Hapludoll, pH 6.2 Nil P fertilizer 8 Singh and Singh
SAP: RP 3 (1993)
27 mg kg1
Aspergillus Ultisol, pH 3.9 RP 24 Goenadi et al.
sp. SAP: (1995)
37 mg kg1
Aspergillus Ultisol SAP: Nil P fertilizer 14 Goenadi (1995)
sp. 0.5 mg kg1 RP 38
Source: Whitelaw (2000). SAP Soil available P, TSP triple superphosphate
16.4.1 Mechanisms of Microbial Pi-Solubilization
Several mechanisms have been proposed to explain the microbial solubilization of

P compounds. The mechanisms include: -(1) release of organic acids produced
during organic residue decomposition (Hameeda et al. 2006; Bar-Yosef et al. 1999),
(2) excretion of protons due to NH4+ assimilation by microorganisms (Whitelaw
2000; Illmer and Schinner 1995; Abd-Alla 1994; Asea et al. 1988; Roos and
Luckner 1984; Kucey 1983), (3) formation of complexes between organic acids/
anions with cations (Al3+, Fe3+, Ca2+) (Welch et al. 2002), and (4) desorption of
Pi-sorbed onto soil clay and/or oxides (Osorio 2008). Nitric and sulfuric acid produced
by Nitrosomonas and Thiobacillus species, respectively, have also been reported to
dissolve Pi-compounds (Azam and Memon 1996). Equally, P compounds may
be solubilized by carbonic acid formed as a result of organic matter decomposition
(Memon 1996). An increase in soil P availability may be caused by several
reactions involving microorganisms that produce organic acids and humic substances
(Stevenson 1986). Presumably, these substances can replace or compete with Pi-
ions for sorption sites.
Kim et al. (1997) found that the production of acidity was a major mechanism
in the solubilization of hydroxyapatite by Enterobacter agglomerans under
in vitro conditions. For comparison, Kim et al. (1997) employed citric acid, oxalic
acid, lactic acid, and HCl at the same pH produced by E. agglomerans. They
found that at pH 4.0–4.1 (and a shaking time of 48–50 h), there were no significant
differences among P solubilization produced by this bacterium and that produced
by the application of citric acid, oxalic, and HCl. However, lactic acid exhibited a
significantly (P0.05) lower capacity for solubilizing hydroxyapatite. IIlmer et al.

(1995) reached the same conclusion while studying AlPO4 solubilization by several
PSM. For instance, A. niger produced organic acids but other PSM species did not
produce detectable amounts of the organic acids. Under in vitro conditions, the pH
of the growth medium decreased as a result of acid production by PSM. Osorio and
Habte (2001) found an inverse relation between culture medium pH and Pi released
from RP by PSM (Mortierella sp.) isolated from Hawaiian soils.
Some of the organic acids (or their respective anions) commonly associated with
microbial solubilization of Pi are gluconic acid (Bar-Yosef et al. 1999; Di-Simine
et al. 1998), oxalic acid, citric acid (Kim et al. 1997, Kucey et al. 1989), lactic acid,
tartaric acid, and aspartic acid (Venkateswarlu et al. 1984). These acids are products
of microbial metabolism, in some cases by oxidative respiration or by fermentation
of carbonaceous substrates (e.g., glucose) (Trolove et al. 2003; Jones et al. 2003;
Gyaneshwar et al. 2002; Prescott et al. 1999; Atlas and Bartha 1997). The reactions
of P solubilization are believed to occur in the rhizosphere where carbonaceous
compounds are released and where solubilized Pi may be taken up by the root or
mycorrhizal system. Amos and Walters (2006) estimated that for maize (Zea mays)
up to 29% of the total carbon fixed by photosynthesis could be excreted into the
rhizosphere. According to Nguyen (2003), on average, 17% of the net C fixed by
photosynthesis is lost by roots and recovered as rhizosphere respiration (12%) and
soil residues (5%). Many rhizosphere microorganisms are heterotrophs and might
use these carbonaceous substrates to produce organic acids. Recently, Hameeda
et al. (2006) found that the type of carbon source affected the effectiveness of RP-
solubilizing bacteria and, for Serratia marcescens and Pseudomonas sp., the more
favorable carbon source for RP solubilization followed the order, glucose > galac-
tose > xylose > mannose¼maltose > cellobiose > arabinose. However, no solubili-
zation of RP was detected with the last carbon source of this series. In addition,
Serratia marcescens and Pseudomonas sp. were capable of solubilizing RP using
different kinds of composted crop residues including rice (Oryza sativa), pigeon
pea (Cajanus cajans), and a grass. Furthermore, Reyes et al. (2006) also compared
the effect of the carbon source on RP solubilization and found that Penicillium sp.
and Azotobacter sp. were more effective when the medium contained sucrose rather
than dextrose. When PSM were inoculated in neutral or alkaline soils, the produc-
tion of acids decreased rhizosphere pH, favoring the solubility of soil native
calcium-phosphate and added RP (Kim et al. 1998a). These results have commonly
been found in temperate-zone soils of Europe and North America (Kucey et al.
1989; Kucey 1983, 1987, 1988) and other countries, like Egypt (Omar 1998) where
calcareous soils are abundant. The following reactions (1)–(3) suggest that the
increase in H+ activity leads to a substantial increase in solubilization of calcium-
phosphates. Moreover, if Ca2+ is chelated by organic anions, the dissolution of both
solids is favored. For example, Welch et al. (2002) found that organic acid/anions
produced by microorganisms were capable of dissolving apatite by forming
a complex with Ca either in solution and/or directly at the mineral surface.
ðDicalcium phosphateÞ CaHPO4 þ Hþ $ H2 PO4 þ Ca2þ K ¼ 100:3 ; ð1Þ

ðHydroxyapatiteÞ Ca5 ðPO4 Þ3 OH þ 7Hþ $ 3H2 PO4 2 þ 5Ca2þ þ H2 O

K ¼ 1014:5 ; ð2Þ
ðFluoroapatiteÞ Ca5 ðPO4 Þ3 F þ 6Hþ $ 3H2 PO4 2 þ 5Ca2þ þ F K ¼ 100:2 : ð3Þ
On the other hand, in highly weathered acidic soils, P solubility is controlled

by other compounds, mainly variscite (AlPO4.2H2O) and strengite (FePO4 2H2O)
(Bohn et al. 1985). In this case, the decrease in soil pH may not increase the
dissolution of strengite and variscite (Lindsay 2001) and, hence, Pi is not released.
This happens because gibbsite [Al (OH)3] and kaolinite [(Al2Si2O5OH)4] can
control the solubility of Al in these soils, while goethite (FeOOH), hematite
(Fe2O3) and soil-Fe(OH)3 control the solubility of Fe. Reductions in the soil
pH would release more Al and Fe ions, which would precipitate Pi. Thus, if soil
pH decreases, Pi-solubility is likely to decrease, as shown in reactions (4)–(8)
(Lindsay 2001).
AlPO4 2H2 O þ H2 O $ H2 PO4 þ AlðOHÞ3 þ Hþ K ¼ 1010:5 ; ð4Þ
AlPO4 2H2 O þ SiO2 ðquartzÞ þ 0:5H2 O $ H2 PO4 þ 0:5Al2 Si2 O5 ðOHÞ4 þ Hþ

K ¼ 109:2 ; ð5Þ
FePO4 2H2 O þ H2 O $ H2 PO4 þ FeðOHÞ3 þ Hþ K ¼ 109:6 ; ð6Þ
FePO4 2H2 O $ H2 PO4 þ FeOOH þ Hþ K ¼ 106:8 ; ð7Þ
FePO4 2H2 O $ H2 PO4 þ 0:5H2 O þ 0:5Fe2 O3 þ Hþ K ¼ 106:9 : ð8Þ
The microbial solubilization of soil P seems to be associated with the presence of

calcium-phosphates. In fact, most of the research on microbial solubilization has
been done with solubilizers of RP (mixture of hydroxy- and fluor-apatites) (Osorio
and Habte 2001; Kim et al. 1998b) or tricalcium phosphate (Ca3PO4) (Vyas et al.
2007; Wani et al. 2007; Banik and Dey 1981a, 1981b, 1981c; Paul and Rao 1971;
Agnihorti 1970; Louw and Webley 1959; Sperber 1957, 1958; Pikovskaia 1948).
However, researchers on PSM no longer accept isolation of PSM using culture
medium with Ca3PO4 because it can supply free Pi. Some authors reported that
in vitro microbial solubilization not only occurred with calcium-phosphate but also
with Al and Fe phosphates. However, the solubilization was higher with calcium
phosphates (IIlmer et al. 1995; Banik and Dey 1983; Rose 1957).
Solubilization of Al and Fe phosphates could be easily observed under in vitro
conditions where no soil minerals interfere with Pi-solubility (9) and (10). IIlmer
et al. (1995) found that A. niger, Penicillium simplicissium, Pseudomonas auran-

tiogriseum, and Pseudomonas sp. were effective in solubilizing AlPO4 under
in vitro conditions via organic acid production or proton excretion due to NH4+
assimilation. Aluminum and Fe ions in solution may be chelated by organic anions,
such as oxalate and citrate (Bolan et al. 1994), favoring the dissolution of Al and Fe
phosphates. However, the role of organic acids released by PSM in solubilizing Pi
from Al- and Fe-phosphate under acidic soil conditions must be thoroughly inves-
tigated.
AlPO4 2H2 O þ 2Hþ $ Al3þ þ H2 PO4 þ 2H2 O K ¼ 102:5 ; ð9Þ
FePO4 2H2 O þ 2Hþ $ Fe3þ þ H2 PO4 þ 2H2 O K ¼ 106:8 : ð10Þ
On the other hand, organic anions produced by PSM can also compete with
phosphate for Pi-sorption sites onto the surface of soil minerals. In this regard, He
and Zhu (1997, 1998) suggested that Pi-sorbed onto the surfaces of some minerals
was displaced when a culture medium was inoculated with soil samples containing
microorganisms (unidentified) that presumably excreted organic acids. Similarly,
Osorio (2008) found that Mortierella sp., a PSF, was capable of desorbing
Pi-sorbed from soil by releasing oxalic acid. The effectiveness of this fungus to
desorb Pi was controlled by the type of soil and followed the order: mollisol >
oxisol > ultisol > andisol. Moreover, the effectiveness of Mortierella sp. to desorb
Pi-sorbed from soil minerals decreased in the order: montmorillonite > kaolinite >
goethite > allophane.
16.4.2 Role of Organic Acids in Pi-Solubilization
Many organic acids have been found effective in solubilizing soil P compounds
(Hue 1991) and other soil minerals (Calvaruso et al. 2006; Welch et al. 2002).
These acids or anions are produced by roots (Corrales et al. 2007; Radersma and
Grierson 2004; Kirk et al. 1999; Marschner 1995) and by microbial activity during
decomposition of organic matter or induced by Pi-deficiency stress (Jones et al.
2003; Fransson et al. 2004; Bohn et al. 1985). In accordance with these, Bolan et al.
(1994) found organic acids or anions in high concentrations in poultry manure,
lesser amounts in the rhizosphere soil, very little in the bulk soil, and trace amounts
in leaf litter. Also, Le Bayon et al. (2006) reported a relatively higher production of
organic anions (citrate, fumarate, and malate) in the rhizosphere of Lupinus albus
under Pi-starvation. Furthermore, to substantiate the role of such organic anions in
P solubilization, Bolan et al. (1994) assessed the influence of monocarboxylic
(acetic, formic, and lactic), dicarboxylic (malic, tartaric, and oxalic), and tricarbox-
ylic (citric) acids/anions in the solubilization and sorption of Pi on an andisol
(Hydric Dystrandept) and an alfisol (Typic Fragiaqualf) of New Zealand. The
Table 16.4 Stability constant of organic anions with aluminum (Log KAl) and calcium (Log KCa)
and their effects on soil Pi-sorption and solubilization of two Pi-fertilizers
Organic acid Log KAla Log KCab Sorbed P Dissolved Dissolved
(mmol kg1 soil) MCPe (%) NCPR f (%)
Water (control) – – 52 2.35 1.28
Formic 1.36 1.43b 47 12.27 11.86
Acetic 1.60 1.18b 45 10.35 12.57
Lactic 2.41 1.63c 46 8.71 13.92
Malic 5.40 2.25c 37 32.65 31.98
Tartaric 5.62 2.80b 35 30.49 32.31
Oxalic 6.16 3.44d 32 36.44 34.02
Citric 7.98 4.87b 18 83.78 86.41
Source: Bolan et al. (1994)
a
Hue et al. (1986); bMINTEQA2 (a model for equilibrium speciacion in geochemical enviroments,
accesed at www.epa.gov on March 15, 2008); cBazin et al. (1995); dFinlayson et al. (1972);
e
Monohydrogen calcium phosphate; fNCRP North Carolina rock phosphate
addition of these organic acids significantly decreased Pi-sorption on allophane

surface. The effectiveness of such acids followed the order: tricarboxylic > dicarbox-
ylic > monocarboxylic. This was explained by the formation of complexes between
Al with the conjugated anion of each organic acid and by their respective stability
constant (Log KAl) (Hue et al. 1986), as depicted in Table 16.4. The addition of
organic acids or anions also favored the solubilization of North Carolina rock phos-
phate (NCRP) and monohydrogen calcium phosphate (MCP), increased dry matter
yield of ryegrass (Lolium rigidium), and enhanced plant Pi-uptake (Bolan et al. 1994).
Hue (1991) obtained similar results on the availability of soil Pi when organic
acids/anions were added to two andisols, an oxisol, an ultisol, and a vertisol of
Hawaii. The effectiveness of the acids to reduce Pi-sorption from a soluble source
(KH2PO4) was higher with malic acid (monohydroxy dicarboxylic), followed by
protocatechuic acid (dihydroxy monocarboxylic), and acetic acid (monocarboxylic).
The effect was higher when the acid was applied first and Pi last. The dry weight of
lettuce (Lactuca sativa) was significantly higher when the soils received organic
acids or anions plus Pi compared to those that received only Pi. The magnitude
of this effect was much higher in the two andisols and in the oxisol whereby plant dry
weight was 5- to 15-fold higher than control plants, whereas in the vertisol the
increase was only 1.3- to 1.72-fold. Such variations in effects were suggested to
be due to differences in clay mineralogy that determines the soil Pi-sorption capacity.
From this study, it was concluded that the efficiency of Pi-fertilizers might
be enhanced if they are added with organic acids or anions or, more practically,
with green manures or animal wastes. Results of recent studies have also shown that
RP could be more effective if it is applied with manures, composts, and crop litters
(Reddy 2007; Bah et al. 2006; Singh et al. 2006).
The phenomenon of Pi-desorption by organic anions is widely accepted by soil
scientists. Recently, Sato and Comerford (2006) used organic anions to model the
Pi-desorption from a Brazilian soil. They concluded that the organic anions can
increase soluble Pi by two process: (1) the desorption of P sorbed onto soil surface
(ligand exchange), and (2) the dissolution of soil P compounds (ligand dissolution)
(e.g., calcium phosphates). Since PSM can release the same organic acids as
reported by Hue (1991) and Bolan et al. (1994), these microorganisms can presum-
ably reduce the activity of Al ions in the rhizosphere, decrease Pi-sorption, and
enhance plant Pi-uptake. Miyasaka et al. (1991) found that the Al tolerance of
plants is associated with the ability of roots to release organic acids/anions (citrate
and oxalate, in particular) into the rhizosphere. Since some soil microorganisms are
able to produce organic acids, it is possible that such microorganisms could help
plants to grow in soils with toxic levels of Al3+. In a series of experiments,
De la Fuente and Herrera (1999) isolated a gene that encodes for citrate synthetase
overproduction in the TCA cycle of a phosphate solubilizing strain of Pseudomonas
aeruginosa. This gene was then transferred to tobacco (Nicotiana tabacum) cells of
Al-intolerant plants. Transgenic plants were able to produce high amounts of citric
acid and citrate, its conjugated anion, and grew in solutions with high concentration
of Al. The process was successfully replicated with papaya (Carica papaya) plants.
Although these experiments were aimed at enhancing Al tolerance of these plants,
they also tested mechanisms proposed for the microbial solubilization of soil Pi.
However, Delhaize et al. (2001) reported that they were not able to repeat the
results obtained by De la Fuente and Herrera (1999) and, hence, suggested that the
expression of P. aeruginosa genes are either sensitive to environmental conditions
or that the observed Al tolerance and improved Pi-nutrition were due to other
factors. Generally, the roles of organic acid/anion production in the rhizosphere
on Pi-desorption and Pi-solubilization has been accepted. However, experimental
results indicate that its efficiency in increasing plant Pi-uptake depends on plant
species, age, physiological state, and soil mineralogy (Trolove et al. 2003). The
concentration of these organic compounds is relatively low in most soils because
they can be precipitated with free ions (e.g., Al3+, Fe3+, Ca2+) or sorbed on soil clay
reactive surfaces. Also, their persistence in rhizosphere soil is very low because
they can be used as carbon sources by soil microorganisms (Jones et al. 2003).
Thus, competent rhizosphere microorganisms capable of producing organic acids
or anions can play an important role in the management of Pi-deficient and high
Pi-sorbing soils.
16.5 Interactive Effect of Arbuscular Mycorrhizal Fungi and

Phosphate-Solubilizing Microbes on Plant Pi-Uptake
The dual inoculation of PSM and AM fungi may overcome the limitations on the
effectiveness of PSM to enhance plant Pi-uptake in soils with high Pi-fixation
capacity. During this interaction, mycorrhizal plants release higher amounts of
carbonaceous substances into the rhizosphere (Rambelli 1973; Linderman 1988)
which is used as a carbon source by PSM (Azcon and Barea 1996). Moreover, the
extensive mycorrhizal hyphae network formed around roots can efficiently take up
Pi released by PSM, thus minimizing its refixation. As long as PSM grow in the
rhizosphere (or mycorrhizosphere), there is a great opportunity to satisfy their
Table 16.5 Effects of E. agglomerans (PSM) and G. etunicatum (AMF) inoculation on growth
and Pi-uptake in tomato plants 75 days after inoculation
Treatment Shoot dry weight Root dry weight Shoot P content
(g per plant) (g per plant) (mg per plant)
Control 42.2 (100)a 4.3 (100) 116.6 (100)
PSM 48.5 (115) 5.1 (118) 125.3 (107)
AMF 47.6 (113) 5.6 (130) 120.9 (104)
PSM+AMF 54.6 (129) 6.8 (158) 134.4 (115)
LSD (P 0.05) 1.96 0.5 9.8
Source: Kim et al. (1998a)
a
Values in parenthesis indicates percentage increase over control.
carbon requirement and deliver Pi into the soil solution. Synergistic effects have
been found in sunflower (Helianthus annus) with the triple inoculation of two PSM
(Azotobacter chroococcum and Penicillium glaucum) and the AM fungus
G. fasciculatum (Gururaj and Mallikarjunaiah 1995). Similar effects were found
in cotton (Gossypium hirsutum L.) with the inoculation of Pseudomonas striata and
Azospirillum sp. (PSM) and the arbuscular mycorrhizal fungi (AMF) G. fascicula-
tum (Prathibha et al. 1995). In rice (O. sativa), favorable effects were also reported
with P. striata (PSM) and Bacillus polymyxa (PSM) and the AM fungus
G. fasciculatum (Mohod et al. 1991). In chili (Capsicum annuum), synergistic
effects werre reported with two AMF, G. fasciculatum or G. macrocarpum, and a
PSM P. striata (Sreenivasa and Krishnaraj 1992). In tomato (Lycopersicon
lycopersicum), beneficial results were found with E. agglomerans and G. etunicatum
(Kim et al. 1998a) (Table 16.5). Moreover, positive results have been ob-
tained in wheat with multiple combinations that included P. striata (PSM) and
G. fasciculatum (AMF), P. putida, P. aeruginosa and P. fluorescens (PSM) with
G. clarum (AMF), P. striata and Agrobacterium radiobacter (PSM) combined
with two AMF, G. fasciculatum and G. margarita (Gaur et al. 1990).
Kopler et al. (1988) indicated that more legume nodulation was obtained with
concurrent inoculation of Rhizobium and Pseudomonas spp. (PSM). Sturz et al.
(1997) found that nodulation by Rhizobium leguminosarum b.v. trifolii in red clover
(Trifolium pratense) was promoted when it was coinoculated with the PSM Bacillus
insolitus, B. brevis or Agrobacterium rhizogenes. Similar results were obtained with
the inoculation of G. mosseae (AMF) and Azorhizobium caulinodans (PSM) for
Sesbania rostrata (Rahman and Parsons 1997). In soybean (Glycine max), the
combination of Bradyrhizobium japonicum (N2 fixer) with P. fluorescens (PSM)
and G. mosseae (AMF) showed equally good results (Shabayey et al. 1996). Lately,
El-Azouni (2008) also found that the dual inoculation of A. niger and P. italicum
significantly increased the plant dry weight and yield of soybean. Such results are
likely to be due to a higher plant Pi-uptake promoted by the combined action of
PSM and AMF, which may satisfy the high Pi requirements of the N2-fixing process
(Azcon and Barea 1996; Young et al. 1990). More recently, Khan and Zaidi (2007)
found synergistic effects with the triple inoculation of two plant growth-promoting
rhizobacteria A. chroococcum and Bacillus sp. and Glomus fasciculatrum on plant
growth, yield and nutrient uptake of wheat plants uinder field conditions.
Peix et al. (2001) found that the N2-fixing bacterium Mesorhizobium

mediterraneum was able to solubilize Ca3(PO4)2 under in vitro and soil conditions.
Inoculation with M. mediterraneum of seeds of chickpea (Cicer arietinum) and
barley (Hordeum vulgare) planted in a Calcic Rhodoxeralf significantly increased
plant growth and total N- and Pi-content in both plants, which were increased even
further when M. mediterraneum and Ca3PO4 were applied simultaneously. Such
enhancement in the overall performance of the tested crops was not only due to the
symbiotic N2 fixation but also to the availability of soluble P to the plants.
Apparently, there is a certain degree of specificity among PSM, AMF, and P source.
Toro et al. (1996) studied the combined effect of AMF (Glomus spp.) and eight
PSM (bacteria) on plant growth and Pi-nutrition of a tropical legume, kudzu
(Pueraria phaseoloides). The PSM were isolated from an oxisol and were char-
acterized by their ability to solubilize RP, Al- and Fe-P compounds. In general, the
combined inoculation of PSM, Rhizobium and AMF increased growth, yield, and
nutritional properties of kudzu plants. However, such response was not observed for
all combinations of AMF and PSM. For instance, the three PSM namely,
Azospirillum sp., Bacillus sp., and Enterobacter sp., had a greater effect when they
were coinoculated with G. mosseae. In contrast, Pseudomonas sp. and an unidenti-
fied isolate demonstrated a better performance when they were combined with
G. fasciculatum. On the other hand, Fe-P solubilizers were more effective if they
were used alone, while Al-P and RP solubilizers performed better when they were
concurrently inoculated with AMF. Reasons for these differences may be due to
the interactions found between the microorganisms. For instance, some of these
PSM could be more effective in stimulating a rapid mycorrhizal colonization,
and enhancing the length, distribution, and/or survival of external fungal myce-
lium. Mycorrhizal fungi might differ in the amount and type of hyphal exudates
released into the mycorrhizosphere. In addition, a high capacity to solubilize
Pi might stimulate plant growth and favor mycorrhizal activity. Kucey (1987)
inoculated a mollisol (pH 7.2) of Canada with P. bilaji, in which either
mycorrhizal or nonmycorrhizal wheat or beans were grown. In the case of
wheat, mycorrhizal inoculation alone increased Pi-uptake significantly by 30%,
while P. bilaji alone did not do so. However, P. bilaji increased Pi-uptake of
mycorrhizal wheat by 10% in the unfertilized soil, but not in the soil fertilized
with RP. In the case of bean, mycorrhizal inoculation alone did not increase
plant Pi-uptake, but P. bilaji alone was able to increase it significantly by 31%.
Dual inoculation, however, did not increase Pi-uptake beyond the level obtained
with P. bilaji. In other words, no synergism was established between the
inoculated microorganisms and, hence, Pi-uptake by bean plants grown in
unfertilized and RP-fertilized soil did not increase. Mollisols usually exhibit a
low Pi-fixation capacity. For this reason, it is not surprising that inoculation with
this PSM alone increased bean Pi-uptake. However, the differences in the effect
on wheat and bean plants could also be due to the release of different types
and amounts of root exudates that otherwise stimulate acid production in the
rhizosphere by PSM. The interactive effect of both PSM and AMF on plant
P-uptake is explained in Fig. 16.1 (Osorio 2008). Once the plant releases
Fig. 16.1 Diagram showing the microbial solubilization of soil and added P by Mortierella sp. and
the mycorrhizal Pi-uptake (Osorio, original drawing)
organic compounds into its rhizosphere, the PSM produces organic acids that
dissolve insoluble calcium phosphate compounds (added o native). Also, such
organic acids can desorb Pi sorbed from soil minerals, which is controlled by
the soil Pi-sorbing capacity. The Pi-release can be efficiently taken up by the
mycorrhizal hyphae favoring in this way the plant growth.
16.6 Microbial Pi-Solubilization in Temperate

and Tropical Soils
Currently, P. bilaji is commercially available in North America under the name of

ProvideTM, which has been successfully tested to enhance plant Pi-uptake of some
crop plants (Whitelaw 2000). However, the tests have been conducted in mollisols
of low Pi-fixation capacity. In contrast, little research on PSM has been conducted
in highly weathered and volcanic ash soils of the tropics with high Pi-fixation
capacity. Toro et al. (1996) isolated various effective PSM from an oxisol of
Venezuela. However, they were not tested in this soil from which they were
originated but in a calcareous soil of Spain in which they were able to improve
plant growth and Pi-uptake of tropical kudzu (Pueraria phaseoloides). Whitelaw
et al. (1997) inoculated an acidic Pi-deficient soil of Australia (pH 4.6) with
P. radicum in combination with several levels of KH2PO4 (P: 0–20 kg ha1). The
inoculation with this PSF increased wheat Pi-uptake by 8% in the unfertilized soil.
When the fungus was coinoculated with Pi-fertilizer, plant Pi increased between
2 and 28%; increase was highest when the rate of added P was 15 kg ha1. Young
Table 16.6 Effect of AM fungus, phosphate-solubilizing microbes and rock phosphate on peanut
yield (kg ha1) in two soils of Taiwan
Treatment Hualain soil (pH 4.2) Yuanchang soil (pH 5.4)
Control 1,875 b (100) 3,667 c (100)
RP (660 kg ha1) 2,250 a (120) 6,167 a (169)
AMF 2,350 a (125) 6,208 a (140)
PSM 2,259 a (120) 6,333 a (173)
AMFþRP 2,367 a (126) 5,125 b (140)
PSMþRP 2,275 a (121) 6,083 ab (166)
Letters indicate mean separation by Duncan’s multiple range test (P 0.05). Values in parenthesis
indicate percentage increase over control.
Source: Young et al. (1990)
Table 16.7 Effect of AM fungus and phosphate solubilizing microbes on growth (g per pot) of
leucaena grown in three soils of Taiwan
Treatment Hinshe (pH 5.0; Wunfun (pH 5.5; Taitung (pH 7.8;
P: 2 mg kg1) P: 3 mg kg1) P: 95 mg kg1)
Control 8.4 c (100) 13.7 b (100) 22.0 b (100)
PSM 10.4 b (124) 12.9 b (94) 30.8 a (140)
AMF 13.1 a (156) 27.3 a (199) 26.8 b (122)
PSMþAMF 12.8 a (152) 26.0 a (190) 23.6 b (107)
Letters indicate mean separation by Duncan’s multiple range test (P 0.05). Values in parentheses
indicate percentage increase over control.
Source: Young et al. (1990)
et al. (1990) found that inoculation with either PSM or AMF significantly increased
peanut (Arachis hypogea) production in two subtropical–tropical acidic soils of
Taiwan. Inoculation with either AMF or PSM in unfertilized soils was as effective
as the addition of RP alone (Table 16.6). Inoculation with AMF or PSM of RP-
fertilized soils did not increase peanut yield above that obtained with AMF or PSM
inoculation in unfertilized soils. Unfortunately, dual inoculation of AMF and PSM
was not evaluated. Interestingly, PSM inoculation alone increased peanut yield by
73% in the less acidic soil (Yuanchang soil), but the increase was only 20% in the
strongly acidic soil (Hualain soil). In addition, Young et al. (1990) found that
the responses to single or mixed inoculations with PSM and/or AMF had variable
effects on plant growth of Leucaena grown in three soils of Taiwan. Inoculation
with PSM was not as effective as AMF inoculation in enhancing plant growth in the
soil with the lowest available Pi-level (Hinshe soil) (Table 16.7). In the Wunfun soil
(also with a low soil available Pi-level), PSM inoculation was ineffective in
increasing plant growth unlike AMF. In the alkaline soil containing the highest
soil available Pi (presumably rich in calcium-phosphates), PSM inoculation alone
significantly increased plant growth (40%) above the AMF inoculation effect,
which did not increase growth.
Effectiveness of PSM inoculation alone to enhance plant Pi-uptake in subtropi-
cal and tropical acidic soils is relatively low and variable. The increases recorded
were 8% (Whitelaw et al. 1997), 13% (Osorio and Habte 2001), and 24–25%
(Young et al. 1990) compared with those reported in less weathered soils (mostly
Mollisols) of the temperate zone, where soil Pi-fixation capacity is low. By contrast,
effectiveness of PSM inoculation to enhance plant Pi-uptake of mycorrhizal plants
grown in tropical or subtropical soils can be relatively higher compared to data
reported in temperate soils (Table 16.7). Mycorrhizal colonization in combination
with PSM is often needed to obtain improvements in plant P uptake in highly
weathered soils, in contrast to results obtained in less weathered soils. In these less
weathered soils that normally exhibit low soil P sorption, the inoculation of PSM
alone has been found effective to increase plant P-uptake of nonmycorrhizal plants
(Peix et al. 2001; Omar 1998; Kucey 1983, 1987, 1988; Asea et al. 1988; Kucey
et al. 1989; Gleddie 1993). Most of the soils used by these authors were mollisols,
calcareous soils, or sandy soils, which are characterized by a low P-sorption
capacity and relatively high soil Ca–Pi content (Cross and Schlesinger 1995).
Therefore, the freshly released Pi by PSM can remain longer in the soil solution
until its absorption by the roots. For instance, Toro et al. (1998) found that the PSM
Enterobacter sp. alone was as effective as the mycorrhizal fungus G. mosseae when
used alone, and increased the P uptake of alfalfa grown in a calcareous soil of Spain
by twofold. Duponnois et al. (2006) found that single inoculation of fungus
Arthrobotrys oligospora increased the P uptake and shoot dry weight of Acacia
holoserica grown in a sandy soil of Senegal by 56 and 46%, respectively. The
increase in plant P uptake and growth were even higher when RP was added with
the PSM (74 and 103%, respectively). The application of RP alone, however, did
not significantly increase growth and plant P uptake. Similar results were observed
by Wakelin et al. (2004a, 2004b) for Penicillum radicum- inoculated wheat grown
in sandy soils in Australia with neutral to alkaline soil reactivity. In these soils,
Wakelin and coworkers observed increases in plant growth between 34 and 76%.
Furthermore, Penicillium thomii has shown a threefold higher increase in plant
P uptake of mint (Mentha piperita), grown in a soil-less medium (vermiculite–
perlite) fertilized with RP compared to those observed for uninoculated plants and
unfertilized control (Cabello et al. 2005). The RP alone was, however, ineffective.
The impressive increase in plant P uptake is understandable given the very low
P sorption on this kind of substrates.
The results obtained recently by Osorio (2008) indicate that the effectiveness
of Mortierella sp., a phosphate-solubilizing fungus (PSF), in enhancing plant
Pi-uptake and growth was controlled by the type of soil, particularly by the
Pi-sorption capacity of the soil (Table 16.8). In a mollisol (low Pi-sorption
capacity) Mortierella sp. alone was capable of increasing shoot dry weight of
Leucaena. However, the effect was significantly higher in the presence of the
mycorrhizal fungus G. fasciculatum. On the other hand, in two oxisols (medium
Pi-sorption capacity), the PSF stimulated dry matter accumulation in shoots only
in the presence of the mycorrhizal fungus. In contrast, the PSF was ineffective
in increasing plant Pi-uptake in an andisol (very high P sorption capacity) even
in the presence of the mycorrhizal association. The P0.2 value (Tables 16.1 and
16.8) seems to be a good predictor of the effectiveness of PSM to increase
plant Pi-uptake via Pi-desorption, RP solubilization, and Pi-uptake by mycorrhi-
zal roots.
Table 16.8 Effect of AM fungus (AMF) and phosphate- solubilizing fungus (PSF) on growth
(g per pot) of leucaena grown in three tropical soils of Colombia
Treatment Mollisol Oxisol Andisol
(P0.2 ¼ 45 mg kg1) (P0.2 ¼ 417 mg kg1) (P0.2 ¼ 2,222 mg kg1)
Control 0.73 0.32 0.26
PSF 1.18 0.30 0.28
AMF 1.36 0.84 0.28
PSF + AMF 1.48 0.97 0.26
LSD 0.09 0.12 0.09
Vertical comparisons (LSD test, P 0.05).
Adapted from Osorio (2008)
16.7 Conclusion
One of the major limiting factors for plant productivity in the tropics is low soil
Pi-availability. Phosphorus is rendered unavailable for plant uptake due to adsorp-
tion onto the surface of soil minerals and precipitation by free Al3+ and Fe3+ ions.
As a result, the efficiency of Pi-fertilization becomes quite low and hence, require
application of high rates of soluble P fertilizer. A viable alternative to overcome
dependance on chemical phosphatic fertilizer is the use of phosphate rocks (RP),
which are locally available and are cheaper. The use of rhizosphere PSM has
received greater attention to increase the agronomic effectiveness of RP. The
most accepted mechanism is the production and release of organic acids (e.g., citric
acid, oxalic acid, gluconic acid, among others). The released protons decrease the
rhizosphere pH, while the organic anions can form complex with calcium favoring
thus the dissolution of RP. In addition, it has been demonstrated that organic anions
produced by PSM can also desorb Pi from soil minerals. However, given the high
Pi-sorption capacity in highly weathered soils of the tropics the effectiveness of
PSM to increase plant Pi-uptake and growth can be low. Synergistic effects of PSM
and AMF on plant nutrition, growth, and yields have been reported. This dual
inoculation seems to be more relevant in the case of soil exhibiting high Pi-sorption
capacity (e.g., oxisols, ultisols, andisols) that are abundant in the tropics. Further
studies are, however, required in tropical soils in order to establish an effective use
of PSM inoculants in a more predictable and sustainable manner.
References
Abd-Alla MH (1994) Use of organic phosphorus by Rhizobium leguminosarum biovar. viceae

phosphatases. Biol Fertil Soils 18:216–218
Agnihorti VP (1970) Solubilization of insoluble phosphates by some soil fungi isolated from
nursery seedbeds. Can J Microbiol 16:877–880
Amos B, Walters DT (2006) Maize root biomass and net rhizodeposited carbon: an analysis of the
literature. Soil Sci Soc Am J 70:1489–1503
Asea PEA, Kucey RMN, Stewart JWB (1988) Inorganic phosphate solubilisation by 2 Penicillium
species in solution culture and soil. Soil Biol Biochem 20:459–464
Atlas R, Bartha R (1997) Microbial ecology. Addison Wesley, New York

Azam F, Memon GH (1996) Soil organisms. In: Bashir E, Bantel R (eds) Soil science. National
Book Foundation, Islamabad, pp 200–232
Azcon C, Barea JM (1996) Interactions of arbuscular mycorrhiza with rhizosphere microorganisms.
In: Guerrero E (ed) Mycorrhiza. Biological Soil Resource. FEN, Bogota, Colombia, pp 47–68
Bache BW (1963) Aluminum and iron phosphates studies relating to soils. I. Solution and
hydrolysis of variscite and strengite. J Soil Sci 14:113–123
Bah A, Zaharah AR, Hussin A (2006) Phosphorus uptake from green manures and phosphate
fertilizers applied in a acid tropical soil. Commun Soil Sci Plant Anal 37:2077–2093
Banik S, Dey BK (1981a) Phosphate solubilizing microorganisms of a lateritic soil. I. Solubiliza-
tion of inorganic and production of organic acids by microorganisms isolated in sucrose
calcium phosphate agar plates. Zentralblatt Bakteriol Parasitenkunde infectionskarankheiten,
hygiene. 2. Naturwiss Miikrobiology Landwirtsch 136:476–486
Banik S, Dey BK (1981b) Phosphate solubilizing microorganisms of a lateritic soil. II. Effect of
some tricalcium phosphate-solubilizing microorganisms on available phosphorus content of
the soil. Zentralblatt Bakteriol. Parasitenkunde infectionskarankheiten, hygiene. 2. Naturwiss
Miikrobiology Landwirtsch 136:487–492
Banik S, Dey BK (1981c) Phosphate solubilizing microorganisms of a lateritic soil. III. Effect of
inoculation of some tricalcium phosphate-solubilizing microorganisms on available phospho-
rus content of rhizosphere soils of rice (Oryza sativa L. cv. IR-20). Zentralblatt Bakteriol.
Parasitenkunde infectionskarankheiten, hygiene. 2. Naturwiss Miikrobiology Landwirtsch
136:493–501
Banik S, Dey BK (1982) Available phosphate content of an alluvial soil as influenced by
inoculation of some isolated phosphate solubilizing microorganisms. Plant Soil 69:353–364
Banik S, Dey BK (1983) Phosphate solubilizing potentiality of the microorganisms capable of
utilizing aluminium phosphate as a sole phosphate source. Zentralbl Mikrobiol 138:17–23
Barber SA (1995) Soil nutrient bioavailability: a mechanistic approach. Wiley, New York
Bar-Yosef B, Rogers RD, Wolfram JH, Richman E (1999) Pseudomonas cepacia-mediated rock
phosphate solubilization in kaolinite and montmorillonite suspensions. Soil Sci Soc Am J
63:1703–1708
Bazin H, Bouchu A, Descotes G, Petit-Ramel M (1995) Comparison of calcium complexation of
some carboxylic acids derived from D-glucose and D-fructuose. Can J Chem 73:1338–1347
Bohn H, McNeal BL, O’connor G (1985) Soil chemistry. Wiley, New York
Bolan NS (1991) A critical review on the role of mycorrhizal fungi in the uptake of phosphorus by
plants. Plant Soil 134:189–207
Bolan NS, Naidu R, Mahimairaja S, Baskaran S (1994) Influence of low-molecular-weight organic
acids on the solubilization of phosphates. Biol Fertil Soils 18:311–319
Brady NC, Weil RR (1999) The nature and properties of soils. Prentice Hall, Upper Saddle River,
New Jersey
Buol S, Hole FD, McCraken RJ, Southard RJ (1997) Soil genesis and classification. Iowa State
University Press, Ames
Cabello M, Irrazabal G, Bucsinszky AM, Saparrat M, Schalamuk S (2005) Effect of an arbuscular
mycorrhizal fungus, Glomus mosseae, and a rock-phosphate-solubilizing fungus, Penicillium
thomii, on Mentha piperita growth in a soilless medium. J Basic Microbiol 45:182–289
Calvaruso C, Turpault MP, Frey-Klett P (2006) Root-associated bacteria contribute to mineral
weathering and to mineral nutrition in trees: a budgeting analysis. Appl Environ Microbiol
72:1258–1266
Canbolat MC, Bilen S, Cakmakci R, Sahin F, Aydin A (2006) Effect of plant growth-promoting
bacteria and soil compaction on barley seedling growth, nutrient uptake, soil properties, and
Cardoso I, Boddington CL, Janssen BH, Oenema O, Kuyper T (2006) Differential acces to
phosphorus pool of an Oxisol by mycorrhizal and non-mycorrhizal maize. Commun Soil Sci
Plant Anal 37:1537–1552
Corrales I, Amenos M, Poschenrieder C, Barcelo J (2007) Phosphorus efficiency and root

exhudates in two contrasting tropical maize varieties. J Plant Nutr 30:887–900
Cross AF, Schlesinger WH (1995) A literature review and evaluation of the Hedley fractionation:
application to the biogeochemical cycle of soil phosphorus in natural ecosystems. Geoderma
64:197–214
De la Fuente JM, Herrera L (1999) Advances in the understanding of aluminum toxicity and the
development of aluminum tolerant transgenic plants. Adv Agron 66:103–121
Delhaize E, Hebb DM, Ryan PR (2001) Expression of a Pseudomonas aerugionosa citrate
synthase gene in tobacco is not associated with either enhanced citrate accumulation or efflux.
Plant Physiol 125:2059–2067
Di-Simine CD, Sayer JA, Gadd GM (1998) Solubilization of zinc phosphate by a strain of
Pseudomonas fluorescens isolated from a forest soil. Biol Fertil Soils 28:87–94
Do Carmo Harta M, Torrent J (2007) Phosphorus desorption kinetics in relation to phosphorus
forms and sorption properties of Portuguese acid soils. Soil Sci 172:631–638
Duponnois R, Kisa M, Plenchette C (2006) Phosphate-solubilizing potential of the nematophagous
fungus Arthrobotrys oligospora. J Plant Nutr Soil Sci 169:280–282
El-Azouni IM (2008) Effect of phosphate solubilizing fungi on growth and nutrient uptake of
soybean (Glycine max L.) plants. J Appl Sci Res 4(6):592–598
Engelstad OP, Terman GL (1980) Agronomic effectiveness of phosphate fertilizers. In:
Khasawneh FE, Sample E, Kamprath E (eds) The role of phosphorus in agriculture. Soil
Science Society of America, Madison, WI, pp 311–332
Finlayson G, Roth R, Bubois L (1972) Calcium oxalate solubility studies in urinary calculi.
In: International Symposium on Renal Stone Research Madrid, Spain, pp 1–7
Fox RL (1979) Comparative responses of field grown crops to phosphate concentrations in soil
solutions. In: Munsell H, Staples R (eds) Stress physiology in crop plants. Wiley, New York,
pp 81–106
Fox RL, Kamprath E (1970) Phosphate sorption isotherms for evaluating phosphorus requirements
of soils. Soil Sci Soc Am Proc 34:902–907
Fransson AM, Valeur I, Wallander H (2004) The wood-decaying fungus Hygrophoropsis
aurantica increases P availability in acid forest humus soil, while N addition hampers this
effect. Soil Biol Biochem 36:1699–1705
Gaur A, Rana J, Jalali B, Chand H (1990) Role of VA mycorrhizae, phosphate solubilizing bacteria
and their interactions on growth and uptake of nutrients by wheat crops. In: Trends in
mycorrhizal research. Proceedings of the National Conference on Mycorrhizae, Hisar, India,
pp 105–106
Gleddie SC (1993) Response of pea and lentil to inoculation with the phosphate-solubilizing
fungus Penicillium bilaii (Provide). In: Proceedings of the Soils and Crops Workshops.
Saskatoon, Saskatchewan, pp 47–52
Goenadi DH (1995) Suitability of selected mixtures of clay minerals with humic substances as
carrier of phosphates-solubilizing microbes. Menara Perkebunan 63:102–113
Goenadi DH, Saraswati R, Nganro NN (1995) Nutrient-solubilizing and aggregate stabilizing
microbes isolates from selected humic tropical soils. Menara Perkebunan 63:60–66
Gururaj R, Mallikarjunaiah R (1995) Interactions among Azotobacter chroococcum, Penicillium
glaucum and Glomus fasciculatum and their effect on the growth and yield of sunflower. Helia
18(23):73–84
Gyaneshwar P, Kumar GN, Parekh LJ, Poole PS (2002) Role of soil microorganisms in improving
P nutrition of plants. Plant Soil 245:83–93
Habte M, Manjunath A (1991) Categories of vesicular-arbuscular mycorrhizal dependency of host
species. Mycorrhiza 1:3–12
Habte M, Osorio NW (2001) Arbuscular mycorrhizas: producing and applying arbuscular
mycorrhizal inoculum. University of Hawaii, Honolulu
Hameeda B, Kumar YH, Rupela OP, Kumar GN, Reddy G (2006) Effect of carbon substrates on rock
phosphate solubilization by bacteria from compost and macrofauna. Curr Microbiol 53:298–302
Hamel C (2004) Impact of arbuscular mycorrhizal fungi on N and P cycling in the root zone. Can
J Soil Sci 84:383–395
Hammond L, Leon L (1992) Evaluation of the North Carolina natural phosphate as a phosphoric
fertilizer. Suelos Ecuat 22:143–150
Havlin J, Beaton J, Tisdale SL, Nelson W (1999) Soil fertility and fertilizers: an introduction to
nutrient management. Prentice Hall, Upper Saddle River, New Jersey
He ZL, Zhu J (1997) Transformation and bioavailability of specifically sorbed phosphate on
variable-charge mineral soils. Biol Fertil Soils 25:175–181
He ZL, Zhu J (1998) Microbial utilization and transformation of phosphate adsorbed by variable
charge minerals. Soil Biol Biochem 30:917–923
Hetrick BAD (1991) Mycorrhizas and root architecture. Experentia 47:355–362
Holdford ICR (1997) Soil phosphorus: its measurement, and its uptake by plants. Aust J Soil Res
35:227–239
Hue NV (1991) Effects of organic acids/anions on P sorption and phytoavailability in soils with
different mineralogies. Soil Sci 152:463–471
Hue NV, Craddock GR, Adams F (1986) Effects of organic acids on aluminum toxicity in subsoils.
Soil Sci Soc Am J 50:28–34
IIlmer P, Barbato A, Schinner F (1995) Solubilization of hardly-soluble AlPO4 with P-solubilizing
microorganisms. Soil Biol Biochem 27:265–270
Illmer P, Schinner F (1995) Solubilization of inorganic calcium phosphates-solubilization
mechanisms. Soil Biol Biochem 27:257–263
Jackman JM, Jones RC, Yost RS, Babcock CJ (1997) Rietveld estimates of mineral percentages to
predict phosphate sorption by selected Hawaiian soils. Soil Sci Soc Am J 61:618–625
Jacobsen I, Abbott LK, Robson AD (1992) External hyphae of vesicular-arbuscular mycorrhizal
fungi associated with Trifolium subterraneum L. New Phytol 120:371–380
Jeffries P, Gianinazzi S, Perotto S, Turnau K, Barea JM (2003) The contribution of arbuscular
mycorrhizal fungi in sustainable maintenance of plant health and soil fertility. Biol Fertil Soils
37:1–16
Jones RC (1981) X-ray diffraction line profile analysis vs. phosphorus sorption by 11 Puerto Rican
soils. Soil Sci Soc Am J 45:818–825
Jones DL, Dennis PG, Owen AG, Van Hees PAW (2003) Organic acid behavior in soils–
misconceptions and knowledge gaps. Plant Soil 248:31–41
Juo A, Ellis B (1968) Chemical and physical properties of iron and aluminum phosphate and their
relation to phosphorus availability. Soil Sci Soc Am Proc 32:216–221
Juo ASR, Fox RL (1977) Phosphate sorption characteristics of some bench-mark soils of West
Africa. Soil Sci 124:370–376
Khan MS, Zaidi A (2007) Synergistic effects of the inoculation with plant growth-promoting
rhizobacteria and a arbuscular mycorrhizal fungus on the perfomance of wheat. Turk J Agr For
31:355–362
Khan MS, Zaidi A, Wani PA (2007) Role of phosphate-solubilizing microorganisms in sustainable
agriculture – a review. Agron Sustain Dev 27:29–43
Kim KY, McDonald GA, Jordan D (1997) Solubilization of hydroxyapatite by Enterobacter
agglomerans and cloned Escherichia coli in culture medium. Biol Fertil Soils 24:347–352
Kim KY, McDonald GA, Jordan D (1998a) Effect of phosphate solubilizing bacteria and vesicu-
lar-arbuscular mycorrhizae on tomato growth and soil microbial activity. Biol Fertil Soils
26:79–87
Kim KY, Jordan D, McDonald GA (1998b) Enterobacter agglomerans, phosphate solubili-
zing bacteria and microbal activity in soil. Effect of carbon sources. Soil Biol Biochem
30:995–1003
Kirk GJD, Santos EE, Findenegg GR (1999) Phosphate solubilization by organic anion excretion
from rice (Orytza sativa L.) growing in aerobic soil. Plant Soil 211:11–18
Kopler J, Lifshitz R, Schroth M (1988) Pseudomonas inoculants to benefit plant production. ISI
Atlas Sci Anim Plant Sci 1:60–64
Kucey RMN (1983) Phosphate solubilising bacteria and fungi in various cultivated and virgin
Alberta soils. Can J Soil Sci 63:671–678
Kucey RMN (1987) Increased phosphorus uptake by wheat and field beans inoculated with a
phosphorus solubilising Penicillium bilaii strain and with vesicular-asbuscular mycorrhizal
fungi. Appl Environ Microbiol 53:2699–2703
Kucey RMN (1988) Effect of Penicillium bilaii on the solubility and uptake of P and micronu-
trients from soil by wheat. Can J Soil Sci 68:261–270
Kucey RMN, Janzen HH, Leggett ME (1989) Microbial mediated increases in plant available
phosphorus. Adv Agron 42:199–228
Le Bayon RC, Weisskopf L, Martinoia E, Jansa J, Frossard E, Keller F, Follmi KB, Gobat JM
(2006) Soil phosphorus uptake by continuously cropped Lupinus albus: a new microcosm
design. Plant Soil 283:309–321
Li X, Ecckhard G, Marschner H (1991) Phosphorus depletion and pH decrease at the root-soil and
hypha-soil interfaces of VA mycorrhizal white clover fertilized with ammonium. New Phytol
119:397–404
Linderman RG (1988) Mycorrhizal interaction with the rhizosphere microflora: The mycorhizo-
sphere effect. Phytopathology 78:366–371
Lindsay WL (2001) Chemical equilibria in soils. Blackburn, Caldwell, New Jersey
Louw HA, Webley DM (1959) The solubilization of insoluble phosphates. V. The action of some
organic acids on iron and aluminium phosphates. NZ J Sci 2:215–218
Lynch JP, Ho MD (2005) Rhizoeconomics: carbon costs of phosphorus acquisition. Plant Soil
269:45–56
Manjunath A, Hue NV, Habte M (1989) Response of Leucaena leucocephala to vesicular-
arbuscular mycorrhizal colonization and rock phosphate fertilization in an Oxisol. Plant Soil
114:127–133
Marschner P, Solaiman Z, Rengel Z (2006) Rhizosphere properties of Poacea genotypes under
P-limiting conditions. Plant Soil 283:11–24
Mattingly GEG (1975) Labile phosphate in soils. Soil Sci 119:369–375
McCully M (1999) Roots in soil: unearthing the complexities of roots and their rhizospheres. Annu
Rev Plant Physiol Plant Mol Biol 50:695–718
Memon KS (1996) Soil and fertilizer phosphorus. In: Bashir E, Bantel R (eds) Soil science.
National Book Foundation, Islamabad, pp 291–314
Miyasaka SC, Habte M (2001) Plant mechanisms and mycorrhizal symbioses to increase phos-
phorus uptake efficiency. Commun Soil Sci Plant Anal 32:1101–1147
Miyasaka S, Buta JG, Howell RK, Foy CD (1991) Mechanisms of aluminum tolerance in
snapbeans. Plant Physiol 96:737–743
Mohod S, Gupta DN, Chavan AS (1991) Effects of P solubilizing organims on yield and N uptake
by rice. J Maharashtra Agric Univ 16:229–231
Msolla MM, Semoka JMR, Borggaard OK (2005) Hard Minjingu phosphate rock. An alter-
native P source for maize production on acid soils in Tanzania. Nutr Cyc Agroecosyst
72:299–308
Msolla MM, Semoka JMR, Szilas C, Borggaard OK (2007) Crop (maize) response to direct
application of local phosphate rock on selected acidic soils of Tanzania. Commun Soil Sci
Plant Anal 38:93–106
Nguyen C (2003) Rhizodeposition of organic C by plants: mechanisms and controls. Agronomie
23:375–396
Nye PH, Tinker PB (1977) Solute movement in the soil-root system. Blackwell, Oxford
Ojo OD, Kintomo AA, Akinride EA, Akoroda MO (2007) Comparative effect of phosphorus
sources for grain amaranth production. Commun Soil Sci Plant Anal 38:35–55
Omar SA (1998) The role of rock-phosphate-solubilizing fungi and vesicular-arbuscular-mycorrhiza
(VAM) in growth of wheat plants fertilized with rock phosphate. World J Microbiol Biotechnol
14:211–218
Osorio NW (2008) Effectiveness of microbial solubilization of phosphate in enhancing plant

phosphate uptake in tropical soils and assessment of the mechanisms of solubilization. Ph.D.
dissertation, University of Hawaii, Honolulu
Osorio NW, Habte M (2001) Synergistic influence of an arbuscular mycorrhizal fungus and P
solubilizing fungus on growth and plant P uptake of Leucaena leucocephala in an Oxisol. Arid
Land Res Manage 15:263–274
Pandey A, Trivedi P, Kumar B, Palni LMS (2006) Characterization of a phosphate solubilizing
microorganism and antagonistic strain of Pseudomonas putida (B0) isolated from a sub-apline
location in the Indian Central Himalaya. Curr Microbiol 53:102–107
Parfitt RL (1989) Phosphate reactions with natural allophane, ferrihydrite and goethite. J Soil Sci
40:359–369
Paul NB, Rao WVBS (1971) Phosphate-dissolving bacteria in the rhizosphere of some cultivated
legumes. Plant Soil 35:127–132
Peix A, Rivas-Boyero AA, Mateos PF, Rodriguez-Barrueco C, Martinez-Molina E, Velasquez E
(2001) Growth promotion of chickpea and barley by a phosphate solubilizing strain of
Mesorrhizobium mediterraneum under growth chamber conditions. Soil Biol Biochem
33:103–110
Pikovskaia RI (1948) Mobilization of phosphates in soil in conection with the vital activities of
some microbial species. Mikrobiologie 17:362–370
Plenchette C, Fortin JA, Furlan V (1983) Growth response of several plant species to mycorrhiza in a
soil of moderate P fertility. I. Mycorrhizal dependency under field conditions. Plant Soil 70:191–209
Prathibha CK, Alagawadi A, Sreenivasa M (1995) Establishment of inoculated organisms in
rhizosphere and their influence on nutrient uptake and yield of cotton. J Agric Sci 8:22–27
Prescott L, Harley J, Klein DA (1999) Microbiology. McGraw-Hill, Boston
Pypers P, Delrue J, Diels J, Smolders E, Merckx R (2006) Phosphorus intensity determines short
term P uptake by pigeon pea (Cajanus cajan L.) grown in soils with different P buffereing
capacity. Plant Soil 284:217–227
Radersma S, Grierson P (2004) Phosphorus mobilization in agroforestry: organic anions, phos-
phatase activity and phosphorus fractions in the rhizophere. Plant Soil 259:209–219
Rahman MK, Parsons JW (1997) Effects of inoculation with Glomus mosseae, Azorhizobium
caulinodans and rock phosphate on the growth of and nitrogen and phosphorus accumulation in
Sesbania rostrata. Biol Fertil Soils 25:47–52
Rambelli A (1973) The rhizosphere of mycorrhyzae. In: Marks GC, Kozlowski TT (eds) Ecto-
mycorrhyzae. Their ecology and physiology. Academic, London, pp 299–343
Randhawa P, Condron LM, Di HJ, Sinaj S, McLenaghen RD (2006) Phosphorus availability in
soils amended with different phosphate fertilizers. Commun Soil Sci Plant Anal 37:25–39
Rao S (1992) Biofertilizers in agriculture. A. A. Balkema, Rotterdam
Rashid M, Khalil S, Ayub N, Alam S, Latif F (2004) Organic acids production and phosphate
solubilization by phosphate solubilizing microorganisms (PSM) under in vitro conditions. Pak
J Biol Sci 7(2):187–196
Redding MR, Shatte T, Bell K (2006) Soil-sorption-desorption of phosphorus from piggery
effluent compared with inorganic sources. Eur J Soil Sci 57:134–146
Reddy DD (2007) Phosphorus solubilization from low-grade rock phosphates in the presence of
decomposing soybean leaf litter. Commun Soil Sci Plant Anal 38:283–291
Reddy MS, Kumar S, Babita K, Reddy MS (2002) Biosolubilization of poorly soluble rock
phosphates by Aspergillus tubigensis and Aspergillus niger. Bioresour Technol 84:187–189
Reyes I, Valery A, Valduz Z (2006) Phosphate-solubilizing microrganisms isolated from rhizospheric
and bulk soils of colonizer plants at an abandoned rock phosphate mine. Plant Soil 287:69–75
Rodriguez H, Fraga R, Gonzalez T, Bashan Y (2006) Genetics of phosphate solubilization and
its potential applications for improving plant growth-promoting bacteria. Plant Soil
287:15–21
Roos W, Luckner M (1984) Relationships between proton extrusion and fluxes of ammonium ions
and organic acids in Penicillium cyclopium. J Gen Microbiol 130:1007–1014
Rose RE (1957) Techniques of determining the effect of microorganisms on insoluble inorganic

phosphates. NZ J Sci Technol 38:773–780
Salih HM, Yahya AI, Abdul-Rahem AM, Munam BH (1989) Availability of phosphorus in a
calcareous soil treated with rock phosphate or superphosphate as affected by phosphate-
dissolving fungi. Plant Soil 120:181–185
Sanchez P, Logan T (1992) Myths and science about the chemistry and fertility of soils in the
tropics. In: Lal R, Sanchez P (eds) Myths and science of soils of the tropics. Soil Science
Society of America, Madison, WI, pp 35–46
Sanchez P, Uehara G (1980) Management considerations for acid soils with high phosphorus
fixation capacity. In: Khasawneh FE (ed) The role of phosphorus in agriculture. Soil Science
Society of America, Madison, WI, pp 471–514
Sato S, Comerford N (2006) Organic anions and phosphorus desorption and bioavailability in
a humid Brazilian Ultisol. Soil Sci 171:695–705
Schachtman DP, Reid R, Ayling SM (1998) Phosphorus uptake by plants: from soil to cell. Plant
Physiol 116:447–453
Schwertmann U, Herbillon AJ (1992) Some aspects of fertility associated with the mineralogy of
highly weathered tropical soils. In: Lal R, Sanchez P (eds) Myths and science of soils of the
Tropics. Soil Science Society of America, Madison, WI, pp 47–60
Shabayey VP, Smolin VY, Mudrik VA (1996) Nitrogen fixation and CO2 exchange in soybeans
inoculated with mixed cultures of different microorganisms. Biol Fertil Soils 23:425–430
Shoji S, Nanzyo M, Dahlgren RA (1993) Volcanic ash soils-genesis, properties, and utilization.
Elsevier, Amsterdam
Shrivastava M, Bhujbal BM, D’Souza SF (2007) Agronomic efficiency of Indian rock phosphate
in acidic soils employing radiotracer A-value technique. Commun Soil Sci Plant Anal
38:461–471
Singh S, Kapoor KK (1999) Inoculation with phosphate-solubilizing microorganisms and a
vesicular-arbuscular mycorrhizal fungus improves dry matter yield and nutrient uptake by
wheat grown in a sandy soil. Biol Fertil Soils 28:139–144
Singh HP, Singh TA (1993) The interaction of rock phosphate, Bradyrhizobium, vesicular
arbuscular mycorrhizae, and phosphate-solubilising microbes on soybean grown in a sub-
Himalayan Mollisol. Mycorrhiza 4:37–43
Singh V, Dhillon NS, Brar BS (2006) Effect of incorporation of crop residues and organic manures
on adsorption/desorption and bioavailability of phosphate. Nutr Cyc Agroecosyst 76:95–108
Smith FW (2002) The phosphate uptake mechanism. Plant Soil 245:105–114
Smith SE, Read DJ (1997) Mycorrhizal symbiosis. Academic, San Diego
Smith FW, Mudge SR, Rae AL, Glassop D (2003) Phosphate transport in plants. Plant Soil
248:71–83
Snoeyink VL, Jenkins D (1980) Water chemistry. Wiley, New York
Sperber JI (1957) Solution of mineral phosphates by soil bacteria. Nature 180:994
Sperber JI (1958) Solution of apatite by soil microorganisms producing organic acids. Aust J Agric
Res 9:782–787
Sreenivasa M, Krishnaraj M (1992) Synergistic interaction between VA mycorrhizal fungi and a
phosphate solubilizing bacterium in chili. Zentralbl Mikrobiol 147:126–130
Stevenson FJ (1986) Cycles of soil. Wiley, New York
Stumm W, Morgan JJ (1995) Aquatic chemistry: chemical equilibria and rates in natural waters.
Wiley, New York
Sturz AV, Christie BR, Matheson BG, Nowak J (1997) Biodiversity of endophytic bacteria which
colonize red clover nodules, roots, stems and foliage and their influence on host growth. Biol
Sylvia D (1999) Mycorrhizal symbioses. In: Sylvia DJ, Fuhrmann J, Hartel P, Zuberer D (eds)
Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ,
pp 408–426
Taylor AW, Gurney EL (1964) Solubility of variscite. Soil Sci 98:9–13
Tinker PB (1980) Role of rhizosphere microorganisms in phosphorus uptake by plants. In:

Khasawneh FE, Sample EC, Kamprath EJ (eds) The role of phosphorus in agriculture. Soil
Science Society of America, Madison, WI, pp 617–654
Toro M, Azcon R, Herrera R (1996) Effects on yield and nutrition of mycorrhizal and nodulated
Pueraria phaseolides exerted by P-solubilizing rhizobacteria. Biol Fertil Soils 21:23–29
Toro M, Azcon R, Barea JM (1998) The use of isotopic dilution techniques to evaluate the
interactive effects of rhizobium genotypes, mycorrhizal fungi, phosphate-solubilizing rhizo-
bacteria and rock phosphate on nitrogen and phosphorus acquisition by Medicago sativa. New
Phytol 138:265–273
Trolove SN, Hedley MJ, Kirk GJD, Bolan NS, Loganathan P (2003) Progress in selected areas of
rhizosphere research on P acquisition. Aust J Soil Res 41:471–499
Veith JA, Sposito G (1977) Reactions of aluminosilicates, aluminum hydrous oxides, and alumi-
num oxide with o-phosphate: the formation of x-ray amorphous analog of variscite and
montebrasite. Soil Sci Soc Am J 41:870–876
Venkateswarlu B, Rao AV, Raina P (1984) Evaluation of phosphorus solubilization by micro-
organisms isolated from Aridisols. J Indian Soc Soil Sci 32:273–277
Vyas P, Rahi P, Chauhan A, Gulati A (2007) Phosphate solubilization potential and stress
tolerance of Eupenicilium parvum from tea soil. Mycol Res 111:931–938
Wakelin SA, Warren RA, Harvey PR, Ryder MH (2004a) Phosphate solubilization by Penicillium
spp. closely associated with wheat roots. Biol Fertil Soils 40:36–43
Wakelin SA, Warren RA, Ryder MH (2004b) Effect of soil properties on growth promotion of
wheat by Penicillium radicum. Aust J Soil Res 42:897–904
Wani PA, Khan MS, Zaidi A (2007) Synergistic effects of the inoculation with nitrogen fixing and
phosphate-solubilizing rhizobacteria on the perfomance of field grwon chickpea. J Plant Nutr
27:599–610
Welch S, Taunton AE, Banfiled JF (2002) Effect of microorganisms and microbial metabolites on
apatite dissolution. Geomicrobiol J 19:343–367
Whitelaw MA (2000) Growth promotion of plants inoculated with phosphate-solubilizing fungi.
Adv Agron 69:99–151
Whitelaw MA, Harden TJ, Bender GL (1997) Plant growth promotion of wheat inoculated with
Penicillium radicum sp. nov. Aust J Soil Res 35:291–300
Xavier LJC, Germida JJ (2003) Bacteria associated with Glomus clarum spores influence mycor-
rhizal activity. Biol Fertil Soils 35:471–478
Young R, Davies C (1980) Phosphate fertilizers and process technology. In: Khasawneh FE,
Sample E, Kamprath E (eds) The role of phosphorus in agriculture. Soil Science Society of
America, Madison, WI, pp 195–226
Young CC, Chen CL, Chao CC (1990) Effect of Rhizobium, vesicular-arbuscular mycorrhiza, and
phosphate solubilizing bacteria on yield and mineral phosphorus uptake of crops in subtropi-
cal-tropical. In: 14th International Congress of Soil Science Transactions, vol. 3. International
Society of Soil Science, Kyoto, Japan, pp 55–60
Yusdar H, Anuar AR, Hanafi MM, Azifah H (2007) Analysis of phosphate rock dissolution
determining factors using principal component analysis in some acid Indonesian soils.
Commun Soil Sci Plant Anal 38:273–282
Index
A Aspergillus, 330, 332

Abiotic, 134, 135, 147, 148 ATPase, 90
Acacia, 229, 230, 236, 237, 239 Azorhizobium, 229, 237
ACC. See 1-Aminocyclopropane-1- A. caulinodans, 292–296, 298
carboxylate Azospirrlum, 243, 245, 246
ACC deaminase, 31, 36, 38, 39, 63, 65,
69–77
Acetylene, 245, 247 B
Actinorhizal nitrogen fixers, 290 Bacillus, 94, 95, 98
Agriculture Bacillus thuringiensis, 186–189, 192, 196,
conventional, 207, 210, 213, 217 197, 199, 201
high-input, 209, 210 Bacteria, 186, 187–189, 192, 195, 198
low-input, 210 Bacteroids, 66, 72, 73, 75, 76
organic, 206, 208, 210, 212, 213, 218 Barley, 340
sustainable, 205–219 Beneficial biofilms, 52–55
Agrobacterium, 236, 237 Bioaccumulation, 91, 94–95
Agro-ecosystem(s), 134, 139, 152, 207, Biochemical pesticides, 194–195
209–215 Bio-control, 28, 31–34, 38, 41, 147
Albizia, 236, 237 Biocontroling agents, 53
Alfalfa, 65, 68, 70, 73, 77 Biodiversity, 135
Alkalinity/salinity, 25 Biofertilizer(s), 135, 138, 140, 151–153,
Allelochemicals, 194 206, 208, 211, 213, 218, 228
Alphaproteobacteria, 229, 248 Biofilm, 95
Aluminum, 326, 336, 337 Biofilmed biofertilizers, 56–60
Amino acids, 69, 71 Biofilm formation, 52
1-Aminocyclopropane-1-carboxylate BIOLOG, 295
(ACC), 139, 140, 147, 151 Biological nitrogen fixation (BNF), 289,
Ammonia, 63, 65, 66, 71 291, 298
Andisol, 326, 336, 337, 343 Biopesticides, 185–202
Antibiotics, 106, 108, 109, 120–122 Bioremediation, 90–98, 100, 151
Arbuscular mycorrhiza (AM), 256–260, 262 Biosorption, 91–94
Arid land, 229 Biotechnology, 134, 152, 153
Arsenic, 87 Biotic stress, 148–150
353
354 Index
BNF. See Biological nitrogen fixation Desertification, 219

Botanicals, 193–194 DGGE. See Denaturing gradient gel
Bradyrhizobium, 65, 69 electrophoresis
Diazotroph(s), 287–298
Diazotrophic, 137
C Diazotrophic bacteria, 56, 59
Cadmium (Cd), 86–88, 90, 94, 96–99 Diversification
Carbon cropping system, 177–178
drain, 212, 213 framing system, 178
sequestration, 206 soil health, 177–178
Carolina phosphate rock, 313 DNA, 231, 235–237, 239, 240, 242,
Chelators, 117 245–247
Chickpea, 340 Drought stress, 242
Chili, 339
Chitinase(s), 135, 140
E
Chrome Azurol S (CAS), 116
Ecosystem(s), 301–318
Chromosomal insertion, 34
Efflux, 90, 94
Citric acid, 333, 334, 336
Endophytes, 245
Citrobacter, 92
Endosymbiont, 292, 298
Clover, 291
Engineering biocontrol agents, 198
Coinoculation, 150
Engineering crop plants, 186, 199
Community level physiological profiles
Ensifer, 229, 236
(CLPP), 107
Enterobacter, 330, 333, 340, 343
Consistency, 138, 141, 152
Entomopathogenic fungi, 190–192
Conventional biofertilizers, 56
Entomopathogenic protozoa and
Cotton, 339
microsporida, 193
Crop rotation(s), 210–213
Essential nutrients, 134
Cropping system, 207, 210, 211, 218
Ethylene, 63–77, 138, 139, 147
carbon sequestration, 171
Exopolysaccharide (EPS), 29
microbial community, 178
Extracellular polymeric substances (EPS),
rhizodeposition, 177
52, 55
soil health, 161–180
Exudates, 138
tailoring, 178
Current agricultural problems
agrochemical(s), 173 F
farmers awareness, 174 Farming
heavy vehicle traffic, 173–174 organic, 210, 218
inappropriate cropping system, 172–173 practices, 210
intensification, 161, 162, 165, 172–173 system, 206, 212, 213, 218
sewage and sludge, 173–174 Farm yard manure (FYM), 314
soil testing protocol, 174 Fatty acid methyl ester (FAME), 108
Cytokinin(s), 66, 138, 139 Fertilizer
biofertilizer(s), 206, 208, 211, 213, 218
chemical, 206, 207, 211
D
inorganic, 210
Decontamination, 91 Flavonoids, 66, 230
Denaturing gradient gel electrophoresis Formulation, 135, 151–152
(DGGE), 306 Four-microbe FRB, 58
Index 355
Functional diversity, 105–124 SOM, 172

Fungal–bacterial biofilm (FBB), 53–58 Intercropping, 212, 213
Fungal–rhizobial biofilm (FRB), 53–58 Ionic ratios, 269
Ionic status, 268–269
Itai-itai, 86
G
Gene density, 315
Gibberellins, 138, 139
K
Gigaspora margarita, 329
Glomus aggregatum, 328 Kaolinite, 335, 336
Glomus fasciculatum, 339 a-Ketobutyrate, 63, 65, 71
Gluconic acid, 334 Kudzu, 340, 341
Glucose dehydrogenase (GDH), 35
Glycine betaine, 239, 242, 243
L
GOD activity, 33
Lead, 87, 88, 94
Goethite, 326, 335, 336
Legumes, 63–77
Granular inoculants, 294
Lettuce, 337
Growth-promoting rhizobacteria (PGPR),
Leucaena, 75, 76, 328, 342û344
105–124
Ligand, 115–117
Lipochitooligosaccharides, 232, 245
H Liquid inoculums, 297
Hemoglobin, 232 Lotus, 230, 236, 242
Heavy metal resistance, 90, 97 Lotus japonicus, 68
Heavy metals, 64, 65, 74, 77, 147, 151 Lupinus, 336
Herb legume, 235 Lytic enzymes, 122–123
Higher order biofilms, 60
Horticultural
M
crop, 207, 214–215
production, 207, 214–215 Macroptilium atropurpureum, 68
Maize, 334
Mechanisms of action, 135–137, 140
I Medicago truncatula, 230
IAA. See Indole-3-acetic acid Mediterranean forest ecosystems, 256, 257
Indian mustard, 118, 123 Mercury, 87, 90, 94
Indole acetamide (IAM) pathway, 113 Meristem, 66, 69
Indole-3-acetic acid (IAA), 64, 71, 140 Mesorhizobium, 65, 72, 229, 234–237, 240,
Induced systemic resistance (ISR), 120–122 241
Inocula, 53 Metabolic cooperation, 52, 58
Inoculants, 134, 135, 140, 141 Metal toxicity, 109
Inoculation, mycorrhizal, 210, 213–215, Microarray(s), 231, 239, 240
217, 218 Microbial community structure, 303–308,
Inoculum 317
mycorrhizal, AM, 211, 216, 219 Microbial diversity, 302–304, 306,
tuning, 215, 216, 218, 219 312, 313
Intensification Microbial pesticides, 186–189
diversity loss, 163, 169, 172 Microbial RNA, 312
production sustainability, 172 Micropropagation, 214
resilience capacity, 172 Mint, 343
356 Index
Mobilization, 135, 137–138 O

Modulation enzyme, 119–120 Ochrobacterium, 123
Mollisol, 330, 332, 336, 340, 341, Organic acids (OA), 27–29, 33, 41, 71
343 Oxalic acid, 334, 336, 344
Monoculture Oxidative stress, 87, 89, 97
aboveground biodiversity, 173 Oxisol, 326, 328, 336, 337, 340, 341, 343
belowground biodiversity, 173
nematode infestation, 173
P
soil sickness, 173 Papaya, 338
vulnerable, 173 Parasponia, 232, 234
Montmorillonite, 336 Pathogen
Mortierella, 332, 336, 341, 343 root, 206, 212
Most probable number (MPN), 304 soil-borne, 208
MPN. See Most probable number Pathogenic, 135, 147
Mycorrhiza, 325–344 PCNB. See Pentachloronitrobenzene
Mycorrhizal, 229, 243, 244 PCR. See Polymerase chain reaction
industry, 215, 217, 219 Peanut, 242
responsiveness, 206–210, 213, 214 Peat, 244, 245
symbiosis, 206, 210–212, 219 Penicillium, 330, 332, 334, 336, 343
Mycorrhizal dependency (MD), 329 Penicillium bilaji, 332
Mycorrhizal fungi, 58 Pentachloronitrobenzene (PCNB), 316
Mycorrhizosphere, 340 Pesticide(s), 207, 211
Mycotrophic, 207, 210, 212 PGPB, 3–4
ACC deaminase, 4, 8, 9, 14
auxin, 4
N direct plant-growth promotion, 3
National Botanical Research Institute P indirect plant-growth promotion, 3
(NBRIP) medium, 26 PGPF, 5
N content, 292, 296 arbuscular mycorrhizae, 5, 10, 14
Nematode(s), 186, 187, 192 ectomycorrhizae, 7, 12, 13
N2 fixation, 135, 137, 138 endophytic fungi, 7
N2-fixing bacteria, 51, 53 PGPR. See Plant growth-promoting
nif. See Nitrogen fixation rhizobacteria
Nitrogen PGRs. See Plant growth regulators
macronutrient, 269–272 Phaseolus, 237, 241
transport systems, 269 Phosphatase(s), 30, 135, 137, 140, 151
Nitrogenase(s), 151, 233, 241–243 Phosphate
Nitrogenase activity, 53 depletion zone, 327, 329
Nitrogen fixation (nif), 231, 269–273, desorption, 328, 333, 337, 338, 343
276 diffusion, 327, 329
N-methyl-N’-nitro-N-nitrosoguanidine fertilization, 327, 344
(NTG), 24 fixation, 326–328, 330, 332, 338, 340,
Nod factor(s), 66, 68, 69, 230, 232 341, 343
Nod gene, 230, 232, 246 fixation categories, 327
Nodulation, 270, 271, 282 Phosphatic fertilizers, 87
Nodule, 64–70, 73, 75–77 Phosphobacterin, 330
Nodule-like structures, 51, 54, 58 Phospholipid fatty acid (PLFA), 108
Nurse plant, 255–262 Phosphorus, 23, 24
Index 357
availability, 206, 208, 209 Rhizobiotoxine, 69

P-deficient, 213 Rhizobium, 63–77
Photosynthesis, 86 Rhizobium leguminosarum, 65
Photosynthetic carbon, 308 Rhizoplane, 135, 307, 309
Phytohormone(s), 69, 76, 107–110, 112, Rhizosphere, 58, 134, 135, 138, 141, 151
135, 138, 139, 151 Ribosomal DNA, 235, 245
Phytoremediation, 90 Rice, 334, 339
Pigeon pea, 334 Rock phosphate (RP), 325, 328, 337, 342
Pisum, 235 RP. See Rock phosphate
Planktonic cells, 52 R:S ratio, 309
Plant growth hormones, 58 Ryegrass, 337
Plant growth-promoting rhizobacteria
(PGPR), 64, 65, 70–72, 74, 77, 133–153
Plant growth regulators (PGRs), 111–113,
S
134, 135, 138
Salinity, 229, 230, 238–244, 248
Plant health-promoting rhizobacteria
effects, 2
(PHPR), 106
ethylene rise, 2, 8
Plant–plant interactions, 262
osmotic stress, 2
Polymerase chain reaction (PCR), 305, 306
plant tolerance, 5, 14
Precursor, 138, 147
Salt stress, 238, 239, 241–244, 246, 248
Proline, 242, 243, 248
Salt tolerance, 230, 239–241, 243, 246
Prosopis, 229, 236, 237
Seeds
Protein, 66, 72, 73, 75
productivity, 268, 274–277
Proteomics, 96–98
protein content, 268, 276–277
Provide TM, 341
Semiarid, 237, 240
Pseudomonas, 65, 72, 74, 90, 94, 95, 330,
Sesbania, 229, 236, 237
334, 336, 338–340
Sesbania rostrata, 68
Pseudonodules, 51, 54, 58
Sex pheromones, 195
PSM mechanisms
Siderophore(s), 64, 76, 95, 106, 108–111,
acid production, 334
113–119, 135, 138, 140, 151
ammonium assimilation, 333, 336
Silver, 90, 94
organic acid, 330, 333, 334, 336, 338,
Sinorhizobium, 75, 229, 230, 234–237, 240
341, 344
Soil
Pi-desorption, 337–338, 343
desertified, 206, 218
P-solubilizing fungi, 51, 53
erosion, 206, 208
Pyrroloquinoline quinone (PQQ), 34, 35
Soil aggregates, 302
Soil fertility, 106, 124, 301, 302, 312, 313,
Q 317, 318
Quorum sensing, 52 Soil health
concept, 163–164
diagnosis, 162, 163
R indicators, 162–169, 174, 179, 180
Reactive oxyzen species (ROS), 87–89 indices (SHI), 168, 176
Reforestation, 255–262 Soil health assessment methodology
Responsiveness data screening, 167
to AM fungi, 206–209, 213, 214, 216 indicator integration, 168
to mycorrhiza, 206–210, 214, 216 MDS validation, 168
Rhizobacteria, 64, 133–153 redundancy, 167
358 Index
Soil health importance Symbiotic nitrogen fixation, 63, 76

animal health, 171 Systemic resistance, 139
crop production, 168–169
crop quality, 169–170
environment health, 171 T
human health, 170 Tobacco, 338
production sustainability, 170 Tomato, 339
Soil health indicator Transgenic, 147
criteria, 164–165 Transmission electron microscopy (TEM),
selection, 164–165 94
significance, 165–167 Tree legume, 229, 235–237
Soil health management Tricalcium phosphate (TCP), 24, 29
cover crops, 176–177, 179 Trichoderma, 28, 33, 38, 40
crop and farm diversification, 178 Trifolium, 329
crop rotation, 175–176
farmer’s friendly SHI, 179
V
government policy, 175
Variscite, 326, 335
legislation, 174–175, 179
Vegetative organs
organic husbandry, 178–179
ion accumulation, 277–281
soil test-based fertilization, 163, 165,
nitrogen nutrition, 277–281
176, 179
plant growth, 277
tillage conservation, 176–177
Vertisol, 337
Soil pH, 307–312
Vicia, 234–236, 244
Soil solution Pi, 325, 330
Viruses, 186, 187, 189–190, 195, 199, 201,
Soybean, 68, 76, 339
202
Stabilization, 90, 91
Strengite, 335
Stress, 134, 139, 140, 147–151 W
Substrate, 134, 135, 138 Wheat, 287–298, 332, 339–341, 343
Sugars, 71 Wild, 227–229, 234–237, 241–243, 248
Sunflower, 339 Woody legume, 237
Sustainability, 133, 134, 139,
141, 152
Symbiotic, 227–242, 244, 245, X
247, 248 Xenobiotic, 134

Pub Microbial Strategies For Crop Improvement

Uploaded by

Copyright:

Available Formats

Pub Microbial Strategies For Crop Improvement

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Pub Microbial Strategies For Crop Improvement

Uploaded by

Copyright:

Available Formats

Microbial Strategies for Crop Improvement

Mohammad Saghir Khan l Almas Zaidi l

Prof. Javed Musarrat

ISBN 978-3-642-01978-4 e-ISBN 978-3-642-01979-1

# Springer-Verlag Berlin Heidelberg 2009

Cover design: WMXDesign GmbH, Heidelberg, Germany

Printed on acid-free paper

Springer is part of Springer Science+Business Media (www.springer.com)

and development of plants. However, bacterial enzyme 1-aminocyclopropane-1-

characteristics and also through positive influence on soil microbiota, especially on

India, March 2009 Mohammad Saghir Khan

1 The Use of Microorganisms to Facilitate the Growth of Plants

2 Recent Advances in Plant Growth Promotion by

3 Developing Beneficial Microbial Biofilms on Roots of Non legumes:

4 Role of 1-Aminocyclopropane-1-carboxylate deaminase

5 Strategies for Crop Improvement in Contaminated Soils

6 Functional Diversity Among Plant Growth-Promoting Rhizobacteria:

7 Plant Growth Promoting Rhizobacteria and Sustainable

8 Soil Health – A Precondition for Crop Production . . . . . . . . . . . . . . . . . . . 161

9 Recent Advances in Biopesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

10 Benefits of Arbuscular Mycorrhizal Fungi to Sustainable

11 Enhancement of Rhizobia–Legumes Symbioses and Nitrogen

12 Monitoring the Development of Nurse Plant Species

13 Pea Cultivation in Saline Soils: Influence of Nitrogen Nutrition . . . . 267

14 Plant Growth-Promoting Diazotrophs and Productivity of Wheat

15 Factors Affecting the Variation of Microbial Communities

16 Strategies for Utilizing Arbuscular Mycorrhizal Fungi and

Mohammad Saghir Khan, Ph.D. is an Associate Professor at the Department of

biochemistry, microbiology, and molecular biology to postgraduate students for the

Munees Ahemad, Department of Agricultural Microbiology, Faculty of

Pervaze Ahmad Wani, Department of Agricultural Microbiology, Faculty of

Abdulaziz A Al Khedhairy, DNA Research Chair, College of Science, King Saud

J. Albrechtová, Charles University in Prague, Faculty of Science, Department of

Arshad Ali, Department of Plant Protection, Faculty of Agricultural Sciences,

Anthony O. Anyia, Alberta Research Council, Bioresource Technologies,

Daniel J. Archambault, Laurentian University, 935 Ramsey Lake Road, Sudbury,

Muhammad Arshad, Institute of Soil and Environmental Sciences, University of

Carols J. Bécquer, Ministerio de la Agricultura, Instituto de Ganaderı́a Tropical,

Graziella Berta, Dipartimento di Scienze dell’Ambiente e della Vita, Università

Rummana A. Choudhury, Department of Plant Protection, Faculty of

B. Dreyfus, IRD. UMR 113. CIRAD/INRA/IRD/SUPAGRO/UM2. Laboratoire

R. Duponnois, IRD. UMR 113. CIRAD/INRA/IRD/SUPAGRO/UM2. Laboratoire

Etelvina Figueira, Centre for Cell Biology, Departamento de Biologia,

A. Galiana, CIRAD. UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2. Laboratoire

Elisa Gamalero, Dipartimento di Scienze dell’Ambiente e della Vita, Università

Bernard R. Glick, Department of Biology, University of Waterloo, Waterloo,

Mitiku Habte, University of Hawai’i at Manoa, College of Tropical Agriculture

M. Hafidi, Equipe Ecologie et Environnement, Faculté des Sciences Semlalia,

Hamdi Hussein Zahran, Botany Department, Faculty of Science, Beni-Suef

Azeem Khalid, Department of Environmental Sciences, PMAS Arid Agriculture

Tariq Mahmood, Department of Environmental Sciences, PMAS Arid

Biswapati Mandal, AICRP on Soil Test Crop Response Correlation, Directorate

Javed Musarrat, Department of Agricultural Microbiology, Faculty of

Nelson-Walter Osorio, Universidad Nacional de Colombia at Medellin, College

L. Ouahmane, Equipe Ecologie et Environnement, Faculté des Sciences

Mohammad Oves, Department of Agricultural Microbiology, Faculty of

K.R.E. Padmathilake, Biological Nitrogen Fixation Project, Institute of

Y. Prin, CIRAD. UMR 113 CIRAD/INRA/IRD/AGRO-M/UM2. Laboratoire des