Soil Science

CARBOXYLATE RELEASE AND PHOSPHORUS
ACQUISITION IN WHEAT AND MAIZE
By
SHAZIA IQBAL
2007-ag-2091
M.Sc. (Hons.) Agriculture
(Soil Science)
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
SOIL SCIENCE
INSTITUTE OF SOIL & ENVIRONMENTAL SCIENCES

FACULTY OF AGRICULTURE,
UNIVERSITY OF AGRICULTURE
FAISALABAD, PAKISTAN
2020
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MY ALLAH and PROPHET MUHAMMAD (PBUH)
The Greatest Social Reformer and a Model for Mankind
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Acknowledgments
All magnificence to Almighty ALLAH, the Most Beneficent and Merciful, whose
exaltation and blessings thriven my determinations and thoughts to have the relish fruit of
my diffident struggle in the form of this thesis. My distinct praise to His Holy Prophet
Muhammad (peace be upon him) who is the flame of everlasting guidance for mankind.
With profound gratitude and a deep sense of devotion, I wish to thank my worthy
supervisor Prof. Dr. Javaid Akhtar, Professor, Institute of Soil and Environmental
Sciences for his guidance, critical review, precious comments and cooperative attitude
during the study period and preparation of this thesis. I also wish to show gratitude to my
committee members, Dr. Zulfiqar Ahmad Saqib, Assistant Professor at Institute of Soil
and Environmental Sciences and Dr. Rashid Ahmad, Professor, Department of
Agronomy for their erudite recommendations and esteemed comments in completion of
this thesis. I would also like to thank Dr. Tayyaba Naz, Lecturer, at the Institute of Soil
and Environmental Sciences, for her guidance and corporation in every step of this
research project.
I am very thankful to Higher Education Commission of Pakistan for providing all

the funds under the Indigenous PhD fellowship Phase 2, Batch-2 and giving me a chance
to fulfill my dream.
I feel proud to mention the feelings of commitments towards my family and

colleagues who helped and encouraged me during my work.
Shazia Iqbal
2007-ag-2091
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3.2.2. Sterilization of seeds 26
3.2.3. Nursery raising and transplantation 26
3.2.4. Growth conditions 26
3.2.5. Root exudates collection 27
3.2.6. Analysis of Carboxylates 27
3.2.7. Plant measurements 28
3.2.8. Plant Analysis 28
3.2.9. Phosphorus determination 29
3.2.10. Phosphorus acquisition parameters measurement 29
3.2.11. Statistical analysis 30
Study 2: Effect of carboxylates (organic acids) on P adsorption
3.3. 30
(Incubation study)
3.3.1. Pre-sowing soil Analysis 30
3.3.2. Experimental procedure for P adsorption 33
3.3.3. Experimental procedure for carboxylate effects on P adsorption 34
3.3.4. Analysis of adsorption data 34
3.3.5. Statistical Analysis 35
Study 3: Influence of soil types and deficient levels of

3.4. phosphorus on the release of carboxylates and phosphorus 36
acquisition characteristics in wheat and maize
3.4.1. Soil preparation 36
3.4.2. Growth conditions 36
3.4.3 Treatment plan for wheat 37
3.4.4. Treatment plan for maize 37

3.4.5. Harvesting 37
3.4.6. Plant measurement 38
3.4.7. Total chlorophyll content (SPAD value) 38
3.4.8. Physiological parameters 38
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3.4.9. Root exudate collection 38
Analysis of carboxylates, pH measurement, Plant analysis, and P

3.4.10 38
determination
3.4.11. P acquisition parameters measurement 38
3.4.12. Root Analysis 39
3.4.13. Statistical analysis 40
Chapter 4 Results and Discussion 41

Study 1: Effects of phosphorus on growth characteristics and
4.1 41
carboxylate release in wheat and maize genotypes
Effects of phosphorus on growth characteristics and release of
4.1.1. 42
carboxylate in wheat genotypes
4.1.1.1. Growth responses of wheat genotypes 42
4.1.1.2. Root P in various wheat genotypes 46
4.1.1.3. Shoot P in various wheat genotypes 46
4.1.1.4. Effects of P on Phosphorus use efficiency (PUE) in various 49

wheat genotypes
4.1.1.5. Effects of P on pH in various wheat genotypes 49
Effects of P on root mass fraction (RMF) in various wheat
4.1.1.6. 52
genotypes
4.1.1.7. Effects of P on citrate release in various wheat genotypes 52
4.1.1.8. Effects of P on oxalate release in various wheat genotypes 55
4.1.1.9. Effects of P on malate release in various wheat genotypes 55
Effects of phosphorus on growth characteristics and release of
4.1.2. 59
carboxylate in maize genotypes
4.1.2.1. Growth responses of maize genotypes 59
4.1.2.2. Root P in various maize genotypes 64
4.1.2.3. Shoot P in various maize genotypes 64
Effects of P on Phosphorus use efficiency (PUE) in various

4.1.2.4. 67
maize genotypes
4.1.2.5. Effects of P on PH in various maize genotypes 67
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Effects of P on root mass fraction (RMF) in various maize
4.1.2.6. 70
genotypes
4.1.2.7 Effects of P on citrate release in various maize genotypes 70
4.1.2.8. Effects of P on oxalate release in various maize genotypes 73
4.1.2.9. Effects of P on malate release in various maize genotypes 73
4.1.3. Discussion 77
4.1.4. Conclusion 82
Study 2: Effect of carboxylates (organic acids) on P adsorption

4.2. 83
(Incubation study)
4.2.1. P adsorption in three different textured soils 84
4.2.1.1. Phosphorus equilibrium concentration (C) 84
4.2.1.2. Adsorbed P 85
Adsorption Isotherms for P (KH2PO4) in three different textured
4.2.1.3. 87
soils
4.2.1.3.1. Freundlich Equation 87
4.2.1.3.2. Langmuir Equation 87
4.2.2. Effect of carboxylates (Organic acids) on phosphorus adsorption. 92

4.2.2.1. Phosphorus adsorption in loam soil 92
4.2.2.2. Phosphorus adsorption in sandy loam soil 94
4.2.2.3. Phosphorus adsorption in sandy clay loam soil 94
Adsorption Isotherms for the interaction of P (KH2PO4) with
4.2.2.4. 95
carboxylates (organic acids) in three different textured soils
4.2.2.4.1. Freundlich equation 95
4.2.2.4.2. Langmuir equation 96
4.3. Study 3: Influence of soil types and deficient levels of P on the

release of carboxylates and P acquisition characteristics in wheat 112
and maize
4.3.1. Influence of soil types and deficient levels of P on the release of 113
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carboxylates and P acquisition characteristics in wheat
4.3.1.1. Growth responses 113

4.3.1.2. Root morphological responses 118
4.3.1.3 Phosphorus acquisition parameters 120
4.3.1.4 Change in rhizosphere pH of wheat 126
4.3.1.5 Carboxylates release by wheat roots 127
4.3.1.6. Physiological responses 129
Influence of soil types and deficient levels of P on the release of
4.3.2. 132
carboxylates and P acquisition characteristics in maize
4.3.2.1. Growth responses 132
4.3.2.2. Root morphological responses 136
4.3.2.3. Phosphorus acquisition parameters 140
4.3.2.4. Change in rhizosphere pH of maize 145
4.3.2.5. Carboxylates release by maize roots 145
4.3.2.6. Physiological parameters 147
Pearson Correlation coefficients between phosphorus acquisition
4.3.2.7. parameters, root characteristics, carboxylate and pH in wheat and 152
maize
4.3.5. General Discussion 163
Chapter 5 Summary 171
References 173
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List of Tables
No. Title
Page
2.1. Rhizosphere acidification in different plant species 12
2.2. Name, chemical and molecular formula and number of carboxyl groups of 12
most common carboxylates
3.1. Physico-chemical properties of soils used in the experiment 32
4.1. Shoot fresh and dry weight and shoot length of wheat genotypes under 43
normal and deficient P
4.2. Root fresh and dry weight, root length and Root: Shoot of wheat genotypes 45
under normal and deficient P
4.3. Classification of wheat genotypes on the basis of % increase in total 58

carboxylate production in root under P deficiency compared to normal P
4.4. Shoot fresh and dry weight and shoot length of maize genotypes under 60
4.5. Root fresh and dry weight, root length and Root: Shoot of maize genotypes 62
under normal and deficient P
4.6. Classification of maize genotypes on the basis of % increase in total 76

4.7. Amount of P in soil solution and P adsorbed 86
4.8. Correlation coefficients (R2) and regression equations for adsorbed P and 86
added P
4.9. P sorption capacity and sorption energy for loam, sandy loam, and sandy 89
clay loam soil.
4.10. Model and linear form of Freundlich Equation 89
4.11. Maximum P adsorption capacity and the constant of the energy of adsorption 89
as determined by the Langmuir model
4.12. Effect of carboxylate (organic acids) application on P adsorption in three 93
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different textured soils
4.13. Freundlich adsorption isotherm parameters for P adsorption affected by 98

carboxylates (organic acids) application in three different textured soils.
4.14. Langmuir adsorption isotherm parameters for P adsorption affected by 99

carboxylates (organic acids) application in three different textured soils.
4.15. Shoot length, shoot fresh and dry weight of wheat in loam, sandy loam, and 114
sandy clay loam soils under different P levels
4.16. Root length, root fresh and dry weight of wheat in loam, sandy loam and 115
4.17. Root mass fraction, Root: Shoot and Root volume of wheat in loam, sandy 117
loam and sandy clay loam soils under different P levels
4.18. Root surface area, Number of root tips and Average root diameter of wheat 119
in loam, sandy loam and sandy clay loam soils under different P levels.
4.19. Rhizosphere soil available P, root P and shoot P of wheat in loam, sandy 121
loam and sandy clay loam soils under different P levels.
4.20. Phosphorus acquisition efficiency (PAE), Phosphorus use efficiency (PUE) 123
and Phosphorus uptake of wheat in loam, sandy loam and sandy clay loam
soils under different P levels.
4.21. Phosphorus physiological efficiency index (PPEI), Phosphorus stress factor 125
(PSF) and pH of wheat in loam, sandy loam and sandy clay loam soils under
different P levels.
4.22. Carboxylates (citrate, malate, and oxalate) release by wheat roots in loam, 128
sandy loam and sandy clay loam soils under different P levels.
4.23. Chlorophyll, Transpiration rate, Photosynthetic rate, and Respiration rate of 131
wheat plants in loam, sandy loam and sandy clay loam soils under different P
levels.
4.24. Shoot length, shoot fresh and dry weight of maize in loam, sandy loam, and 133
sandy clay loam soils under different soils under P application
4.25. Root length, root fresh and dry weight of maize under different soil types at 135
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varying level of P application
4.26. Root mass fraction, Root: Shoot and Root volume of maize in loam, sandy 137
loam and sandy clay loam soils under different P levels
4.27. Root surface area, Number of root tips and Average root diameter of maize 139
in loam, sandy loam and sandy clay loam soils under different P levels.
4.28. Rhizosphere soil available P, Root and shoot P of maize in loam, sandy loam 141
and sandy clay loam soils under different P levels.
4.29. Phosphorus acquisition efficiency (PAE), Phosphorus use efficiency (PUE) 143
and Phosphorus uptake of maize in loam, sandy loam and sandy clay loam
4.30. Phosphorus physiological efficiency Index (PPEI), Phosphorus stress factor 146
(PSF) and pH of maize in loam, sandy loam and sandy clay loam soils under
different P levels.
4.31. Carboxylates (citrate, malate, and oxalate) release by maize plant roots in 148
loam, sandy loam and sandy clay loam soils under different P levels.
4.32. Chlorophyll, Transpiration rate, Photosynthetic rate, and Respiration rate 151
of maize plants in loam, sandy loam and sandy clay loam soils under
different P levels
4.33. Pearson Correlation coefficients between P acquisition parameters, Root 153
characteristics, Carboxylate and pH in wheat
4.34. Pearson Correlation coefficients between P acquisition parameters, Root 154
characteristics, Carboxylate and pH in maize
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List of figures
No. Title age
2.1. Mechanism of increased carboxylates production under P deficiency 18
2.2. Mechanism of carboxylate secretion from the root 20

2.3. Mechanism of Phosphorus mobilization by carboxylates in the 20
rhizosphere
4.1. Impacts of P application and deficiency on root P 47
4.2. Impacts of P application and deficiency on shoot P 48
4.3. Impacts of P application and deficiency on PUE 50
4.4. Impacts of P application and deficiency on pH 51
4.5. Impacts of P application and deficiency on RMF 53
4.6. Impacts of P application and deficiency on citrate release in the root 54
4.7. Impacts of P application and deficiency on oxalate release in the root 56
4.8. Impacts of P application and deficiency on malate release in the root 57
4.9. Impacts of P application and deficiency on root P 65
4.10. Impacts of P application and deficiency on shoot P 66
4.11. Impacts of P application and deficiency on PUE 68
4.12. Impacts of P application and deficiency on pH 69
4.13. Impacts of P application and deficiency on RMF 71
4.14. Impacts of P application and deficiency on citrate release in the root 72
4.15. Impacts of P application and deficiency on oxalate release in the root 74
4.16. Impacts of P application and deficiency on malate release in the root 75
4.17. Total carboxylates (citrate, malate, and oxalate) released by wheat and 80
maize in P+ (a) and P- (b) treatments.
4.18. Graphical presentation of Freundlich adsorption isotherm for 90
phosphorus in loam, sandy loam, and sandy clay loam soils
4.19. Graphical presentation of Langmuir adsorption isotherm for 91
phosphorus in loam, sandy loam, and sandy clay loam soils
4.20(a) Graphical presentation of Freundlich adsorption isotherm in loam soil 100
for P adsorption
4..20(b) Graphical presentation of Freundlich adsorption isotherm in sandy 101
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loam soil for P adsorption
4.20(c) Graphical presentation of Freundlich adsorption isotherm in sandy clay 102
4.21(a) Graphical presentation of Langmuir adsorption isotherm in loam soil 103
for P adsorption
4.21(b) Graphical presentation of Langmuir adsorption isotherm in sandy loam 104
soil for P adsorption
4.21(c) Graphical presentation of Langmuir adsorption isotherm in sandy clay 105
4.22. Total carboxylates (citrate, malate, and oxalate) exuded by wheat and 160
maize roots
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ABSTRACT
Phosphorus (P) is one of the 17 essential nutrients and a key element for the development
of the plant. Although soils are rich in total P, its large portion remains unavailable to
plant due to fixation with calcium (Ca+2) and magnesium (Mg+2) in alkaline soils. This
results in the increased application of P fertilizers to improve the acquisition of P. It is
vital to improve the efficient phosphorus use and acquisition of P, to get high yields of the
crop to meet up the worldwide demand of food. To check the growth and physiological
response of plants at lower levels of P application, this research work was planned. It was
hypothesized that a lower level of P application might optimize the growth of a plant by
the release of carboxylates and the adoption of acquisition characteristics that ultimately
may reduce the fertilizer cost. Firstly, five maize and wheat genotypes were screened on
the basis of carboxylate release and P acquisition characteristics in solution culture
technique under normal and deficient P application. Wheat genotype, SARC-1, and maize
genotype Pioneer-32F10 performed better than other genotypes in the release of
carboxylates and improving root to shoot ratio, root mass fraction and phosphorus use
efficiency. In 2nd study, the effect of carboxylates on adsorption of P in loam, sandy loam,
and sandy clay loam soils was studied. Three important carboxylates, citric acid, malic
acid, and oxalic acid were used in two concentrations (0.5 mM and 1.0 mM) in three-
gram soils at five P application levels 100, 200, 300,400 and 500 mg kg-1 and then data
were fitted to Langmuir and Freundlich models. Phosphorus adsorption decreased with
carboxylates application and a higher level of carboxylates application proved better in
the reduction of P adsorption. Among carboxylates, citric acid was more effective in the
reduction of P adsorption. Data were fitted best to both Freundlich and Langmuir model.
Among soils, in sandy loam soil, lower adsorption was observed compared to loam and
sandy clay loam soil. The maximum P sorption capacity (Pmax) was lower for sandy loam
soil at 1 mM citric acid application and higher was measured in sandy clay loam soil at
0.5 mM malic acid application. In a 3rd study, selected genotypes of wheat and maize
from study-1 were grown in three different textured soils at five P application rates lower
than the recommended dose of each crop. At flowering, plants were harvested and
carboxylates from rhizosphere were collected. Other necessary parameters were
measured. The lower level of P application increased carboxylates exudation through
roots compared to the higher P application rate in all three soils. Malate was the dominant
carboxylate released by both wheat and maize. The concentration of exuded carboxylates
was higher for maize crop compared to wheat. Rhizosphere available P and proton release
also increased with increasing carboxylate exudation, but it did not affect the root and
shoot P. Root and shoot P increased with increasing P level but at 33.75 and 45 mg P
application kg-1 higher shoot P was observed than 45 and 60 mg P application kg-1 in
wheat and maize, respectively. In the case of soils, plants in sandy loam soil released a
low concentration of carboxylates and in sandy clay loam soil, the release was higher than
other soils in both wheat and maize. At a lower level of P application, metabolite
partitioning was observed that resulted in increased root growth and decreased shoot
growth. At P application level lower than the recommended dose (33.75 and 45 mg P
application kg-1 for wheat and maize), an increase in P acquisition was observed. It is
concluded that plants released more carboxylates under low P and ultimately increased
the rhizosphere soil available P. It is also concluded that despite the higher release of
carboxylate, improved rhizosphere available P and root growth at 0 mg P application kg-1,
it was not an effective P application level for crop growth. Phosphorus applications at
33.75 mg kg-1 for wheat and 45 mg kg-1 for maize in sandy loam soil is an economical
xviii
and effective level for optimized growth and higher acquisition compared to 45 mg P
application kg-1 (wheat) and 60 mg P application kg-1 (maize).
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CHAPTER 1
INTRODUCTION
Green revolution boosted up agricultural yield in developing countries by familiarizing
rice and wheat dwarf genotypes efficient to fertilizer response without lodging. There is a
need for another green revolution to enhance crop yields grown in less fertile soils. Low
soil fertility is due to low access of farmers to fertilizers as well as the low availability of
applied fertilizers in most parts of the world. The main aim of the second green revolution
will be the improvement of breeding techniques for low nutrient tolerant crops (Lynch
and Brown, 2001; Lynch, 2007).
Phosphorus (P) is a vital nutrient (Gyaneshwar et al., 2002; Dorahy et al., 2004) and it
contributes in metabolic processes during energy transformation and plays crucial role in
root development, seed and flower formation, strengthening of stem and stalk, nitrogen
fixation in legumes and disease resistance in many crop species (Gyaneshwar et al., 2002;
Hao et al., 2002; Achal et al., 2007). P deficiency is the major limiting factor for growth
as well as the productivity of crops (Raghothama and Karthikeyan, 2005). Globally, about
90% arid to semi-arid soils are undersupplied to available P (Mehrvarz and Chaichi,
2008). Despite high soil P contents (Abou El-Yazeid and Abou-Aly, 2011) its availability
to plants is quite low because of the chemical nature of soils (Hussain et al., 2013).
Among the macronutrients, P is mostly studied by the researchers due to low solubility in
the soils and immobility from soil to plants.
Phosphorus in the soil is classified as organic P and inorganic P. However, a little of total
P (0.1% or 1 ppm) is available for plants because of lower solubility and high P adsorbing
capacities (Mahidi et al., 2011). Plants uptake P as anions of phosphate (HPO4-2 or H2PO4-
) from the soil solution but these phosphate anions are reactive and become inaccessible
for plants. When P fertilizers are applied in soil, they often become intricate due to the
complex formation with cations in low pH soils (Hao et al., 2002; Dorahy et al., 2004),
and calcium and magnesium in high pH soil (Khan et al., 2007; Mittal et al., 2008).
Overall P utilization efficiency is lower than optimum P utilization efficiency in Pakistani
soils (Vance, 2001).
In calcareous soils, high calcium content is the reason for low P recovery (Aziz et al.,
2006). Managing P fertilization deprivation by chemical means typically distresses the
soil health and affects the production cost (Gyaneshwar et al., 2002). Continuous and
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imprudent use of P fertilizers results in (1) reduction in soil fertility (Gyaneshwar et al.,
2002), (2) reduced agronomic crop yield (3) interruption in metabolic activities and
diversity of microbes (Khan et al., 2009). Whereas, the key objective of management of
soil-applied P should be to get optimum crop growth and improve the mobility of P
(Fasim et al., 2002).
Under these conditions, there is a need to develop and grow those crops that are tolerant
to low P. Some plants have natural adaptability to low soil P concentrations. For survival,
these plants have many mechanisms to compensate for P deficiency. Therefore, they are
well-thought-out as a model to study plant under P deficiency (Hu et al., 2010). Some
plants are known for using different pools of soil P due to either carboxylate exudation or
proton release (Hinsinger, 2001; Richardson et al., 2009b). A variety of plants can
withstand low P in soil by developing special characteristics such as, release of large
quantities of carboxylates in the rhizosphere, release of acid phosphatases and
modifications of rhizosphere pH (Skene, 2003; Hu et al., 2010), and formation of
specialized roots (cluster roots, duciform roots) (Shane et al., 2004). Along with these
characteristics, plants also showed many physical changes (Linkohr et al., 2002).
Phosphorus tolerant plants also showed physiological changes that include high internal
phosphorus use efficiency (PUE) that is measured by the low requirement of internal P
and efficient remobilization of P from older tissues. Under low P availability, some
species of Lupinus produces specialized cluster roots and secrete carboxylates that help in
enhancing P acquisition (Lambers et al., 2013). Plant species diverge in the volume of
root exudates (P-mobilizing) and differ in the distribution of these exudates in the root
zones (Dakora and Phillips, 2002). The release of carboxylates especially malate and
citrate also vary from monocots to dicots in the rhizosphere, as dicots are more efficient
in the release of carboxylates as compared to monocots (Raghothama, 1999).
Carboxylates release through plant roots is the main process for the P solubilization in
soil solution when P is marginally soluble. Carboxylates are organic anions among the
organic acids with varying chain length, having a different number of carboxyl groups in
their structure (Jones, 1998). Most common carboxylates are citrate, malate, oxalate,
fumarate, succinate, etc. They are categorized as mono, di, and tri carboxylates based on a
number of carboxyl groups. Tri-carboxylates have more affinity for sorption sites than di-
carboxylates and di-carboxylates have more affinity than mono-carboxylates.
Carboxylates compete with phosphate for sorption sites and have a better affinity for
2
sorption sites. Carboxylates bind to the sorption site that results in P release to soil
solution, ultimately relieving plant from P stress (Jones et al., 2003). Availability of other
(zinc, Ca, iron) nutrients also improved due to these carboxylates (Ryan et al., 2001). In
more efficient cluster roots, carboxylates release is enhanced and accessibility of P
around root surface also enhanced (Shane and Lambers, 2005). Metabolic enzyme
Phosphoenol Pyruvate Carboxylase (PEPC) is important in the release of carboxylates.
Increased PEPC activity caused higher carboxylates exudation in shoots of rape plants
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under P deprivation conditions (Hoffland, 1992). In white lupin shoot and root C-
labeling study, it was observed that almost 30 percent of roots released carbon (in the
form of carboxylates, mainly citrate) comes from the dark reaction in which PEPC has an
important role (Johnson et al., 1996).
Phosphorus availability due to mobilization by carboxylates and induction of carboxylates

due to the deficiency of P have been tested in many plant species, e.g. in chickpea,
exudation is an essential trait with fewer P benefits. Phosphorus deprivation in mature
cluster roots of Lupinus and Banksia species caused a release of high concentrations of
carboxylates (Shane and Lambers, 2005). However, this kind of behavior is not seen in
many other species (Wouterlood et al., 2005). The release pattern of carboxylates differs
between species. Carboxylates (main malonate) release by chickpea not only helped to
improve P acquisition but also reduced the microbial degradation of carboxylates
(Wouterlood et al., 2004, 2005). Changes in the degree of carboxylates ionization
changed soil chemistry due to the fluctuations in rhizospheric pH (Hinsinger et al., 2003).
Rhizospheric acidification noticed by the enhanced H+ (Proton) extrusion from the L.
albus roots in low P supply (Sas et al., 2001). Reason behind the proton extrusion from
plant roots is to sustain electro neutrality inside plant, under fewer anions’ intake than
cations (Hinsinger et al., 2003). Several plant species, rape, tomato, and chickpea released
protons under P deficiency and acidify their rhizosphere (Sas et al., 2001). In white lupin,
increased citrate exudation and proton release by cluster roots were recorded during P
deficiency (Shen et al., 2005). Faba beans released protons in calcareous soil and
increased P use in cereal (Li et al., 2007).
Wheat (Triticum aestivum L.) is a dietary food. Pakistan is ranked as 4th among the
wheat-producing countries of the world and as the main source of diet for the people of
Pakistan with average per capita consumption is about 125 kg of wheat (GOP, 2015).
Keeping in mind the importance of wheat crops in Pakistan, there is a need to work on the
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yield reducing factors such as P unavailability to plants. In Pakistani soils, due to high
calcium contents, an applied chemical P fertilizer gets fixed and become inaccessible.
This result in increased fertilizer application rate and ultimately the cost of production
also increased. In this situation, like many other crops, wheat also has the ability to adapt
mechanisms that efficiently help the plants in utilizing fixed or unavailable P. Release of
carboxylates especially malate in wheat (C3 metabolism) was observed (Jones, 1998). In
Buckwheat, under low P availability, proton excretion is improved that helped in P
acquisition (Bekele et al., 1983). Phosphorus utilization efficiency in wheat is associated
with high root length (Manske et al., 2000) and a high R/S ratio (Bhadoria et al., 2001).
Along with wheat, maize is also a major food grain crop in Pakistan and dwells in a
substantial place in the agricultural economy of the country. It is ranked as the 3rd most
important cereal crop (Chaudhary, 1983). Being a highly responsive crop, maize needs
high nutrients fertilization especially P. In Pakistani soil’s conditions, P availability to
maize is low which results in stunted growth and maize leaves show purple discoloration
on margins. Under low P condition, maize roots release exudates for solubilizing fixed P
in soil from the rhizosphere. Because of C4 metabolism, maize is highly efficient in
nutrient utilization efficiency especially nitrogen and P (Ghannoum et al., 2008; Vogan
and Sage, 2011). Plant metabolism also affects internal carboxylates release and C4 plants
released more carboxylates by roots as compared to C3 plants (Johansson et al., 2009;
Hajlaoui et al., 2010; Nabais et al., 2011). Higher release of malate in wheat and of citrate
in maize has been observed under P stress environment (Zhang et al., 2001).
Keeping in view the above information, research was conducted on carboxylate release
and P acquisition in wheat and maize. The main objectives of this research were
➢ To explore the genotypic variability of wheat and maize for the potential of
carboxylate release under low P availability.
➢ To check the influence of carboxylate on P availability in different types of soils

(loam, sandy loam, and sandy clay loam).
➢ To study different levels of P application on P acquisition characteristics, plant

growth and release of carboxylates in the rhizosphere of wheat and maize in
different types of soils.
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➢ To study the relation between the increase in proton release along with
carboxylates in the rhizosphere of wheat and maize at P levels and to optimize the
level of P for optimum growth of wheat and maize.
Problem statement and thesis outline
Pakistani soils are alkaline and rich in calcium content because of arid to a semiarid
climate which results in low availability of many nutrients especially P. Under alkaline
soils, the P form insoluble complexes with calcium with increased soil pH and reduced
the P availability. PUE of applied P is only 15-20%. In calcareous soils, the major reason
for the low recovery of applied P is the calcium content (Aziz et al., 2006). Plants living
in these depleted soils must be able to acquire and utilize P more efficiently (Lambers et
al, 2006). There is now a requisite for another Green Revolution to enhance crop growth
in less fertile soils. Plants adapt to a low P environment by releasing carboxylates through
roots in the rhizosphere. These carboxylates compete with phosphate ion for adsorption
sites in the soil. It was hypothesized that carboxylate release by plant root might be an
effective approach to cope with limiting P availability and acquiring P efficiently at the
lower P application level.
The first study consisted of two parts: In the first part, the genotypic variability of five
wheat genotypes on the basis of carboxylate release in P deficient condition was studied
and one genotype with higher relative carboxylate releasing ability in P deficient
condition was screened out. In the second part, the genotypic variability of five maize
genotypes on the basis of carboxylate release in P deficient condition was studied and one
genotype with higher relative carboxylate releasing ability and p acquisition
characteristics was screened out. In second study, focus was on checking the role of
carboxylates on P adsorption in a controlled lab environment. The third study focused on
the response of selected wheat and maize genotypes to different P levels in three different
soils. The release of carboxylates and P acquisition characteristics at different P levels in
three different soils was checked out and the relation of these carboxylates with low P
acquisition characteristics was studied.
5
CHAPTER 2
REVIEW OF LITERATURE
Rock P, a source of P, is expected to end in the next 60 to 80 years (Smil, 2000; Cordell
et al., 2009). Phosphorus is crucial for optimal plant growth as well as completing life
cycle of the plant. As a macronutrient, P is required in large amounts by crops (Lal,
2002). Phosphorus in the soil is chemically reactive. Inorganic P in the soil largely occurs
as precipitated or adsorbed P. Moreover, the organic P form constitutes 80% of total
surface soil P (Vance et al., 2003; Trolove et al., 2003). A minimal percentage of (0.1~10
μM) is in the plant available form, as phosphate ions (Frossard et al., 2000). Phosphorus
shares part as a basic structural and functional unit of the cell, like nucleic acids, energy
transporters, and phospholipids. Phosphorus regulates the activities of enzymes (Kuo and
Chiou, 2011), involves in signal transduction chains and in pathways of energy
metabolism (Péret et al., 2011). Phosphorus is the yield determining mineral nutrient of
crops (Chaudhary et al., 2003). In Pakistani soils, due to high calcium (Ca) contents and
high pH, the availability of P is a major issue. At high pH (> 8), phosphate ion forms
compounds with calcium that makes P unavailable for plant uptake (Ahmad et al., 2003;
Rashid et al., 2005). So, it is a need of the hours to improve the plant's P availability for
high yield. To gain high yield, the management of chemical fertilizers is very important.
But Managing P fertilization deprivation through chemical methods typically distresses
the soil health and affects the cost of (Gyaneshwar et al., 2002). Phosphorus deficiency
can slow down nucleic acid and protein synthesis that results in tissue accumulation of
soluble nitrogen compounds and finally affects cell growth. Phosphorus deficiency
symptoms include lowering in plant height, reduction in dry matter, delayed leaf
emergence, secondary root development, and a number of tillers and decreased seed
production (Hoppo et al., 1999).
2.1. Problem of P availability in Pakistani soils
The concentration of soil available inorganic P rarely goes above 10 milli molars
(Bieleski, 1973), far lesser than the P in tissues that is up to 20 milli molar inorganic P
(Raghothama, 1999). The major forms of P amendments are mono-calcium phosphate
and mono-potassium phosphate. When mono-calcium phosphate is applied to the soil, di-
calcium phosphate (DCP) and many protons are released (Benbi and Gilkes, 1987). Then
DCP is converted to octa-calcium phosphate and hydroxyapatite (HAP), which are more
6
stable forms and less plant available at basic pH (Arai and Sparks, 2007). In calcareous
soils, > 50% of inorganic P comprised of HAP, is due to long term fertilizer experiments.
At this stage, a decrease in pH increased HAP dissolution (Wang and Nancollas, 2008).
Soil mineral P adsorption mostly exists in the outer zone because of comparatively high
inorganic P concentration in that zone (Moody et al., 1995).
2.2. Strategies to improve P availability
For alkaline and calcareous soils, several P fertilizer management strategies are used to
improve P nutrition for plants, namely: 1) Use of complexed P fertilizer, 2) high rates of P
fertilizer, 3) cation complexing P fertilizer, 4) balancing P with other nutrients, 5) slow
release P fertilizer, and 6) seasonal application of P fertilizer. For plants in calcareous and
alkaline soils, these strategies can be used in combination to supply P efficiently.
2.3. Plant adaptation to low P availability
Various Plants developed a variety of mechanisms and special features for acclimatizing
to lower P. These mechanisms include biochemical, physiological, and morphological
changes. For example, rhizosphere pH change through the release of protons (Hu et al.,
2010), partitioning of dry mass, a higher amount of carboxylates in root areas, release of
phosphatase enzymes (Skene, 2003), and the development of specialized cluster roots
(Shane et al, 2004). These changes are collectively called the Pi starvation response
(PSR) (Niu et al., 2013). Under P deficiency, these responses are a result of a
manifestation of PSI (Pi-starvation inducible) genes. This involves in sustaining and
efficient utilization of inner phosphate or consenting better contact with soil phosphate.
Thus, phosphate availability is increased by these modifications in the rhizosphere that
ultimately results in increased phosphate uptake and use (Richardson et al., 2009a;
Plaxton and Tran, 2011). White lupin successfully adapts these P starvation mechanisms
(Cheng et al., 2011).
2.3.1. Morphological adaptations of plants
2.3.1.1. Root architecture
Morphological traits of roots are helpful in P acquisition because of the movement of P in

soil (Hodge, 2004). In response to P starvation, root architecture is changed in plants.
Changes in root architecture include an increase in R/S ratio, change in metabolite
distribution patterns, root elongation, root hairs, root topsoil foraging, root morphology,
and topology. Formation of proteoid roots also occurs in some species (Vance, 2008;
7
Fernandez and Rubio 2015). Gravitropism in roots controls the root architecture and it
adjusts the topsoil root system. Genotypes or species with shallow root systems should be
chosen for P uptake because of high labile P concentration in the surface soil (Gamuyao
et al., 2012). In upper soil layers, high root length in wheat is an important characteristic
in improving P acquisition in P stress (Manske et al., 2000).
P deprivation results in the enhancement of root length and it also causes primary root
growth reduction (Lopez et al., 2003; Desnos, 2008). Many species also showed the
capability to grow more determinative and dense lateral roots with more root hair
(Lambers et al., 2006; Vance, 2008). So, the structural design of root shows a significant
part in enhancing P uptake (Lynch, 1995). Some root system adjustments in P deprivation
are recognized in Arabidopsis (Lambers et al., 2006; Desnos, 2008; Rouached et al.,
2010) and maize (Postma et al., 2014; Miguel et al., 2015). These adaptive changes under
P deprivation include alteration in carbohydrates distribution among shoots and roots. P
efficient common bean genotypes showed shallower roots in upper layer of soil as
compared to the layer where relatively more P contents are present (Lynch and Brown,
2008).
Root morphology differs in low P supply. Different native Australian grasses showed a
positive correlation between root length, mass, root hair cylinder volume and P uptake at
a different level of P supply, while in the case of B. hordeaceus, reduction in root length
significantly reduced the expected phosphor uptake at low P. Root hair length, specific
root length and root mass ratio ranged from 1.5-2.7 folds between low and high P supply
(Waddell et al., 2017).
2.3.1.2. Root biomass
When P is restrictive for plant growth, many plant species contribute more biomass to
roots than shoots (Hermans et al., 2006). Variation in biomass allocation among plants
that are grown with a low and high P supply may be due to their genetic makeup (Kemp
and Blair, 1994). Though, P application participates in partitioning of biomass without
their genetic variability (De Groot et al., 2001). Many species of lupinus plant are
considered as P efficient does not show a significant change in the partitioning of biomass
under P deprivation. This little change is proving in many plants that are even P efficient
(Pearse et al., 2006b).
8
2.3.1.3. Root to shoot ratio
In P deficiency, plant assigns more core carbon to roots that result in enhanced root
growth, upsurge in root hairs length and number, and more root formation laterally (Liao
et al., 2001; Hermans et al., 2006; Postma et al., 2014; Lynch, 2015). These processes
lead to an increased area of roots that helps in extra soil exploration (Postma et al., 2014;
Lynch, 2015). More soil exploration increases the acquisition of P. When P efficient
genotypes are exposed to P deficient condition, these genotypes generally maintain high P
content in roots as compared to shoots and results in improvement of root system for
efficient nutrients uptake (Akhtar et al., 2008; Lynch, 2015; Miguel et al., 2015;
Schneider et al., 2017).
2.3.1.4. Root hairs
Root hairs are lateral extensions of roots. Growth of hairs of root is a significant strategy
that plants adapt under little P (Bates and Lynch, 2001; Li et al., 2016). Root hairs
intercept with P diffusing towards roots. Then they enhance the roots depletion zone by
exploring more areas for P (Ma et al., 2001; Hill et al., 2010; Zhu et al., 2010). Out of the
total surface area, up to 70% is contributed by root hairs, however, root length differs in
inter as well as intraspecies (Jungk, 2001). In legumes increased root hair length and
density are reported (Yan et al., 2004). The development of root hairs is very important in
response to soil P availability. Length of root hairs of tomato rape and spinach has
antagonistic effects on P concentration in the soil solution (Foehse and Jungk, 1983).
Species having longer root hairs are more efficient at acquiring inorganic P from soils e.g.
Lolium perenne than those species lacking these characteristics like Podocarpus totara,
and these species show less response towards plant growth (Clarkson, 1981). The
genotype of Hordeum vulgare tended to yield better in low P environment due to the
formation of longer root hairs as compared to genotypes with less root hair growth
(Gahoonia and Nielsen, 2004). A. thaliana has shown the variation in root hair
development due to ethylene and low Pi availability. Low P status leads to increased
numbers of root hair-bearing epidermal cells, increased cortical cell number and
decreased the size (Zhang et al., 2003). Root hairs require to shoot derived signals to
increase their length. Shoot intellects low P supply and translocate signals to root. Low P
status of the root can also trigger the root hair length signals (Jungk, 2001).
9
2.3.2. Physiological adaptations of plants
In P deprivation, plants adapt to a wide array of physiological strategies to ‘chemically

mine’ the P from soil reserves. These physiological adaptations include the release of
protons, organic anions (carboxylates) secretion, phosphatase enzymes secretion, and a
lot more compounds.
2.3.2.1. Proton release
Reason behind the extrusion of a proton from plant roots is to sustain electro neutrality
inside plant, because of fewer anions’ intake than cations intake (Hinsinger et al., 2003).
Several plant species, tomato, chickpea, and rape reported to release more protons under
P deficient conditions and acidify their rhizosphere (Sas et al., 2001; Richardson et al.,
2011; Lopez-Arredondo et al., 2014). P deprivation in white lupin arouses citrate
exudation and proton release by specialized cluster roots (Shen et al., 2005). Faba bean
released more protons in calcareous soil. This release of protons is noteworthy in
increasing P attainment in cereals (Li et al., 2007).
Rhizosphere acidification does not always increase P availability in all types of soil. The
effectiveness of rhizosphere acidification is related to the presence of acid-soluble forms
of P (calcium phosphate). Differences in dominant forms of P reduce the effectiveness of
some mechanisms in acidic soils that help in improving P uptake in calcareous soil. A
reduction in rhizosphere pH may enhance solubility of Ca-P complexes while in low pH
soils, dominating Al and Fe phosphates solubility improved with increasing pH (Li et al.,
2010). Rhizosphere acidification is because of the proton extrusion by plant roots
improves the rock phosphate utilization efficiency (Sas et al., 2002) but its role in P
mobilization from low quantities of acid-soluble P in highly weathered soil is not clear. In
strongly weathered soils correlation between rhizosphere acidification and uptake of P is
not obvious (Hedley et al., 1994). Forms of nitrogen applied, affect rhizosphere pH.
Assimilation of the ammonical form of nitrogen is linked with rhizosphere acidification,
while Nitrate form of nitrogen is related to alkalization effect in the rhizosphere (Tang
and Rengel, 2003; Tang et al., 2004; Shen et al., 2005). Rhizosphere acidification
induced by root can lower pH 2 to 3 units in rhizosphere comparative to bulk soil which
results in considerable solubilization of frugally available form of P in soil (Marschner,
1995). Legumes acidify the rhizosphere by absorbing more cations as compared to anions
(Sas et al., 2002). B. napus retained alkaline rhizosphere with an average pH of 7.4 at 300
10
µM of P while Lupinus species decreased pH from 6.7 to 4.3 in P deficient environment
(Pearse et al., 2006a).
An interesting aspect of cluster roots is their capacity to produce immense quantities of

carboxylates in P deficiency and acidify the rhizosphere where cluster roots in white lupin
reduced the pH 7.5 to 4.8 in high calcium soil i.e. 20% CaCO3 (Dinkelaker et al., 1989).
Rhizosphere pH can be reduced up to pH 3.6 by cluster roots. Rhizosphere acidification
by cluster roots is because of the huge concentration of anions being excreted by the root,
predominantly as malic and citric acid (Keerthisinghe et al., 1998). Highest rate of export
of organic acids by cluster roots is found in plants. The highest exudation activity is found
in mature cluster roots (Watt and Evans, 1999). Rootlets are so closely situated, leads to
acids accumulation and leading to high concentrations of carboxylates (Yan et al., 2004).
2.3.2.2. Carboxylates (Organic anions) release
Carboxylates are organic anions with varying chain length, having carboxyl groups.
These are secreted by the plant roots due to either P deficiency or accumulation of
aluminum (Aono et al., 2001; Wang et al., 2007; Carvalhais et al., 2010). By another
definition, carboxylates are organic anions, which are organic acids minus the proton(s).
Citrate is the result of de-protonation of citric acid (Lambers et al., 2015). Some
carboxylates, malate, oxalate, and citrate are an essential part of the primary metabolism
of plants (Wouterlood et al., 2004).
Carboxylates include citrate, malate, oxalate, acetate, pyruvate, fumarate, iso-citrate,

succinate, etc. These carboxylates help in plant metabolism, in increasing availability of
micronutrients, macronutrients, and metallic ions, and in soil aggregates stabilization
(Gerke et al., 2000a; Baise et al., 2006). These carboxylates also carry out many
physiological activities like solubilization of P, therefore enhance the availability of P.
Phosphorus is liberated in alkaline soil from Ca-P complexes and in acidic soils, from Al-
P complexes by the action of these carboxylates (Ryan et al., 2001; Yang et al., 2013).
Aluminum toxicity is reduced by the displacement of toxic aluminum in the plant roots
with organic anions in P deficiency (Chen et al., 2009; Arunakumara et al., 2013).
11
Table 2.1. Rhizosphere acidification in different plant species
Species Rhizosphere acidification Reference

White lupin 7.5-4.8 Dinkelaker et al., 1989
White lupin 7.0-3.6 Li et al., 1997
19891989
T. aestivum 6.9-6.8 Pearse et al., 2006a
V. faba 6.7-6.5 Pearse et al., 2006a
B. napus 7.5-7.2 Pearse et al., 2006a
L. culinaris 6.5-6.5 Pearse et al., 2006a
C. arietinum 6-6.2 Pearse et al., 2006a
P. sativum 6.1-6 Pearse et al., 2006a
L. luteus 5.1-4.7 Pearse et al., 2006a
L. pilosus 4.7-4.3 Pearse et al., 2006a
L. albus 5.3-5.1 Pearse et al., 2006a
L. atlanticus 6-5.7 Pearse et al., 2006a
L.angustifoliu 5.8-5.7 Pearse et al., 2006a
s L. mutabilis 5.85-5.9 Pearse et al., 2006a
L. cosentinii 5.5-5.3 Pearse et al., 2006a
Table 2.2. Name, chemical and molecular formula and number of carboxyl groups
of most common carboxylates
Carboxylates Formula Molecular -COOH
functional
weight (g/mol)
groups
Citrate Tri-carboxylate C6H8O7 192.124 3

Malate Di-carboxylate C4H6O5 134.078 2
Oxalate Di-carboxylate C2H2O4 90.03 2
Maleate Di-carboxylate C4H4O4 116.06 2
Tartrate Di-carboxylate C4H6O6 150.06 2
Glutamate Di-carboxylate C3H8O4 132.12 2
Succinate Di-carboxylate C4H6O4 118.09 2
Pyruvate Mono-carboxylate C3H4O3 88.06 1
Lactate Mono-carboxylate C3H6O3 90.08 1
Butyrate Mono-carboxylate C4H8O2 88.011 1
12
Carboxylate concentration varies from plant species to species, growth stages, soil type,
microorganism’s activity in the rhizosphere, various stress factors, nutrient availability,
mechanical impedance, environmental conditions (light intensity and temperature, etc.),
and type of plant metabolism (C3, C4, CAM) (Brimecombe et al., 2007; Damin et al.,
2010).
Carboxylates release by Banksia prionotes (Proteaceae) in the rhizosphere and its role in
P solubilization was observed. Citrate accumulation was high, and it was up to 72 μM g-1
soil. Such high concentrations of any carboxylates are enough to solubilize P in the
rhizosphere and it mobilized ‘available’ P up to 250% and total P of about 44% in the soil
(Pate and Watt, 2002). For substantial P solubilization, higher concentrations of
carboxylates, greater than 10 μmol g-1 soils are vital (Gerke et al., 2000a, b). Apart from
some cases, a small concentration of carboxylates in white lupin was released by plant
roots. Moreover, carboxylates can rapidly take up by soil micro flora. Hence, the role of
carboxylates in the acquisition of P is still not clear (Jones, 1998). Considerably higher
secretion of citric and oxalic acid was noticed in roots of P. Australis grown under saline
conditions created by seawater. The nature and extent of carboxylates release also depend
on the physiological stage and growth medium of plants (Rocha et al., 2015).
Carboxylates like citric, malic, and oxalic acids application alone or in combination
solubilized P in its habitat from the intensive P sorbing soil. Sand-binding root
specialization also improved P uptake in other habitats when P accessibility was very low
in non-mycorrhizal cactus (Abrahao et al., 2014). The chemical form of carboxylate
released by roots also affects the plant’s capacity to solubilize. Effect of citric acid and
potassium citrate on P solubilization and then acquisition by the plant was studied. K-
citrate was effective in increasing the availability of P than citric acid. Phosphorus uptake
was also higher when K-citrate was applied (Palomo et al., 2006).
2.3.2.2.1. Effect of P stress on carboxylate release
Plant alteration and persistence in a low nutrient environment are basically determined by
an individual’s ability to attain capitals (Aerts, 1999). Root system performs a major part
in resource acquisition (Lynch and Brown, 2001). The proclamation of organic
complexes by roots is the main feature of some plants in the acquisition of nutrients as
well as in facilitating plant and microbial interaction (Pierret et al., 2007).
13
Roots of plants react to different sources of different nutrients, as in P deprivation, cluster
roots are developed that releases carboxylate in the rhizosphere to improve P
solubilization (Shu et al., 2007). Carboxylates (especially di and tri carboxylates) release
in P deprived environment is increased by plant roots in dicotyledonous plant species
relative to enough P supply. These carboxylates mobilized P in P fixing soils. For
mobilizing fixed P, citrate was more efficient as compared to oxalate and then malate.
Quantitative effects of carboxylate on P release and its acquisition can only be proved
when rhizosphere chemistry and increased release of carboxylates are studied together
(Neumann and Ro¨ mheld, 2007).
Under a low P environment, many species showed the ability of carboxylate release
(Pearse et al., 2006a). Among all species, white lupin was considered more efficient
(Dinkelaker et al., 1989). Alfalfa and white lupin mainly secreted citrate (Watt and
Evans, 1999), while for wheat and maize; malate was shown to be the main carboxylate
(Jones, 1998). Piscidate and oxalate have also shown the P mobilizing ability in low P
environment and hence helps in inorganic P procurement by pigeon pea and sugar beet,
respectively. The main role played by carboxylates is mobilizing P from binding sites by
replacing phosphate ions (Gerke et al., 2000a, b). In general, carboxylates such as citrate,
as having three carboxyl groups, are more effective at releasing inorganic P because of
their high affinity soil adsorption sites than di and mono-carboxylates (Jones et al., 2003).
Root specializations (cluster roots) effectively solubilize and uptake P by releasing large
concentrations of carboxylates under low P availability in the rhizosphere. Disco cactus
placentiformis (Cactaceae), campos rupestres in a nutrient-poor environment, revealed its
high carboxylate exudation ability in the hydroponic environment as the level of applied
P was decreased. Among carboxylates, oxalate concentration was dominant. Malonate,
fumarate, citrate, lactate, and malate were also identified (Abrahao et al., 2014). Low
plant P status is also the triggering force for the increasing carboxylate exudation. Under
low P availability, different species respond differently in the release of carboxylates both
quantitatively and qualitatively. Quantitatively rhizosphere carboxylate concentrations
were lower for L. culinaris, V. faba, B. napus and T. aestivum relative to other species.
Species bearing cluster roots exuded more carboxylates. Species, as well as genus
variation, exist in the release of proton and carboxylates. Species also differed in the
qualitative release of carboxylates as C. arietinum and L. Culinaris mainly released
14
malonate and citrate while other species were dominant in the secretion of malate and
citrate (Pearse et al., 2006a).
Soil P level and plant development stage also had an effect on carboxylate release and
acquisition of P in chickpea cultivars. Chickpea cultivar Heera showed more carboxylate
concentrations in the rhizosphere as compared to cultivar Tyson. Carboxylate release was
also influenced by the root surface distance and development status of roots. Increased
carboxylate exudation was noted as the distance was increased from the apex of the root
in the soil. This might occur due to the mature part of the root as they exuded more
(Wouterlood et al., 2004).
The concentration of exuded carboxylates and extraction of P increased

disproportionately and simultaneously in four plant species. The species-specific response
was noticed for carboxylate release. Two herbaceous (Brassica napus, Orychophragmus
violaceus) plants and two woody (Morus Alba, Broussonetia papyrifera) plants were
grown under P deficiency. Carbon cost for exuded carboxylates is considered negligible,
as it was imitative from photosynthetic products of the plant. In calcareous soil, O.
Violaceus used lesser carboxylates and extracted more P than B. Napus. Similarly, in
calcareous soil, B. Papyrifera used lesser carboxylates and extracted more P than M.
Alba. Malate and oxalate showed more P extraction in Moraceae plants, while in
herbaceous cruciferous plants, citrate accounted for higher increment in P extraction.
Strongest P extraction ability at lost carbon cost was exhibited by O. Violaceus and B.
Papyrifera. More rapid response to P deficiency was exhibited by O. Violaceus, while the
slowest response was shown B. Napus. High extracting ability and rapid response to P
deficiency at low carbon cost may be considered the adaptability of O. Violaceus and B.
Papyrifera to a low P environment (Zhao and Wu, 2014).
P deficiency affected the release of carboxylate and proton in the rhizosphere in white
lupin plant. Proton extraction and carboxylate release (10% malate and 90% citrate) were
highly related to P supply. An increase in cluster root formation occurred as the shoot
growth decreased in the low P environment. The decrease in proton extrusion with an
increase in plant development was noticed. On the equi-molar basis, carboxylate release
by roots was two to three times lower than proton extrusion in P deficient condition.
Increased carboxylate release is due is associated with (proton) H+ extrusion under P
deficiency but it subsidizes only a part of total acidification (Sas et al., 2001). White lupin
is adapted by changing root architecture and the physiology to acquire sparingly soluble
15
inorganic P. In a solution culture, P deficiency of only 14 days, enhanced production and
release of carboxylates in shoot and root (Muller et al., 2014).
Carboxylate release is changed by a form of P applied. Phosphorus was applied at

different P levels (10, 20, 40 or 100 mg P kg–1 of sand) and sources were aluminum
phosphate Al (PO4) and potassium phosphate. Wheat accumulated fewer carboxylates
however acquired P efficiently. Rhizosphere pH of wheat increased but of Lupinus did
not change with level as well as a form of applied P (Pearse et al., 2006b). Increased
exudation of citrate was measured in the iron-phosphate supplemented boxes compared to
other P sources (Shu et al., 2007).
Carboxylate concentration and composition can be affected by intercropping and P form

as well as P supply in the rhizosphere. All these effects are species-specific. Maize, white
lupin and faba bean were grown alone and as an intercropping pattern. All species were
supplied with P sources FePO4 and KH2PO4 @ of 0–250 mg P. Carboxylate composition
in white lupin and maize differed by a form of P (FePO4 or KH2PO4), no effect was
noticed in faba bean. Phosphorus uptake was similar in intercropping and mono-cropping.
Carboxylate concentration was increased at a high KH2PO4 supply (>100 mg P kg−1) in
the rhizosphere of maize (Li et al., 2010).
Carboxylate makes adsorbed P available by replacing phosphate from adsorption sites as

bearing more affinity for these sites. Lupinus albus L. were grown with anion-exchange
resins. The soil was also amended with Al–P or Fe–P. Rapid complexation of
carboxylates in the P amended soil was sanctioned. Citrate was the main carboxylate
detected. The little amount of fumarate and malate was also measured. Qualitative
measurements of carboxylates were not affected by P source. However, citrate
concentrate decreased as the shoot P status improved. Citrate (79%) and malate (50%)
recovery were measured (Shane et al., 2008).
2.3.2.2.2. Mechanism of increased carboxylates production under P deficiency
Under P deficiency, the production of carboxylates increased in cells of roots that result
in increased release of these carboxylates into the rhizosphere (Figure 2.1) (Neumann et
al., 1999). Many enzymes of glycolytic pathways are involved in this process, but the
main enzyme is important in the increased production of carboxylates in phosphoenol
pyruvate carboxylase. Studies on roots and shoots of white lupin and rape plants showed
that increased enzyme activity enhanced carboxylates mainly citrate production up to
16
30% in dark CO2 fixation (Hoffland, 1992; Johnson et al., 1996). Consequently, roots
boosted gene, enhanced activities of respiration enzymes in vitro (sucrose synthase,
fructo-kinase, phospho- glucomutase) (Massonneau et al., 2001), and increased the
biosynthesis of carboxylate producing enzymes (Citrate synthase, malate dehydrogenase,
PEPC) (Uhde-Stone et al., 2003).
Under P deprivation, plants change their mechanisms in order to save internal P that is
stored in plant cells. This alteration includes the use of metabolic bypass reactions that are
not dependent on Pi, but they are dependent on inorganic pyrophosphate (PPi) and
phospholipids subsitution by galacto and sulfo-lipids in bio-membranes (Plaxton and
Tran, 2011). One bypasses reaction is glycolysis (PPi-dependent) that maintains the flow
of carbon to the citric acid (Krebs’s cycle). Additionally, an alternative oxidase (AOX) is
another bypass reaction of the mitochondrial electron transport chain. This is a non-
energy conserving pathway. So, at low levels of Pi and ADP, the respiration process can
be sustained. The AOX respiratory pathway, replacement of phospholipids and initiation
of PPi-dependent glycolysis reaction is well-documented adjustments found in P efficient
plants (Uhde-Stone et al., 2003; Wang et al., 2014). TCA reactions in P efficient plants
proceed very another way from TCA reactions in P deficient plants. Enhanced synthesis
of citrate occurred but the conversion of citrate to iso-citrate is inhibited due to decreased
activity of the enzyme involved in this conversion. This outcome in more accumulation of
citrate in the cell, that is then released in the rhizosphere (Neumann and Martinoia, 2002;
Kihara et al., 2003). Plants showed a reduced level of P related metabolites such as myo-
inositol-phosphate glucose-6-phosphate, glycerol-3-phosphate, and fructose-6-phosphate
(Muller et al., 2014).
2.3.2.2.3. Mechanism of carboxylate secretion from the root
Root cap and root hairs mainly participate in the secretion process by roots (Figure 2.2)
(Pineros et al., 2002; Nguyen, 2003). Root hairs are epidermal cell extensions and
comprise about 77% of the total surface area of the root. Root caps are the tips of root
hairs. These tips come in direct contact with nutrients and water and the absorption and
acquisition process starts with these caps. More root hairs mean more surface area of
roots. This increase in surface area helps in more exploration of soil for mining of
chemicals that are less available near plant roots. These root hairs established a main
contact point between plant and rhizosphere from where takes water and mineral nutrients
for its growth and development (Parker et al., 2000). They are the part of fundamental
17
Figure 2.1. Mechanism of increased carboxylates production under P deficiency
(Neumann and Martinoia, 2002)
rhizospheric reactions like the uptake of nutrients and water and anchorage of the plant to
the soil (Fan et al., 2001; Grierson et al., 2001; Michael, 2001). In the secretion process,
root hair cells are involved. Root cells other than root hair and root cap cells, also take
part in the process of root secretion. Maize plant roots secrete citrate in the rhizosphere,
and it was observed that this secretion was dominant at 5cm above the root cap showing
that steller cells and cortex cells are involved (Pineros et al., 2002). The release of
carboxylate is generally constitutive, but this secretion process may be the result of
abiotic as well as biotic stress. A passive process involves in the plant roots secretion that
is facilitated by 3 different passages like vesicle transport, ion channels, and diffusion
18
(Bertin et al., 2003). Through the diffusion process, uncharged and small polar molecules
are moved across membranes due to cytosolic pH (Marschner, 1995) and permeable
nature of membrane (Sanders and Bethke, 2000). Carboxylate as anions, moved across
the membranes through the use of the protein along an electrochemically mediated
gradient. These electrochemical gradients allow these molecules to pass from milli-molar
range in the cytoplasm of root cells to micro-molar range in the soil (Samuel et al., 1992).
Carboxylate exudation is linked to the extrusion of protons. This proton extrusion lowers
the pH of soil that helps in enhanced availability of P if the pH of the soil is high (Roelofs
et al., 2001). The diffusion potential of cytosolic potassium and protons extrusion via
ATPase creates a +vely charged gradient that stimulates carboxylate anions release
(Neumann and Martinoia, 2002).
It is well known that the export of protons from plant cells to the soil is done across
plasma membrane (PM) (Sze et al., 1999). H+ ATPase serves as a principal transporter of
protons for releasing protons outside the cell. As a result of this export electric potential
and pH differences are created across the PM. The main functions of H+ ATPase are,
therefore, to supply the driving force of various substances for membrane transport and
maintain H+ homeostasis of cells. H+ ATPase may be specifically essential for cluster
roots, which released citric acid. On the one side, a high concentration of H+ must be
released during the synthesis of citric acid to save the cell from acidosis process. On the
other hand, the overall charge balance for citrate release can be maintained through H+
release by H+ ATPase as they act as counter ions (Zhu, 2004).
Membrane integrity is an important factor in the release of carboxylates. Factors affecting

membrane integrity may stimulate the release of carboxylates (Jones and Darrah, 1995).
The release of compounds by roots is controlled by anion channels (Sakaguchi et al.,
1999). For every compound released from roots, there is a specific transporter (Hussain et
al., 2004; Hirner et al., 2006; Grabov, 2007; Svennerstam et al., 2007). It was studied that
malate release is affected by malate transporter (AtALMT1) in Arabidopsis plants
(Kobayashi et al., 2007).
2.3.2.2.4. Mechanism of P mobilization by carboxylates in the rhizosphere
Principal mechanisms for release of adsorbed P is, increase in carboxylate production

(citrate, malate, oxalate, acetate, lactate, glycolate, tartrate, and succinate) and release
through roots in the rhizosphere.
19
Figure 2.2. Mechanism of carboxylate secretion from the root
(Badri and Vivanco, 2009)
Figure 2.3. Mechanism of P mobilization by carboxylates in the rhizosphere
(Neumann and Martinoia, 2002)
20
These carboxylates strive with P for binding to adsorption sites on soil particles and
calcium content and liberate P due to the chelation with iron, aluminum oxides (Figure
2.3) in acid soil (Puente et al., 2004) and calcium in alkaline soil (Gerke et al., 2000a, b).
Organic acids having three carboxyl groups called tri-carboxylates, for example, citrate,
are more efficient in releasing P due to stronger ligand binding compared to mono-
carboxylates (having one carboxyl attached to their structure) and di- carboxylates
(having two carboxyls attached to their structure) for example, malate. Tri carboxylates
have a high affinity for soil adsorption sites and for divalent and trivalent metals (Jones et
al., 2003). Citrate effect was calculated in chelating of calcium from hydroxyapatite
particles in alkaline soil. Citrate dissolves the calcium-phosphate bond proficiently by
weakening the stability of hydroxyapatite nanoparticle and by the control of free calcium
availability; in that way, the control of the rate of nucleation (Martins et al., 2008).
Consequently, carboxylate release by roots can increase P acquisition by plant, due to
increased P solubility of stable pools in soil that is essential to enhance P availability to
alkaline and calcareous soils (Gang et al., 2012).
2.3.2.2.5. Genotypic variation in carboxylate release under low P availability
Genotypic variation in many crops in the release of root exudates under P stress also
exists. The difference in root exudation by barley genotypes also noticed under P stress
(Asmar et al., 1995). Release of protons and organic acids was higher in P non-sensitive
genotype (JX17) comparative to P sensitive rice genotypes (ZYQ8) in P stress
environment (Ming et al., 2002). Due to the high capacity of acidifying the rhizosphere,
Andean genotypes of common bean more P from Ca-P compared to Mesoamerican
genotypes (Yan et al., 1996).
Phosphorus availability is also affected by root secretions both high and lower molecular
weights carboxylates. In common bean genotypes, increased secretion of carboxylates by
roots was recorded in P efficient genotypes that result in enhanced P uptake as compared
to the genotypes with less activity of P solubilization (Shen et al., 2002), while in tobacco
more uptake of P was recorded (Han et al., 2014). Also, among soybean genotypes, Pi-
efficient soybean genotypes exuded more malic acid, that ultimately caused improved P
sustenance and enhanced aluminum toxicity tolerance of plant (Liao et al., 2006).
Similarly, the qualitative, as well as quantitative modifications in carboxylate release
under P stress among the green gram and maize genotypes was recorded that showed both
intra and inter-species variations in carboxylates exudation, root and shoot P content and
21
P uptake do occur among green gram and maize. Overall, green gram had more
carboxylate exudation as compared to maize. Both green gram and maize genotypes
showed genetic variability in carboxylates exudation (Singh and Pandy, 2003).
Carboxylate concentrations improved in the rhizosphere as plant developed, in cultivar
Heera, but it was not increased for cultivar Tyson in chickpea (Wouterlood et al., 2004).
Clusters root forming, Lupinus albus and Lupinus pilosu were planted at different P levels
(0, 7.5 mg P, 15 mg P or 40 mg P kg-1) for thirty two, forty two and sixty two days.
Phosphorus supply @ 15 mg kg-1 of P gave major effects on the allocation of biomass to
cluster root in L. atlanticus compared to other species and hence released more
carboxylates (Wang et al., 2013). Chickpea cultivar Heera showed more carboxylate
concentrations in the lateral roots rhizosphere as compared to cultivar Tyson (Wouterlood
et al., 2004).
2.3.2.2.6. Role of plant metabolism in carboxylate release under low P availability
Root exudation also depends on plant metabolism. All plants are categorized into three
categories: C3 plant metabolism, C4 plant metabolism, CAM plant metabolism. C3 plants
have more carbohydrate in their roots as comparative to C4 plants although more organic
acids in roots of C3 plants are present compared to C4 plants (Johansson et al., 2009;
Jimenez et al., 2011). Carboxylates released internally vary by plant metabolism. Malate,
a carboxylate is stored temporarily by the CAM plants as a source of carbon. C3 plants
secrete and transport malate and citrate to mitochondria for backing the respiration
process (Meyer et al., 2010). With a similar experimental setup, in spite of plant
metabolism, there are many other factors that cause the exudation from roots e.g. all the
environmental factors (Lesuffleur et al., 2007). If all the factors that can influence root
exudation like root architecture, type of collection medium, mechanical impedance, plant
sterility, and age are not considered as the reason for exudation, (Johansson et al., 2009;
Toyama et al., 2011) C3 may release a low amount of total organic carbon per unit mass
of plant roots compared to C4 plant (Yoshitomi and Shann, 2001; Baudoin et al., 2003).
Dominant compounds percentage in exudates, especially organic acids, usually vary

according to the kind of plant metabolism. In the case of water-soluble root exudation, the
proportion of organic acids is the same in C4 plants, but this proportion of organic acids
is not the same in C3 plants (Lesuffleur et al., 2007; Shu et al., 2007; Chang et al., 2008).
Broad spectrum of organic acids was studied in root exudates of C3 plants. In C3 plants
dominant acids were malonic, α-ketoglutaric, pyruvic, formic, acetic, gluconic, lactic,
22
butyric, propionic, and formic acid. On the other side, trans-aconitic and fumaric were
identified as the leading organic acids of C4 plants (Sun et al., 2010; Shi et al., 2011;
Yang et al., 2011).
Different treatment gave a prominent effect on C3 than C4 for root exudation. Exudation
response of plants to different environmental factors: nutrient supply (Jelani et al., 2010),
chemical or mechanical stress, collection medium and cultivar is affected greatly by plant
metabolism (Mucha et al., 2005; Chang et al., 2008; de Andrade et al., 2011; Garzón et
al., 2011; Li et al., 2011).
Exudates are re uptaken by plants after a certain time period. This reuptake mechanism
may differ in plant species depending on their metabolism. Experiments on maize plants
(C4) were performed. Reuptake of organic acids was 10% of total organic acids (Jones et
al., 2005; Biernath et al., 2008), while C3, Triticum aestivum along with eleven grasses
recovered 70% of the low molecular weight compounds (Falkengren-Grerup et al., 2000;
Michonneau et al., 2004; Ge et al., 2009; Andresen et al., 2011). pH of root exudates of
C4 plants is also different from the pH of C3 crops (Tao et al., 2004) and mineralization
rates are also dissimilar (Formanek and Ambus, 2006). The difference of carbon exudate
release results in specific desirability of A. lipoferum by C4 and Azospirillum brasilence
C3 roots (Azevedo et al., 2005). A C3 plant shows more discrimination against 13C as
compared to C4 plants. Rhizodeposition of d13C in C4 ranged –10 to –14 and in C3 from
20 to 35 (Gillson et al., 2004).
2.3.2.2.7. Effect of soil texture on carboxylate release under low P availability
Change in pH measurement in the rhizosphere is also an indicator of the release of OH–

/HCO3 and H+ by roots. This depends on organic matter contents, calcium carbonate
contents and type and nature of clay minerals. In the three different textured soils
containing 2–6% CaCO3, no reduction in pH was noticed while it was observed in other
soils (0.73 to 1.49) (Hinsinger et al., 2003).
Carboxylate release varies with soil type and within species. The difference in
carboxylate exudation in white lupin and chickpea was noticed. While lupin released
more carboxylates compared to chickpea. Chickpea grown in pots in eleven soils, with
low P concentrations showed variation in all P acquisition characteristics and carboxylate
release. Minor changes in root morphological traits were observed while P content varied
almost five-fold and shoot, and root mass varied more than two-fold. Phosphorus uptake
23
was increased with these adaptations. A positive relation was found amongst P uptake and
rhizosphere carboxylates concentration. Carboxylate release was higher in soils with low
sorbing capacity as there was higher available P. However, plant P contents were not
positively correlated to bicarbonate extractable P in any soil type. Carboxylates increased
P availability to plant though, the factors affecting root carboxylate release were not
known. White lupin cultivars had no significant differences in rhizosphere concentrations
of carboxylates when they were grown in the same six Western Australian soils. Variation
in the rhizosphere carboxylates concentration of chickpea did not same with that of white
lupin across the same six soils. However, the proportion of malate in both species
increased and of citrate decreased at lower soil pH (Veneklaas et al., 2003).
Conclusion
It is obvious that P availability is soil specific as well as crop specific. Ample estimates of
the P bioavailability in the soils call for the acquaintance with P acquisition features of
the plant species, soil texture, and the chemical nature of soil P. The knack of plant
species to acquire less available P by the carboxylate’s exudation had studied
comprehensively for many other crops. However, this kind of information is missing for
wheat and maize. On the other hand, P adsorption on the basis of soil texture had studied
broadly but minute consideration had given to the role of soil texture in the release of
carboxylates by crops under P deficiency. The issue is whether the wheat and maize
plants display mechanisms for acquiring P, like other species, under different levels of P
supply and soil textures. Such data is fundamental for building up a reasonable P fertility
management plan for long haul wheat and maize production. This research thesis aims to
recognize P acquisition characteristic (especially carboxylates release) of wheat and
maize in different textured soils, and also compare P acquisition strategies of wheat and
maize.
24
CHAPTER 3
MATERIALS AND METHODS
The proposed research was planned to check the carboxylate release and P acquisition in
wheat and maize at deficient P levels. A series of experiments were conducted to assess
the carboxylate release by plants in response to P deficiency and its effect on P
acquisition. A brief description of studies conducted in this research is given below
3.1. Studies Conducted
The research work given in this thesis is divided into the following studies.
3.1.1. Study 1
➢ Screening of wheat and maize genotypes on the basis of carboxylate release in

solution culture
➢ Measuring the P acquisition characteristics (Root length, R/S ratio, phosphorus

use efficiency and Root mass fraction, etc.) and carboxylate release ability of all
genotypes under P deficient environment
➢ Selection of P efficient genotype of wheat and maize for the next study
3.1.2. Study 2
➢ Selection of soils on texture basis
➢ Carboxylates effect on P sorption (Incubation Study)
➢ To check the P adsorption behavior in loam, sandy loam, and sandy clay loam soil
➢ To evaluate the effect of carboxylates (organic acids) in soil P adsorption

capacities.
➢ To check the influence of carboxylate on P availability in soil
3.1.3. Study 3
➢ Influence of soil types and deficient levels of P on the release of carboxylates
➢ Assessment of release of carboxylates as a P acquisition strategy at deficient

levels of P (less than recommended dose) in different textured soils in wheat and
maize (Pot Experiment)
➢ Evaluation of P acquisition strategies
25
➢ Evaluation of P levels for optimum growth of wheat and maize
3.2. Study 1
Effect of phosphorus on growth characteristics and carboxylate release in wheat and

maize genotypes
3.2.1. Seed Source
Five wheat and five maize genotypes from Saline agriculture research labs, ISES, UAF
were used in this study. Wheat genotypes Kohinoor-83, B4-5711, SARC-1, SARC-2 and
SARC-3 and maize genotypes Cargel-6525, Syngenta-8711, Pioneer-33H15, Monsanto-
6525 and Pioneer-32F10 were used in this study.
3.2.2. Sterilization of seeds
Seeds of wheat and maize were immersed for 5 min in sodium hypochlorite (1%) and then
thoroughly washed several times with deionized water.
3.2.3. Nursery raising and transplantation
Seeds of selected genotypes were sown in iron trays with 2-inch sand layer. For seed
germination and seedling establishment, moisture was sustained with distilled water.
Fifteen days old seedlings were shifted to (6 L for maize and 3 L for wheat) holes
plugged with foam, in polystyrene sheets floating over ½ strength Hoagland’s solution
(Hoagland and Arnon, 1950) in plastic tubs. Each genotype was replicated 3 times in
separate tubs. The pots arranged randomly, according to factorial under CRD
(Completely randomized design).
3.2.4. Growth conditions
This experiment was carried out in glasshouse of Institute of Soil and Environmental
Sciences, University of Agriculture Faisalabad Pakistan. Plastic tubs were aerated by
aeration pumps for about 8 h every day. Solution pH was daily maintained at 6-6.5 with
NaOH and HCl. The solution was changed every 5th day or a week. After growing plants
for 7 days in normal ½ strength Hoagland solution, treatments were applied. Normal P
treatment (Control) received complete nutrients of half-strength Hoagland solution while
in P deficient treatment P was omitted from nutrient solution. In P deficient treatment, the
source of P, KH2PO4 was replaced by KNO3 to maintain the potassium level in the
treatment. According to lower demand of plant for P and slow initiation of deficiency
26
responses by the plant, described by Marschner, (1995) and Nagy et al., (2006), P
deficiency period was set to 15 days.
3.2.5. Root exudates collection
Root exudates were collected from all treatments fifteen days after treatment application.
For root exudates collection, two hours after onset of light period plants were transferred
to 250 ml vials containing 0.2 mM CaCl2, in which root exudates were collected for 6 hrs.
This extract was used for carboxylate determination on HPLC (High performance liquid
chromatography) (Cawthray, 2003).
3.2.6. Analysis of carboxylates
Mobile phase preparation
For mobile phase preparation (0.1% Phosphoric acid), 1ml of HPLC grade phosphoric
acid was taken and mixed into 999 ml of double deionized water and this is then filtered
through syringe membrane filter assembly.
Sample preparation
For carboxylates determination on HPLC, samples were prepared by using mobile phase.
For this purpose, 1ml of root exudate extract and 7 ml of mobile phase was added in this
centrifuge tube. These samples were centrifuged at 1600 rpm for 25 minutes at 20 ºC
temperature. After centrifugation, the supernatant was taken and filtered through a 0.2 µm
syringe filter in 1ml cryovials for HPLC analysis.
Analysis of HPLC
The carboxylates from extracts were analyzed by HPLC [LC-10AT pump, plus manual
sampler and UV-VIS-SPD-10AV detector, Shimadzu, Japan] using Shim-Pack CLC-ODS
(C-18) reverse-phase column (Cawthray, 2003) with flow rate 1ml/min at room
temperature. Working standards were malic, citric and oxalic acids. These organic acid
standards were purchased from Merck. Firstly, working standards were run on HPLC and
their retention times were determined by chromatogram. Then samples were run on
HPLC.
Qualitative Analysis
Qualitative determinations of carboxylates were done by equating the retention times of

the samples on chromatogram and retention times of standards. When retention times
27
corresponded with those of carboxylate standards, identity confirmed by paralleling the
absorption spectra of the samples at 215 nm with the spectra of the corresponding
carboxylates.
Quantitative Analysis
The quantitative determination in each sample was done by comparing peak heights of
standards with the peak heights of samples.
pH measurement
Immediately after the root exudates collection in 250 ml vial, the pH of this extract was
measured.
3.2.7. Plant measurement
After 30 days of sowing, plants were harvested, and physical parameters were measured.
To measure dry matter, the harvested plants were kept in an air-dried oven at 65 ± 5 ◦C.
After that, dry biomass was measured by an analytical balance.
3.2.8. Plant Analysis
Sample preparation
Dried samples were ground with a mechanical grinder to powder form and stored in
plastic bags
Wet digestion
Dried, finely ground plant sample i.e. root, and the shoot was separately digested. For full
recovery of P from plant samples, the procedure described by Jones and Case, 1990
was used. Dried root (0.25 g) and shoot samples (0.5 g) were taken in a 50 mL
conical flask. 7.5 mL of di-acid (HNO3: HClO4 in 3:1 ratio) was added in root
samples and 15 mL in shoot samples. These flasks were kept overnight and the next
day, samples were placed on the hot plate at 350 oC till the material was clear. After
that samples were cooled, diluted by adding de-ionized water to 50 mL and then
filtered by filter paper No. 42.
28
3.2.9. Phosphorus determination
Phosphorus determination in plants is based upon the light absorbance at wavelength 880
nm by standard or sample placed in the light path.
Standard preparation
For the preparation of 100 ppm stock solution, 0.1 g of P from source salt (KH2PO4) was
liquified in 1 L of distilled water.
Working standards were prepared by the stock solution. 0, 0.5, 1, 2, 3, 4, 5 mL was taken
from stock solution for making working standards of concentrations 0,1, 2, 4, 6, 8, 10
ppm.
Sample preparation
In 100 ml volumetric flask, 10 mL of root and shoot plant digest was pipetted. Then 10
mL of Barton reagent was added in samples as well as standards and made volume up to
mark with distilled water. After preparation, samples and standards were retained for half
an hour, and then standards were run on a spectrophotometer at 430 nm and reading was
noted. Similarly, sample readings were recorded. By using standards, a calibration curve
was drawn. Phosphorus concentrations in samples were determined from the calibration
curve (Chapman and Pratt, 1961).
Preparation of Barton reagent
For the preparation of Barton reagent, method by Ashraf et al., (1992) was used.
3.2.10. Phosphorus acquisition parameters measurement
Phosphorus use efficiency (PUE)
For determining PUE, shoot/root dry matter was divided by shoot/root P concentration
(Siddiqi and Glass, 1981).
Root mass fraction (RMF)
For RMF, root dry matter and shoot dry matter was used
(Ryan et al., 2012)
29
Root: Shoot (R/S ratio)
It was calculated as:
(Lyu et al., 2016)
3.2.11. Statistical analysis
Data were compared between the groups with Phosphorus (P+) and without Phosphorus
(P-) application. The statistical analysis of data was done with software Package SPSS
Statistics 19.0 (Hussain et al., 2018). The data was accomplished by the standard analysis
of variance (TWO WAY ANOVA) (Steel et al., 1997) and mean values were compared
by Duncan’s least significant difference (LSD) at 5% significance level (Duncan, 1955).
Microsoft Office Excel 2010 was used for graphical presentation. Multiple comparison
tests (descriptive) were used to compare the genotypes.
3.3. Study 2
Effect of carboxylates (organic acids) on P adsorption (Incubation

study)
3.3.1. Pre-sowing soil analysis
Collection and preparation of the soil
Soil samples were collected from Proka research farm, Agronomy Research Area,
University of Agriculture, Faisalabad and from cultivation fields of Chak # 122 Jb,
Faisalabad. Soils were air-dried and powdered to gain a 2 mm size by passing through the
sieve and was mixed to get a homogenized mixture. From these samples, a portion of the
soil was used to analyze physical and chemical characteristics (Table 3.1).
Soil texture determination
Particle size analysis
Particle size was analyzed by the Hydrometer method (Bouyoucos, 1962).
Calculations were made as given below;
30
Saturation percentage (SP)
The saturation percentage of each soil was determined by preparing the saturated soil
paste. After paste preparation, pastes were transferred to a tarred china dish. The
weight of a china dish plus pastes was noted. That pastes in china dish were then
oven dried (105 °C) till persistent weight. formula is:
pH of saturated soil paste (pHs)
Soil paste of each soil was made by 250 grams of each soil in a separate beaker and
added distilled water to saturate the soil. After one hour, the pH of these pastes was
determined by using pH meter (Method 21a, U.S. Salinity Lab. Staff, 1954).
Electrical conductivity (ECe)
The electrical conductivity of extract of each soil paste was measured with
conductivity meter paste (Method 3a and 4b, U.S. Salinity Lab. Staff, 1954).
Cation Exchange Capacity (CEC)
Five grams of each soil was extracted with 1N Sodium acetate. Sodium in extract was
analysed with the help of a flame photometer. CEC was calculated:
CEC (Cmolc Kg-1) =
31
Table 3.1. Physico-chemical properties of soils used in the experiment
Sandy clay
Characteristics Units Loam Sandy loam
loam
Sand % 49.8 61.8 49.5
Silt % 33.6 24 26.6
Clay % 16.6 14.2 23.9

Saturation
% 33.0 29.0 44.0
percentage
pH (1:5) 8.1 8.0 8.2
ECe dS m-1 1.62 1.59 1.73
CaCO3(%) % 6.2 4.9 9.0
Organic matter % 0.56 0.62 0.48
Total nitrogen % 0.048 0.055 0.05
CEC cmolc kg-1 18.0 15.0 32.0
Available P mg kg-1 7.3 10.8 5.7
Available K mg kg-1 239 247 130
Cu mg kg-1 0.5 0.9 0.6
Mn mg kg-1 1.2 1.6 1.7
Zn mg kg-1 0.5 0.6 0.1
Fe mg kg-1 0.2 0.1 6.1
32
Calcium carbonate (CaCO3: lime)
One gram of each soil was taken in Erlenmeyer flask. Hydrochloric acid (1 N) was added
to it. The next day, 2-3 drops of indicator (phenolphthalein) and 100 mL of distilled water
were added in each flask. Then Samples were titrated against 1 N NaOH and the end
point was faint pink (Allison and Moodie, 1965).
Organic matter
For the determination of soil organic carbon, the method given by Moodie et al., (1959)
was used. Two-gram soil samples were taken with 40 mL of concentrated sulfuric acid
and 20 mL of 1 N solution of K2Cr2O7 were added and thoroughly mixed. Then 50 mL of
0.5 N ferrous sulphate and 300 mL of distilled water were added and titrated against 0.1N
potassium permanganate till the pink end point.
Total nitrogen
Soil nitrogen determination was carried out by Ginning and Hibbard’s method of H2SO4
digestion and macro Kjeldhal’s was used for distillation purposes (Jackson, 1962).
Available P
Soil available P was determined by the method described by Watanabe and Olsen, (1965).
Extractable potassium
Extraction was done with ammonium acetate (1N of pH 7.0) and potassium was
determined by using Flame photometer (Method 1la, U.S. Salinity lab. staff, 1954).
Plant available micronutrients
Di-ethylene tri-amine penta-acetic acid (DTPA) test (Lindsay and Norvell, 1978) was
used to measure Mn, Zn, Cu and Fe in soil samples.
3.3.2. Experimental procedure for P adsorption
Three-gram soil samples from each soil were taken into a centrifuge tube (50 mL). A
range of P concentrations (0, 100, 200, 300, 400 and 500 ppm) was prepared. Potassium
dihydrogen phosphate was used as a P source. For solution preparation, Milli-Q water
containing 0.01 M CaCl2 was used. Calcium chloride acts as an electrolyte. In each
centrifuge tube, 30 mL aliquots of the solutions were added to give 0, 100, 200, 300, 400
33
and 500 mg P kg-1 of soil. These samples were shaken for 24 hrs on a shaker at 216 rpm
and then centrifuged at 20000×g for 20 min. After that, the supernatant was filtered
through a 0.2 µm syringe filter. The filtrate was analyzed for available P by the method
described by Olsen et al., (1977). The amount of P adsorbed was calculated from the
difference between added P and P present in the solution. The adsorbed P was calculated
as mg kg-1 soil. The P in the control (no P) treatment solution was taken into account.
3.3.3. Experimental procedure for carboxylate effects on P adsorption
To measure the P extraction ability of exogenously applied organic acids (carboxylates),

solution of citric acid (CA), oxalic acid (OA), and malic acid (MA) were made. The two
concentrations of organic acids: 0.5 mM and 1 mM were prepared with the ranges of P
concentration (0, 100, 200, 300, 400 and 500 mg Kg-1). Potassium dihydrogen phosphate
was used as a P source. For solution preparation, Milli-Q water containing 0.01 M CaCl2
was used. Calcium chloride acts as an electrolyte. Three-gram soil samples from each soil
were taken into centrifuge tubes (50 mL). Further, 30 mL of each prepared solution was
added in each centrifuge tube according to the treatment plan. These centrifuge tubes with
samples were stirred for 2 hrs every day for a week on a shaker at 216 rpm. After one
week of incubation, these samples were centrifuged at 20000×g for 20 min. After that, the
supernatant was separated through a syringe filter (0.2 µm) and then analyzed the
availability of P followed by Olsen et al., (1977) method. The amount of adsorbed P was
determined from the difference method between added P and the P present in the solution.
The adsorbed P was calculated as mg kg-1 soil. The P in the control (no P) treatment
solution was taken into account.
3.3.4. Analysis of adsorption data
An adsorption isotherm defines the adsorption of solute by the solid surface at constant
pressure and temperature and it also tells about the adsorbed quantity of solute. Desorbed
solute quantity is described as a “function of the equilibrium concentration of solute in
solution”.
The P adsorption data for the organic acids in three different textured soils were fitted to
the following adsorption equations:
34
Langmuir adsorption equation:
where,
c = solution equilibrium concentration of P (μg P mL-1)
x/m= Q = mass of P adsorbed per unit mass of soil (mg kg-1)
k = constant related to bonding energy of P to the soil and its value is calculated from
intercept (1/kb)
b =Pmax= maximum P adsorption capacity (mg kg-1) and its value is calculated from the
slope (1/b)
Freundlich adsorption equation:
By reordering the above equation;
where,
x/m= Q = mass of adsorbed P per unit of soil mass (mg kg-1)
c = solution equilibrium concentration of P (μg P mL-1)
a = it is a constant and its value is calculated from intercept (log a)
b = it is a constant and its value is calculated from the slope (b)
A straight line of plot of log (x/m) against log c gives a clue for the fitness of the
adsorption process fit the model.
3.3.5. Statistical Analysis
The analysis of data was done by using analysis of variance technique (ANOVA) (Steel et
al., 1997) with Factorial under CRD (three factors) for the 2nd part of the study and CRD
two factor factorial for the 1st part of the study. For this purpose, computer software
Statistix 8.1 was used with comparison means by the least significant difference (LSD)
test (Duncan, 1955). For Freundlich and Langmuir model parameters determination and
graphical presentation, Microsoft office Excel 2010 was used.
35
3.4. Study 3
Influence of soil types and deficient levels of P on the release of carboxylates and P
acquisition characteristics in wheat and maize
A pot experiment was conducted at wirehouse of Institute of Soil and Environmental

Sciences, University of Agriculture, Faisalabad to examine the effect of a low level of P
application on root exuded carboxylates and plant P concentration of wheat and maize
plant. Two separate studies for wheat and maize were conducted under the same
experimental conditions.
3.4.1. Soil preparation
Three types of soils (based on texture) were air dried and passed through 2mm sieve and
then used for the experiment.
3.4.2. Growth conditions
The experiment was carried out in the glass house of the institute of soil and
environmental sciences, University of Agriculture, Faisalabad. Four kg (Wheat) and eight
kg (Maize) of each of three types of sieved soil were filled in plastic bags that are
perforated at the bottom for leaching and proper aeration. These bags were placed in
plastic pots. In the soil, half of nitrogen fertilizer dose and a full dose of potassium were
mixed. Remaining half dose of nitrogen was applied one month after the sowing of seeds.
Initially, four seeds were sown and thinned to two plants per pot after germination. Soil
moisture was sustained at 50% of field capacity by applying water three times a week. To
supply other nutrients, the nutrient solution was applied every 15 days. Composition of
nutrient solution was: Macronutrient (mg kg-1 soil); MgSO4=0.19, K2SO4=0.17 and Ca
(NO3)2·4H2O=2.65 and micronutrients (mg kg−1 soil) MnSO4·4H2O=0.6, H3BO3=0.5,
CuSO4·5H2O=2.0, Na2MoO4·2H2O=0.5, Fe-Na-EDTA=0.4, ZnSO4·7H2O=2.2 and
CoCl2·6H2O=0.4 (Li et al., 2010).
Other agronomic practices, weeding, and hoeing was done according to requirement. At
the flowering stage, plants were harvested.
36
3.4.3. The treatment plan for wheat
The study design was Factorial under Completely Randomized Design (CRD) with three
replications.
The experimental plan included the following treatments:
T1= 0 mg P application kg-1 soil
T2= 11.25 mg P application kg-1 soil
T5= 45 mg P application kg-1 soil (recommended dose) (Control)
These treatments were repeated for three different soil types (loam, sandy loam, and
sandy clay loam soil).
3.4.4. The treatment plan for maize
The study design was Factorial under Completely Randomized Design (CRD) with three
replications.
The experimental plan included the following treatments:
T5= 60 mg P application kg-1 soil (recommended dose) (Control)
These treatments were repeated for three different soil types (loam, sandy loam, and
sandy clay loam soil).
3.4.5. Harvesting
At flowering plants were harvested with intact root and rhizosphere soil in order to collect
root exudates. For that purpose, plastic bags containing soil plus root system were taken
out of plastic pots, cut open and placed on the clean bench. For separating roots from the
soil, the soil was pressed softly and then the root system was gently winched up out of the
37
soil. Roots were shaken carefully so that excess soil can be removed. Soil clumps were
taken out of roots. The soil loosely adhered to roots was termed as ‘rhizosphere soil’.
3.4.6. Plant measurement
At the flowering stage, plants were harvested and physical parameters were measured
3.4.7. Total chlorophyll content (SPAD value)
Leaf chlorophyll contents (SPAD value) were determined one day before harvesting
using a hand-held SPAD- 502 (Minolta, Osaka, Japan). Chlorophyll readings were taken
from the young fully expanded leaf. Three values were taken from a single leaf and then
the average of these three values was taken.
3.4.8. Physiological parameters
Physiological parameters, photosynthetic rate (A), transpiration rate (E) and respiration
rate (Ce) were determined by using Infrared Gas Analyzer (IRGA).
3.4.9. Root exudate collection
The root system was then shifted to a beaker of the appropriate size of the root system. In
that beaker, roots were washed off by 0.2 mM CaCl2 solution. Calcium chloride (0.2 mM)
was used to ensure membrane integrity. Beaker was gently shaken for 90 seconds. The
roots were then removed from the soil extract and representative subsamples of the roots
were subjected to image analysis to determine root parameters. Soil extract was filtered,
and a subsample of extract was taken and filtered through 0.22 μm syringe filters (Pal
Gelman Acrodisc syringe filters) into 1 mL HPLC vials. These vials were frozen at -21
o
C. The rest of the rhizosphere soil extract was frozen (Veneklaas et al., 2003).
3.4.10. Analysis of carboxylates, pH measurement, Plant analysis, and P

determination
Same as in Study 1 (Section 3.2.6-3.2.10)
3.4.11. Phosphorus acquisition parameters measurements
Root to shoot ratio (R/S ratio)
It was calculated as
(Lyu et al., 2016)
38
Phosphorus uptake
For shoot/root P uptake, P concentration was multiplied by dry matter
(Hunt, 2003)
For determining PUE, formula used by Siddiqi and Glass, (1981).
Phosphorus stress factor (PSF)
(Hunt, 2003)
P physiological efficiency index (PPEI)
(Ghulam et al., 2005)
Phosphorus Acquisition Efficiency (PAE)
Phosphorus acquisition efficiency was calculated
(Lyu et al., 2016)
Root mass fraction (RMF)
For RMF, root dry matter and shoot dry matter was used
(Ryan et al., 2012)
3.4.12. Root Analysis
Root volume
For root volume determination, the gravimetric technique was used. For that purpose,
roots were thoroughly washed and blotted dry. Electrical Balance was tarred and set
reading at zero. Plastic tub with water inside was placed on an electrical balance and
again tarred the balance to zero. Then the root system was submerged in that plastic tub
39
until the water surface was 2 mm above the root system. At this point, weight was
recorded in ml. Repeated measurements were taken in order to reduce errors (Harrington
et al., 1994).
Root Scanning
After harvesting, root parameters root surface area, average root diameter, number of tips
and root surface area were measured by scanning the roots. For the root system
morphology, the Delta T scan image analysis system was used. For root morphology
(Bouma et al., 2000) the definition of 300 (dpi) was used and then the root characteristics
were determined (Magalhaes et al., 2011).
3.4.13. Statistical analysis
We compared data between the P levels and soil type groups. The statistical analysis of
the data was performed with software Package SPSS Statistics 19.0 was used (Hussain et
al., 2018). The data was accomplished by the standard analysis of variance (TWO WAY
ANOVA) (Steel et al., 1997) and means were compared by Duncan’s least significant
difference (LSD) at the 5%, 1% and 0.1% level of significance (Duncan, 1955). Microsoft
Office Excel 2010 was used for graphical presentation. Multiple comparison tests
(descriptive) were used to compare P levels and soil types.
40
CHAPTER 4
RESULTS AND DISCUSSION

4.1. Study 1
Effects of phosphorus on growth characteristics and carboxylate release
in wheat and maize genotypes
Genetic differences in P nutrition occur among crops. These differences are demonstrated
as physiological and morphological expressions. Under low P stress, most plant acquires
P by changing morphology and physiology of roots (Shen et al., 2011). Release of
protons and organic acids was higher in P non-sensitive genotype (JX17) relative to P
sensitive genotypes (ZYQ8) of rice in P stress environment (Ming et al., 2002)
Carboxylates concentration differs considerably among maize genotypes. Malate, citrate
and trans-aconitase comprised of about 80% of total carboxylates released by all maize
genotypes (Gaume et al., 2001). Therefore, amount and composition of root released
carboxylates fluctuate with nutritional status, species, and genotypes (Zhao and Wu,
2014). Chickpea cultivar Heera showed more carboxylate concentrations in the lateral
roots rhizosphere as compared to cultivar Tyson (Wouterlood et al., 2004).
Low input sustainable agriculture (LISA) has increased attentiveness among researchers
for the selection of crop genotypes that are easy-going to mineral stress in the soils. In a
low nutrient environment, the ability of plant genotype to absorb, use, accumulate and
translocate mineral elements is important. These characteristic variations among
genotypes are accountable for failure or survival in a low nutrient environment (Ahmad et
al., 1998). A commonly established goal is to develop such varieties that grow and give
better yield in low extractable P soils (Lynch, 2007). Developing and selection of P
efficient genotypes can be a conceivable way for decreasing P demand. Many studies
have revealed that variances in stress tolerance of low P occur in genotypes within crop
species (Richardson et al., 2011; Simpson et al., 2011; Cordell and White, 2015).
Keeping in view these points, following objectives were made
➢ To screen out wheat and maize genotypes on the basis of carboxylate release
under P deficiency
➢ To study rhizosphere acidification under deficient P
➢ To check growth responses of wheat and maize genotypes under deficient P
41
Results
Part 1
4.1.1. Effects of phosphorus on growth characteristics and release of carboxylate in

wheat genotypes
4.1.1.1. Growth responses of wheat genotypes
In this experiment, the normal and deficient phosphorus (P) application has significant
effects on shoot fresh weight (SFW) in different wheat genotypes (p ≤ 0.05) (Table 4.1).
B4-5711 showed a significantly higher SFW and the lowest SFW was observed in
genotype SARC-3 compared with other genotypes under normal P treatment. In P
deficiency treatment, the highest SFW was recorded in the B4-5711 genotype relative to
other wheat genotypes. However, in P deficiency treatment, all five genotypes respond to
P deficiency by decreasing their SFW. Reduction in SFW in SARC-1 (15.6%) was the
lowest which shows the tolerance to P deficiency compared with other wheat genotypes
while SARC-2 reduced its SFW up to 29.1% compared to P normal treatment which was
the highest among all genotypes. The SFW was reduced by 20.1%, 28.7%, and 23.2%
respectively in Kohinoor-83, B4-5711, and SARC-3 genotypes as compared to normal P
treatment.
Shoot dry weight (SDW) affected significantly by normal and deficient P treatment in
wheat genotypes except Kohinoor-83 and B4-5711 genotypes (p ≤ 0.05) (Table 4.1). The
highest and the lowest SDW were recorded in Kohinoor-83 and SARC-3 respectively
compared with genotype under normal P treatment. However, the reduction in SDW was
observed in all genotypes under P deficient than normal P application. With P deficient
treatment, reduction in SDW was the highest (31.4%) in the SARC-3 genotype than other
genotypes. The lowest decrease in SDW was noted in SARC-2 (4.5%) than the rest. The
decrease in SDW in Kohinoor-83 (10.7%), B4-5711 (6.1%), and SARC-1 (7.3%) were
recorded under P deficiency than normal P.
Shoot length (SL) also showed significant variation among all wheat genotypes due to P
application except SARC-1 and SARC-3 that gave an insignificant effect (p ≤ 0.05)
(Table 4.1). Under normal P application, the highest SL was observed in Kohinoor-83 and
the minimum SL was recorded in SARC-3.
42
Table 4.1. Shoot fresh and dry weight and shoot length of wheat genotypes under
Shoot fresh Shoot dry
Shoot length
Genotypes Phosphorus weight weight
(cm)
(g plant-1) (g plant-1)
P+ 0.85±0.014b 0.068±0.003a 31.36±1.23a
Kohinoor-83
P- 0.68±0.01c 0.060±0.006ab 29.96±0.97a
P+ 1.00±0.01a 0.0675±0.002a 30.00±0.84a

B4-5711
P- 0.72±0.015c 0.063±0.004ab 26.05±0.37b
P+ 0.45±0.03e 0.043±0.001c 23.66±0.69b-d

SARC-1
P- 0.38±0.01f 0.040±0.003cd 22.66±0.19cd
P+ 0.79±0.03b 0.057±0.001b 26.05±0.60b

SARC-2
P- 0.56±0.013d 0.054±0.003b 24.55±0.89bc
P+ 0.27±0.007g 0.032±0.001d 22.71±1.14cd

SARC-3
P- 0.20±0.02h 0.022±0.002e 21.19±0.85d
Phosphorus 223.91*** 8.48** 12.55**
F value Genotypes 437.02*** 62.14*** 36.22***
Interaction 16.19*** 0.56: ns 0.99: ns
43
In P deficiency treatment, SARC-1 performed better and showed reduction in SL by 4.2%
as compared to normal P. Shoot length was decreased in Kohinoor-83 (4.4%), B4-5711
(13.1%), SARC-2 (5.7%) and SARC-3 (6.6%) with respect to their SL when normal P
was applied, respectively.
Root fresh weight (RFW) showed significant (p ≤ 0.05) variation among all wheat
genotypes under normal and deficient P treatments (Table 4.2). Under normal P
treatment, Kohinoor-83 performed better regarding RFW among all wheat genotypes. The
lowest RFW was noted in the SARC-3 genotype. In P deficient treatment, Kohinoor-83
also gave the highest RFW. The lowest RFW was recorded in SARC-3. The P deficiency
treatment significantly increased the RFW in SARC-1 (42.7%) as compared to normal P
application and it was better than all other genotypes. SARC-3 did not perform well and
gave a non-significant increase of 5.6% in RFW among all genotype as compared to
normal P
Root dry weight (RDW) was significantly variable among different genotypes (p ≤ 0.05)
(Table 4.2). Various phosphorus treatments also altered the RDW and P deficiency
showed a significant effect on all wheat genotypes used in this study. Under normal P,
wheat genotypes Kohinoor-83 and SARC-1 showed significantly higher RDW. The
lowest RDW was noted in B4-5711 as compared to other genotypes. Under P deficient
treatment, increase in RDW in all genotypes was noticed as compared to normal P.
Significantly higher root dry was noticed in SARC-1(45%) in P deficient treatment while
SARC-3 had the lowest RDW as compared to normal P. Other genotypes, Kohinoor-83
(5.7%), B4-5711 (7.3%), SARC-2 (7.07%) and SARC-3 (7.4%) also increased its RDW
comparative to normal P.
Root length (RL) was affected in all wheat genotypes by P treatments (Table 4.2).
Genotypes had not varied significantly to P treatment (p ≤ 0.05). However, the treatment
effect was significant. The highest and the lowest RL was recorded in Kohinoor-83 and
SARC-3 respectively under normal P. Phosphorus deficient treatment had a positive
effect in the case of RL of four genotypes except Kohinoor-83. The only Kohinoor-83
showed a non-significant reduction of 0.45%. SARC-1 performed best and gave an
increase (34.1%) in RL as compared to normal P treatment. The RL was increased in B4-
5711 (12.69%), SARC-2 (19.1%), and SARC-3 (33.4%) respectively in P deficient
treatment comparative to normal P.
44
Table 4.2. Root fresh and dry weight, root length and Root: Shoot of wheat
genotypes under normal and deficient P
Root fresh Root dry
Root length
Genotypes Phosphorus weight weight Root: Shoot
(cm)
P+ 0.71±0.006c 0.043±0.001b 27.6±0.61a 0.64±0.019b
Kohinoor-83
P- 0.93±0.024a 0.05±0.0009a 27.5±1.03a 0.78±0.092b
P+ 0.65±0.014d 0.04±0.0003c 24.9±0.40b 0.60±0.017b

B4-5711
P- 0.76±0.014b 0.04±0.0003b 28.1±0.54a 0.69±0.042b
P+ 0.52±0.007e 0.03±0.0007f 09.3±0.36e 0.73±0.021b

SARC-1
P- 0.74±0.021c 0.05±0.0007a 12.4±0.67d 1.16±0.096a
P+ 0.28±0.008g 0.04±0.0002e 14.0±0.26d 0.63±0.009b

SARC-2
P- 0.32±0.009f 0.038±0.001d 16.7±0.54c 0.72±0.058b
P+ 0.13±0.005h 0.023±0.001g 07.0±0.05f 0.71±0.032b

SARC-3
P- 0.13±0.008h 0.024±0.001g 09.3±0.305e 1.13±0.095a
Phosphorus 210.39*** 129.0*** 42.21*** 39.14***
F value Genotypes 993.08*** 301.04*** 535.8*** 12.1***
Interaction 28.84*** 32.22*** 3.15* 4.58**
45
Root to shoot ratio (R/S ratio) ad not varied significantly between genotypes but variation
between P treatments was significant (p ≤ 0.05) (Table 4.2). In normal P treatment, R/S
ratio was ranged from 0.59-0.73. The highest and the lowest R/S ratio verified in SARC-1
and B4-5711 under normal P treatment. In the case of P deficient treatment, R/S ratio was
range was 0.68-1.15 with the highest value for SARC-1 and the lowest for B4-5711 than
normal P. In this treatment, all genotypes increased R/S ratio as compared to normal P
treatment. Increase in R/S ratio was the highest in SARC-1 (58.24%) and it was the
lowest in SARC-2 (13%) under P deficiency than normal P. While in other three
genotypes, SARC-3 (58.8%), Kohinoor-83 (21%), and B4-5711 (15%) increase in R/S
ratio was also noticed.
These variations in growth characteristics were clearly mentioning the surviving abilities
to phosphorus application in all wheat genotypes. These differences could be due to the
genetic nature of these wheat genotypes to show resistance against any nutrient (P)
deficiency.
4.1.1.2. Root P in various wheat genotypes
Root has direct contact with soil and plays a key role in mineral uptake. Phosphorus (P)
uptake was significantly different within all wheat genotypes (p ≤ 0.05) (Figure 4.1). In
this experiment, the maximum root P concentration was noticed in SARC-1 while the
minimum concentration was in SARC-3 under normal P treatment. In P deficient
treatment, the minimum root P was noticed in SARC-3 and the maximum was obtained
by SARC-1. SARC-2 showed the highest percent of reduction (11.1%) in shoot P
concentration while the minimum reduction (2.5%) in root P concentration was detected
in SARC-1 as compared to its P concentration in P normal treatment.
4.1.1.3. Shoot P in various wheat genotypes
The variation among genotypes for P uptake in shoot was observed among different
wheat genotypes (Figure 4.2). The response of P application on shoot P concentration was
significantly different in all wheat genotypes except Kohinoor-83 and SARC-1 (p ≤ 0.05).
In P normal treatment, higher P concentration was observed in SARC-2 while B4-5711
gave the lowest P concentration among all genotypes. Under P deficiency, all genotypes
exhibited a reduction in shoot P concentration and their P concentration was different
from each other. Shoot P concentration was decreased and the maximum decrease
(15.9%) in shoot P concentration was recorded in SARC-1 with respect to the
46
Figure 4.1. Impacts of P application and deficiency on root P
47
Figure 4.2. Impacts of P application and deficiency on shoot P
48
P concentration of SARC-1 in P normal treatment. Reduction of 10.2% was shown by
Kohinoor-83 which was the minimum among genotypes while B4-5711 (12.9%), SARC-
2 (11.6%) and SARC-3 (12.5%) also showed a reduction in P concentration comparative
to normal P, respectively.
4.1.1.4. Effects of P on Phosphorus use efficiency (PUE) in various wheat genotypes
Phosphorus use efficiency showed significant variability in wheat genotypes and it was
non-significant between normal and deficient P (p ≤ 0.05) (Figure 4.3). In normal P
treatment, the difference in PUE was observed by different genotypes. Phosphorus use
efficiency was higher for B4-5711 (0.37 g2 SDM mg-1 P) and the lowest PUE was shown
by SARC-3 (0.14 g2 SDM mg-1 P). In P deficient treatment, B4-5711, SARC-1, and
SARC-2 showed an increase in their PUE while Kohinoor-83 and SARC-3 reduced PUE
comparative to normal P treatment. The increase in PUE by SARC-1 was 16.7%
presenting the highest increase among all genotypes. The lowest increase was given by
B4-5711 (7.5%). Kohinoor-83 and SARC-3 showed a reduction of 0.24% and 21.3%,
respectively compared to PUE of these genotypes in normal P treatment.
4.1.1.5. Effects of P on pH in various wheat genotypes
In this study, all genotypes showed variation in their proton release in the root system
(Figure 4.4). Under P normal application, the SARC-1, and SARC-3, and Kohinoor, B4-
5711 and SARC-2 showed the non-significant difference in root medium pH (p ≤ 0.05).
Phosphorus deficiency activates the carboxylate release in the root system to create
homeostasis in a plant system. These carboxylates caused increase release of protons than
normal P condition. In P deficient treatment, SARC-3 showed significantly variable
results than rest genotypes. More protons release reduced pH due to P deficiency. The
maximum and the minimum reduction in pH were recorded in SARC-1 (11.2%) and
Kohinoor (2.7%) relative to normal P. Other wheat genotypes, B4-5711, SARC-2, and
SARC-3 decreased the pH by 4.2%, 6.5%, and 5.4%, respectively, compared with normal
P.
49
Figure 4.3. Impacts of P application and deficiency on PUE
50
Figure 4.4. Impacts of P application and deficiency on pH
51
4.1.1.6. Effects of P on root mass fraction (RMF) in various wheat genotypes
The phosphorus application also affects the RMF (Figure 4.5). In this experiment, P
application and deficiency showed significant differences in RMF (p ≤ 0.05). B4-5711
(0.37 g g-1) showed the highest RMF value and SARC-1 (0.42 g g-1) g g-1 showed the
lowest value under normal P application. However, in P deficient condition, RMF was
increased from 0.40-0.53 g g-1 with the highest value for SARC-1 and the lowest for B4-
5711. The increase in RMF was not the same relatively in all genotypes. SARC-1 and
SARC-3 proved better than other genotypes as these both genotypes showed an increase
of 26.7% and 27% in RMF, respectively, as compared to normal P. SARC-2 did not
perform well and gave an increase of 7.4% in RMF compared to normal P. Kohinoor-83
and B4-5711 increased RMF by 11.11 and 8.7% respectively, as compared to normal P
treatment.
4.1.1.7. Effects of P on citrate release in various wheat genotypes
All the wheat genotypes showed different responses in the release of citrate in P normal
and deficient conditions (Figure 4.6). All wheat genotypes showed the ability to release
citrate in both P normal and deficient treatment. In contrast with normal P treatment, the
deficient P gave a positive response to citrate release in root especially in Kohinoor-83,
B4-5711, SARC-1, and SARC-3. In deficient P condition, the release of citrate by these
genotypes was increased except in B4-5711 and this behavior revealed the P tolerance
potential of genotypes towards low P. The highest increase in citrate concentration was
noticed in SARC-1 (101%) under deficient P condition than normal P.
52
Figure 4.5. Impacts of P application and deficiency on RMF
53
Figure 4.6. Impacts of P application and deficiency on citrate release in the root
54
4.1.1.8. Effects of P on oxalate release in various wheat genotypes
Genotypic difference in the release of oxalate is evident from the data in both P normal
and deficient treatment (Figure 4.7). Where normal P was added, oxalate concentration in
wheat genotypes was ranged from 39 to 164 µM g-1 root DW. All the genotypes showed a
release of oxalate in control treatment. The maximum oxalate release capacity was shown
by B4-5711 while the minimum oxalate concentration was noticed in SARC-3. In a
treatment where P was deficient, genotypes of wheat showed an increase in oxalate
release with respect to the release of oxalate in normal P treatment except B4-5711 in
which oxalate was not detectable. Among the other four genotypes, SARC-1 proved
better as it exhibited an increase of 290% in oxalate concentration with respect to oxalate
released in normal P treatment while genotype SARC-3 showed the minimum potential
(34%) to release oxalate in P deficient condition as compared to control.
4.1.1.9. Effects of P on malate release in various wheat genotypes
Data showing the effect of P treatments on the release of malate by all genotypes are
represented in Figure 4.8. Malate exudation was affected significantly by genotype, P
treatments and their interaction (p ≤ 0.05). Among wheat genotypes, SARC-2 showed a
significantly higher result for the release of malate in normal P treatment. Although all
genotypes released malate in normal P treatment except SARC-3 genotype, in which
release of malate by root was not in the detectable range. Results have shown that all
genotypes had significantly variable potential to release malate in response to P
deficiency. In a P deficient treatment, different genotypes responded to P deficiency by
enhancing malate exudation. Out of five, three genotypes showed that behavior.
Kohinoor-83 (105.2%), B4-5711 (119%) and SARC-1 (104.4%) released more malate in
P deficient treatment compared to normal P treatment. Malate concentration was not
detectable in SARC-3 in P deficient treatment.
55
Figure 4.7. Impacts of P application and deficiency on oxalate release in the root
56
Figure 4.8. Impacts of P application and deficiency on malate release in the root
57
Table 4.3. Classification of wheat genotypes on the basis of % increase in total
Total Total
Genotypes Phosphorus Carboxylate carboxylate Classification
(µMg-1 RDW) increase (%)
P+ 293.77
Kohinoor-83 80.72 2
530.90
P-
P+ 640.80
20.81
B- 7112 4
774.12
P-
P+ 1005.78
116.02
SARC-1 1
P- 2172.70
P+ 582.93
SARC-2 -29.37 5
P- 411.72
P+ 144.32
SARC-3 48.99 3
P- 215.03
Here treatments are normal P (P+) and deficient P (P-).
58
4.1.2. Part 2
Effects of phosphorus on growth characteristics and release of carboxylate release to

maize genotypes
4.1.2.1. Growth responses of maize genotypes
All maize genotypes respond differently to both normal and deficient P treatment (Table
4.4). Phosphorus, genotypes and their interaction affected shoot fresh weight (SFW)
significantly while genotypic was not significant in Monsanto-6525 and Pioneer-32F10
(P<0.05). In normal P treatment, Syngenta-8711 gave significantly higher fresh weight as
compared to all other genotypes. The lowest fresh weight was shown by Cargel-6525. In
P deficient treatment, all the genotypes showed variation in their fresh weight. These
genotypes respond according to their capacity to show their SFW. Out of five, four
genotypes, Syngenta-8711(16.07%), Pioneer-33H15(16.2%), Monsanto-6525 (17.78%)
and Pioneer-32F10 (32.82%) showed decrease in SFW as compared to normal P
treatment while one genotype Cargel-6525 showed an increase (4.15%) in SFW as
compared to normal P. Among all genotypes, Pioneer, 32F10 performed better.
Shoot dry weight (SDW) exhibited a significant variation among different maize
genotypes except among Cargel-6525 and Syngenta-8711 (Table 4.4). Both genotypes
showed statistically similar results. Genotypes, P and interaction effects were significant
(P<0.05). In P normal treatment, Pioneer-33H15 showed a significantly higher SDW as
compared to all other genotypes. Lowest dry weight observed in Pioneer-32F10. In P
deficient treatment, Cargel-6525 performed better and gave the highest SDW relative to
other genotypes. All genotypes reacted to P deficiency by minifying their SDW. Pioneer-
33H15 reduced its SDW up to 36.7% which was the highest reduction among all
genotypes. The minimum decrease of 4.6% was ascertained by Cargel-6525. Shoot dry
weight was also reduced in Syngenta-8711 (20.1%), Monsanto-6525 (28.7 %) and
Pioneer-32F10 (23.2%) as compared to normal P treatment.
59
Table 4.4. Shoot fresh and dry weight and shoot length of maize genotypes under
Shoot fresh weight Shoot dry weight Shoot length
Genotypes Phosphorus
(g plant-1) (g plant-1) (cm)
P+ 08.00±0.19e 1.07±0.01d 39.90±0.38ab
Cargel-6525
P- 08.34±0.67e 1.02±0.02e 32.10±1.33d
P+ 14.77±0.68b 1.20±0.02b 42.70±2.31a

Syngenta-
8711
P- 12.40±0.70cd 0.87±0.01h 33.37±0.99cd
P+ 18.51±0.60a 1.49±0.01a 38.31±1.42a-c

Pioneer-
33H15
P- 15.51±0.61b 0.94±0.02f 35.10±4.00b-d
P+ 11.70±0.29d 1.14±0.01c 38.70±1.07a-c

Monsanto-
6525
P- 09.61±0.83e 0.80±0.008i 37.73±1.70a-d
P+ 13.84±0.60bc 0.93±0.01g 36.67±1.88a-d

Pioneer-
32F10
P- 09.30±0.72e 0.61±0.02j 35.90±1.01b-d
Phosphorus 36.37*** 1383.46*** 14.12***
F value Genotypes 58.92*** 277.90*** 0.59: ns
Interaction 4.18* 83.87*** 2.24: ns
60
Shoot length (SL) data showed that all genotypes of maize showed differential response
to P deficient treatment in hydroponics (Table 4.4). For SL, genotypes did not respond
significantly to P treatment (p ≤ 0.05) however P effect was significant (p ≤ 0.05). In
normal P treatment, Syngenta-8711 showed a higher SL compared to all other varieties.
While the Pioneer-32F10 showed the lowest SL among all genotypes. In P deficient
treatment, almost all genotypes showed a reduction in SL as compared to normal P
although that decrease in SL was not significant among genotypes. Reduction in SL by
Syngenta-8711 was the highest (27.9%) than other genotypes. Pioneer-32F10 performed
better and gave a decrease of only 2.13% in SL as compared to normal P treatment. Other
three genotypes, Cargel-6525 (24.2%), Pioneer-33H15 (9.21%) and Monsanto-6525
(2.65%) also showed decrease in SL compared to normal P treatment.
Root fresh weight (RFW) for all five maize genotypes was measured in two P treatments
(Table 4.5). Phosphorus treatment, genotypic effect as well as interaction effects on RFW
were significant (p ≤ 0.05). In normal P treatment, the variable response appeared in all
genotypes though which was not significant. Cargel-6525 showed the highest potential
for RFW production among all genotypes and Monsanto-6525 gave the lowest RFW. In P
deficient treatment, Syngenta-8711 gave the highest RFW while the lowest RFW
appeared in Monsanto-6525. Pioneer-32F10 performed better in P deficient treatment by
showing the increase of 80.22% among all genotypes as compared to normal P treatment.
A significant increase in RFW was also given by Syngenta-8711 (21.22%) and Pioneer-
33H15 compared to normal P treatment. Reduction in RFW was recorded in Cargel-6525
(23.5%) and Monsanto-6525 (6.8%) comparative to normal P treatment.
Root dry weight (RDW) of all the five maize genotypes is represented in Table 4.5. Non-
significant genotypic difference in RDW is evident in P normal as well as in P deficient
treatment. However significant variation appeared in genotypes when the comparative
effect of P normal and P deficient treatment was evaluated. RDW was significantly
variable among genotypes especially in P deficient treatment relative to normal P
treatment. In normal P treatment, Pioneer-33H15 showed a higher RDW as compared to
all other genotypes but the difference in RDW among genotypes was not significant. The
lowest RDW was observed in Monsanto-6525.
61
Table 4.5. Root fresh and dry weight, root length and Root: Shoot of maize
genotypes under normal and deficient P
Root fresh Root dry
Root length
Genotypes Phosphorus weight weight Root: Shoot
(cm)
Cargel- P+ 6.68±0.36a 0.32±0.01ab 26.90±0.34d 0.30±0.01e
6525
P- 6.60±0.11a 0.31±0.02ab 26.60±0.72d 0.31±0.01e
Syngenta- P+ 5.11±0.02b 0.29±0.01bc 32.90±2.66bc 0.22±0.01f

8711
P- 3.88±0.32cd 0.33±0.01a 35.40±0.44ab 0.49±0.01b
Pioneer- P+ 5.59±0.20b 0.32±0.01a 33.17±0.60bc 0.17±0.01g

33H15
P- 3.61±0.41cd 0.34±0.02a 33.77±0.96bc 0.42±0.01c
Monsanto- P+ 6.77±0.25a 0.25±0.01d 33.13±0.58bc 0.14±0.02h

6525
P- 3.10±0.36d 0.29±0.01c 31.67±0.88c 0.36±0.01d
P+ 4.08±0.40c 0.28±0.01c 23.70±0.79d 0.31±0.01e

Pioneer-
32F10
P- 5.58±0.30b 0.33±0.01a 37.33±1.20a 0.83±0.02a
Phosphorus 35.30*** 25.44*** 17.94*** 2147.48***
F value Genotypes 18.29*** 16.04*** 14.15*** 418.76***
Interaction 22.41*** 3.29* 15.09*** 221.96***
62
In P deficient treatment, a comparative improvement in RDW was pointed out in all
genotypes except in Cargel-6525. RDW was reduced in Cargel-6525 while in Syngenta-
8711, Pioneer-33H15, Monsanto-6525 and Pioneer-32F10, it was increased. Reduction in
RDW in Cargel-6525 was 1.7% while in Syngenta-8711, Pioneer-33H15, Monsanto-
6525, and Pioneer-32F10, increase in RDW was 12.1, 4.9, 12.7 and 15.29% respectively,
with respect to normal P. The highest increase (15.29%) in RDW was observed in
Pioneer-32F10 compared to normal P.
Root length (RL) showed significant results by P treatment (p ≤ 0.05). The interaction
effect was also highly significant (p ≤ 0.05). All the maize genotypes in normal P
treatment showed different responses in their RL (Table 4.5). Pioneer-33H15 showed
remarkably higher RL compared to all other genotypes. The minimum RL was noticed in
Pioneer-32F10 although statistically, no significant difference appeared in any genotypes
for RL. In P deficient treatment, the highest RL was observed by Pioneer-32F10 and the
lowest was in Cargel-6525. Out of five, three genotypes Syngenta-8711, Pioneer-33H15
and Pioneer-32F10 showed an improvement in RL while a decrease in RL appeared in
Cargel-6525 and Monsanto-6525. The decrease of 1.23% and 4.43% in RL was exhibited
by Cargel-6525 and Monsanto-6525, respectively, as compared to P normal condition.
The maximum increase was ascertained by Pioneer-32F10 (61.8%). The increase of
7.59% and 1.81% was noticed by Syngenta-8711 and Pioneer-33H15 respectively,
comparative to normal P treatment.
Data related to root to shoot ratio (R/S ratio) is marked in Table 4.5. Statistical results
depicted significant P treatment and genotypic differences (p ≤ 0.05). In normal P
treatment, significant differences in the R/S ratio appeared between genotypes. R/S ratio
was ranged from 0.31-0.54 with Cargel-6525 and Pioneer-32F10 having the lowest and
the highest value respectively. In P deficient treatment, as the RDW was higher than
normal P treatment, the R/S ratio was also increased in all these genotypes. The increase
in the R/S ratio was not the same relatively in all genotypes. Pioneer-32F10 proved better
than other genotypes as it showed an increase of 79.6% in the R/S ratio as compared to
normal P treatment. The increase in root to shoot ratio by Cargel-6525 was 3.2% as
compared to normal P which was the lowest than other genotypes. Syngenta-8711,
Pioneer-33H15 and Monsanto-6525 increased R/S ratio by 58.14%, 65.90% and 61.18%
respectively, as compared to normal P treatment.
63
4.1.2.2. Root P in various maize genotypes
Genotypes and P treatment affected root P concentration significantly and the interaction
effect was not significant (p ≤ 0.05) (Figure 4.9). At normal P treatment, higher root P
concentration was obtained in Pioneer-33H15 while Monsanto-6525 showed the lowest
root P concentration among all genotypes. At the deficient level of P, the highest root P
was in Pioneer-32F10 and the lowest root P concentration was obtained by Monsanto-
6525. Due to P deficiency, genotypes exhibited a reduction in root P concentration. The
maximum decrease in root P concentration was recorded in Monsanto-6525 and that a
decrease in root P concentration was 29% with respect to its root P concentration in P
normal treatment. A reduction of 2.82% was shown by Pioneer-32F10 which was the
minimum among genotypes while Cargel-6525 (28.9%), Syngenta-8711(14.15%) and
Pioneer-33H15 (9.59%) showed a decrease in root P concentration as compared to normal
P treatment.
4.1.2.3. Shoot P in various maize genotypes
Shoot P concentration of all maize genotypes is represented in figure 4.10. Shoot P

concentration was affected significantly by genotype, P treatment while the interaction
effect was not significant (p ≤ 0.05). All other genotypes showed similar results
statistically. In the case of P, both treatments gave statistically significant variation in
results. At normal P treatment, the maximum P concentration was noticed in Monsanto-
6525 while the minimum concentration was in Pioneer-33H15. At P deficient treatment,
shoot P concentration decreased overall. However, the percent decrease was different in
all genotypes. Pioneer-32F10 showed the highest percent (49%) of reduction in shoot P
concentration and the minimum reduction in P concentration was noticed in Syngenta-
8711(45.6%) as compared to its P concentration in normal P treatment.
64
Figure 4.9. Impacts of P application and deficiency on root P
65
Figure 4.10. Impacts of P application and deficiency on shoot P
66
4.1.2.4. Effects of P on Phosphorus use efficiency (PUE) in various maize genotypes
Phosphorus use efficiency for all genotypes in P deficient and normal P treatment is
presented in figure 4.11. Treatment, genotypic and interaction effect was significant (p ≤
0.05). In normal P treatment, genotypic variability was evident, but this variability was
not significant. Pioneer-33H15 was more efficient (0.67 g2 SDM mg-1 P) in using P as
compared to other genotypes. Phosphorus use efficiency demoed by Pioneer-32F10 (0.32
g2 SDM mg-1 P) was less than other genotypes. In P deficient treatment, all the genotypes
used P efficiently and depicted a sharp increase in PUE compared to their efficiencies in
normal P treatment. The maximum PUE was calculated in Pioneer-33H15 (0.81 g2 SDM
mg-1 P) and the minimum was in Monsanto-6525 (0.56 g2 SDM mg-1 P).
4.1.2.5. Effects of P on pH in various maize genotypes
Root medium pH was significantly (p ≤ 0.05) affected by genotypes except in genotypes

Syngenta-8711 and Pioneer-33H15 that shared the same letter and were not statistically
different (Figure 4.12). The effect of P treatments and interaction was highly significant
(p ≤ 0.05). In normal P treatment, pH was not variable among genotypes. While in P
deficient condition, results differed significantly among genotypes. All genotypes showed
a reduction in pH in P deficient treatment as compared to normal P treatment. In P
deficient condition, reduction in pH was higher in Pioneer-32F10 (19.7%) while the
minimum reduction was observed by Cargel-6525 (1.5%) as compared pH of the same
genotypes in normal P treatment. Reduction in pH of 10.1%, 7.9%, and 11.4% were
noticed by Syngenta-8711, Pioneer-33H15, and Monsanto-6525, respectively as
compared to pH for these genotypes in normal P treatment.
67
Figure 4.11. Impacts of P application and deficiency on PUE
68
Figure 4.12. Impacts of P application and deficiency on pH
69
4.1.2.6. Effects of P on root mass fraction (RMF) in various maize genotypes
Data regarding the RMF is represented in figure 4.13. Root mass fraction differed
significantly between genotypes as well as between P treatments (p ≤ 0.05). In normal P
treatment, RMF was ranged from 0.17-0.23 g g-1. The highest RMF was noticed in
Pioneer-32F10 while the lowest was in Pioneer-33H15. In P deficient treatment, all
genotypes increased RMF as compared to normal P treatment. Root mass fraction was
ranged from 0.23-0.35 g g-1 with the highest value for Pioneer-32F10 and the lowest for
Cargel-6525. An increase in RMF was the highest for Pioneer-32F10 (51.4%) and Cargel-
6525 showed the lowest increase (2.43%) in RMF with respect to normal P treatment.
Syngenta-8711, Pioneer-33H15, and Monsanto-6525 increased RMF by 42.18%, 48.68%
and 45.02% respectively, as compared to normal P treatment.
4.1.2.7. Effects of P on citrate release in various maize genotypes
Fig 4.14 represents the data for the effect of P deficiency on the release of citrate by all
genotypes. Phosphorus treatment and genotype, as well as their interaction, showed a
significant effect on citrate release (p ≤ 0.05). In normal P treatment, all genotypes
released citrate, though their potential to release citrate was variable. In normal P
treatment, the highest citrate release was noticed in Pioneer-33H15. Pioneer-32F10
released the minimum amount of citrate among all genotypes. In P deficient treatment, an
increase in citrate release was recorded by Cargel-6525 (5.45%), Syngenta-8711 (127.4
%) and Pioneer-32F10 (2771%) and the maximum release of citrate was recorded by
Pioneer-32F10 as compared to normal P treatment. Pioneer-33H15 and Monsanto-6525
reduced citrate release by 62% and 4% when exposed to P deficiency. Monsanto-6525
revealed the lowest potential to release citrate in P deficient condition.
70
Figure 4.13. Impacts of P application and deficiency on RMF
71
Figure 4.14. Impacts of P application and deficiency on citrate release in the root
72
4.1.2.8. Effects of P on oxalate release in various maize genotypes
Data showing the effect of P deficiency on the release of oxalate by all genotypes are
represented in Fig 4.15. The effect of P treatment and genotypes on oxalate release by
plant root was significant (p ≤ 0.05). Among all maize genotypes, only two genotypes
Monsanto-6525 and Pioneer-32F10 revealed their potential of oxalate exudation at both
normal P as well as P deficient treatment. The other three genotypes Cargel-6525,
Syngenta-8711, and Pioneer-33H15 were not efficient enough to exude oxalate in normal
P as well as P deficient treatments. Under P deficiency, Monsanto-6525 and Pioneer-
32F10 both showed an increase in their exudation of oxalate. Monsanto-6525 showed
16.54% and Pioneer-32F10 showed 32.20% more oxalate release in P deficient treatment
comparative to normal P treatment that represents the potential of these genotypes to cope
to low P environment.
4.1.2.9. Effects of P on malate release in various maize genotypes
Maize genotypes showed different responses in the release of malate in P deficient

treatment as well as in P normal treatment (Figure 4.16). From the statistical result, it was
evaluated that treatment effect and genotypic response to P deficiency for the release of
malate was significant (p ≤ 0.05). In P normal treatment, Pioneer-32F10 showed a
significantly higher malate release compared to all other genotypes. In P deficient
treatment, malate release by Pioneer-32F10 was the highest among all genotypes. An
increase in malate release by Pioneer-32F10 was the highest (101.8%) in P deficient
condition as compared to normal P among genotypes in P deficient condition. Syngenta-
8711, Pioneer-33H15, Pioneer-32F10 and Cargel-6525 increased malate release 93.45%,
28.15%, 101.8% and 54.26% in P deficient treatment that depicted adoption of these
genotypes to low P condition. However, Monsanto-6525 gave a decrease of 20.02% in
malate release by roots in P deficient treatment as compared to normal P.
73
Figure 4.15. Impacts of P application and deficiency on oxalate release in the root
74
Figure 4.16. Impacts of P application and deficiency on malate release in the root
75
Table 4.6. Classification of maize genotypes on the basis of % increase in total
Total Total
Genotypes Phosphorus Carboxylate carboxylate Classification
(µM g-1 RDW) increase (%)
P+ 210.44
Cargel-6525 54.55 3
P- 325.23
P+ 207.78
Syngenta-8711 93.87 2
P- 402.83
P+ 931.97
Pioneer-33H15 27.53 4
P- 1188.58
476.90
P+
Monsanto-
-20.01 5
6525
P- 381.48
P+ 1519.18
Pioneer-32F10 102.44 1
P- 3075.49
Here treatments are normal P (P+) and P deficiency (P-)
76
4.1.3. Discussion
Phosphorus (P) is considered a limiting nutrient as its availability is low in Pakistani soils.
To meet the P demand for crops, additional P is added to soils. This additional P exerts
economical pressure. So, there is a need to find out genotypes that can grow well under
low P availability. Some plants under low P availability change their physiology and
release carboxylates by roots. Therefore, this study was planned to screen P efficient
wheat and maize genotypes in hydroponics culture on the basis of their root released
carboxylate concentration.
Five wheat genotypes (Kohinoor-83, B4-5711, SARC-1, SARC-2, and SARC-3) and five
maize genotypes (Cargel-6525, Syngenta-8711, Pioneer-33H15, Monsanto-6525, and
Pioneer-32F10) were screened at P normal and deficient levels. Significant differences
were observed in different wheat and maize genotypes. Normal P application significantly
improved the growth characteristics. However, P deficiency significantly reduced the
growth characteristics but increased the carboxylate released in the root.
Root length significantly increased under P deficient compared with P normal application
in both wheat and maize genotypes. It is an important parameter in terms of the release of
carboxylates and efficient in utilization of P under deficient conditions. Results indicated
a significant increase in RL by both wheat and maize genotypes in P deprivation
comparative to P availability. Parallel outcomes were testified by many scientists, roots
growth was more in P limiting environment because of non- mobility of P in soil
(Marschner, 1995; Gill et al., 2002; Kosar et al., 2002). Under P deprivation, more
metabolites mobilized towards roots to increase RL and biomass for exploring more
volume to get P (Lynch and Brown, 2001; Liao et al., 2001). High RL in wheat is an
imperative characteristic to improve P acquisition in P stress (Manske et al., 2000).
Root dry weight is an important parameter in the selection of P efficient genotypes. Root
dry mass increased significantly in all genotypes as P application is decreased. Genotypes
that showed comparatively more increase in RDW in P deficient condition as compared
to P sufficiency are considered tolerant to low P (Yaseen and Malhi, 2009). In P
deficiency response, more carbon allocated to roots that result in enhanced root growth
that may lead to enhanced root dry matter (Lynch and Brown, 2001; Liao et al., 2001).
Similar results between T222 and T149 genotypes of maize in root growth were noticed
by Yi-Kai et al., (2013).
77
In this study, four genotypes of wheat and five maize genotypes showed an upsurge in
R/S ratio in lower P condition as compared to P normal condition. But the percent
increase was different for all genotypes. Root mass fraction was high for all genotypes in
P deficient conditions. Both RMF and R/S ratio is related to root and shoots dry weight.
So, the genotypes that showed high RDW also showed an increased R/S ratio and RMF
(Ramaekers et al., 2010). Our outcomes were in accordance with Zhu, (2004) who
reported that the R/S ratio increased by 39% at low P availability in maize efficient
genotype.
Shoot P concentration at deficient P level was lower as compared to P normal level in all
genotypes. These differences varied from genotype to genotype. This shows genotypes
with more accumulated shoot P in a deficient medium are considered tolerant of P
deficiency (Yaseen and Malhi, 2009). The reason for this behavior is that under low P
condition, plants start using internal P sources to overcome the P deficiency which may
reduce the plant P contents. Haynes et al., (1991) also reported parallel outcomes. He
reported that higher shoot P concentration in P efficient genotypes were related to
improved P uptake efficiency in terms of higher R/S ratio at low P relative to P sensitive
genotypes (Balemi and Schenk, 2009).
In this experiment, P deficiency showed a decrease in root P in all genotypes. This

decrease in root P was significantly different in maize genotypes. Phosphorus efficient
genotypes normally gave the minimum reduction in its root P concentration. These P
efficient genotypes also maintain relatively higher P content in root as compared to shoot
so that the root system can be improved. So, these improved root systems can efficiently
uptake nutrients (Akhtar et al., 2008; Yi-Kai et al., 2013). Marked variances in P
utilization efficacy between genotypes of both wheat and maize were noticed. The
substantial difference in the ability of wheat and maize genotypes in uptake and
utilization of P was observed in low P conditions.
In this experiment, PUE increased in almost all wheat and maize genotypes under P
deficient treatment. An important factor that caused an increase in PUE might be due to
the intrinsic characteristics of different genotypes. Similarly, Osborne and Rangel, (2002)
Chen et al., (2009) and DoVale and Fritsche-Neto, (2013) also reported variation in maize
and wheat genotypes in the PUE in the nutrient solution.
Genotypic variation in growth characteristics such as SL, SFW, and SDW of both maize
78
and wheat was observed in normal P as well as deficient P condition. In normal P
condition, variation in SL may be due to the intrinsic ability of each genotype. All the
genotypes differ in their growth response to the same treatment. In P deficient condition,
each genotype responded variably to P normal condition. Reduction in SL, fresh and dry
weight in P deficient condition was observed which might be due to the translocation of
carbon compounds from shoot to roots. Similarly, Yi-kai et al., (2013) reported
noteworthy variances in biomass of shoot of maize genotype at deficient P level.
The most important plant's response to P deficient condition is, exudation of carboxylates
by roots in this study. A significant variation in P deficient as well as in normal P was
observed in the root released carboxylates among genotypes of both maize and wheat. In
some genotypes, carboxylate release was not in detectable range, while many genotypes
responded to P deficient condition by the release of carboxylates. Genotypes responded to
P deprivation by the release of carboxylates are well-thought-out as efficient genotypes
under P deficiency. The reason behind this increased carboxylate exudation is the change
in the TCA cycle. TCA reaction in P efficient plants proceeds very another way from
TCA reaction in P deficient plants. Enhanced synthesis of citrate occurred but the
conversion of citrate to iso-citrate is inhibited due to the decreasing activity of the enzyme
involved in this conversion. This results in increased accumulation of citrate in the cell,
which is then released in rhizosphere (Kihara et al., 2003). Our outcomes are also in
accordance with the results of Shen et al., (2002). They testified increased secretion of
carboxylates by roots in P efficient common bean genotypes that caused an enhanced P
uptake comparative to P inefficient genotypes of less P solubilizing activity. Parallel
results were reported by Ming et al., (2002). They described carboxylate release was
more in P tolerant genotype (JX17) comparative to P sensitive genotypes (ZYQ8) of rice
in a P stress environment.
Carboxylates released by roots are strongly associated with increased growth especially
RL. In this study, an increase in carboxylate release is linked to RL, R/S ratio and RDW.
As the RL and dry weight increased, carboxylates release was also increased. This
increase in root parameters might help the plants to increase root exudation through
increasing root surface area. Our outcomes were in accordance with Shu et al., (2007).
They testified that roots length, RDW, RMF, and R/S ratio enhanced in response to P
deficiency. This increase in the root system may be due to the carboxylates that help in P
solubilization in the soil.
79
Figure 4.17. Total carboxylates (citrate, malate, and oxalate) released by wheat and mai
ze in P+ (a) and P- (b) treatments. Genotype numbering on X- shows wheat (Kohinoor-
83= 1; B4-5711= 2; SARC-1= 3; SARC-2= 4; SARC-3= 5) and maize genotypes (Cargel-
6525= 1; Syngenta-8711=2; Pioneer-33H15= 3; Monsanto-6525= 4; Pioneer-32F10=5).
80
In this study, it was found that the type and concentration of carboxylates release vary
within species (Zhao and Wu, 2014). Genotypic variation in the carboxylate release in
barley genotypes is also reported by Asmar et al., (1995). Similar observations were
observed in soybean genotypes. Pi-efficient soybean genotypes exuded more malic acid,
which ultimately caused improvement in plant P nutrition (Liao et al., 2006). Genotypic
variation in carboxylates exudation by roots of green gram and maize genotypes were
noted in solution culture where P concentrations were ranged from 0 - 100 micro molars
(Singh and Pandy, 2003). An increased carboxylate exudation with a decreasing supply of
P was noticed, indicating that P deprivation had roused carboxylate release (Abrahao et
al., 2014).
In this study, the amount of carboxylate exudation was different in wheat genotypes
compared with maize genotypes. The total carboxylates release in maize genotypes was
more as compared to wheat genotypes (Figure 4.17). This difference might occur due to
the difference in plant metabolism. If all the factors that can influence root exudation like
root architecture, type of collection medium, mechanical impedance, plant sterility, and
age are not considered as the reason for exudation, (Aulakh et al., 2001; Skene, 2003; Tu
et al., 2004; Johansson et al., 2009; Toyama et al., 2011) C4 plants may release a high
amount of total organic carbon per unit mass of plant roots compared to C3 plant
(Yoshitomi and Shann, 2001; Baudoin et al., 2003).
Our results are supported by many investigators previously (Haase et al., 2007; Johansson
et al., 2009; Hajlaoui et al., 2010; Nabais et al., 2011) as they reported that C4 (maize)
showed more carboxylate release than C3 (wheat). Wheat, being a C3 crop, released
fewer carboxylates as compared to maize that has C4 metabolism.
Both wheat and maize genotypes showed a reduction in pH in deficient P treatment as

compared to normal P treatment. Genotypes that showed more reduction in pH can be
considered P efficient genotypes. This lowering of pH might be due to more proton
released by roots of P efficient genotypes. This proton release occurs because of released
carboxylates. When carboxylates are released, protons help to maintain charge neutrality
inside the plant (Hinsinger et al., 2003). Our results are supported by Keerthisinghe et al.,
(1998) and Neumann et al., (1999), who reported that acidification of the rhizosphere,
might be resulted because of increased proton release.
81
On the basis of a percent increase in total carboxylates in P deficient condition compared
to normal P, we categorized the tolerance approach of wheat (Table 4.3) and maize (Table
4.6) genotypes against deficient P. According to this, wheat genotypes SARC-
1>Kohinoor-83>SARC-3>B4-5711>SARC-2 and maize genotypes, Pioneer-
32F10>Syngenta-8711> Cargel-6525> Pioneer-33H15>Monsanto-6525 were categorized.
4.1.4. Conclusion
In the present hydroponic study, plant physiological responses, carboxylate release,

proton release, R/S ratio, RMF, PUE were significantly enhanced under P deficient
condition compared to normal P. However, genotypes differ in their response to deficient
P condition. From all the above results evaluation and discussion, it is concluded wheat
genotype SARC-1 and maize genotypes Pioneer-32F10 showed better adaptability and
are efficient and responsive to the deficient P environment. Efficient genotypes selection
was done on the basis of a high percent increase in carboxylate release, decrease in pH,
PUE, RL, RMF, and R/S ratio. Both genotypes were selected for further study. This
useful genetic difference that exists among wheat and maize genotypes can be well
utilized for identification and assortment of high yielding P efficient genotypes for P
deficient soils through the breeding process in the future.
82
4.2. Study 2
Effect of carboxylates (organic acids) on P adsorption (Incubation
study)
Due to high calcium (Ca) contents and high pH, the availability of P is a major issue. At
high pH (> 8), phosphate ion forms compounds with calcium that makes P unavailable for
plant uptake (Ahmad et al., 2003; Rashid et al., 2005). So, it is a need of the hours to
improve the plant's P availability for high yield. To gain high yield, the management of
chemical fertilizers is very important. But Managing P fertilization deprivation through
chemical methods typically distresses the soil health and affects the cost of (Gyaneshwar
et al., 2002).
Organic increments are well known for improving availability of P in high P fixing soils
(Gichangi and Mnkeni, 2009). Carboxylates are organic anions with varying chain length,
having carboxyl groups. These are secreted by the plant roots due to either P deficiency or
accumulation of aluminum (Aono et al., 2001; Wang et al., 2007; Carvalhais et al.,
2010). By another definition, carboxylates are organic anions, which are organic acids
minus the proton(s). Citrate is the result of de-protonation of citric acid (Lambers et al.,
2015).
Organic acids showed a necessary part in sorption and desorption of soil P. Organic acids
might enhance P in the soil solution upto 1000 times (Fox and Comerford, 1990). Organic
acids contend for exchange sites with P in soil and make P available (Yamamura et al.,
2004; Hoffland et al., 2006). Therefore, application of organic acids might be helpful in
desorption of adsorbed P. Such type of desorption using carboxylates (organic acids) in
Pakistani soils has not been reported extensively. The precise system or nature of
collaboration among organic acids and inorganic P has not been settled (Wang et al.,
2008). Experiments to study carboxylates assisted phosphate desorption would be
beneficial in understanding the role of carboxylates in P desorption (Mihoub et al.,
2016b). Keeping in view the significance of P in the soil system, a study was planned
with the objectives
➢ To check P adsorption behavior of three different textured soil
➢ To evaluate the role of carboxylates in P desorption in three different textured

soils
83
This study was divided into two parts: In the first part, the P adsorption capacity of three
different textured soils was studied and data were evaluated by using adsorption
equations. In the second part, adsorption of P in the presence of carboxylates in these
soils was studied.
Results
4.2.1. P adsorption in three different textured soils
4.2.1.1. P equilibrium concentration (C)
Data regarding P equilibrium concentration after treating all soils with different
concentrations of P are represented in Table 4.7. Phosphorus concentration was different
in all three different textured soils at similar levels of applied P in soil solution. All soils
at different levels of applied P were substantially diverse (p ≤ 0.05). In loam soil, the P
concentration in soil solution increased as, amount of P increased. Percent decline in P
equilibrium concentration was negatively related with addition of P. Highest P
equilibrium concentration was observed in 500 mg P application kg-1 soil showing the
lowest percent of added P adsorption (77.8%). The minimum equilibrium P concentration
was found at 100 mg P kg-1 soil application showing the highest percent of added P
adsorption (95%).
In sandy loam soil, increase in equilibrium P concentration along with added P was
evident. Phosphorus equilibrium concentration was significantly different at all P applied
levels (p ≤ 0.05). The maximum P equilibrium concentration was detected at 500 mg P
kg-1 but this showed the minimum percent decrease in added P (71.8%) in equilibrium
concentration. The lowest P equilibrium concentration was found 100 mg kg-1 added P in
soil, with highest percent decrease in added P (91%).
In sandy clay loam soil, significant variations (p ≤ 0.05) in equilibrium P concentration

values were noticed. The increasing level of P improved equilibrium P concentration
relatively. At 100 mg kg-1 added P in soil, the amount of P that remained in soil solution
was 0.09 µg mL-1 which was the minimum but in terms of percent of added P, it was the
maximum (99.1%). The maximum equilibrium P concentration was observed at 500 mg P
application kg-1 soil, it was the minimum in terms of percent of added P (92.9%). From
all three different textured soils, more equilibrium P concentration was noticed in sandy
loam soil at all levels of applied P when compared with other soils. Sandy clay loam soil
showed the lowest equilibrium P concentration.
84
4.2.1.2. Adsorbed P
P adsorption data in the soil after treating all soils with different concentrations of P are
represented in Table 4.7. Significant variances in adsorbed P values were observed at all
P applied levels as well as in soil types (p ≤ 0.05).
In loam soil, the amount of P adsorption improved with an increase in level of P addition.
The maximum P adsorption (390 µg g-1) was observed at 500 mg kg-1 of added P which
was lowest in terms of percent of added P adsorbed (78% of applied P) while at 100 mg
kg-1 soil added P, the minimum P adsorption of 95 µg g-1 in soil was noticed which was
the highest in terms of percent of added P adsorbed (95% of applied P). In sandy loam
soil, a linear relation between adsorbed P and added P was evident. At 100 mg kg-1 added
P in soil, the minimum amount (91 µg g-1) of P adsorbed was observed which was the
highest in terms of percent of added P adsorption (91% of applied P) while at 500 mg kg-1
P, the maximum P adsorption of 359 µg g-1 (71.8% of applied P) was observed.
In sandy clay loam soil, increase in P adsorbed was linearly related with added P (Table
4.7). At 100 mg kg-1 added P in soil, the minimum P adsorption of 99.1µg g-1 (99.1% of
applied P) in soil was noticed. At 500 mg P application kg-1of soil, the highest P
adsorption was 464.6 µg g-1 (92.92% of applied P) was recorded.
A comparison of three different texture soils indicated more P adsorption in sandy clay
loam soil at all P applied levels when compared with loam and sandy loam soils. Sandy
loam soil showed the lowest P adsorption. The highest P adsorption was observed in
sandy clay loam soil when it was treated with 500 mg kg-1 P. While the lowest adsorption
of P was observed in sandy loam soil when this soil was treated with 100 mg P kg-1 of
soil.
85
Table 4.7. Amount of P in soil solution and P adsorbed
P Added Phosphorus remained Phosphorus adsorbed

(mg Kg-1 in soil solution (µg mL-1) C in soil (µg g-1) Q
soil)
Loam Sandy Sandy Loam Sandy Sandy
loam clay loam loam clay loam
0 0 0 0 0 0 0
100 0.5k 0.9j 0.09m 95n 91o 99.1m
200 1.9i 2.1h 0.24l 181k 179l 197.6j
300 3.1g 3.3f 0.92j 269h 267i 290.8g
400 3.9d 5.9c 1.89i 361d 341f 381.1c
500 11.1b 14.1a 3.54e 390b 359e 464.6a
Values are means of three replicates (n=3). Mean values with the same letter do not differ
significantly at (p ≤ 0.05).
Table 4.8. Correlation coefficients (R2) and regression equations for adsorbed P and
added P
Soil Regression equation R2 Value

Loam Y=13.429+8.1029x 0.7842
Sandy loam Y=18.095+7.5229x 0.7987
Sandy clay loam Y=5.8524+9.3206x 0.8434
86
4.2.1.3. Adsorption Isotherms for P (KH2PO4) in three different textured soils
P adsorption data were plotted and fitted to Freundlich and Langmuir equations according
to Langmuir and Freundlich equations for all soils. Data from these equations were used
to estimate P sorption energy, the maximum P adsorption capacity, and constant of the
energy of adsorption and sorption capacity in all three different textured soils. The
goodness of fit to Freundlich and Langmuir was estimated by correlation coefficient R2.
4.2.1.3.1. Freundlich Equation
For fitting adsorption data to the Freundlich equation, log C (Equilibrium P

concentration) was plotted against Log Q (Adsorbed P) (Figure 4.18). The Freundlich
parameter, P sorption capacity and P sorption energy for all the three different textured
soils were calculated by plotting P equilibrium concentration against P adsorbed (Table
4.9). Data of all three different textured soils for P adsorption showed the goodness of fit
to the Freundlich equation as the values of R2 were 0.91, 0.87, 0.96 for loam, sandy loam,
and sandy clay loam soil, respectively (Table 4.9). These correlation coefficient values
apparently demonstrated a good conformation of adsorption data to Freundlich model.
Commonly, at all P equilibrium concentrations, the Freundlich model seemed fit.
P sorption capacity was ranged from 117.06-295.72 µg g-1. Sandy clay loam soil showed
more P sorption capacity (295.72 µg g-1). The p sorption capacity of sandy loam soil was
(117.06 µg g-1) lower than loam and sandy clay loam soils.
P sorption energy in all soils was ranged from 0.4004-0.5098 mL g-1. The highest
sorption energy was noticed in sandy loam soil (0.5098 mL g-1) while loam soil showed
0.4919 mL g-1 sorption energy and sandy clay loam soil showed 0.4004 mL g-1 sorption
energy which was the lowest than loam and sandy clay loam soils. Linear and model form
of Freundlich is presented in Table 4.10.
4.2.1.3.2. Langmuir Equation

For the Langmuir equation, adsorption data were plotted as 1/C against 1/Q. (Figure
4.19). From the linear Langmuir equation, maximum adsorption capacity and the constant
of the energy of adsorption of soil for P were calculated (Table 4.11). The fitness of data
to Langmuir model was confirmed by the R2 value of all three soils. Adsorption data of
three different textured soils appeared fit to the model. R2 values for loam, sandy loam,
and sandy clay loam soil were 0.96, 0.98 and 0.99 (Table 4.11). Sandy clay loam
appeared best fitted to Langmuir equation followed by sandy loam soil. Different textured
87
soils showed variation in their sorption characteristics. From results, it is evident that all
three different textured soils have high sorption sites because these soils had the
maximum P sorption capacity ranging from 120 to 1428 mg kg-1. Among all different
textured soils, the highest sorption capacity (1428 mg kg-1) was in sandy clay loam soil
followed by loam soil (238 mg kg-1). Constant of the energy of adsorption was ranged
from 0.304 to 4.88 with the maximum constant of the energy of adsorption in sandy loam
soil and the minimum (0.304) in sandy clay loam soil.
88
Table 4.9. P sorption capacity and sorption energy for loam, sandy loam, and sandy
clay loam soil.
Sorption Capacity P sorption energy
Soil R2 Value
(µg g-1) (a) (mL g-1) (b)
Loam 143.21 0.4919 0.9156
Sandy loam 117.06 0.5098 0.8705
Sandy clay loam 295.72 0.4004 0.9678
Table 4.10. Model and linear form of Freundlich Equation
Freundlich equation Freundlich equation

Soil
Model form Linear form
Loam Q= 143.21C0.4919 Y= 0.4919x+2.156
Sandy loam Q= 117.06C0.5098 Y= 0.5098x+2.0684
Sandy clay loam Q= 295.72C0.4004 Y= 0.4004x+2.4709
Table 4.11. Maximum P adsorption capacity and the constant of energy of

adsorption as determined by the Langmuir model
Maximum P
Constant of energy
adsorption capacity
Soil of adsorption (k) R2
(mg kg-1) (a)
Loam 238 1.75 0.96

Sandy loam 120 4.88 0.98
Sandy clay loam 1428 0.304 0.99
89
Figure 4.18. Graphical presentation of Freundlich adsorption isotherm for P in loam,
sandy loam and sandy clay loam soils
90
Figure 4.19. Graphical presentation of Langmuir adsorption isotherm for P in loam,
sandy loam and sandy clay loam soil
91
4.2.2. Effect of carboxylates (Organic acids) on P adsorption.
4.2.2.1. P adsorption in loam soil
Phosphorus adsorption in loam soil treated with organic acids along with different P
concentrations is shown in Table 4.12. Significant differences among applied P levels and
among the different concentrations of organic acids were observed.
Citric acid, when applied in loam soil at different concentrations of P, a significant

reduction in P adsorption in soil was recorded as compared to soils where P was added
but no organic acid was applied. Citric acid application at the rate of 1 mM showed less
adsorption when compared with 0.5 mM citric acid addition at all concentrations of P.
The highest reduction of 11.5% in P adsorption was noticed where applied P level was at
500 mg kg-1. Minimum reduction in P adsorption (1.7%) was noticed at P addition of 300
mg kg-1 soil.
In treatments where oxalic acid was applied, reduction in adsorption was noted as
comparative to treatments when P applied without organic acids. 1 mM oxalic acid
addition at 500 mg P kg-1 of soil application showed 10.5% less adsorption while 1.4 %
less adsorption was noted at 0.5 mM oxalic acid application in 300 mg P kg-1 soil. Decline
in adsorption was further high when oxalic acid at a rate of 1 mM was applied as
compared to oxalic acid addition at a rate of 0.5 mM, at all levels of applied P.
Malic acid application (1 mM) caused more reduction in adsorption comparative to

separate P application and 0.5 mM malic acid application. The minimum reduction in P
adsorption (0.85%) was noticed at P application of 300 mg kg-1 soil and the maximum
reduction (7.23%) was observed in 1 mM malic acid application at 500 mg P kg-1 soil.
From all the applied organic acids, citric acid proved more efficient in the reduction of P
adsorption in the soil as compared to other organic acids while 1 mM organic acid
application was more efficient in the reduction of P adsorption comparative to 0.5 mM of
organic acids application.
92
Table 4.12. Effect of carboxylate (organic acids) application on P adsorption in three different textured soils
P
Add Loam Sandy loam Sandy clay loam
ed
mg C.A C.A O.A O.A M.A M.A C.A C.A O.A O.A M.A M.A C.A C.A O.A O.A M.A M.A
kg-1 (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1) (0.5) (1)
0 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D 0D
91.5 90.8 92.2v 91 92.9 91.3 85.7 81.6 86.9 84.5 88.2 84.4 98.6 97.9 98.6 98 98.71 98.2
100
wx x w x v wx A C z B y B u u u u u u
177 171.4 177.3 171.6 179 172r- 172.5 171 173.4 173 177.7 175.1 192. 191 193.7 191.6 194.3 193.4
200
o st o st n t q-s t q qr o p 8l m kl m k kl
264.2 262.7 265.1 263 265.6 263.8 258.8 251.3 259.9 252.1 264.3 254.6 286. 281.7 289.5 283 289 285.8
300
ef g de g d fg h j h j ef i 3a c Z b Z a
354.2 340.3 355.3 343.3 357.9 346.7 332.6 321.2 336.9 323.7 339.2 324.3 378. 366.1 378.3 367.9 378.9 369.4
400
N ST N R M P V Y U X T X 3G K G J G I
358.8 344.9 362.4 348.8 374.1 361.8 340.6 330.8 347.2 332.4 355 343.8 430. 410.1 442.9 415.2 448.6 428.2
500
M Q L O H L S W P V N QR 7C F B E A D
*LSD = 0.33
Letters sharing the same letter do not differ significantly (p ≤ 0.05) according to LSD test with multiple comparisons. Lettering starts with capital A-Z then small a-z
and then Italic capital A-Z. C. A indicates citric acid, O. A indicates oxalic acid and M. A indicates malic acid.
93
4.2.2.2. P adsorption in sandy loam soil
Data regarding the P adsorbed on the sandy loam soil after incubation with a series of
concentrations of P and organic acids (Citric acid, Malic acid, and Oxalic acid) is
presented in Table 4.12. The amount of adsorbed P on the sandy loam soil was different
at all levels of added organic acids.
At 100 mg kg-1 P application, the highest decrease (10.32%) in P adsorption was recorded
at 1 mM citric acid application as compared to treatment with same P application and no
organic acid while at 400 mg kg-1 P application, 2.46% lowest decline in P adsorption
was noticed at 0.5 mM citric acid application as compared to treatment with the same P
application and no organic acid. Adsorption was low when citric acid was at a rate of 1
mM as compared to treatments where citric acid applied at a rate of 0.5 mM at all levels
of applied P.
In case of oxalic acid, the highest decrease in adsorption (7.4%) at 1 mM oxalic acid was
observed at 500 mg P kg-1 comparative to treatments with separate P applications.
Decrease in P adsorption by the application of 0.5 mM oxalic acid was the lowest (1.2%)
-1
at 400 mg P kg application. Overall 1 mM oxalic acid reduced adsorption at a higher
rate as compared to 0.5 mM oxalic acid as well as separate addition of P.
Malic acid at a rate of 1 mM caused the maximum reduction in adsorption (7.3%) in

treatment where 100 mg P kg-1 was applied while at 0.5 mM malic acid the minimum
reduction (0.5%) was noticed at 400 mg P kg-1.
At both concentrations of all organic acid, citric acid showed less adsorption compared to
the other two organic acids. Overall pattern of reduction in adsorption was Citric
acid>Oxalic acid>Malic acid.
4.2.2.3. P adsorption in sandy clay loam soil
In sandy clay loam soil, P adsorption was reduced by the application of organic acids as
compared to adsorption by separate P application (Table 4.12). Overall, citric acid proved
better in reducing the adsorption relative to other organic acids. Higher level (1 mM) of
organic acids performed better compared to the lower level (0.5 mM) of carboxylate
application.
94
Citric acid at a higher level (1 mM) reduced adsorption up to 11.7% at 500 mg P kg-1 soil
and the minimum reduction (0.50%) in P adsorption was noticed by 0.5 mM citric acid
application at 100 mg P kg-1 soil as compared to separate P application.
Oxalic acid (1 mM) decreased adsorption up to 10.63% at 500 mg P kg-1 soil while 0.5
mM oxalic acid caused the minimum reduction in adsorption (0.44%) at 300 mg P kg-1
soil with respect to the separate application of P.
Malic acid reduced adsorption up to 7.8% at 500 mg P kg-1 soil with respect to treatment
where no organic acids were applied. 0.5 mM malic acid showed the lowest decrease in
adsorption (0.4%) compared to the separate P application.
Reduction in adsorption due to organic acids was in Citric>Oxalic>Malic order. When

compared two levels of organic acids for a reduction in P, the order of decrease was
Oxalic>Citric>Malic.
4.2.2.4. Adsorption Isotherms for the interaction of P (KH2PO4) with carboxylates

(organic acids) in three different textured soils
P adsorption data were plotted and fitted to Freundlich and Langmuir equations for all
soils. These data were used to estimate P sorption energy, the maximum P adsorption
capacity, buffering capacities of soil for P, constant of the energy of adsorption and
sorption capacity in all three different textured soils. The goodness of fit to Freundlich
and Langmuir was estimated by correlation coefficient R2.
4.2.2.4.1. Freundlich Equation
For fitting adsorption data to the Freundlich equation, log C (Equilibrium P

concentration) was plotted against Log Q (Adsorbed P) (Figure 4.20: (a) for loam soil, (b)
for sandy loam soil, (c) for sandy clay loam soil). Effect of organic acids application on P
sorption capacity and P sorption energy of soil was calculated by plotting P equilibrium
concentration against adsorbed P. The goodness of fit of the Freundlich model was
proved by correlation coefficient values (Table 4.13). Data for P adsorption with all three
organic acids application were fitted well to the Freundlich equation as it is evident from
coefficient correlation R2 values (Table 4.13). Phosphorus adsorption data of sandy clay
loam soil was best fitted to the Freundlich equation followed by loam soil and then sandy
loam soil.
95
Data of P adsorption at 0.5 mM malic acid in sandy clay loam soil was best fitted to the
Freundlich equation followed by sandy clay loam soil at 1 mM malic and least conformity
was found in sandy loam soil at 1 mM citric acid.
For all organic acid’s application, P sorption capacity was ranged from 109.1-126.85 µg
g-1 for loam soil. The highest P sorption capacity (126.85 µg g-1) was obtained at 0.5 mM
malic acid among all organic acids and the lowest (109.1 µg g-1) was noticed at 1 mM
citric acid. For sandy loam soil, P sorption capacity was 77.16-104.47 µg g-1 at all levels
of organic acids with the highest value (104.47 µg g-1) at 0.5 mM malic acid application.
For sandy clay loam soil, the maximum P sorption capacity (251.02 µg g-1) was obtained
at 0.5 mM malic acid application. At a higher level of organic acids application (1 mM),
P sorption capacities were lower as compared to a lower level (0.5 mM) of organic acids
and no organic acid application in all soils (Table 4.9). Order of decrease in P sorption
capacity in all soil was sandy clay loam>loam>sandy loam soil. Organic acids showed a
reduction in P sorption capacity as Malic acid>Oxalic acid>Citric acid. Overall higher
level (1 mM) of citric acid in sandy loam soil performed better in case of P sorption
capacity among all treatments.
P sorption energy was ranged from 0.5043-0.5342 mL g-1 for loam soil with the highest in
treatment where 1 mM malic acid was applied. In sandy loam soil, 1 mM citric acid
application showed the maximum (0.6029 mL g-1) P sorption energy while the minimum
(0.5493 mL g-1) was given by 0.5 mM malic acid. In sandy clay loam soil, P sorption
energy was ranged from 0.4012-0.4289 mL g-1. The highest P sorption energy was
obtained by 0.5 mM malic acid and the minimum was showed by 1 mM citric acid.
Phosphorus sorption energy in soils was decreased in order of sandy loam> loam> sandy
clay loam soil.
4.2.2.4.2. Langmuir Equation

Adsorption data were fitted to Langmuir equation by plotting 1/C against 1/Q (Figure
4.21: (a) for loam, (b) for sandy loam soil, (c) for sandy clay loam soil). Langmuir
parameters (Maximum P adsorption and Constant energy of adsorption) were calculated
from the linear regression equation (Table 4.14). Adsorption behavior was described by
the linearized Langmuir equation. The conformity of adsorption data to the Langmuir
model was ascertained by R2 values. Data for P adsorption with all three organic acids
application was fitted well to the Langmuir equation as it is evident from coefficient
correlation R2 values (Table 4.14). Sandy clay loam soil at 1 mM malic acid showed the
96
highest conformity of data to the Langmuir model followed by loam soil at 0.5 mM citric
acid.
The maximum sorption was ranged from 120-158 mg kg-1 for loam soil at all levels of
organic acids. In treatments where citric, oxalic and malic acid were added, the maximum
sorption value was lower as compared to no organic acid application (Table 4.11). In
loam soil, the maximum sorption (158 mg kg-1) for 0.5 mM malic acid was the highest
and 1 mM citric acid showed 120 mg kg-1, the maximum sorption which was the lowest
among all treatments. In sandy loam soil, 1 mM citric acid and 0.5 mM malic acid gave
the minimum and the maximum values for P maximum adsorption. Sandy clay loam
showed the highest value for P maximum sorption among all soils with the highest value
of 1000 mg kg-1 for 0.5 mM malic acid and the lowest value of 625 mg kg-1 for 1 mM
citric acid as well as for 1 mM oxalic acid. Maximum P sorption capacities were higher in
sandy clay loam followed by sandy loam and then loam soil. In treatments where citric,
oxalic and malic acids were added, Constant of the energy of adsorption was higher as
compared to no carboxylate application in all soil (Table 4.11). For all organic acids
application, Constant of the energy of adsorption was ranged from 3.15-4.33 in loam soil,
9.5-27.42 in sandy loam and 0.4-0.61 in sandy clay loam soil. Constant of the energy of
adsorption was higher for sandy loam soil followed by loam soil and the lowest was
calculated for sandy clay loam soil. A higher level of carboxylate (1 mM) application
showed more value for the constant of the energy of adsorption as compared to the lower
level of organic acids (0.5 mM).
97
Table 4.13. Freundlich adsorption isotherm parameters for P adsorption affected by carboxylates (organic acids) application in
three different textured soils
Organic
Loam Sandy loam Sandy clay loam
acids
Sorption P Sorption P Sorption P Sorption P Sorption P Sorption
Capacity energy R2 capacity energy R2 capacity energy R2
(µg g-1) (a) (mL g-1) (b) (µg g-1) (a) (mL g-1) (b) (µg g-1) (a) (mL g-1) (b)
C.A
118.8 0.5125 0.8369 90.452 0.5723 0.8192 231.51 0.4026 0.9483
(0.5 mM)
CA
109.1 0.5043 0.8566 77.165 0.6029 0.8025 198.4 0.4012 0.9575
(1 mM)
O.A
122.5 0.5062 0.8483 94.19 0.5723 0.8349 244.98 0.4221 0.9586
(0.5 mm)
O.A
110.14 0.5095 0.8554 85.94 0.5654 0.8343 204.11 0.4042 0.9581
(1 mM)
M.A
126.85 0.5157 0.8742 104.47 0.5493 0.8321 251.02 0.4289 0.9751
(0.5 mM)
M.A
110.32 0.5342 0.873 86.74 0.5819 0.8356 218.86 0.4094 0.9642
(1 mM)
Mean values were used for fitting in Freundlich adsorption isotherm. C. A indicates citric acid, O. A indicates oxalic acid and M.A indicates malic acid.
98
Table 4.14. Langmuir adsorption isotherm parameters for P adsorption affected by carboxylates (organic acids) application in three
different textured soils.
Organi
Loam Sandy loam Sandy clay loam
c acids
Maximum P Constant of the R2 Maximum P Constant of the R2 Maximum P R2
energy of energy of Constant of the
adsorption adsorption adsorption
adsorption (k) adsorption (k) energy of
capacity (mg kg- capacity (mg kg- capacity (mg kg-
1) (a) 1) (a) 1) (a) adsorption (k)
C.A
(0.5 mM)
128 4.33 0.9806 69 13.18 0.9628 909 0.42 0.9606
C.A
(1 mM)
120 3.95 0.9711 52 27.42 0.9188 625 0.61 0.9769
O.A
(0.5 mm)
142 3.68 0.9784 76 10.91 0.9737 909 0.44 0.9694
O.A
(1 mM)
123 3.85 0.9689 64 12.83 0.9495 625 0.61 0.9783
M.A
(0.5 mM)
158 3.15 0.9806 87 9.5 0.965 1000 0.4 0.9726
M.A
(1 mM)
126 3.95 0.9669 64 15.4 0.9242 714 0.58 0.9881
Mean values were used for fitting in Langmuir adsorption isotherm. C. A indicates citric acid, O. A indicates oxalic acid and M. A indicates malic acid.
99
100
101
102
103
104
105
4.2.3. Discussion
Soil Physico-chemical properties
Three soils, loam, sandy loam, and sandy clay loam were used in this study. Texture is
important in nutrient and water retention and it serves as a medium for plant rooting
(Enwezor et al., 1990). Organic matter in all soils was extended from 0.38-0.62% that is
less than the normal range of organic matter. These outcomes were in agreement with
Mihoub et al., (2016a).
The three different textured soils were moderately calcareous due to the calcium content
that was in the range of 4.9-9%. Soils having greater than 3% of calcium carbonate
contents are considered as calcareous. Parallel results were described by Mihoub et al.,
(2016a). Potassium was ranged from 130 to 239 mg kg-1. These are in adequate range as
was reported by Obaid-ur-rehman et al., (2004) and Sarfaraz et al., (2009). Available P
contents were 7.3 mg kg-1, 10.8 mg kg-1 and 5.7 mg kg-1 in sandy clay loam, sandy loam
and loam soils, respectively. These P contents are below the adequate range of P. Low
availability in our soil may be due to the alkaline reaction and calcareous nature of soils
(Mihoub et al., 2016a).
In the case of micronutrients, a high amount of available iron was found in sandy clay
loam which was 6.1 mg kg-1 and in sandy loam soil, these values were 0.1 mg kg-1.
Threshold values ranged from 2.5-4.5 mg kg-1 for crop production. Iron values in studied
soils fall below the adequate range for crop growth. Manganese was ranged 1.2-1.7 mg
kg-1 in all soils under study. Critical levels for crop production of available manganese are
1-50 mg kg-1. So, manganese concentration in soil is adequate. Sims and Johnson, (1991)
reported similar results.
P adsorption in three different soil types
In the first study, where different concentrations of P were added in a 0.01 mM CaCl2
solution, the equilibrium P concentration increased substantially in all soils with an
increase in added P except in sandy clay loam soil that had not shown a noteworthy
increase in equilibrium P concentration. This non-significant result for equilibrium P
concentration may be because of more soil capacity for adsorption. In treatment where
500 mg P kg-1 was applied, equilibrium P concentration was high in sandy loam soil that
shows low ability of P adsorption of this soil. These high equilibrium P concentrations
possibly come because of CaCl2 solution that may have extracted little more of the
106
available P from the sandy soil. At the same treatment, the lowest equilibrium
concentration was obtained from sandy clay loam soil that may happen because of the
calcareousness and high calcium content in this soil. Among all soils, sandy clay loam
soil showed lower equilibrium concentration relative to loam and sandy loam soil at all
levels of applied P. As sandy clay loam soil contains more calcium content that has the
ability to combine with phosphate and make insoluble Ca-phosphate compounds. So, this
may be the reason for the low P equilibrium concentration. Another reason that can
hinder the equilibrium P concentration is the presence of high clay content that can reduce
the available form of P. Organic matter can reduce P complexation and improve P
variability by producing organic acids to some extent as they bind with positive charges
like calcium in soil (Leytem and Mikkelsen, 2005).
The amount of P adsorbed in all types of soils declined with an increasing P addition
level. In the treatment where 500 mg of P was added, the maximum adsorption was
noticed while the lowest adsorption was observed at 100 mg of applied P. Although there
were -ve relationships among added and adsorbed P at 500 mg added P decrease in
adsorption was not significant from 400 mg of added P in all soils. The soil is a complex
matrix in which many factors like calcium, clay contents, pH and organic matter may
interact and ultimately influence adsorption of P. Phosphate adsorption is related to the
presence of exchange sites in the soil. There is a likelihood of the event that with
increasing P addition, exchange sites are occupied by phosphate ions and further addition
may decrease adsorption of phosphate due to decreased availability of exchange sites.
Reduced affinity for P adsorption with an increase in the P addition has already been
testified (Barrow, 1978). In another study by MacAlister, (2016) reported that the %age
of P adsorbed in soil declined with an increasing P addition signifying that available
binding sites for P were decreasing. The results of this study are also in accordance with
earlier conclusions presented by Abedin and Saleque, (1998) and Akhtar et al., (2003)
that revealed that significant increase in adsorption is associated with increased levels of
added P and the %age of adsorption of added P decreased with an increasing P addition
level.
Adsorption pattern in soil was sandy clay loam> loam >sandy loam soils. Low adsorption
of P in sandy loam soil, have occurred because of low clay content in soil comparative to
other two soils. High clay value reduced the adsorption of phosphate by serving as an
alternate binding site for positive ions in soils. These outcomes were in agreement with
107
outcomes presented by Samadi and Gilkes, (1999), Samadi, (2001; 2006). They reported
that calcium content was associated with the extent of P adsorption in calcareous soils.
Regression analysis of all the three different textured soils indicated that association
between adsorbed and equilibrium P and between added and adsorbed P was linear. The
rate of increase in adsorption was higher in sandy clay loam while it was the lowest in
sandy loam soil. The R2 values at 0.05 level of probability were significant in all three
different textured soils.
Adsorption isotherm was made by plotting Freundlich equation and Langmuir equation
linearly (Zhou and Li, 2001). The data fitness to model was calculated by R2 values
which showed a good conformity of adsorption data to both models. Adsorption data
fitted best to Langmuir adsorption model over Freundlich which was also testified by
Khan et al., (2010a). Freundlich isotherm is associated with saturation percentage, clay
contents and CaCO3 content of soils (Hussain et al., 2003). Phosphorus sorption capacity
is termed as the amount of adsorbed P obligatory to sustain a unit P concentration in
solution. Phosphorus sorption energy value represents the rate of P adsorption (mL g−1)
(Bhal and Toor, 2002). Phosphorus sorption energy value was lower for sandy loam soil
while higher values were noticed in sandy loam soil. Phosphorus sorption capacity was
lower for sandy loam soil that’s why this soil showed low adsorption out of all soils while
P sorption capacity value was higher for sandy clay loam soil indicating high adsorption
of P in this soil. Similarly, Siemans et al., (2004) reported that P sorption capacities are
affected by soil texture and clay mineralogy. According to Kuo and Lotse, (1974), P
sorption capacity value depends on P concentration in a solution that might be the reason
in P sorption changes from soil to soil
Constant energy for adsorption and maximum capacity for P adsorption represent the
strength of P binding to exchange sites. Sandy loam soil had lower capacity for P
adsorption but higher constant of the energy for adsorption while sandy clay loam soil
showed higher capacity for P adsorption and lower constant of the energy of adsorption.
It is possible that soils comprising huge amounts of clay (more exposed surface area) will
fix more P than those containing small amounts of clay (small surface area exposed)
(Tisdale et al., 1985).
108
Effects of carboxylate (Organic acids) on P adsorption and desorption.
Equilibrium P concentration increased in all soils when organic acids were added as
compared to no addition of organic acids. Order of increase in equilibrium P
concentration was by citrate>oxalate>malate. Organic acids strive with P for adsorption
sites because of high affinity for adsorption sites. This phenomenon might have increased
the P equilibrium concentration (Mihoub et al., 2016b). Our outcomes are reinforced by
Cajuste et al., (1996) that a higher level of all organic acids proved more operative in
improving equilibrium P concentration. Zhang et al., (2009) and Zhuo et al., (2009)
testified this possibility that organic acids addition triggers dissociation of Ca–P bond in
alkaline soil that ultimately increases level of P available for plant uptake.
In this study, adsorbed P was reduced in all soils when organic acids were applied. Citrate
proved more effective in decreasing adsorption while the least decrease in adsorption was
observed where 0.5 mM malate was applied. Citrate is a tri-carboxylate, having three
carboxyl groups and has more affinity for adsorption sites than other organic acids (Jones
et al., 2003). Tsado et al., (2012) conveyed analogous findings. They said that citrate
makes a strong bonding with calcium in the soil as compared to malate and oxalate.
Increased levels of all organic acids caused more decrease in adsorption as 1 mM of all
organic acids was more effective compared to 0.5 mM of these organic acids. These
findings are verified by results of Cajuste et al., (1996) that with the increase in loading of
citrate and oxalate, P adsorption was decreased.
The adsorption isotherm made by plotting Freundlich equation and the Langmuir
equation linearly. From the Freundlich equation, parameters like P sorption capacity and
sorption energy were obtained. From the Langmuir equation, Pmax and constant energy of
adsorption were obtained. High adsorption data conformity with both modes was
measured. Our data for P adsorption with the organic acid application was best fitted to
both Freundlich and Langmuir models. P adsorption data were best fitted to Freundlich
adsorption model and it was also testified by Khan et al., (2010b). Phosphorus sorption
capacity (a) is termed as the amount of adsorbed P essential to sustain a unit P
concentration in solution. Soil with larger ‘a’ value has more adsorbing capacity than soil
with smaller ‘a’ value (Assimakopoulos et al., 1986). The decrease in P sorption
capacities was noticed when carboxylate was applied in all soils compared to P sorption
capacities of these soils in the first part of the study. The decrease in P sorption capacities
might be the result of P replacement with organic acids from binding sites. A similar
109
finding was reported by Von Wandruszka, (2006). They reported that organic acids
reduced the P sorption capacities. However, citrate was better than all organic acids in
reducing P sorption capacity of all soils.
Results showed that lower P sorption capacity was observed with citrate as compared to
oxalate. Sandy loam soil in all organic acids showed the lowest P sorption capacities and
the highest was obtained in sandy clay loam soil. This outcome proved by outcomes
reported by Kuo and Lotse, (1974) and Assimakopoulos et al., (1986). They stated that
the value of P sorption capacity varied from soil to soil because it is associated to soil
solution P concentration.
Constant of energy for adsorption and Pmax are related to desorption of P, like these, both
parameters represent the degree of strength of bonding. Organic acids in sandy clay loam
soil showed a lower constant of energy for adsorption and higher Pmax compared to
organic acids in the other two soils. Organic acids in sandy loam showed a higher
constant of energy for adsorption and lower Pmax comparative to organic acids in the other
two soils. This might happen because of tight bonding of P with higher calcium content in
sandy clay loam whereas, in sandy loam soil, low calcium content might have caused
weak P bonding (Hedley and Stewart, 1982). Comparable results were reported by
Ahmed et al., (2008). They described that Pmax and constant energy of adsorption were
higher in Griffith soil compared to Narrabri soil.
All the above parameters that are calculated from Freundlich and Langmuir adsorption
isotherm can be utilized to estimate the adsorption properties of different soil.
110
4.2.4. Conclusion
Understanding the P behavior in the soil is necessary for better P fertilizer management
that may ultimately help in better PUE. From the above results, it is concluded that
organic acids are useful in enhancing P availability as they showed a reduction in
adsorption of P. Adsorption data was best fitted to Freundlich and Langmuir adsorption
equation. When both equations were compared, Langmuir was best as data was fitted
better than the Freundlich equation. Among organic acids citrate was more efficient in
reducing adsorption of P in all soils. Citrate application at a higher rate showed the lowest
P sorption and the highest Pmax in all soils. In sandy loam soil, adsorption rate was slow
compared to loam and sandy clay loam soil. Adsorption was reduced in pattern of sandy
loam<loam<sandy clay loam soil. Overall, citrate application at 1 mM in sandy loam soil
performed best.
111
4.3. Study 3
Influence of soil types and deficient levels of P on the release of
carboxylates and P acquisition characteristics in wheat and maize
Plants can only take P from soil solution. Concentration of P in the soil solution is
tremendously truncated comparative to the prerequisite of plants regardless of its high
total soil concentrations (Barber, 1995). Various Plants developed a variety of
mechanisms and special features for acclimatizing to lower P. These mechanisms include
biochemical, physiological, and morphological changes. For example, partitioning of dry
mass, a higher amount of carboxylates in root areas, release of phosphatase enzymes
(Skene, 2003), rhizosphere pH change through the release of protons (Hu et al., 2010),
and the development of specialized cluster roots (Shane et al, 2004). These changes are
collectively called the Pi starvation response (PSR) (Niu et al., 2013). This involves in
sustaining and efficient utilization of inner phosphate or consenting better contact with
soil phosphate. Thus, phosphate availability is increased by these modifications in the
rhizosphere that ultimately results in increased phosphate uptake and use (Richardson et
al., 2009a; Plaxton and Tran, 2011). White lupin successfully adapts these P starvation
mechanisms (Cheng et al., 2011).
Under low P availability, a plant species in order to well-adapted to this environment need
to develop various approaches to improve P acquisition. The growth of species that are
poorly-adapted to P deprivation will depend on P fertilizer. So, a large input of P fertilizer
needed to optimize growth. Such efforts to find and classify P efficient species at low P
levels and their adaptive strategies will improve the productivity of plants in low P soils
with less application of P fertilizer (Pearse et al., 2006b).
So, this study was planned with the following objectives
➢ effect of different levels of P on the release of carboxylates in the rhizosphere
➢ effect of P levels and soil texture on rhizosphere acidification
➢ effect of soil texture on plant growth and carboxylate release
➢ effect of carboxylates, released by plant roots, on rhizosphere soil available P
112
Results
4.3.1. Influence of soil types and deficient levels of P on the release of carboxylates
and P acquisition characteristics in wheat
4.3.1.1. Growth responses
Shoot length (SL) was affected non-significantly by P level as well as by soil type (loam,
sandy loam, and sandy clay loam) and their interaction (p ≤ 0.05) (Table 4.15). An
increase in P increased the SL up to 33.75 mg P application kg-1 soil and then declined. At
45 mg P application kg-1 soil, reduction in SL was observed compared to 33.75 mg P
application kg-1 soil. In sandy loam and loam soil, SL results were according to this trend.
Plants in sandy clay loam soil showed a continuous increase in SL and the maximum SL
was noticed at P application of 45 mg kg-1 soil. Plants in sandy loam soil showed higher
SL as compared to the other two soils at all P levels. When P was applied at 33.75 mg kg-
1
of sandy loam soil, the highest SL (68.2 cm) was observed and at P application of 0 mg
kg-1 sandy clay loam soil, the lowest SL (63.1 cm) of wheat was recorded.
Shoot fresh weight (SFW) was found non-significant (p ≤ 0.05) statistically at the
interaction of soil and soil applied P levels however the separate effect of P and soil type
was significant (Table 4.15). Increase in P level increased SFW up to 33.75 mg P
application kg-1 soil in loam and sandy loam soil. Sandy clay loam soil showed a gradual
increase in SFW with an increasing level of P and the highest SFW was recorded at 45
mg P application kg-1 soil. Overall, the maximum SFW (33.1 g plant-1) was recorded
when P was applied @ 33.75 mg kg-1 of sandy loam soil and the lowest SFW (19.5 g
plant-1) was recorded at P application of 0 mg kg-1 sandy clay loam soil.
Shoot dry weight (SDW) of the wheat plant in different soil types at different P levels was
recorded (Table 4.15). Non-significant (p ≤ 0.05) effect was observed for soil type as well
as for P and soil interaction but P level affected SDW significantly (p ≤ 0.05). An
increase in applied P level increased SDW. An increase in SDW was linearly related to
increasing P level but this pattern was not clear at 45 mg P application kg-1 soil. In loam
and sandy loam soil, the highest SDW was observed at 33.75 mg P application kg-1 soil
and in sandy clay loam soil, the highest SDW was measured at 45 mg P application kg-1
soil. SDW was the highest (6.98 g plant-1) when P was applied @ 33.75 mg kg-1 of sandy
loam soil and the lowest (4.70 g plant-1) was obtained in sandy clay loam soil with 0 mg P
application.
113
Table 4.15. Shoot length, shoot fresh and dry weight of wheat in loam, sandy loam,
and sandy clay loam soils under different P levels
Shoot fresh Shoot dry
Phosphorus Shoot length
Soil Types weight weight
(mg P kg-1) (cm)
0 65.0±1.74ab 25.1±1.71cd 4.82±0.15f
11.25 66.7±1.59ab 27.8±1.56b-d 5.53±0.87b-f
Loam 22.50 67.0±1.29ab 30.6±0.94ab 6.38±0.06a-e
33.75 67.7±1.83ab 30.9±0.78ab 6.73±0.27a-c
45 65.8±0.76ab 28.9±1.84a-c 6.21±0.73a-e
0 66.0±1.15ab 27.1±0.59b-d 5.15±0.41ef
11.25 67.0±0.55ab 31.1±1.41ab 5.48±0.23c-f
Sandy loam 22.50 67.7±0.64ab 31.8±0.48ab 6.83±0.59ab
33.75 68.2±1.79a 33.1±1.96a 6.98±0.07a
45 66.3±0.81ab 30.6±0.78ab 6.56±0.55a-d
0 63.1±1.73b 19.5±1.52e 4.70±0.08f
11.25 64.4±1.65ab 24.0±1.69d 5.26±0.04def
Sandy clay
22.50 65.7±1.56ab 25.8±1.74cd 5.84±0.23a-f
loam
33.75 66.3±1.17ab 27.6±1.12b-d 6.28±0.13a-e
45 66.6±1.10ab 29.6±1.81a-c 6.89±0.07a
Phosphorus 1.59: ns 10.49*** 11.24***
F value Soil Types 2.35: ns 18.95*** 1.34: ns
Interaction 0.32: ns 1.16: ns 0.66: ns
114
Table 4.16. Root length, root fresh and dry weight of wheat in loam, sandy loam and
Root fresh Root dry
Phosphorus Root length
Soil Types weight weight (g
(mg P kg-1) (cm)
(g plant-1) plant-1)
0 58.0±0.58cd 2.67±0.04a-c 1.40±0.15ab
11.25 56.0±1.20d 2.57±0.08a-d 1.34±0.13a-c
Loam 22.50 57.0±1.53d 2.54±0.19a-d 1.06±0.08a-c
33.75 48.8±0.60ef 2.30±0.09c-f 0.97±0.09a-c
45 46.0±1.53fg 1.95±0.06fg 0.93±0.01a-c
0 43.0±0.58gh 2.46±0.08b-d 1.32±0.16a-c
11.25 41.7±1.20h 2.30±0.05c-f 1.25±0.12a-c
Sandy loam 22.50 35.2±1.01i 2.16±0.40d-g 1.02±0.04a-c
33.75 32.0±1.15i 2.04±0.07e-g 0.85±0.24c
45 33.3±1.45i 1.75±0.05g 0.86±0.02bc
0 66.7±0.88a 2.91±0.06a 1.44±0.19a
11.25 62.8±0.93b 2.86±0.05ab 1.37±0.41a-c
Sandy clay
22.50 61.2±1.01bc 2.76±0.02ab 1.26±0.01a-c
loam
33.75 52.7±1.53e 2.64±0.05a-c 1.11±0.05a-c
45 50.7±1.45e 2.15±0.08d-g 1.14±0.05a-c
Phosphorus 71.01*** 12.6*** 4.24*
F value Soil Types 470.61*** 20.38*** 2.08: ns
Interaction 3.11* 1.15: ns 0.09: ns
115
Soil type, P and interaction effects were significant (p ≤ 0.05) for root length (RL) (Table
4.16). RL in three different soils decreased markedly with the increase in the P
application level. RL decreased from 0 to 45 mg P application kg-1 soil and gave the
lowest RL at 45 mg P kg-1. At 33.75 and 45 mg P application kg-1 soil, a decrease in RL
was not much pronounced as compared to the other three levels of applied P. In the case
of different textured soils, results for RL varied significantly (p ≤ 0.05) among soils.
Plants in sandy clay loam soil gave higher RL as compared to loam and sandy loam soil.
Following sandy clay loam soil, plants in loam soil performed better. The maximum RL
(66.7 cm) was recorded when P was applied at 0 mg kg-1 of sandy clay loam soil and the
minimum RL (32.0 cm) was measured at P application of 45 mg kg-1 sandy loam soil.
The integrated effect of soil type and P on root fresh weight (RFW) was non-significant
(p ≤ 0.05) (Table 4.16) but the separate effect of soil type and P was significant (p ≤
0.05). With the increase in the level of P, a decrease in RFW was recorded. Different
textured soils showed a significantly different response in RFW at different levels of P
application. Plant roots in sandy clay loam soil had more RFW as compared to loam and
sandy loam soil. The maximum RFW (2.91 g plant-1) was measured when P was applied
@ 0 mg kg-1 of sandy clay loam soil and the lowest root (1.75 g plant-1) fresh weight was
recorded at P application of 45 mg kg-1 sandy loam soil.
There was no significant effect on root dry weight (RDW) for soil type and for the
interaction of soil with P (p ≤ 0.05) (Table 4.16). The effect of P was significant (p ≤
0.05). The wheat plant responded to P deficient conditions by increasing their RDW.
With the increase in P level, RDW decreased but at a higher level of P (45 mg P
application kg-1 soil), RDW in sandy loam and sandy clay loam soil was higher than
33.75 mg P application kg-1 of soil. The maximum RDW (1.44 g plant-1) was measured
when P was applied @ 0 mg kg-1 of sandy clay loam soil and the minimum RDW (0.85 g
plant-1) was measured at P application of 33.75 mg kg-1 sandy loam soil.
Phosphorus effect on root mass fraction (RMF) was significant but soil type and
interaction effect were not significant (p ≤ 0.05) (Table 4.17). An increase in the P
application level decreased RMF in different soils. As RMF was linked to root growth so
as the RDW decreased with the increase in the level of P application, RMF also
decreased. But at 45 mg P application kg-1 soil, this pattern was not followed and RMF
was higher than P application of 33.75 mg kg-1 in loam and sandy loam soil. Plants in
116
Table 4.17. Root mass fraction, Root: Shoot and Root volume of wheat in loam,
sandy loam and sandy clay loam soils under different P levels
Phosphorus Root mass Root volume
Soil Types Root: Shoot
(mg P kg-1) fraction (g g-1) (cm3)
0 0.231±0.02ab 0.29±0.04a 15.67±0.67ab
11.25 0.201±0.03a-d 0.26±0.05ab 15.00±0.58a-c
Loam 22.50 0.142±0.01c-f 0.17±0.02bc 13.83±0.73a-d
33.75 0.126±0.02d-f 0.15±0.01bc 11.17±0.92de
45 0.134±0.02c-f 0.16±0.02bc 11.50±1.04c-e
0 0.206±0.03a-c 0.26±0.05ab 14.93±1.02a-c
11.25 0.185±0.02a-e 0.23±0.02a-c 14.33±0.33a-d
Sandy loam 22.50 0.132±0.01c-f 0.15±0.02bc 13.17±1.36b-d
33.75 0.106±0.03f 0.12±0.03c 9.50±0.28e
45 0.118±0.01ef 0.13±0.02c 12.83±1.92b-d
0 0.234±0.03a 0.31±0.045a 17.00±1.52a
11.25 0.200±0.10a-d 0.26±0.08ab 15.33±0.33ab
Sandy clay
22.50 0.178±0.01a-f 0.22±0.01a-c 15.33±1.45ab
loam
33.75 0.150±0.004b-f 0.18±0.01bc 14.00±1.15a-d
45 0.142±0.004c-f 0.17±0.01bc 13.83±0.72a-d
Phosphorus 9.67*** 9.36*** 8.12***
F value Soil Types 2.29: ns 2..07: ns 5.78**
Interaction 0.13: ns 0.08: ns 0.59: ns
117
sandy clay loam soil decreased its RMF from 0 to 45 mg P application kg-1 soil linearly.
The lowest RMF (0.106 g g-1) was noticed in sandy loam soil when P was applied at @ of
33.75 mg kg-1 soil. The maximum RMF (0.234 g g-1) was calculated in the treatment
where 0 mg P was applied kg-1 of sandy clay loam soil.
Root to shoot ratio (R/S ratio) affected non-significantly by soil types and interaction
effect of soil and P but P application showed a significant effect (p ≤ 0.05) (Table 4.17).
A negatively linear pattern for the R/S ratio with an increasing level of P was found. With
the increase in applied P level, R/S ratio reduced but this trend changed at 45 mg P
application kg-1 soil, as this level showed a minute increase in its R/S ratio from 33.75 mg
P application kg-1 in loam and sandy loam soil. R/S ratio in sandy clay loam soil
decreased up to 45 mg P application kg-1 soil. R/S ratio was lower in (0.12) treatment
where 33.75 mg P application kg-1 in sandy loam soil was done while the highest R/S
ratio (0.31) was obtained at 0 mg P application kg-1 of sandy clay loam soil.
4.3.1.2. Root morphological responses
The volume of roots of the wheat plant in different soil types with different levels of P
application is presented in Table 4.17. Statistical analysis depicted that there was a
significant (p ≤ 0.05) effect of P and soil on root volume but the interaction effect was not
significant. An increase in P level decreased root volume up to 33.75 mg P application kg-
1
of loam and sandy loam soil. In sandy clay loam soil, root volume decreased gradually
with increasing P levels and the lowest root volume was observed at 45 mg P application
kg-1 soil. The wheat plant had higher root volume in sandy clay loam soil at all levels of
applied P followed by loam soil. The highest root volume (17 cm3) was recorded when P
was applied @ 0 mg kg-1 of sandy clay loam soil and the lowest root volume (9.50 cm3)
was recorded at 33.75 mg P application kg-1 sandy loam soil.
The average root diameter of wheat roots affected significantly (p ≤ 0.05) by soil type, P
levels as well as by the interaction of soil type and P (Table 4.18). With the increase in
applied P amount, the average root diameter increased gradually up to 33.75 mg P
application kg-1of loam and sandy loam soil. Plants in sandy clay loam soil represented
the highest value for average root diameter relative to loam and sandy loam soil at all P
levels. The maximum average root diameter (0.261 cm) was recorded in sandy clay loam
soil when P was applied at 45 mg kg-1 soil and the minimum average root diameter (0.067
cm) was recorded at P application @ 0 mg kg-1 loam soil.
118
Table 4.18. Root surface area, Number of root tips and Average root diameter of
wheat in loam, sandy loam and sandy clay loam soils under different P
levels
Phosphorus Root surface Number of root Average root
Soil Types
(mg P kg-1) area (cm2) tips diameter (cm)
0 68.39±0.48d 403±1.73b 0.067±0.001i
11.25 49.32±1.52f 380±1.15d 0.093±0.001g
Loam 22.50 49.91±0.75f 302±2.00g 0.133±0.002d
33.75 29.14±1.45i 133±2.00l 0.252±0.008a
45 39.84±1.37g 261±1.50i 0.135±0.002d
0 82.50±0.88b 390±2.64c 0.071±0.003hi
11.25 46.51±0.87f 351±1.00f 0.079±0.004h
Sandy loam 22.50 33.85±1.45h 262±1.50i 0.098±0.001fg
33.75 22.8±0.80j 90±2.88m 0.203±0.007b
45 33.23±1.93h 171±1.15j 0.110±0.001e
0 94.72±1.04a 469±1.52a 0.092±0.001g
11.25 77.10±0.91c 385±2.08cd 0.106±0.001ef
Sandy clay
22.50 74.40±1.99c 363±2.08e 0.157±0.003c
loam
33.75 60.60±1.53e 288±2.64h 0.204±0.003b
45 57.99±1.83e 148±1.56k 0.261±0.003a
Phosphorus 495.12*** 11102.43*** 1102.77***
F value Soil Types 709.71*** 2071.11*** 305.71***
Interaction 22.98*** 121.61*** 27.31***
119
A significant variation in the root surface area of the wheat plant was observed (Table
4.18). Different levels of P, soil types and their interaction gave significantly (p ≤ 0.05)
variable results for root surface area. With the increase in the level of P application, the
root surface area decreased gradually. Sandy clay loam soil gave a more pronounced
increase in root surface area as compared to loam and sandy loam soil. The highest root
surface area (94.72 cm2) was recorded when P was applied at 0 mg kg-1 of sandy clay
loam soil and the lowest root surface area (22.80 cm2) was recorded at P application of
33.75 mg kg-1 sandy loam soil.
The comparative effect of soil type and P application on a number of root tips in wheat
soil is presented in Table 4.18. Phosphorus levels, soil types and interaction of P with soil
types affected a number of root tips significantly (p ≤ 0.05). The number of root tips
significantly decreased with increasing P levels in sandy clay loam soil while in loam and
sandy loam soil, a decrease in the number of root tips did not appear at 45 mg P
application kg-1 soil. Among all soils, sandy clay loam soil ranked first in which plant
roots gave the maximum number of root tips comparative to loam and sandy loam soil.
Overall, P application @ 0 mg kg-1 of sandy clay loam soil gave the highest number of
root tips (469) however the lowest number of root tips (90.2) was recorded in sandy loam
soil when P was applied @ 33.75 mg kg-1 soil.
4.3.1.3. Phosphorus acquisition parameters
Rhizosphere soil available P
Rhizosphere soil available P in the wheat rhizosphere was significantly (p ≤ 0.05)

affected by P application, soil type as well as their interaction (Table 4.19). With the
increase in the level of P, the decrease in rhizosphere available P was noticed. Different
textured soils showed significantly different responses in the rhizosphere available P at
different levels of P application. All three different textured soils (loam, sandy loam, and
sandy clay loam) showed better results at 0 mg P application kg-1 soil comparative to
other levels of applied P. Sandy clay loam soil was significantly better in improving
rhizosphere soil available P when compared with loam and sandy loam soil under low P
application. The maximum rhizosphere soil available P (8.5 mg kg-1 in sandy clay loam
soil) was analyzed at 0 mg P application kg-1 soil and the minimum rhizosphere soil
available P (3.5 mg kg-1 in sandy clay loam soil) was measured at 45 mg P application kg-
1
soil.
120
Table 4.19. Rhizosphere soil available P, Root and shoot P of wheat in loam, sandy
loam, and sandy clay loam soils under different P levels
Rhizosphere
Phosphorus
Soil Types soil available P Root P (mg g-1) Shoot P (mg g-1)
(mg P kg-1)
(mg kg-1)
0 5.8±0.04f 0.15±0.003g 0.97±0.01f
11.25 5.8±0.05f 0.16±0.002e-g 1.05±0.03e
Loam 22.50 5.6±0.04g 0.17±0.001cd 1.25±0.02bc
33.75 4.0±0.06i 0.18±0.003a-c 1.31±0.02ab
45 5.3±0.03h 0.18±0.003a 1.28±0.01ab
0 7.4±0.05b 0.15±0.002g 0.96±0.01f
11.25 6.6±0.07d 0.16±0.003ef 1.07±0.03e
Sandy loam 22.50 6.3±0.03e 0.17±0.001bc 1.29±0.03ab
33.75 5.8±0.03f 0.18±0.003ab 1.34±0.04a
45 5.7±0.04fg 0.18±0.002a 1.32±0.01a
0 8.5±0.03a 0.13±0.001h 0.83±0.01g
11.25 7.2±0.03c 0.15±0.001fg 0.99±0.01f
Sandy clay
22.50 6.2±0.05e 0.16±0.007de 1.18±0.02d
loam
33.75 5.6±0.06g 0.16±0.003de 1.19±0.03cd
45 3.5±0.05j 0.17±0.003bc 1.21±0.02cd
Phosphorus 1670.52*** 72.98*** 184.25***
121
Root P
Root P value was significantly affected by P application and soil types however
interaction effect was non-significant (p ≤ 0.05) (Table 4.19). An increase in P application
caused an increase in root P content in the wheat plant. In all types of soils, a similar
pattern was observed the however the rate of increase in root P was variable among soils.
Root P values in sandy loam and loam soils were not significantly variable while root P in
sandy clay loam soil was different significantly from sandy loam and loam soil. Plant in
sandy clay loam soil showed the lowest root P content relative to plants in loam and
sandy loam soil. The maximum root P content (0.18 mg g-1) was noticed by plants grown
at 33.75 and 45 mg P application kg-1 sandy loam and loam soil and the lowest root P
content (0.13 mg g-1) was recorded at P application of 45 mg kg-1 sandy clay loam soil.
Shoot P
Phosphorus content in the shoot of the wheat plant was affected significantly by P
application and soil types while the interaction effect was non-significant (p ≤ 0.05)
(Table 4.19). An increase in shoot P content in a wheat plant with increasing P levels in
different soils was observed. Wheat plant in sandy clay loam soil showed this behavior in
response to P application while in loam and sandy loam soil, P content increased with
increasing P levels from 0 to 33.75 mg P application kg-1 soil and 45 mg P application kg-
1
soil showed a reduction in P content. When the performance of the wheat plant in
different soils was compared plant in sandy loam soil performed better with respect to
shoot P content. Sandy clay loam soil showed the lowest shoot P at all levels of applied P
comparative to loam and sandy loam soil. Overall sandy loam soil at 33.75 mg P
application kg-1 soil gave the highest shoot P content (1.34 mg g-1) and the lowest shoot P
content (0.83 mg g-1) was recorded at P application of 0 mg kg-1 sandy clay loam soil.
Phosphorus acquisition efficiency (PAE)
Phosphorus acquisition efficiency, the ability of the plant to acquire P, is presented in

Table 4.20. Phosphorus acquisition efficiency was affected significantly (p ≤ 0.05) by P
application, soil type, and their interaction. Phosphorus application increased PAE up to
45 mg P application kg-1of loam, sandy loam soil and sandy clay loam soil. The
maximum PAE was observed at 45 mg P application kg-1 soil. Different soils had a
significant effect on the ability of the plant to acquire P. Plants in sandy loam acquire
more P as compared to plants in the other two soils.
122
Table 4.20. Phosphorus acquisition efficiency (PAE), Phosphorus use efficiency
(PUE) and Phosphorus uptake of wheat in loam, sandy loam and sandy
clay loam soils under different P levels.
PUE
Phosphorus PAE P Uptake
Soil Types (g2 SDM mg-1
(mg P kg-1) (mg P cm-1 RL) (mg plant-1)
P)
0 0.019±0.0001fg 4.96±0.12a 4.67±0.20ef
11.25 0.021±0.0001fg 5.26±0.81a 5.82±1.00de
Loam 22.50 0.025±0.001de 5.10±0.10a 7.97±0.08a-c
33.75 0.031±0.0010c 5.13±0.14a 8.82±0.49ab
45 0.031±0.0011c 4.85±0.58a 7.95±0.92a-c
0 0.025±0.0012d 5.37±0.11a 4.93±0.40ef
11.25 0.029±0.0014c 5.13±0.20a 5.87±0.30de
Sandy loam 22.50 0.041±0.0011b 5.31±0.10a 8.77±0.57ab
33.75 0.046±0.0022a 5.21±0.01a 9.35±0.19a
45 0.047±0.0020a 4.95±0.30a 8.69±1.00ab
0 0.014±0.0001h 5.67±0.11a 3.90±0.11f
11.25 0.018±0.0001g 5.31±0.03a 5.20±0.10ef
Sandy clay
22.50 0.022±0.0004ef 4.95±0.22a 6.89±0.41cd
loam
33.75 0.026±0.0013d 5.28±0.13a 7.47±0.22bc
45 0.027±0.0010cd 5.69±0.20a 8.32±0.10a-c
Phosphorus 104.86*** 0.19: ns 37.32***
F value Soil Types 284.04*** 1.21: ns 6.34**
123
Plants in sandy clay loam soil showed low PAE comparative to plants in the other two
soils. Plants showed the maximum PAE (0.047 mg P cm-1 RL) at 45 mg P application kg-1
sandy loam soil. The minimum PAE (0.014 mg P cm-1 RL) was shown by plants @ 0 mg
P application kg-1 sandy clay loam soil.
Phosphorus use efficiencies were not significantly affected by different levels of applied
P and soil types as well as by the interaction of P and soil types (p ≤ 0.05) (Table 4.20).
All P levels showed statistically similar values however differences were obvious
numerically. Higher PUE (5.69 g2 SDM mg-1 P) was observed by plants in a treatment
where P was applied @ 45 mg kg-1 sandy clay loam soil and the lowest PUE (4.85 g2
SDM mg-1 P) was recorded at P application of 45 mg kg-1 loam soil.
In loam soil, PUE increased from 0 to 11.25 mg P application kg-1 soil and then decreased
up to 45 mg P application kg-1 soil. The highest PUE was at 11.25 mg P application kg-1
soil (5.26 g2 SDM mg-1 P) and the lowest was at 45 mg P application kg-1 soil (4.85 g2
SDM mg-1 P). In sandy loam soil, no specific trend appeared with respect to P levels. In
sandy clay loam soil, PUE decreased from 0 to 22.50 mg P application kg-1 soil and then
increased up to 45 mg P kg-1 soil. The maximum PUE (5.69 g2 SDM mg-1 P) was found at
45 mg P application kg-1 soil and the minimum (4.95 g2 SDM mg-1 P) was observed at
22.50 mg P application kg-1 soil. Plants in sandy clay loam soil were the highest in their
ability to utilize P efficiently compared to loam and sandy loam soil at all P levels.
Phosphorus uptake
The effects of P application and soil types on P uptake by wheat plants were highly
significant (p ≤ 0.05) (Table 4.20) however interaction effect was non-significant (P ≤
0.05 mg plant-1). Increasing P level from 0 to 33.75 mg P application kg-1 of loam and
sandy loam soil increased P uptake and after that, up to 45 mg P application kg-1 soil,
declining behavior in P uptake by the plant was observed. Phosphorus uptake in sandy
clay loam increased with increasing P application from 0 to 45 mg kg-1 soil. Among soils,
plants in sandy loam soil showed the highest P uptake and in sandy clay loam soil gave
the lowest P uptake. The minimum P uptake (3.9 mg plant-1) was recorded at 0 mg P
application kg-1 sandy clay loam soil and the maximum (9.35 mg plant-1) was at 33.75 mg
P application kg-1 sandy loam soil.
124
Table 4.21. Phosphorus physiological efficiency index (PPEI), Phosphorus stress
factor (PSF) and pH of wheat in loam, sandy loam and sandy clay
loam soils under different P levels.
Phosphorus PPEI PSF
Soil Types pH
(mg P kg-1) (mg g-1) (%)
0 1.03±0.01b 20.51±8.16ab 7.48±0.094b-d
11.25 0.95±0.03c 11.07±7.22a-c 7.52±0.113b-d
Loam 22.50 0.80±0.01de -5.89±13.31bc 7.54±0.139cd
33.75 0.76±0.01e -11.99±16.38c 7.65±0.131d
45 0.78±0.01e .00±0.00bc 7.66±0.091d
0 1.04±0.01b 19.08±14.24a-c 7.41±0.089a-d
11.25 0.93±0.02c 15.50±5.76a-c 7.41±0.070a-d
Sandy loam 22.50 0.78±0.02e -7.32±19.06bc 7.44±0.056a-d
33.75 0.75±0.01e -7.88±8.83bc 7.50±0.042b-d
45 0.76±0.02e 0.00±0.00bc 7.49±0.041b-d
0 1.20±0.02a 31.60±1.95a 7.18±0.088a
11.25 1.01±0.01b 23.53±0.87ab 7.23±0.078ab
Sandy clay
22.50 0.85±0.01d 15.04±4.19a-c 7.26±0.117a-c
loam
33.75 0.84±0.02d 8.67±2.78a-c 7.34±0.068a-c
45 0.83±0.01d .00±0.00bc 7.49±0.021b-d
Phosphorus 202.89*** 5.03** 2.35: ns
F value Soil Types 46.36*** 3.07: ns 11.69***
Interaction 2.67* 0.34: ns 0.26: ns
125
Phosphorus physiological efficiency index (PPEI)
Phosphorus physiological efficiency index (PPEI) indicates the ability of a plant to

absorb, accumulate and utilize P for maintaining growth (Table 4.21). Soil type, P levels,
and interaction effect were significant for PPEI (P ≤ 0.05). PPEI decreased linearly with
increasing P levels. Phosphorus application at the rate of 0 mg P application kg-1 soil gave
higher PPEI. But Plant PPEI values in loam and sandy loam soil were not statistically
variable at 33.75 and 45 mg P application kg soil-1. The lowest PPEI values were obtained
by plants in sandy loam soil. In sandy clay loam soil P, the physiological efficiency index
in plants was the highest at all applied P levels comparative to plants in the other two
soils. Plants in sandy loam and loam soil did not show statistically different PPEI values.
The highest PPEI (1.20 mg g-1) was measured in plants when P was applied @ 0 mg kg-1
of sandy clay loam soil and the lowest PEI (0.75 mg g-1) was measured at P application of
33.75 mg kg-1 sandy loam soil.
The phosphorus stress factor indicates the tolerance percentage of the plant at deficient P
levels (Table 4.21). The phosphorus effect was significant while soil and interaction
effect was not significant for PSF (p ≤ 0.05). The stress factor decreased gradually as the
P application increased. The lowest values for PSF were observed at 33.75 mg P
application kg-1 soil compared to all other P levels. The maximum PSF was noted at 0 mg
P application kg-1 soil which was 20.51%, 19.08%, and 31.59 % higher than 45 mg P
application kg-1 of loam, sandy loam, and sandy clay loam soil, respectively. In sandy
clay loam soil, P stress was greater comparative to loam and sandy loam soil at all levels
of P application.
4.3.1.4. Change in rhizosphere pH of wheat
Change in pH was measured in the rhizosphere of wheat plants (Table 4.21). Phosphorus
application and interaction effect were non-significant, but soil type affected pH
significantly (p ≤ 0.05). With the decrease in P levels, an increase in the release of the
proton was observed that lowered the pH of the rhizosphere. Among soils, the highest
unit decrease was noted in sandy clay loam soil followed by loam soil. Sandy loam soil
showed the lowest decrease in pH. Increasing P application decreased the proton release
and hence increased pH. The highest pH (7.66) was observed in loam soil where P was
126
applied @ 45 mg kg-1 soil while the lowest pH (7.18) was noted in sandy clay loam soil at
P application of 0 mg kg-1 soil.
4.3.1.5. Carboxylates release by wheat roots
Citrate release
Citrate released by plant roots at a different level of P in different textured soils is

represented in Table 4.22. Phosphorus levels, soil type, and their interaction significantly
affected the citrate release (p ≤ 0.05). In loam and sandy loam soil, citrate release was
decreased from 0 to 33.75 mg P kg-1 soil and increased after that treatment while in sandy
clay loam soil, citrate released by plant roots decreased from 0 to 45 mg P application kg-
1
soil. Soil texture affected citrate release significantly and the highest citrate exudation
was released by plant roots in sandy clay loam soil at all levels of applied P compared to
the other two soils. Among all the treatments, P application @ 0 mg kg-1 of sandy clay
loam soil severely affected plant root and caused the maximum exudation (1.45 µM g-1
RDW) of citrate. Sandy loam soil gave the lowest citrate release (Not detectable) at 33.75
mg P application kg-1 of soil which was 100% less than 0 mg P application kg-1 soil.
Malate release
P application, soil type, and their interaction significantly affected malate release by
wheat plant roots in the rhizosphere (p ≤ 0.05) (Table 4.22). Phosphorus deficiency
affected malate exudation significantly by stressing the plant and caused the enhanced
release of malate in the rhizosphere. In loam and sandy loam soil, malate release was
decreased from 0 to 33.75 mg P kg-1 soil and increased after that treatment while in sandy
clay loam soil, malate released by plant roots decreased from 0 to 45 mg P application kg-
1
soil. Significant differences by plant roots in malate release were noticed in response to
soil texture when different rates of P were applied. Sandy clay loam soil responded to soil
texture by exuding a higher concentration of malate relative to loam and sandy loam soil.
The maximum malate concentration (81.00 µM g-1 RDW) was noticed by plant roots in
sandy clay loam soil at 0 mg P kg-1 soil while the lowest malate release (40.10 µM g-1
RDW) appeared in treatment where 33.75 and 45 mg P was applied kg-1 sandy loam soil.
127
Table 4.22. Carboxylates (citrate, malate, and oxalate) release by wheat roots in
loam, sandy loam and sandy clay loam soils under different P levels.
Soil Types Phosphorus Citrate Malate Oxalate
(mg P kg-1) (µM g-1 RDW) (µM g-1 RDW) (µM g-1 RDW)
0 1.13±0.080b 76.84±1.29b 20.00±0.47b
11.25 1.05±0.049b 60.69±1.09e 17.27±0.39c
Loam 22.50 0.43±0.024gh 54.56±1.10f 12.27±0.40ef
33.75 0.39±0.031d-f 49.19±0.88g 10.61±0.56gh
45 0.42±0.027d-f 50.56±1.21f 11.18±0.70fg
0 0.48±0.033d 64.17±0.85df 15.00±0.93d
11.25 0.16±0.009g 52.28±0.55f 13.09±0.40e
Sandy loam 22.50 0.30±0.030f 43.64±1.38h 10.82±0.51fg
33.75 0.00±0.000h 40.10±0.74i 08.40±0.49h
45 0.02±0.004gh 40.10±1.12i 09.50±0.64gh
0 1.44±0.121a 81.00±1.46a 24.00±0.71a
11.25 1.01±0.042c 68.60±1.18c 20.48±0.61b
Sandy clay
22.50 0.88±0.037d 66.87±0.82cd 16.86±0.36c
loam
33.75 0.53±0.034ef 66.87±0.81cd 15.50±0.85cd
45 0.37±0.046bc 59.25±0.52f 10.57±0.41fg
Phosphorus 147.06*** 243.29*** 139.46***
Interaction 16.28*** 9.45*** 6.41***
128
Oxalate release
Oxalate release by wheat plant roots at different P levels is presented in Table 4.22.
Oxalate release was affected significantly by different rates of P, soil types and their
interaction (p ≤ 0.05). The decrease in P application increased the oxalate exudation and it
goes to the maximum value at 0 mg P application kg-1 soil. Phosphorus application at the
rate of 33.75 mg per kg of soil showed the lowest release of oxalate by wheat roots in
loam and sandy loam soil. In sandy clay loam soil, the lowest release was observed at 45
mg P application kg-1 soil. Among soils, plants in sandy clay loam soil released higher
concentration of oxalate while plants in sandy loam soil was the lowest in oxalate release
at all levels of P. The highest oxalate concentration (24 µM g-1 RDW) was released by
plant roots at 0 mg P application kg-1 of sandy clay loam soil while the lowest oxalate
release was noticed in sandy loam (8.40 µM g-1 RDW) at 33.75 mg P application kg-1
soil.
4.3.1.6. Physiological parameters
Wheat leaf chlorophyll content showed a positive response to the increasing level of P
(Table 4.23). Phosphorus and soil type affect was significant while the interaction effect
was not significant (p ≤ 0.05). In these two treatments, the chlorophyll value was not
statistically different. The highest value for chlorophyll was obtained at 45 mg P
application Kg-1 of soil (47.9, 50.5 and 50.2 for loam, sandy loam and sandy clay loam
soil, respectively). At 0 mg P application Kg-1 of soil, the lowest value of chlorophyll for
loam (45.83), sandy loam (46.73) and sandy clay loam (34.73) soil was recorded. Among
soils, sandy loam soil performed significantly better compared to the other two soils at all
P levels.
Low P application had a negative impact on the transpiration efficiency of plants (Table
4.23). Phosphorus levels, soil types and interaction effect were significant for the
transpiration rate (p ≤ 0.05). The transpiration rate was positively related to P addition.
The increased value of P significantly increased the transpiration rate. The lowest
transpiration rate in loam (2.9 mm H2O m-2 s-1), sandy loam (3 mm H2O m-2 s-1) and
sandy clay loam soil (2.7 mm H2O m-2 s-1) was measured at 0 mg P application kg-1 of
soil. Higher values of transpiration rate in loam (3.7 mm H2O m-2 s-1) and sandy loam (3.8
mm H2O m-2 s-1) were obtained at 33.75 mg P application kg-1 of soil and in sandy clay
loam (3.58 mm H2O m-2 s-1) at 45 mg P application kg-1 of soil. Plants transpiration rate
129
was statistically variable in all soils. As plant growth was good in sandy loam soil, the
transpiration rate was higher in plants grown in sandy loam soil as compared to loam and
sandy clay loam soil at all P levels.
Photosynthesis was affected by detrimentally when the P supply was low (Table 4.23).
Photosynthesis was affected significantly by soil type, P and their interaction (p ≤ 0.05)
Decrease in P addition caused a significant reduction in photosynthesis of plants. At 0 mg
P application kg-1 of soil, the lowest photosynthetic rate 4, 4.21 and 3.9 µmol CO2 m-2 s-1
in loam, sandy loam, and sandy clay loam were noted, respectively. At highest level of P
application (45 mg P application kg-1 of soil) photosynthetic rate in sandy clay loam soil
was 5.3 µmol CO2 m-2 s-1 while in loam and sandy loam soil, the highest photosynthesis
(5.6 µmol CO2 m-2 s-1 and 5.75 µmol CO2 m-2 s-1, respectively) was recorded at 33.75 mg
P application kg-1 which was greater than all other levels of P. The photosynthetic rate
was significantly variable in different textured soils. Plant in sandy loam soil performed
better as compare to loam and sandy clay loam soil.
The decrease in P application impacted negatively and caused a reduction in soil

respiration rate (Table 4.23). Phosphorus and soil effects were significant while
interaction effect was non- significant (p ≤ 0.05). A gradual decrease in respiration was
observed with a decreasing level of P application. Respiration rate decreased up to 0.11
μmol CO2 s–1, 0.1 μmol CO2 s–1 and 0.08 μmol CO2 s–1 at 0 mg P application kg-1 loam,
sandy loam, and sandy clay loam soil, respectively. In loam, sandy loam and sandy clay
loam soil, respiration rate was significantly variable. In sandy loam soil, higher
respiration rate was found (0.42 μmol CO2 s–1) followed by loam (0.4 μmol CO2 s–1) at
33.75 mg P application kg-1 soil and in sandy clay loam soil (0.38 μmol CO2 s–1) it was
recorded at 45 mg P application kg-1 soil. Overall, the maximum respiration (0.42 μmol
CO2 s–1) rate was observed at 45 mg P application kg-1 of sandy loam soil and the
minimum (0.08 μmol CO2 s–1) was recorded at 0 mg P application kg-1 of sandy clay loam
soil.
130
Table 4.23. Chlorophyll, Transpiration rate, Photosynthetic rate, and Respiration
rate of wheat plants in loam, sandy loam and sandy clay loam soils
under different P levels.
Transpiratio Photosynthesi
Chlorophyll Respiration
Soil Phosphorus n s
(SPAD (μmol CO2
Types (mg P kg-1) (mm H2O m-2 (µmol CO2 m-
value) s–1)
s-1) 2 s-1)
0 45.83±0.50c 2.90±0.050k 4.00±0.04h 0.10±0.01g

11.25 45.47±1.63c 3.08±0.015hi 4.25±0.02g 0.18±0.02ef
Loam 22.50 46.8±0.90bc 3.3±0.016f 4.47±0.03f 0.26±0.02d
33.75 47.13±1.30bc 3.7±0.017c 5.60±0.01e 0.40±0.01a
45 47.9±0.70bc 3.5±0.023d 5.00±0.05e 0.31±0.02c
0 46.73±2.28bc 3.0±0.050ij 4.21±0.04i 0.11±0.01g
11.25 46.23±2.03bc 3.1±0.010h 4.43±0.02f 0.20±0.02e
Sandy
22.50 48.3±0.20bc 3.4±0.025e 4.90±0.05e 0.29±0.01cd
loam
33.75 52.5±1.53ab 4.0±0.025a 5.75±0.04a 0.42±0.03a
45 50.5±0.76ab 3.8±0.026b 5.50±0.05c 0.35±0.01b
0 48.2±1.65bc 2.7±0.020l 3.90±0.03j 0.08±0.02g
11.25 49.0±0.45a-c 2.93±0.030jk 4.17±0.02g 0.15±0.01f
Sandy
22.50 49.4±0.81a-c 3.2±0.041g 4.4±0.04f 0.21±0.02e
clay loam
33.75 49.7±0.64a-c 3.4±0.040e 4.90±0.03e 0.29±0.01cd
45 50.2±1.30a 3.58±0.044d 5.30±0.02d 0.38±0.01ab
Phosphorus 2.74* 429.6*** 1033.56*** 224.34***
F value Soil Types 7.81** 118.21*** 211.91*** 18.14***
Interaction 0.98: ns 5.50*** 8.08*** 0.60: ns
131
4.3.2. Influence of soil types and deficient levels of P on the release of carboxylates
and P acquisition characteristics in maize
4.3.2.1. Growth responses
Shoot length (SL) of maize plant in response to P application in loam, sandy loam, and
sandy clay loam soil was measured at flowering (Table 4.24). Shoot length was
significantly (p ≤ 0.05) affected by P levels, soil types as well as by their interaction.
From 0 to 45 mg P application kg-1 soil, SL increased but the reduction was observed at
60 mg P application kg-1 soil. This pattern was followed by loam and sandy loam soil. But
in sandy clay loam soil, SL increased gradually from 0 to 60 mg P per kg of soil. The
highest SL (110.0 cm) was recorded when P was applied @ 45 mg kg-1 of sandy loam soil
and the lowest SL (85 cm) was recorded at P application of 45 mg kg-1 loam soil. Plants
in sandy loam soil performed best compared to plants in loam and sandy clay loam soil.
Shoot fresh weight (SFW) of maize plants at flowering in response to the different rates
of P in three different textured soils were recorded (Table 4.24). Soil types, P application,
and interaction effect were highly significant (p ≤ 0.05). An increase in SFW was linear
with P application and at 45 mg P application kg-1 soil the highest SFW was noted after
that treatment, SFW started to reduce non-significantly. This pattern was followed by
plants in sandy loam and loam soil. Plants in sandy clay loam soil showed a gradual
increase in SFW with an increasing level of P from 0 to 60 mg P application kg-1 soil.
Plants in sandy clay loam soil were the lowest in SFW value among soils. Plants in sandy
loam soil gave the highest SFW among all three different textured soils at all P levels.
Overall the maximum SFW (164.5 g plant-1) was recorded in sandy loam soil at 45 mg P
application kg-1 soil and the minimum (95.7 g plant-1) was in sandy clay loam soil at 0 mg
P application kg-1 soil.
Maize plants responded to P deficient conditions by decreasing their shoot dry weight
(SDW) (Table 4.24). There was a highly significant (p ≤ 0.05) effect of P application and
soil types on SDW but the interaction effect was not significant. Improved P application
caused an increase in SDW but at a higher level (60 mg P application kg-1 soil) of P,
SDW was low comparative to 45 mg P application kg-1 soil. Plants in sandy clay loam
soil did not follow this pattern and gave a gradual increase with increasing P level.
132
Table 4.24. Shoot length, shoot fresh and dry weight of maize in loam, sandy loam,
Shoot dry
Phosphorus Shoot length Shoot fresh weight
Soil Types weight
(mg P kg-1) (cm) (g plant-1)
(g plant-1)
0 85.3±0.88d 98.7±1.7g-i 20.1±0.45d
15 90.8±0.93c 103.4±1.5fg 21.0±0.96d
Loam 30 93.6±2.39bc 107.1±1.7f 21.6±0.80d
45 98.7±1.45b 112.1±1.7e 24.6±0.87c
60 85.0±2.30d 102.1±1.7f-h 21.0±1.30d
0 95.5±1.04bc 145.0±0.36c 25.2±0.60c
15 106.3±2.03a 152.0±1.7b 25.2±0.52c
Sandy loam 30 108.2±1.69a 156.9±1.8b 28.8±0.55b
45 110.0±2.30a 164.5±2.8a 31.5±0.37a
60 98.7±1.45b 125.4±2.9d 26.1±0.56c
0 91.0±2.30c 95.7±1.45i 15.2±0.57f
15 97.7±2.30b 97.7±1.5hi 17.4±0.47e
Sandy clay
30 95.7±0.88bc 99.7±0.9g-i 17.5±0.85e
loam
45 105.8±2.26a 102.7±1.4f-h 17.7±0.39e
60 104.7±1.86a 122.03±1.2d 16.1±0.88ef
Phosphorus 23.62*** 66.87*** 17.43***
Interaction 4.84** 27.9*** 4.06*
133
Among all soil, plants in sandy loam soil gave the highest SDW followed by loam soil
and then sandy clay loam soil at all levels of applied P. The highest SDW (31.5 g plant-1)
was measured in sandy loam soil at P application of 45 mg kg-1 soil. Plants in sandy clay
loam soil showed the lowest SDW (15.2 g plant-1) at 0 mg P application kg-1 of soil.
Maize plants increased their rooting system under low P availability (Table 4.25). Soil
type, P and interaction effects were significant (p ≤ 0.05) for root length (RL). Plant roots
increased their length as the P availability reduced. The decrease in RL with the increase
in P level was linearly related in both loam and sandy clay loam soil. Plants in sandy loam
soil had different response patterns. The maximum RL (95.0 cm) was recorded when P
was applied @ 0 mg kg-1 of sandy clay loam soil and the minimum RL (71.0 cm) was
measured at P application of 45 mg kg-1 loam and sandy loam soil. Maize plant RL was
higher in sandy clay loam soil as compared to loam and sandy loam soil.
Soil type and P application affected root fresh weight (RFW) significantly (Table 4.25).
But the Interaction effect was non-significant. With the increase in the level of P addition,
a decrease in RFW was recorded. In sandy clay loam, the highest root fresh (26.47 g
plant-1) weight was recorded at 0 mg P application kg-1 soil and in loam soil, the lowest
RFW (10.31 g plant-1) appeared at 60 mg P application kg-1 soil. Different textured soils
showed a significantly different response in RFW at different levels of P application.
Plants roots in sandy clay loam soil had more RFW and plants in loam soil had the lowest
RFW as compared to sandy loam and sandy clay loam soil.
Root dry weight (RDW) decreased gradually as P application increased in all three
different textured soils (Table 4.25). Phosphorus and soil effects were highly significant
(p ≤ 0.05) and interaction effect was non-significant. The decrease in RDW was linear
with increasing P level but this pattern was not evident at 45 and 60 mg P application kg-1
soil. Both treatment levels showed the same RDW in all three different textured soils
numerically as well as statistically. Among all soils, the maize roots had more RDW in
sandy clay loam soil followed by loam soil. Sandy loam soil had the lowest place among
soils for RDW at all levels of applied P. The maximum RDW (7.34 g plant-1) was
observed in a treatment where P was applied @ 0 mg kg-1 of sandy clay loam soil and the
minimum (3.89 g plant-1) was recorded at P application of 45 and 60 mg kg-1 sandy loam
soil.
134
Table 4.25. Root length, root fresh and dry weight of maize in loam, sandy loam and
sandy clay loam soils under different P levels.
Root fresh
Phosphorus Root length Root dry weight
Soil Types weight
(mg P kg-1) (cm) (g plant-1)
(g plant-1)
0 86.3±2.33b 15.90±1.96bc 5.71±0.56cd
15 80.5±0.87cd 15.98±1.95bc 5.08±0.01de
Loam 30 75.7±0.88e 15.55±1.75bc 4.71±0.05e
45 71.2±0.96f 13.52±1.07ab 3.90±0.03f
60 71.0±1.15f 10.31±1.34a 3.90±0.30f
0 81.0±2.08c 20.85±1.42d 5.37±0.02cd
15 69.3±1.33f 19.17±0.70cd 4.68±0.015e
Sandy loam 30 78.0±0.58c-e 17.53±1.27b-d 3.95±0.01f
45 76.3±0.88de 14.8±0.74a-c 3.89±0.02f
60 71.0±1.53f 15.13±0.89cd 3.89±0.003f
0 95.0±1.15a 26.46±2.03e 7.34±0.09a
15 87.0±1.00b 20.96±1.63d 6.39±0.01b
Sandy clay
30 81.7±1.45c 18.40±0.63cd 5.9±0.15bc
loam
45 79.7±1.76c-e 16.40±1.12b-d 5.76±0.49bc
60 78.7±2.02c-e 14.56±1.90a-c 5.79±0.01bc
Phosphorus 41.27*** 13.43*** 32.25***
135
There were significant effects found on root mass fraction ( RMF) by different levels of
applied P, and soil types but the interaction effect was non- significant (p ≤ 0.05) (Table
4.26). With the increase in applied P level, RMF decreased but this trend changed at 60
mg P application kg-1 soil, as this level showed an increase in its RMF. There were
significant differences found in RMF between soils. The highest RMF was found in sandy
clay loam soil followed by loam soil and then sandy loam soil at all levels of P.
Phosphorus application @ 0 mg kg-1 soil gave the maximum RMF (0.327 g g-1) in sandy
clay loam soil while sandy loam soil @ 45 mg P application kg-1 soil gave the minimum
RMF (0.110 g g-1).
Root to shoot ratio (R/S) was affected significantly by soil types and P application but the
interaction effect was non-significant (p ≤ 0.05) (Table 4.26). A negatively linear pattern
for the R/S ratio with an increasing level of P was found. As the R/S ratio was linked to
root growth and SDW so with the increase in RL and RDW, R/S ratio decreased as the
level of P application is increased. All soils depicted significant variation in their response
to R/S ratio at all levels of applied P. Plants in sandy clay loam soil gave higher R/S ratio
and sandy loam soil showed the minimum R/S ratio at all P levels. R/S ratio was lower
(0.12) in treatment where 45 mg P was applied in sandy loam soil while the highest R/S
ratio (0.49) was obtained at 0 mg P application kg-1 of sandy clay loam soil.
4.3.2.2. Root morphological responses
Root morphological response was observed by scanning the plant roots grown under
different levels of P. Root parameters like average root diameter, root surface area and
number of root tips were measured. Root volume was measured by the volumetric
method. Root volume was significantly (p ≤ 0.05) affected by P levels, soil type and
interaction (Table 4.26). An increase in P levels decreased the root volume. The decrease
in root volume with the increase in P level was observed from 0 to 45 mg P application
kg-1 and at 60 mg P application kg-1 soil, root volume increased comparative to 45 mg P
application kg-1 in loam and sandy loam soil but it was not much higher than other P
levels. In sandy clay loam soil, root volume decreased linearly with the increasing level of
P from 0 to 60 mg P application kg-1 soil. Maize plant had higher root volume in sandy
clay loam soil at all levels of applied P followed by loam soil.
136
Table 4.26. Root mass fraction, Root: Shoot and Root volume of maize in loam,
sandy loam and sandy clay loam soils under different P levels.
Phosphorus Root mass Root volume
Soil Types Root: Shoot
(mg P kg-1) fraction (g g-1) (cm3)
0 0.221±0.020cd 0.29±0.031cd 24.0±1.8b
15 0.195±0.006de 0.24±0.011de 19.5±0.3cd
Loam 30 0.179±0.004 ef 0.22±0.006ef 17.5±0.9de
45 0.136±0.003gh 0.16±0.005gh 11.0±0.8g
60 0.158±0.010fg 0.19±0.010fg 14.0±0.6fg
0 0.176±0.003ef 0.21±0.004ef 20.5±0.8cd
15 0.157±0.003fg 0.19±0.004fg 16.0±1.3ef
Sandy loam 30 0.121±0.002h 0.14±0.003gh 16.0±0.2ef
45 0.110±0.001h 0.12±0.002h 11.0±1.5g
60 0.130±0.002gh 0.15±0.003gh 14.0±0.4fg
0 0.327±0.011a 0.49±0.023a 29.0±1.2a
15 0.269±0.005b 0.37±0.010b 21.8±0.9bc
Sandy clay
30 0.253±0.010b 0.34±0.020b 18.1±1.1de
loam
45 0.245±0.021bc 0.33±0.043bc 18.6±0.8de
60 0.266±0.010b 0.36±0.021b 16.0±1.0ef
Phosphorus 29.07*** 25.33*** 56.85***
Interaction 1.31: ns 1.6: ns 3.53**
137
The maximum root volume (24 cm3) was measured when P was applied @ 0 mg kg-1 of
sandy clay loam soil and the minimum root volume (11 cm3) was recorded at P
application of 45 mg kg-1 loam soil and sandy loam soil.
The average root diameter was affected significantly by both P application and soil types
(Table 4.27) however interaction effect was not significant (p ≤ 0.05). A positive linear
relation among P levels and average root diameter were found, as the level of P
application increased from 0 to 45 mg kg-1 loam and sandy loam soil, average root
diameter also increased but in sandy clay loam soil, average root diameter increased from
0 to 60 mg P application kg-1 soil. Plants in sandy loam soil showed higher average root
diameter at all levels of applied P, compared to plants in the other two soils. The average
root diameter in sandy loam soil at 45 mg P application kg-1 soil was the highest (0.148
cm) as compared to all other soils and all P levels while the lowest average root diameter
(0.83 cm) was observed at 0 mg P application kg-1 of sandy clay loam soil.
P application, soil type and interaction effects on a number of root tips were significant (p
≤ 0.05) (Table 4.27). No of root tips decreased with the increase in applied P level in all
types of soils at all levels of applied P, a significant difference in a number of root tips
was appeared except at 45 and 60 mg P application kg-1 of loam and sandy loam soil. The
number of root tips in loam and sandy loam soil was the lowest at 45 mg P application kg-
1
soil but in sandy clay loam soil, it was the lowest at 60 mg P application kg-1 soil. The
maximum number of root tips (360.5) was recorded in a treatment where P was applied
@ 0 mg kg-1 of sandy clay loam soil and the minimum number of root tips (166) was
recorded when P was applied @ 45 mg kg-1 loam soil.
Highly significant (p ≤ 0.05) effect of P level, soil type, and their interaction were
observed on the maize plant root surface area (Table 4.27). There was a negative relation
found between the P level and root surface area. With the increase in the applied P
amount, the root surface area decreased. In sandy clay loam soil, the pattern of decrease
in root surface area was linear with the increase in P but loam and sandy loam soil
showed variation to this pattern at the increasing level of P. Root surface area varied
significantly between soils. Plants in sandy clay loam soil showed significantly higher
root surface area comparative to plants in other soils at all levels of P.
138
Table 4.27. Root surface area, Number of root tips and Average root diameter of
maize in loam, sandy loam and sandy clay loam soils under different P
levels.
Phosphorus Root surface Number of root Average root
Soil Types
(mg P kg-1) area (cm2) tips diameter (cm)
0 60.3±1.41b 310.2±1.5c 0.087±0.002de
15 49.7±0.64de 275.2±1.7e 0.090±0.001de
Loam 30 46.8±0.06e 245.0±2.9g 0.10±0.0003c-e
45 43.2±0.64f 166.0±1.2m 0.123±0.010b
60 47.7±1.90e 230.5±0.9h 0.117±0.010bc
0 58.1±0.96b 345.3±1.5b 0.107±0.003b-e
15 42.1±0.60f 209.3±0.7i 0.109±0.004b-d
Sandy loam 30 41.4±1.38f 204.5±2.3k 0.110±0.0002b-d
45 38.4±0.63g 178.7±0.7l 0.148±0.010a
60 39.7±0.90fg 183.0±1.2l 0.116±0.010bc
0 68.2±0.45a 360.5±0.9a 0.083±0.020e
15 54.1±1.40c 293.0±1.7d 0.088±0.010de
Sandy clay
30 51.8±1.09cd 267.5±1.4f 0.094±0.010c-e
loam
45 47.4±1.90e 214.0±0.6i 0.096±0.010c-e
60 46.6±1.7e 213.2±1.7ij 0.115±0.010ab
Phosphorus 125.31*** 4480.07*** 11.78***
Interaction 4.5*** 317.95*** 0.95: ns
139
Root surface area (68.2 cm2) was higher at 0 mg P application kg-1 of sandy clay loam soil
and the lowest root surface area (38.4 cm2) was recorded at P application of 45 mg kg-1 sandy
loam soil.
4.3.2.3. Phosphorus acquisition parameters
Rhizosphere soil available P
Rhizosphere soil available P was significantly (p ≤ 0.05) affected by P application and soil
type as well as their interaction (Table 4.28). A negative linear relation was noticed between
added P and rhizosphere available P. With the increase in the level of P, the decrease in
rhizosphere P recorded. Different soils showed a significantly different response in
rhizosphere available P at different levels of P application. All soils showed better results at 0
mg P application kg-1 soil comparative to other levels of applied P. The maximum
rhizosphere soil available P (11.2 mg kg-1 in sandy clay loam soil) was analyzed at 0 mg P
application kg-1 soil and the minimum rhizosphere soil available P (5.1 mg kg-1 in sandy loam
soil) was measured at 45 mg P application kg-1soil. Sandy clay loam soil was significantly
better in improving rhizosphere soil available P when compared with loam and sandy loam
soil.
Root P
The comparative effect of different levels of P application and soil type on maize plant root P
content was measured (Table 4.28). Phosphorus and soil type significantly affected the root
P, but the interaction was non-significant (p ≤ 0.05). The overall increase was observed in
root P content as the P application level increased from 0 to 45 mg P application kg-1 of loam,
sandy loam, and sandy clay loam soil. Phosphorus application @ 60 mg kg-1 soil showed a
decrease in root P compared to lower levels of P application. Plants in sandy loam soil
showed the highest value for root P content among all soils. Sandy clay loam soil gave the
lowest root P content. The maximum root P content (0.97 mg g-1) was analyzed when P was
applied @ 45 mg kg-1 of sandy loam soil and the lowest root P content (0.68 mg g-1) was
recorded at P application of 0 mg kg-1 of loam and sandy clay loam soil.
140
Table 4.28. Rhizosphere soil available P, Root and shoot P of maize in loam, sandy loam
Phosphorus Rhizosphere Root P Shoot P
Soil Types
(mg P kg-1) soil P (mg kg-1) (mg g-1) mg g-1)
0 9.0±0.07b 0.68±0.02k 1.32±0.15hi
15 6.7±0.06h 0.77±0.01hi 1.44±0.02fg
Loam 30 6.5±0.05i 0.83±0.01fg 1.55±0.015de
45 6.0±0.05j 0.93±0.01b 1.66±0.03a-c
60 6.1±0.04j 0.88±0.015c-e 1.60±0.02b-d
0 8.8±0.05c 0.75±0.02ij 1.36±0.21h
15 5.3±0.04 m 0.81±0.03gh 1.48±0.02ef
Sandy loam 30 5.8±0.02k 0.86±0.01ef 1.59±0.02cd
45 5.1±0.06n 0.97±0.02a 1.70±0.01a
60 5.6±0.02l 0.91±0.02b-d 1.65±0.01a-c
0 11.2±0.08a 0.68±0.02k 1.27±0.02i
15 8.5±0.04d 0.72±0.01jk 1.39±0.04gh
Sandy clay
30 7.8±0.05e 0.79±0.02g-i 1.50±0.04ef
loam
45 7.2±0.03f 0.92±0.01bc 1.61±0.04b-d
60 7.0±0.06g 0.84±0.017d-f 1.67±0.03ab
Phosphorus 2732.12*** 141.57*** 109.61***
141
Shoot P
Phosphorus content in the shoot at different levels of P in loam, sandy loam, and sandy
clay loam soil was analyzed (Table 4.28). Significant (p ≤ 0.05) effect on shoot P content
was given by different levels of applied P as well as by different types of soils while
interaction effect was non-significant. With the increase in P level from 0 to 45 mg P
application kg-1 soil, increase in shoot P content was appeared but after that level, shoot P
content start to decline in loam, and sandy loam soil but in sandy clay loam soil it
remained increasing. The pattern of response by plants in loam and sandy loam was the
same to shoot P value at different P levels statistically. Plants in sandy loam soil
performed better in shoot P content followed by loam soil. Phosphorus application @ 45
mg kg-1 in sandy loam soil gave higher shoot P content (1.70 mg g-1) in maize plants and
the minimum (1.27 mg g-1) was analyzed at 0 mg P application kg-1 sandy clay loam soil.
Phosphorus acquisition efficiency (PAE)
Phosphorus acquisition efficiency (PAE), the ability of the plant to acquire P, is presented
in Table 4.29. Plants acquire P at a significantly different rate at all levels of applied P in
all soil types and their interaction was also significant (p ≤ 0.05). Phosphorus application
increased PAE. However, PAE at 45 and 60 mg P application kg-1 soil did not differ.
Plants showed the maximum PAE (0.036 mg P cm-1 RL) at 45 mg P application kg-1 loam
and 60 mg P application kg-1 sandy loam soil. The minimum PAE (0.021 mg P cm-1 RL)
was shown by plants @ 0 mg P application kg-1 sandy clay loam soil.
Phosphorus use efficiencies were significantly affected by different levels of applied P

and soil types however interaction effect was not significant (p ≤ 0.05) (Table 4.29). PUE
decreased with the increase in P level but the pattern of the increase was not linear. In
loam soil, PUE was higher at 0 mg P application and then abrupt changes appeared at
increased P application. In sandy loam soil, the highest PUE was obtained at 45 mg P
application kg-1 soil. In sandy clay loam soil, PUE was higher at 15 mg P application kg-1
soil. Plants in sandy loam soil were the better in their ability to utilize P efficiently
compared to loam and sandy clay loam soil. Higher PUE (18.50 g2 SDM mg-1 P) was
observed by plants in a treatment where P was applied @ 0 and 45 mg kg-1 sandy loam
soil and the
142
Table 4.29. Phosphorus acquisition efficiency (PAE), Phosphorus use efficiency
(PUE) and Phosphorus uptake of maize in loam, sandy loam and sandy
clay loam soils under different P levels.
Phosphorus PAE PUE P Uptake
Soil Types
(mg P kg-1) (mg P cm-1 RL) (g2 SDM mg-1 P) (mg plant-1)
0 0.023±0.004a 15.15±0.24de 26.55±0.84gh
15 0.027±0.002cd 14.6±0.50b 30.32±1.73fg
Loam 30 0.031±0.003b 13.93±0.39b 33.51±1.49ef
45 0.036±0.002a 14.84±0.54b 40.90±1.77cd
60 0.035±0.001a 13.09±0.71c 33.57±2.45ef
0 0.026±0.001d 18.5±0.17a 34.24±1.33ef
15 0.033±0.001b 17.01±0.55a 37.22±0.57de
Sandy loam 30 0.031±0.006b 18.10±0.50a 45.71±0.54b
45 0.035±0.001a 18.52±0.22a 53.51±0.75a
60 0.036±0.001a 15.79±0.25b 43.02±1.19bc
0 0.021±0.003f 11.96±0.56cd 19.27±0.57i
15 0.024±0.003e 12.53±0.11c 24.24±1.26h
Sandy clay
30 0.028±0.001c 11.67±0.70de 26.21±1.24gh
loam
45 0.032±0.002b 11.00±0.32e 28.51±1.08g
60 0.032±0.001b 9.66±0.69e 26.82±1.01gh
Phosphorus 210.34*** 11.19*** 51.13***
Interaction 7.23*** 1.71: ns 3.5**
143
lowest PUE (9.65 g2 SDM mg-1 P) was recorded at P application of 60 mg kg-1 sandy clay
loam soil.
Phosphorus uptake
P uptake, a measure of aggregated acquisition mechanism effects, was affected

significantly by P application, soil type as well as by the interaction of P with soil (p ≤
0.05) (Table 4.29). A positive linear trend in P uptake was found with increasing P levels.
But this pattern was followed up to 45 mg P application kg-1 soil and after that at 60 mg P
application kg-1 soil decrease in P uptake was measured. Significant differences were
given by different textured soils for P uptake. In sandy loam soil, plants P uptake was
higher as compared to loam and sandy loam soil. Sandy clay loam soil was the lowest
among soils in P uptake ability. The highest P uptake (53.51 mg plant-1) was shown by
plants grown in sandy loam soil when P was applied @ 45 mg kg-1 soil and the lowest P
uptake (19.26 mg plant-1) was shown by plant @ 0 mg P application kg-1 sandy clay loam
soil.
Phosphorus physiological efficiency index (PPEI)
Phosphorus Physiological Efficiency Index (PPEI) indicates the ability of a plant to

absorb, accumulate and utilize P for maintaining growth. Variation in PPEI by the plant to
P application was recorded (Table 4.30). PPEI decreased linearly with increasing P levels
from 0 to 45 mg P application kg-1 of loam and sandy loam soil but a higher level of P
application (60 mg P application kg-1 soil) did not follow this pattern. At 60 mg P
application kg-1 soil, an increase in PPEI was calculated relative to the treatment of 45 mg
P application kg-1 loam and sandy loam soil. In sandy clay loam soil, PPEI decreases
sharply from 0 to 60 mg P application kg-1 soil. The highest PPEI (0.79 g mg-1) was
measured in plants when P was applied @ 0 mg kg-1 of sandy clay loam soil and the
lowest PPEI (0.59 g mg-1) was measured at P application of 60 mg kg-1 sandy loam soil.
Plants in sandy clay loam and loam soil did not show statistically different P
physiological efficiency index values.
P stress factor indicates the tolerance percentage of the plant at deficient P levels (Table
4.30). The application of P significantly affected PSF while soil and interaction effect was
not significant (p ≤ 0.05). The phosphorus stress factor decreased gradually as the P
application increased. The lowest values for PSF were observed at 45 mg P application
144
kg-1 soil compared to all other P levels. The maximum PSF was noted at 0 mg P
application kg-1 soil which was 4.34%, 3.45% and 5.67 % for loam, sandy loam, and
sandy clay loam soil, respectively. In sandy clay loam soil, P stress was greater
comparative to loam and sandy loam soil.
4.3.2.4. Change in rhizosphere pH of maize
Change in rhizosphere pH was measured in maize plants (Table 4.30). pH was affected
significantly by soil types and P levels as well as by interaction between P and soil types
(p ≤ 0.05). Increasing P application decreased the proton release and hence increased the
pH. With the decrease in P levels, an increase in the release of the proton was observed
that lowered the pH of the rhizosphere. The maximum pH (8.1) was analyzed in loam soil
where P was applied @ 45mg kg-1 soil. The minimum pH (7.55) was noted in sandy clay
loam soil when P was applied @ 0 mg kg-1 soil.
4.3.2.5. Carboxylates release by maize roots
Citrate release
Citrate release by maize roots grown at different soils under different P levels was
analyzed at flowering (Table 4.31). Soil types, P application, and their interaction effect
were highly significantly (p ≤ 0.05) on plant roots in the release of citrate. Phosphorus
deficiency increased stress on plants and caused more exudation of citrate release. The
highest concentration of citrate (3.0 µM g-1 RDW) was released by plants in sandy clay
loam soil when no P was applied while the lowest citrate (0.27 µM g-1 RDW) release was
noticed by plant root in loam soil at 60 mg P application kg-1 soil. Among soils, plants in
sandy clay loam soil showed the highest citrate release compared to all other soils and in
loam soil, the lowest citrate release was observed at all levels of P.
Malate release
Malate release by maize root affected by P application in different soils is represented in

Table 4.31. Plants' response to P levels, soil type, and their interaction was highly
significant (p ≤ 0.05). Increase in P levels from 0 to 45 mg P application kg-1 of loam and
sandy loam soil caused a reduction in malate release and at 60 mg P application kg-1 soil,
malate release was higher than 45 mg P application kg-1 soil.
145
Table 4.30. Phosphorus physiological efficiency index (PPEI), Phosphorus stress
factor (PSF) and pH of maize in loam, sandy loam and sandy clay loam
Phosphorus PPEI PSF
Soil Types pH
(mg P kg-1) (g mg-1) (%)
0 0.76±0.008b 3.81±7.61a 7.80±0.013d
15 0.69±0.008de -0.15±2.40a 7.90±0.011e
Loam 30 0.65±0.006fg -3.22±6.18ab 7.94±0.009e-g
45 0.60±0.010hi -17.44±4.41bc 8.01±0.012h
60 0.63±0.005gh 0.00±0.00a 8.10±0.0521i
0 0.74±0.011bc 3.19±4.37a 7.55±0.019a
15 0.68±0.010ef 3.59±0.76a 7.65±0.004b
Sandy loam 30 0.63±0.005gh -10.93±3.73a-c 7.72±0.013c
45 0.59±0.003i -21.68±4.11c 7.96±0.013f-h
60 0.61±0.003hi 0.00±0.00a 7.81±0.005d
0 0.79±0.009a 4.35±8.41a 7.63±0.005b
15 0.72±0.020cd -9.16±8.53a-c 7.81±0.010d
Sandy clay
30 0.67±0.015ef -8.82±3.63a-c 7.89±0.009e
loam
45 0.62±0.016gh -10.41±3.89a-c 7.90±0.011ef
60 0.60±0.010hi 0.00±0.00a 7.96±0.009gh
Phosphorus 119.07*** 8.25*** 143.60***
F value Soil Types 12.21*** 0.18: ns 153.85***
Interaction 1.66: ns 0.96: ns 3.56**
146
Plant roots released malate at a decreasing rate in sandy clay loam soil with increasing P
application up to 60 mg P application kg-1 soil. The highest release of malate (515.6 µM
g-1 RDW) appeared in sandy clay loam soil at 0 mg P application kg-1 soil and the lowest
(221.2 µM g-1 RDW) was observed at 45 mg P application kg-1 sandy loam soil. The
release of malate by plant roots was affected significantly when they are grown in
different textured soil (loam, sandy loam, and sandy clay loam soil). Stress was appeared
to be the highest in sandy clay loam soil because of the highest release of malate by plant
roots in rhizosphere compared to other soils. Plants in sandy loam soil appeared to be the
lowest in malate release comparative to plants in the other two soils at all P levels.
Oxalate release
Oxalate released by the root of maize plant grown in different soils at different levels of
applied P is represented in Table 4.31. Oxalates released by root were significantly (p ≤
0.05) affected by different P applications but P application at the rate of 45 and 60 mg P
application kg-1 soil did not show a significant difference. Soil type and interaction effects
were highly significant (p ≤ 0.05). The overall response of plants to P application in the
release of oxalate showed a linear decrease in oxalate concentration with increasing P
application rate however 45 and 60 mg P application kg-1 soil showed the same
concentration statistically. The highest oxalate concentration (205.42 µM g-1 RDW) was
recorded at 0 mg P application kg-1 sandy clay loam soils. The lowest value of oxalate for
sandy loam (56.75 µM g-1 RDW) was measured at 60 mg P application kg-1 soil. Sandy
loam soil showed the lowest oxalate concentration relative to loam and sandy clay loam
soil at all levels of P.
4.3.2.6. Physiological parameters
Maize leaf chlorophyll content showed a positive response to the increasing level of
phosphorus (Table 4.32). Phosphorus and soil type affect were significant while the
interaction effect was not significant. Chlorophyll value did not increase significantly
with increasing P level in all soil types. This was the case for 33.75 and 45 mg P
application Kg-1 of soil. In these two treatments, the chlorophyll value was not
statistically different. The highest value for chlorophyll was obtained at 60 mg P
application Kg-1 of loam soil (44.4) and sandy loam soil (44.47) and sandy clay loam soil
(43.57). At 0 mg P application Kg-1, the lowest value of chlorophyll for loam (35.93),
147
Table 4.31. Carboxylates (citrate, malate, and oxalate) release by maize plant roots
in loam, sandy loam and sandy clay loam soils under different P levels
Soil Types Phosphorus Citrate Malate Oxalate
(mg P kg-1) (µM g-1 RDW) (µM g-1 RDW) (µM g-1 RDW)
0 1.83±0.22cd 484.08±6.6ab 158.19±8.5b
15 1.81±0.04cd 461.84±13.1gh 97.77±4.5de
Loam 30 1.17±0.25ef 380.67±9.1a 100.00±7.7de
45 0.27±0.04h 337.95±8.3i 65.48±4.5f
60 0.61±0.02de 339.95±6.6b-d 65.00±8.3f
0 1.47±0.04de 368.29±14.8bc 125.75±9.7c
15 1.17±0.05ef 291.38±9.4fg 64.87±8.2f
Sandy loam 30 1.02±0.21f 265.65±15.0ij 62.63±5.3f
45 0.91±0.02fg 221.29±7.1h 56.75±7.7f
60 0.32±0.1h 233.98±12.3cd 57.49±7.0f
0 3.00±0.17a 515.62±10.5jk 205.42±6.9a
15 2.31±0.1b 466.95±15.6de 140.55±8.1bc
Sandy clay
30 1.97±0.1bc 444.10±18.2h 102.15±7.0d
loam
45 1.12±0.11ef 429.82±11.8k 78.94±7.5ef
60 0.60±0.02gh 405.08±8.2ef 78.00±4.5ef
Phosphorus 82.11*** 67.11*** 90.61***
Interaction 5.57*** 2.56* 4.57**
148
sandy loam (38.33) and sandy clay loam (34.73) soil was recorded. Among soils, sandy
loam soil performed significantly better compared to the other two soils at all P levels.
Low P application had a negative impact on the photosynthesis of plants (Table 4.32).
The photosynthetic rate was positively related to P addition. Increased value of P addition
significantly increased the photosynthetic rate. The maximum transpiration rate in loam
(14.4 µmol CO2 m-2 s-1) and sandy loam (15.5 µmol CO2 m-2 s-1) soil was observed at 45
mg P application kg-1 soil and sandy clay loam (11.7 µmol CO2 m-2 s-1) was measured at
0 mg P application kg-1 soil. Lower values in loam (5.22 µmol CO2 m-2 s-1), sandy loam
(5.5 µmol CO2 m-2 s-1) and sandy clay loam (5.5 µmol CO2 m-2 s-1) were obtained at 0 mg
P application kg-1 soil. Plants transpiration rate was statistically variable in all soils. As
plant growth was good in sandy loam soil, the transpiration rate was higher in plants
grown in sandy loam soil as compared to loam and sandy clay loam soil at all P levels.
Transpiration rate was affected detrimentally when the P supply was low (Table 4.32).
Soil type, P application, and their interaction significantly affected the transpiration rate
of the plant (p ≤ 0.05). The decrease in P addition caused a significant reduction in
photosynthesis of plants. At 0 mg P application kg-1 soil, the lowest photosynthetic rate
3.2, 3.5, and 3.1 mm H2O m-2 s-1 in loam, sandy loam, and sandy clay loam was noted,
respectively. At 45 mg P application kg-1 soil, the maximum transpiration rate was 6.4
mm H2O m-2 s-1and 6.5 mm H2O m-2 s-1 in loam, and sandy loam while in sandy clay
loam it was maximum (6.1 mm H2O m-2 s-1) at 60 mg P application kg-1, respectively,
which was greater than all other levels of P. Transpiration rate was significantly variable
in different textured soils. Plant in sandy loam soil performed better as compare to loam
and sandy clay loam soil.
Lower levels of P application impacted plants negatively and caused a reduction in soil
respiration rate (Table 4.32). Phosphorus, soil type and their interaction significantly
affected the respiration rate (p ≤ 0.05). A gradual decrease in respiration was observed
with a decreasing level of P application. Respiration rate was reduced up to 0.25 μmol
CO2 s–1, 0.29 μmol CO2 s–1 and 0.21μmol CO2 s–1 at 0 mg P application Kg-1 of loam,
sandy loam, and sandy clay loam soil, respectively. In loam, sandy loam and sandy clay
loam soil, respiration rate was significantly variable. In sandy loam soil, a higher
respiration rate was found (0.70 μmol CO2 s–1) followed by loam (0.67 μmol CO2 s–1) at
45 mg P kg-1 soil and in sandy clay loam soil (0.58 μmol CO2 s–1) at 60 mg P kg-1 soil.
Overall, the maximum (0.70 μmol CO2 s–1) respiration rate was observed in sandy loam
149
soil at 45 mg P application kg-1 soil while the minimum (0.21 μmol CO2 s–1) was
observed in sandy clay loam soil at 60 mg P application kg-1 soil.
150
Table 4.32. Chlorophyll, Transpiration rate, Photosynthesis rate and Respiration
rate of maize plants in loam, sandy loam and sandy clay loam soils
under different P levels.
Photosynthesi
Transpiration Respiration
Phosphorus Chlorophyll s
Soil Types (mm H2O m-2 s- (μmol CO2
(mg P kg-1) (SPAD value) (µmol CO2 m-2
1) s–1)
s-1)
0 35.93±1.8bc 3.2±0.04n 5.2±0.56ef 0.25±0.01j
15 38.5±1.42b-d 4.5±0.02k 8.5±0.68c-f 0.48±0.001g
Loam 30 38.5±1.43b-d 5.3±0.03h 10.3±1.33b-e 0.56±0.005e

45 44.0±0.83a 6.4±0.03b 14.4±1.75ab 0.67±0.001b
0.59±0.006c
60 44.4±0.50a 5.9±0.03e 13.9±3.99ab
d
38.83±0.31b-
0 3.5±0.02m 5.5±0.66d-f 0.29±0.001i
d
15 40.57±2.42a-c 4.7±0.02j 9.7±1.48b-f 0.46±0.004h
Sandy loam 30 42.86±1.78ab 5.5±0.02g 10.5±1.76a-d 0.54±0.004f
45 44.23±0.43a 6.5±0.02a 15.5±1.85a 0.7±0.005a
60 44.47±0.55a 6±0.05d 14±1.00ab 0.6±0.003c
0 34.73±0.76d 3.1±0.01o 5.1±0.95f 0.21±0.002k
15 36.43±1.96cd 4.4±0.03l 8.4±1.21c-f 0.24±0.01j
Sandy clay 30 40.57±2.41a-c 5.2±0.06i 9.2±0.66b-f 0.46±0.004h
loam
0.55±0.005e
45 42.77±1.59ab 5.7±0.02f 11.1±0.41a-c
f
60 43.57±0.88ab 6.1±0.03c 11.7±1.21a-c 0.58±0.003d
Phosphorus 15.41*** 4902.99*** 14.65*** 3209.54***
F value Soil Types 4.28* 161.94*** 2.05: ns 797.61***
Interaction 0.64ns 3.36** 0.31: ns 84.04***
151
4.3.2.7. Pearson Correlation coefficients between P acquisition parameters, root
characteristics, carboxylate and pH in wheat and maize
The relationship between different P acquisition parameters, decrease in pH and total

carboxylates in wheat and maize are presented in Table 4.33 and Table 4.34, respectively.
In wheat P acquisition parameters except for PUE and average root diameter gave
significant correlation with a decrease in pH and total carboxylates. Negative but
significant correlation of shoot P, root P, and P uptake was recorded with pH and total
carboxylates. RMF was positively and significantly correlated with both pH and total
carboxylates. A positive and significant correlation was also found between pH and total
carboxylates. PUE showed negative and non-significant correlation with shoot P, root P,
P uptake, PAE and RMF and with all other parameters, PUE showed non-significant but
positive relation. Rhizosphere soil available P was a positively and significantly
correlated decrease in pH, non-significantly with total carboxylate while it was negatively
and significantly related to shoot P, root P, and P uptake.
In maize, P acquisition parameters were positively and significantly correlated with a

decrease in pH except for the root and shoot P which was negatively correlated and with
R/S ratio and PUE which were non-significant. The significantly positive correlation of
total carboxylates was found with all acquisition parameters except root and shoot P,
PAE, P uptake, and average root diameter which were negatively significant. A
significant correlation of PUE with RDW, R/S ratio, P uptake, and total carboxylates was
observed. With all other parameters, the relation of PUE was non-significant. The non-
significant but positive correlation of PUE was found with total carboxylates, P uptake,
and RDW. Rhizosphere soil available P was positively and significantly correlated with a
decrease in pH and total carboxylate, but it was negatively and significantly correlated
with root and shoot P.
152
Table 4.33. Pearson Correlation coefficients between P acquisition parameters, Root characteristics, Carboxylate and pH in wheat
RL RDW ARD RSA RT RV RMF R/S PUE PU PAE PPEI pH T.C Shoot Root P
p
RDW 0.7346
**
ARD -0.1076 -0.5056
ns *
RSA 0.7444 0.8570 -0.3633
** *** ns
RT 0.6617 0.8820 -0.7451 0.8063
** *** ** ***
RV 0.7211 0.9104 -0.5489 0.8754 0.8861
** *** * *** ***
RMF 0.6761 0.9715 -0.6268 0.8382 0.9162 0.8818
** *** * *** *** ***
R/S 0.6793 0.9634 -0.6085 0.8343 0.8984 0.8555 0.9982
** *** * *** *** *** ***
PUE 0.1944 0.3597 0.1973 0.3864 0.0830 0.2989 0.2483 0.2630
ns ns ns ns ns ns ns ns
PU -0.6267 -0.9386 0.6556 -0.8422 -0.9116 -0.8490 -0.9841 -0.9880 -0.2432
* *** ** *** *** *** *** *** ns
PAE -0.9318 -0.8844 0.2644 -0.8329 0.8022 -0.8233 -0.8279 -0.8388 -0.2492 -0.8073
*** *** ns *** *** *** *** *** ns ***
PPEI 0.6038 0.9080 -0.5660 0.8495 0.8365 0.8195 0.9476 0.9583 0.4499 -0.9634 -0.7611
* *** * *** *** *** *** *** ns *** **
pH 0.8333 0.7855 -0.3791 0.8114 0.8114 0.8076 0.7609 0.7663 0.2279 -0.7480 -0.7850 0.7433
*** *** ns *** *** *** *** ** ns ** *** **
T.C 0.8660 0.8912 -0.2676 0.9019 0.7614 0.8122 0.8811 0.8863 0.3465 -0.8539 -0.9143 0.8605 0.8226
*** *** ns *** *** *** *** *** ns *** *** *** ***
Shoot -0.6253 0.9421 0.5896 -0.8595 -0.8727 -0.8509 -0.9731 -0.9779 -0.3934 0.9862 0.8044 -0.9899 -0.7380 -0.8685
P * *** * *** *** *** *** *** ns *** *** *** ** ***
Root P -0.6718 0.9205 0.5267 -0.9086 -0.8788 -0.8897 -0.9263 -0.9277 -0.4594 0.9324 0.7924 -0.9638 -0.8517 -0.8778 0.9571
** *** * *** *** *** *** *** ns *** *** *** *** *** ***
Rhizo 2371 0.5509 -0.7432 0.6006 0.7410 0.5648 0.6279 0.6430 0.1916 -0.7011 -0.3345 0.7227 0.6619 0.4352 -0.6894 -0.7446
P ns * ** * ** * * * ns ** ns ** ** ns ** **
Pearson correlation coefficient values are indicated: significant: ∗ significant at P < 0.05; ∗∗ significant at P < 0.01; ∗∗∗ significant at P < 0.001,
ns: not significant. RL: Root length, RDW: Root dry weight, ARD: Average root diameter, RSA: Root surface area, RT: Number of root tips,
RV: Root volume, RMF: Root mass fraction, R/S: Root to shoot ratio, PUE: Phosphorus use efficiency, PU: Phosphorus uptake, PAE:
Phosphorus acquisition efficiency, PPEI: Phosphorus physiological efficiency index, T.C: Total carboxylates, Rhizo P: Rhizosphere soil available
P.
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Table 4.34. Pearson Correlation coefficients between P acquisition parameters, Root characteristics, Carboxylate and pH in maize
Shoot
RL RDW ARD RSA RT RV RMF R/S PUE PU PAE PPEI pH T.C Root p
P
0.8718
RDW ***
-0.7650 -0.7114
ARD *** **
0.8545 0.8322 -0.7114
RSA *** *** **
0.8104 0.7659 -0.7119 0.9456
RT *** *** ** ***
0.8885 0.8710 -0.7702 0.9205 0.9056
RV *** *** *** *** ***
0.7936 0.9642 -0.6866 0.7602 0.6478 0.7805
RMF *** *** ** ** ** ***
0.8071 0.9621 -0.6740 0.7599 0.6390 0.7862 0.9964
R/S *** *** ** ** * *** ***
-0.3305 -0.5899 0.3258 -0.3192 -0.1397 -0.2976 0.7774 -0.7628
PUE ns * ns ns ns ns *** ***
0.6808 -0.8851 0.6959 -0.7997 -0.7092 -0.7920 0.9302 -0.9094 0.7543
PU ** *** ** *** ** *** *** *** **
-0.9262 -0.8479 0.8229 -0.9153 -0.9349 -0.9533 -0.7294 -0.7281 0.1975 0.7296
PAE *** *** *** *** *** *** ** ** ns **
0.767 0.7216 -0.7141 0.9037 0.9361 0.9159 0.5772 0.5788 -0.0220 -0.6518 -0.9345
PPEI *** ** ** *** *** *** * ** ns ** ***
0.6621 0.5975 -0.8011 0.7850 0.7998 0.7744 0.4500 0.4477 0.0552 0.9061 -0.8920 0.8003
pH ** * *** *** *** *** ns ns ns *** *** ***
0.8648 0.8988 -0.849 0.9130 0.8328 0.8847 0.8754 0.8637 0.5451 -0.9061 -0.8920 0.8003 0.7452
T.C *** *** 6*** *** *** *** *** *** * *** *** *** **
Shoot 0.7411 -0.7067 0.7243 -0.8838 -0.9318 -0.9057 -0.5621 -0.5599 0.0162 0.6545 0.9277 -0.9972 -0.8683 -0.7921
P ** ** ** *** *** *** * * ns ** *** *** *** ***
0.7470 -0.7325 0.7256 -0.8638 -0.9099 -0.8965 -0.6217 -0.6121 0.1277 0.7219 0.9324 -0.9593 -0.8146 -0.8179 0.9663
Root p ** ** ** *** *** *** ** * ns ** *** *** *** *** ***
Rhizo 0.9082 0.8866 0.6757 0.9755 0.9025 0.9196 0.8177 0.8236 -0.3755 -0.7953 -0.9095 0.8487 0.7118 0.9033 -0.8240 -0.8101
P *** *** ** *** *** *** *** *** ns *** *** *** ** *** *** ***
Pearson correlation coefficient values are indicated: significant: ∗ significant at P < 0.05; ∗∗ significant at P < 0.01; ∗∗∗ significant at P < 0.001,
ns: not significant. RL: Root length, RDW: Root dry weight, ARD: Average root diameter, RSA: Root surface area, RT: Number of root tips,
RV: Root volume, RMF: Root mass fraction, R/S: Root to shoot ratio, PUE: Phosphorus use efficiency, PU: Phosphorus uptake, PAE:
Phosphorus acquisition efficiency, PPEI: Phosphorus physiological efficiency index, T.C: Total carboxylates, Rhizo P: Rhizosphere soil available
P.
154
4.3.3. Discussion
Plant growth (SFW, SDW, SL) is considered to improve in soils that have higher
nutrients (Kuang et al., 2005; Li et al., 2011). The improvement in growth parameters of
both maize and wheat plants was observed with increasing P availability however plant
growth was greatly affected by soil texture and species types. In sandy loam soil, growth
was better that might be caused by the low P retention ability of this soil. Sandy clay loam
soil retains more P because of clay and calcium content and showed lower growth relative
to loam and sandy loam soil. However, sandy clay loam soil responded better due to the
plant’s ability to adapt to this low P environment (High carboxylate concentration, better
root growth parameters, etc.) (Ahmed et al., 2008). Reduction in SFW due to low P might
have occurred because of the reduction in photosynthesis (Veronica et al., 2017). Another
reason for the reduction in fresh and weight and SL in P deficient environment might be
the translocation of carbon compounds from shoot to roots to compete for low P
availability and hence reduced P uptake and growth (Rasul, 2016). Variation in the
allocation of biomass among plants that are grown with a low and high P supply might be
genetic (Niklas, 1994). Though, there is correspondingly clear mark about P supply that
plays a role in the partitioning of biomass independent of genetics (De Groot et al., 2001).
Our findings are reinforced by the previous discoveries of Chalapathi et al., (1999) who
described an upsurge in the growth of stevia plant in sandy loam soils, as the nutrient
application increased. Similarly, Rasul, (2016) found the highest growth of wheat plant at
250 kg ha-1 application of P2O5. Alam et al., (2005) testified that improving application of
P in the soil enhanced wheat plant height. However, Maistry et al., (2013, 2015a)
indicated that a normal apportionment of capitals to progressions, that are important at
that point in time, enables plants to grow relatively well under low nutrient conditions.
Root parameters (RL, RDW, ARD, RSA, NRT, and RV) are important P acquisition
parameters. Phosphorus deficiency improved the RL gradually from higher to lower
added P in all types of soils. However, the increase in RL was different between different
soil textures at all levels of applied P. The reason for the increased RL might be the low P
availability. When P stress alleviated, it must allow plants to respond to this situation by
increasing their RL (Lyu et al., 2016). To acquire P, plant roots explore more soil volume
under P deficiency (Nuruzzaman et al., 2005; Pearse et al., 2006b; Shen et al., 2011; Liu
et al., 2016). Schroeder and Janos, (2005), Lyu et al., (2016) and Pang et al., (2010) also
testified that an upsurge in RL is an essential response of almost all plant species to P
155
deficiency. Increase in RL strongly correlated with an increase in rhizosphere
acidification (Table 4.33 and 4.34). This is also proved by the findings described by Jing
et al., (2010), who informed that more rhizosphere acidification in calcareous soil and
enhanced root proliferation that ultimately results in increased P uptake and crop growth
by the application of ammonium and phosphates.
Root dry weight showed a negative response with respect to applied P. In our experiment,
RDW increased with decreasing P application in all types of soil. Both species wheat and
maize showed a similar trend of RDW in response to varying P applications. Our
outcomes are in correspondence with Abbas et al., (2016), who stated that root dry matter
of wheat plants in P deficient (-) medium was higher (0.68 g plant-1) than plants grown in
adequate P (0.52 g plant-1).
In this study, Root: shoot increased in both wheat and maize at lower P application rate
because plants under low P distribute low biomass to shoot growth. R/S ratio is positively
related to the root (Table 4.33 and 4.34) and shoots growth so plants that showed high
RDW at low P also showed an increased R/S ratio (Ramaekers et al., 2010). These
outcomes were reinforced by the results of Nuruzzaman et al., (2005); Pearse et al.,
(2006a, 2007) and Calderón-Vázquez et al., (2009). They found that under a lower level
of P application, R/S ratio increased in Triticum aestivum and Zea mays significantly and
Triticum aestivum showed an increase in RL. Balemi, (2009) reported that the R/S ratio at
low P was two-fold higher than at high P.
In this experiment, RMF decreased with increased P application. The decrease in RMF is
correlated to a decrease in RDW (Table 4.33 and 4.34) and a decrease in SDW. With the
increase in RDW, root mass ratio increased. Similar findings were noticed by Pearse et
al., (2006a) when high P (300 µM) reduced root mass ratio in C. arietinum, T. aestivum,
L. culinaris and B. napus.
In P deficient soils, upsurge in soil exploration by better root growth and proliferation
mainly by increasing roots number with smaller root diameter is main acquisition
mechanism (Lynch and Ho, 2005). In this study, for both maize and wheat, surface area,
number, and volume of roots increased at lower P application compared to higher P
application in all soils. These characteristics are positively and significantly correlated
with RL and carboxylates release (Table 4.33 and 4.34). The greater root exploration and
growth may be connected to improved P acquisition. Similarly, Imada et al., (2008)
156
reported that for improving p absorption, root surface area is increased. Gaume et al.,
(2001) and Magalhaes et al., (2011) also informed about a larger root system that helped
the P efficient maize genotype to survive in a low P environment. Haling et al., (2016)
reported marginal acclimation of average root diameter to low P.
Plant P improved with applied P level up to a certain level and decreased after that. Plants
at a higher level of applied P might not acquire a high P proportion because of high
fixation rate. This situation might result in low PUE (P use efficiency), PAE (P
acquisition efficiency) and P Uptake at higher level of P. These findings are in line with
the results of Sultani et al., (2004), Alam et al., (2005), Rahim et al., (2010), and
Maniruzzaman et al., (2017) who reported that plants used little proportion of P at higher
P application due to the high fixation that leads to low PUE and P uptake. Rasul, (2016)
reported low P efficiency at high P application rates in wheat. Marschner, (1995) also
recorded similar results and she found that higher P holding by plant roots at a low level
of P might have increased PUE. Maniruzzaman et al., (2017) stated that P uptake
increased up to normal level and a further increase in P application decreased P uptake in
stevia plant. In this study, P acquisition efficiency in wheat and maize increased with
increasing P level but at highest level of P application, no significant increase was
observed. Maniruzzaman et al., (2017) reported the same results in stevia plant where
PAE improved with P fertilizer addition up to 100 mg P kg-1 and a further increase in P
application decreased PAE. In this experiment, Phosphorus Physiological Efficiency
Index (PPEI) was the highest for 0 mg P and lowest was obtained at the higher level of P
application. PPEI indicates all mechanisms associated with P acquisition, accumulation,
then use of this P for plant growth. PPEI was affected by P. At low P supply, plants
adapted to more physiological mechanisms that result in higher PPEI. Our outcomes are
agreed to results of Ghulam et al., (2005) and Rasul, (2016), who reported a decrease in
PPEI with increasing P fertilizer application rate. Phosphorus stress factor (PSF) is a
suitable indicator of plant tolerance to P stress levels (Gill et al., 2002; Ahmad et al.,
2001). In our experiment, PSF was higher at 0 mg P and it decreased with P levels, but
higher levels of P application also showed an increase in PSF. Remarkable differences in
PSF values at different P levels indicated variability for adaptation to P deficient
environments. Phosphorus levels at which plants showed low PSF values can be suitable
levels for plants in P deficient soils (Shahbaz et al., 2006).
157
Species variation was observed in P content and P uptake and these values were higher in
maize respective to wheat. These results were strengthened by the fact that maize was
better than wheat in most of P acquisition characteristics like root length, root diameter,
volume, surface area, R/S ratio, and carboxylate release. These morphological features of
roots are essential in P uptake and a strong relationship between these root characteristics
and P uptake was found (Table 4.33 and 4.34). These morphological features are related
to the enhanced aptitude of roots to expand in more soil volume and ultimately enhancing
exploring the potential of plant root for P in soil. Similar results were reported by
MacAlister, (2016). Higher leaf P value in Pol. myrtifolia was noticed because of the total
surface area and length of roots, R/S ratio, and citric acid exudation compared to other
species. Species variation in PUE was also reported by MacAlister, (2016). Pod.
calyptrata had two to eight times higher PUE than other species under a P deficient
environment.
In this experiment, variation in the rhizosphere chemistry of plants was found. It was
found that wheat and maize both released higher carboxylate concentration at the lower
level of applied P. With an increasing deficiency of P, the concentration of carboxylate
release was increased. The reason behind the release of higher carboxylates under P
deficiency is the production of carboxylates increased in cells of roots that resulted in the
exudation of these carboxylates to rhizosphere (Neumann et al., 1999). Carboxylate
makes adsorbed P available by replacing phosphate from adsorption sites as bearing more
affinity for these sites (Shane and Lambers, 2005). Similar outcomes were testified by
many researchers. Ryan et al., (2001), Shu et al., (2007) and Yang et al., (2013) reported
the higher concentration of carboxylates released by plant roots to adjust to these low P.
Sas et al., (2001) found carboxylate release (10% malate and 90% citrate) was highly
related to P supply. Hinsinger, (2001) stated that root released carboxylates under P
deficiency that help in P solubilization in low P soil. Many species, such as Fagopyrum
esculentum (buckwheat), Brassica napus (oilseed rape) and various legumes have been
found to be very efficient at utilizing inorganic P because of organic acids release. In a
study by Lipton et al., (1987) on Medicago sativa (alfalfa), it was found that plants
increased their citrate exudation to rhizosphere two-fold because of P deficiency. In a
study by Pang et al., (2010), more organic acids were measured in the rhizosphere of
plants grown under P deficient conditions and citric acid was predominantly exuded,
though the exudation range peaked at about 130 μmol g-1 root DW whereas the plants in
158
this study peaked at over 5000 μmol g-1 root DW at 10 mg P kg-1 sand. This is most likely
due to the different methods used in the collection of organic acids.
It was observed (Table 4.22 and 4.31) that root exudation of carboxylates varied with
plant species. Both wheat and maize showed the ability to release carboxylates under P
deficiency but the quantitative difference in both species was obvious (Figure 4.22).
These outcomes were in accordance with the conclusions of Pearse et al., (2006b). They
described that under a low P environment, many species showed the ability of carboxylate
release. Different species responded differently in the release of carboxylates both
quantitatively and qualitatively under low P availability. As shown in fig 4.22, maize
released more carboxylates than wheat. This dominance of maize in carboxylate release
might have occurred due to differences in their metabolism. Maize having the C4
metabolism released more carboxylates compared to wheat (C3 crop). In a study by
MacAlister, (2016) it was found that Pol. myrtifolia was superior in terms of citrate and
exuded about two folds more citrate than A. linearis and Pod. Calyptrate
Quantitative analysis showed that malate was the dominating carboxylate under P
deficiency. Similar findings were presented by Jones, (1998) and Neumann and Römheld,
(2002). They reported that dominant carboxylate secreted by roots of rape, tomato,
oilseed, maize, and wheat was malate. Our results are contradictory to the finding of
Rocha et al., 2015 who reported considerably higher secretion of citric and oxalic acid in
the roots of P. australis.
Root exudation of carboxylates varied with soil type. Sandy clay loam soil exuded a
higher concentration of carboxylates compared to loam and sandy loam soil although
plants P concentration in soil was comparatively low. This might be because of high clay
and calcium content in sandy clay loam soil. Because these soils adsorb more P that might
have increased the extent of P deficiency compared to other soils and ultimately plant
roots produced and excreted higher concentration of carboxylates.
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Figure 4.22. Total carboxylates (citrate, malate, and oxalate) exuded by wheat and
maize roots
Our outcomes are supported by MacAlister, (2016) who testified high root exudation of
citric, oxalic and succinic acid in soil site (Rhodes Memorial) that have higher calcium
content than the soil site (Silvermine) with the low calcium content.
Qualitative differences in carboxylates were minute across soil types. This was also
proved by MacAlister, (2016). Wouterlood et al., (2004) reported that chickpea in
Naybing soil (Sandy loam) exuded lower concentration of carboxylates than Northam soil
because of high P retention index and higher diffusion due to more sand percentage in
Naybing soil. Veneklaas et al., 2003 testified differences in release of carboxylates by
chickpea, and wheat, grown in six types of soils having the different sorbing capacity.
The rhizosphere pH affects root physiology, rhizosphere micro-organisms and especially

bioavailability of many nutrients (Hinsinger et al., 2003). Results displayed a rhizosphere
pH drop of 0.17 to 4.2 units in wheat as the amount of applied P was decreased while in
maize 0.75 to 4.2 unit reductions in pH was noticed with the gradual decrease in applied P
in all soils. As pH in rhizosphere decreased but it did not go below neutral pH value in
both species. Decline in pH is due to increased excretion of the proton by roots in
association with carboxylates release under P deficiency. A positive correlation between
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pH decrease and total carboxylate release was found (Table 4.33 and 4.34). Similar
findings were described by Hinsinger et al., (2003, 2006) and Rengel and Marschner,
(2005). They suggested that to maintain the charge neutrality, plants release proton in
association with the release of carboxylates. Similarly, Neumann and Römheld, (2002)
and Sas et al., (2001) reported that several plant species, rape, tomato and chickpea roots
released protons under P deficient conditions and acidify their rhizosphere. Shen et al.,
(2005) described that P deficiency in white lupin arouses citrate exudation and proton
release by cluster roots. Li et al., (2010) testified that in calcareous soil, faba bean
released protons and decrease in rhizosphere pH may have enhanced solubility of Ca-P
complexes. Change in pH was variable between wheat and maize. The difference in pH
changes among species was reported in the previous study by Pearse et al., (2006a). Our
outcomes are contradictory with results of Tang et al., (2009), who testified that P
deficiency did not boost the proton extrusion in soybean roots.
Available P in wheat and maize rhizosphere was ranged from 3.5 to 8.5 and 5.1 to 11.2
mg kg−1 respectively. Maize crop showed comparatively more available P than wheat
crop in all three different textured soils. With the increase in P level, rhizosphere soil
available P decreased. At low P, more percent of available P is may be due to enhanced
exudation of carboxylates that may compete for sorption site with soil P. These results
were in accordance with Solaiman et al., (2007), they described that P addition did not
show an increase in rhizosphere available P in wheat and canola. More root length with
increased root surface area may have secreted more carboxylates in roots that resulted in
increased soil available P. This increase in rhizosphere P is positively related to root
length of the plant (Table 4.33 and 4.34). Similarly, Erel et al., (2016) testified that P in
rhizosphere of low added P was high as compared to high P treatment.
When P supply is reduced, reduction in plants, physiological parameters like

photosynthesis, respiration, total chlorophyll, and transpiration was evident in this study.
The reduction in plant photosynthesis might have occurred due to a reduction in
rejuvenation of ribulose-1,5-bisphosphate rather than slowing down the Rubisco
activation phenomena (Rao and Terry, 1989). Another reason for reduced photosynthesis
under low P might be the reduction in triose phosphate transport from chloroplast stroma
to the cytoplasm for Pi exchange (Stitt and Quick 1989). Phosphorus deficiency showed a
reduction in transpiration rate. This reduction may be attributed to stomatal limitations.
Stomata limitations result in reduced substomatal conductance that might have reduced
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the transpiration efficiency. Our findings were in accordance with Veronica et al., (2017),
they testified that at the reproductive and tillering stage the maximum transpiration rate
and photosynthesis were observed in rice genotypes at 45 kg P application ha-1
comparative to 0 kg P ha-1 soil.
The decrease in plant respiration is a clear indication of P deficiency. Reduced respiration

rate might be attributed because of lower transport of electron that ultimately results in
compromised ATP and NADPH production (Lauer et al., 1989). Chlorophyll in plant leaf
of both species wheat and maize decreased as the P application level increased and higher
chlorophyll values were obtained at higher applied P. The results corroborate with the
finding of Alam and Shereen (2002), Biljana and Stojanovic (2005) and Rasul, (2016).
They reported that the chlorophyll contents improved by different levels of P fertilizer
application.
4.3.4. Conclusion
It is concluded from this research work that carboxylates, as considered as the most
imperative adaptations of plants in low P, showed an increase in rhizosphere available P
and it did give an advantage to plants in P acquisition parameters (PUE, PAE, P Uptake,
R/S ratio, RMF, PPEI). It was observed that root morphological traits such as root
volume, RL, RSA, RMF, R/S ratio, number of root tips, average root diameter, and
carboxylate release are principal mechanisms in P acquisition in both wheat and maize,
however, the most efficient species at P uptake, PUE, PPEI, and PAE was maize, most
probably due to its rooting arrangements. Both wheat and maize grow well in sandy loam
soil compared to other soils. Plants in sandy clay loam soil also gave results better than
expected but not better than other soils. For optimized growth, higher P acquisition
characteristics, improved root and shoot P, P application at the rate of 33.75 mg kg-1 of
soil (wheat) and 45 mg kg-1 of soil (maize) performed better and gave results better than
the recommended dose of P for these crops. It is concluded that these levels can be
beneficial from the economic point of view as well as for utilizing plants' ability to
acquire P at low available P in soil and sandy loam soil can be beneficial for both wheat
and maize in acquiring P at low P application.
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4.3.5. General Discussion
The aim of this research was to get information about the adaptation of plants to low P
through physiological mechanisms and improvement in P acquisition parameters (PUE,
PAE, P Uptake, R/S ratio, RMF, PPEI). Three studies were conducted to assess the
genotypic variability in changing physiology (carboxylate release) under low P, role of
carboxylates on P desorption in lab and optimization of P level in order to get optimized
growth through adaptation to low P. Studying the variation in root traits and their
physiological functions with regards to enhanced P acquisition is fundamental in
identifying species in the Pakistani soil as a possible germplasm source with improved P
acquisition and improving the production of economically important species by growing
them at P levels lower than recommended dose.
Carboxylate and Proton release
The carboxylates exudation was affected by genotypes, by P application, soil types and
species as was observed in the solution culture (study-1) and pot experiment (study-3).
Genotypic variability in both wheat and maize in the release of carboxylates, citrate,
malate, and oxalate was observed. Under P deficiency carboxylate release was higher as
compared to normal P. Wheat genotype SARC-1 and maize genotype Pioneer-32F10
performed better and was considered P efficient as they both released more carboxylates
in deficient P compared to normal P (study-1). Maize was superior in terms of
carboxylate exudation over wheat (Figure 4.17). This genotypic variability was also
reported previously (Aulakh et al., 2001; Skene, 2003; Tu et al., 2004; Johansson et al.,
2009; Toyama et al., 2011).
In study-3 where wheat genotype SARC-1 and maize genotype Pioneer-32F10 were
grown in three different textured soils at five P levels, differences in carboxylate release
were also found. Carboxylate release was high at low P compared to a higher level of P
application (Table 4.22 and 4.31). At 0 mg P application, the maximum carboxylate
release was measured in sandy clay loam in both wheat and maize. However, the P
application level of 33.75 and 45 for wheat while 45 and 60 mg P application for maize
showed not a significant difference in release of carboxylates. Reason behind the release
of higher carboxylates under low P is the production of carboxylates increased in cells of
roots that result in increased release of these carboxylates into the rhizosphere (Neumann
163
et al., 1999). Many enzymes of glycolytic pathways are involved in this process, but the
main enzyme play role in the increased production of carboxylates is phosphoenol
pyruvate carboxylase (Hoffland, 1992). Increased activity of this enzyme enhanced
carboxylates (mainly citrate) production but the conversion of these carboxylate to next
products in the TCA (Tri-carboxylic acid) cycle is inhibited due to decreased enzyme
activity in these conversions. Thus an increased accumulation of carboxylates occurred in
cell, that are then released to rhizosphere (Kihara et al., 2003). In a study by Lipton et al.,
(1987) on Medicago sativa (alfalfa), it was found that plants increased their citrate
exudation to rhizosphere up to two-fold because of P deficiency. In a study by Pang et al.,
(2010), it was found that there were more organic acids in plant’s rhizosphere under P
deficient. Carboxylate exudation was higher in sandy clay loam, followed by loam and
then sandy loam soil (Table 4.22 and 4.31). High clay and calcium content in sandy clay
loam soil might be the reason (Table 3.1). Because these soils adsorb more P (Table 4.7)
that might have increased the extent of P adsorption compared to other soils and
ultimately plant roots produced and excreted higher concentration of carboxylates
(Gahoonia and Nielsen, 2003; Lambers et al., 2008). MacAlister, (2016) also reported
variation in carboxylate release within different textured soils.
Another important finding in study-1 and 3 was the difference in the qualitative analysis
of carboxylates. It was observed that malate was the dominant carboxylate released by
both wheat and maize roots. Jones, (1998) and Neumann and Römheld, (2002) also
reported that dominant carboxylate secreted by roots of rape, tomato, oilseed, maize, and
wheat was malate. Our outcomes are opposing the conclusions of Lipton et al., (1987)
and MacAlister, (2016). They reported citrate as a dominant carboxylate released by plant
roots.
Species variation was observed in the release of carboxylates (study-3). Maize was
superior in terms of carboxylate exudation over wheat (Figure 4.17). The dominance of
maize in carboxylate release might be due to differences in their metabolism. Maize
having the C4 metabolism released more carboxylates compared to wheat (C3) crop.
Veneklaas et al., (2003) and Nuruzzaman et al., (2005) testified that composition and rate
at which these are exuded are highly variable and influenced by the species.
The release of the proton is associated with carboxylate release under P deficiency (Table
4.33 and 4.34). In study-1, both wheat (Figure 4.4) and maize (Figure 4.12) genotypes
164
showed a reduction in pH in deficient treatment as compared to normal P treatment.
Genotypes that showed more reduction in pH is considered P efficient genotypes.
In study-3, rhizosphere pH showed a reduction of 0.17 to 4.2 units in wheat as the amount
of applied P was decreased while in maize 0.75 to 4.2 unit reductions in pH was noticed
with the gradual decrease in applied P in all different textured soils. In sandy clay loam,
pH decrease was higher compared to other soils as the carboxylate release was also higher
in this soil. pH in the rhizosphere decreased but not beyond the neutral pH value in both
species. This decrease in pH might be due to increased secretion of the proton by roots in
association with carboxylates release under P deficiency to maintain charge neutrality.
Similar findings were stated by Keerthisinghe et al., (1998) and Neumann et al., (1999).
They reported correspondence of acidification of the rhizosphere by roots with organic
acids, resulting in increased proton release. Similarly, Sas et al., (2001) reported that
several plant species, such as tomato, white lupin, rape, and chickpea possess ability to
release protons under P deficient conditions and acidify their rhizosphere.
Phosphorus adsorption and Rhizosphere available P
Organic acids are highly operative at making chelates not only with calcium but also with
iron and aluminum and therefore displacing inorganic P which then becomes available to
plants (Braum and Helmke, 1995; Jones, 1998; Ryan et al., 2001; Hinsinger, 2001). To
check the effect of carboxylates (organic acids) on adsorption of P in different textured
soils, an incubation study carried out (study-2). All the organic acids (citrate, malate, and
oxalate) reduced the extent of P adsorption. The citric acid (1.0 mM) was more efficient
in reducing the adsorption of P in soil than oxalate and malate.
Rhizosphere soil available P in the pot experiment (study-3) varied between soils and
between species, (Table 4.19, 4.28) showing a positive relationship between exudation
and soil P availability, however, it was not related to applied P levels and root and shoot P
concentration and showed negative relation with it (Table 4.33 and 4.34). At low P, more
available P might be due to enhanced exudation of carboxylates that could have competed
for sorption site with soil P. These are supported with the prior results by Solaiman et al.,
(2007), for wheat and canola, where P addition did not show an increase in rhizosphere
available P.
An important finding from study-3 was that plants were able to grow differentially in
different textured soils and the two species had similar levels of response in three
165
different textured soils. These observations suggested that the most limiting factors for
plant growth in those areas were soil chemical characteristics. Lemaire et al., (2015). In
sandy loam soil, growth was better that might be caused by low P adsorption capacity in
sandy loam soil compared to other soils under study (study-2). Sandy soil with clay had
higher P adsorption capacity because of high clay and calcium and showed lower growth
performance relative to soils with low clay material (study-2). Sandy clay loam soil
responded better than expected even though it had the lowest plant available P. Better
growth might have occurred due to the plant’s ability to adapt to these low P environment
(High carboxylate concentration, better root growth parameters, etc.) (study-3). Higher
carboxylates release in this soil might have released P in soil solution that helped in
improving growth (study-2). In this regard, Maistry et al., (2015a, b) suggested a
balanced allocation of capitals for mineral acquisition in retort to demand as a
physiological mechanism in the plants for growth in the nutrient-poor nutrient soils.
Phosphorus acquisition characteristics
P acquisition by plants varied within genotypes, with soil types, P application levels and
between species (Orians and Milewski, 2007; Lambers et al., 2008). As was observed in
study-1, root and shoot P concentration at deficient P level was lower compared to P
normal level in all genotypes (Figure 4.1, 4.2, 4.9, 4.10). These differences varied from
one genotype to other genotypes. The reason for this behavior is that under low P
condition, plants start using internal P sources to overcome the P deficiency which may
reduce the plant P contents. Phosphorus efficient genotypes SARC-1 and Pioneer-32F10
normally gave the minimum reduction in its root P concentration. This indicates that
SARC-1 and Pioneer-32F10 gathered high shoots P in P deficient medium hence these
were tolerant of low P (Yaseen and Malhi, 2009). These P efficient genotypes also
maintained relatively higher P content in root as compared to shoot so that the root
system can be improved. So, these improved root systems can efficiently uptake nutrients
(Akhtar et al., 2008; Yi-Kai et al., 2013). Haynes et al., (1991) also observed similar
responses. They reported that higher shoot P concentration in P efficient genotypes were
related to improved P uptake efficiency in terms of higher R/S ratio at low P relative to P
sensitive genotypes (Balemi and Schenk, 2009). Gill et al., (2004) studied PUE of wheat
(30 genotypes). Significant differences for PUE and biomass were testified among
genotypes.
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Shoot and root P also affected by applied P levels (study-3). This was proved by the
findings of study-3 where P increased with applied P level up to a certain level and
decreased after that (Table 4.19, 4.28). Plants at a higher level of applied P might not
acquire a high P proportion because of high fixation rate (study-1). This situation might
result in low PUE (P use efficiency), PAE (P acquisition efficiency) and P Uptake at a
higher level of P (study-3). These findings in line with the conclusions of Sultani et al.,
(2004), Alam et al., (2005), Rahim et al., (2010), and Maniruzzaman et al., (2017). They
reported that plants used a little proportion of P at higher P applications due to the high
fixation that leads to low PUE and P uptake. Rasul, (2016) reported low P efficiency at
high P application rates in wheat. Marschner, (1995) also found that higher P holding by
plant roots at a low level of P might have increased PUE. Maniruzzaman et al., (2017)
testified that P uptake increased up to a normal level and a further P application decreased
P uptake in stevia plant.
P acquisition efficiency in wheat and maize improved with increased P level but at the
maximum P level, no significant increase was observed (study-3). Maniruzzaman et al.,
(2017) informed similar outcomes in the stevia plant where PAE augmented with P
fertilizer addition up to 100 mg P kg-1 and a further increase in P application decreased
PAE.
Phosphorus physiological efficiency index (PPEI) indicates all mechanisms related to P

acquisition, accumulation and then utilization of this P for plant growth. PPEI was
affected by the P application. At low P application, PPEI was higher. At low P supply,
plants might have adapted to more physiological mechanisms that result in higher PPEI.
Our results agreed with conclusions of Ghulam et al., (2005) and Rasul, (2016). They
reported a decrease in PPEI with an increased P fertilizer application rate.
Phosphorus stress factor (PSF) is a suitable indicator of plant tolerance to P stress levels
(Gill et al., 2002; Ahmad et al., 2001). In our experiment (study-3), PSF was high at 0 mg
P and it decreased with high P level, but higher levels of P application also showed an
increase in PSF. Remarkable differences in PSF values at different P levels indicated
variability for adaptation to different P deficient environments. Phosphorus levels at
which plants showed low PSF values can be suitable levels for plants in P deficient soils
(Shahbaz et al., 2006).
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Species variation was observed in P content and P uptake and these values were higher in
maize respective to wheat. These results were strengthened by the fact that maize was
better than wheat in most of P acquisition characteristics like root length, root diameter,
volume, surface area, R/S ratio, and carboxylate release. These morphological features
are related to the enhanced aptitude of roots to expand in more soil volume and ultimately
enhancing exploring the potential of plant root for P in soil. Similar results were reported
by MacAlister, (2016). Higher leaf P value in Pol. myrtifolia was noticed because of the
total surface area and length of roots, R/S ratio, and citric acid exudation compared to
other species. Species variation in PUE was also reported by MacAlister, (2016). Pod.
calyptrata had two to eight times higher PUE than other species under a P deficient
environment.
Shoot growth
Shoot growth (SL, SFW, and SDW) is considered to improve in nutrient-rich soils,
especially P (Kuang et al., 2005; Li et al., 2011). The improvement in growth parameters
of both maize and wheat plants was observed with increasing P availability however plant
growth was greatly affected by soil texture and species types as well as genotypes. In
study-1, all the wheat (Table 4.1) and maize (Table 4.4) genotypes showed an increase in
shoot growth in normal P treatment relative to shoot growth in deficient P treatment.
Variation in the allocation of biomass among plants that are grown with a low and high P
supply might be genetic (Niklas, 1994). While in study-3, soil texture affected the shoot
growth. In sandy loam soil, growth was better (Table 4.7 and 4.24) which might be
caused by low P retention ability of this soil (Mihoub et al., 2016a). Reduction in SFW
due to low P might have occurred because of the reduction in photosynthesis (Veronica et
al., 2017). Another reason for the reduction in fresh and dry weight and SL in P deficient
environment might be the translocation of carbon compounds from shoot to roots to
compete for low P availability and hence reduced P uptake and growth (Rasul, 2016).
Though, there is also a clear substantiation that P supply is important in the partitioning of
biomass independent of genetics (De Groot et al., 2001). Our findings are in accordance
with the results of Chalapathi et al., (1999), reported an upsurge in stevia plant growth in
sandy loam soils, as the nutrient application increased.
168
Root characteristics
Phosphorus in available form is mostly strenuous in top layers of soils and plants that
have an abundance of roots in the surface layers (Manske et al., 2000; Lynch and Brown,
2001) as well as the ability to flourish roots growth in nutrient rich areas (Hodge, 2010)
are more effective in P acquisition. In addition to this, the production of dense rooting
systems is an effective means of increasing P acquisition as well as adjusting soils
holding low P. When there is a deficiency of P, plants allocate more photosynthates into
underground plant parts and explore more soil volumes (Lynch et al., 1991). In study-1,
wheat genotypes SARC-1 and maize genotype Pioneer-32F10 showed more percent
increase in RL, RDW in P deficient environment compared to normal P. This can also be
observed in study-3 where a greater root length, higher RDW, higher root surface area
with decreased average root diameter, increased number of root tips, higher root volume
was observed at lower P application compared to higher P application in all soils. These
characteristics are considered the main traits required for a more superior PUE and PPEI
(Walt, 2003), as was observed in both maize and wheat. These characteristics are
positively correlated with carboxylates release (Table 4.33 and 4.34). The greater root
exploration and growth may be connected to improved P acquisition. Haling et al., (2016)
reported marginal acclimation of average root diameter to truncated P. Root hair length,
and RMF ranged from 1.5-2.7 folds between lower to higher P supply (Waddell et al.,
2017). Magalhaes et al., (2011) also reported a larger root system that helped the P
efficient maize genotype to survive in a truncated P environment. Imada et al., (2008)
reported that for improving P absorption, root surface area is increased. Wen et al., (2017)
concluded that root morphological responses showed a growing tendency with declining
shoot P (1.1–1.3 mg g−1) concentration. Maize was superior in all these characteristics
over wheat (study-3). C3 are generally considered to be poorly adapted for acquiring P
from sparingly soluble sources due to their lack of root biomass accumulation (Power et
al., 2010) and this assertion is supported by findings in chapter 1 and 3 where the wheat
had significantly lower R/S ratio and all other root morphological characteristics relative
to maize, possibly associated with plant metabolism (Markham and Zedeveld, 2007).
Future research
Plants tolerant to low P can act as a potentially economical crops for future cropping
schemes. Imminent research activities should focus on identification of P tolerant genes
that intricate in development and improvement of P acquisition characteristics, and on
169
transfer of these types of genes to sensitive species. Potential for developing ‘new’
tolerant species should be studied. It is extremely doubtful that carboxylates released in
the soil remains till the next crop is mounting. this needs further investigation. Another
aspect that needs to be studied is the release of phosphatase enzyme both by
microorganisms and roots and their effect on availability of P to plants.
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CHAPTER 5
SUMMARY
Plants under low P availability have ability to release carboxylates and adapt different
acquisition strategies that help plants to access unavailable P. In order to check the
behavior of maize and wheat plants in releasing carboxylates and improving P acquisition
characteristics at low levels of P application, this research was planned. A series of lab,
hydroponic and pot studies were conducted. The results of the studies are summarized
below
Screening of five wheat (Kohinoor-83, B4-5711, SARC-1, SARC-2, and SARC-3) and
maize genotypes (Cargel-6525, Syngenta-8711, Pioneer-33H15, Monsanto-6525,
Pioneer-32F10) was done on the basis of their potential to produce and release more
carboxylates in P deficient condition in hydroponics. Two treatments, deficient P and
normal P were applied. After harvesting carboxylates were collected and analyzed by
high performance liquid chromatography. Phosphorus concentration in plant shoot and
root and growth-related parameters were measured. Wheat genotype SARC-1 and maize
genotype Pioneer-32F10 were selected as these genotypes showed more potential to
release carboxylates in low P environment as well as higher percent increase in RL, R/S
ratio, PUE and the lowest decrease in SL in P deficient condition compared to normal P
condition.
Then the effect of carboxylates (organic acids) was studied on three different textured
soils (Lab condition). In six P levels, 0, 100, 200, 300, 400 and 500 mg P kg-1, two
concentrations (0.5 mM and 1.0 mM) of all the three carboxylates (citric acid, malic acid,
and oxalic acid) were added in three different textured soils. Samples were incubated at
28±5 oC for a week with two hours daily shaking at 216 rpm. Available and adsorbed P
(equilibrium P) in the filtrate was analyzed. With the increase in added P, a decrease in P
adsorption was observed and the minimum percent P adsorption was observed at 500 mg
P application kg-1. Sandy loam soil showed the lowest P adsorption among all three
different textured soils, followed by loam soil and then sandy clay loam soil. The
decrease in adsorption was observed with carboxylates addition as compared to P
adsorption in the 1st part of the study. 1 mM carboxylate concentration gave better results
compared to 0.5 mM carboxylate application in all soils. Citric acid performed better than
the other two carboxylates and it gave the maximum percent decrease (10.32%) in
171
adsorption @ 1 mM application with 100 mg P application kg-1 in sandy loam soil.
Adsorption data from all three different textured soils at all carboxylate applications were
fitted best to both Freundlich and Langmuir model.
The influence of soil types and deficient levels of P on the release of carboxylates and P
acquisition characteristics in wheat and maize was studied. Selected genotypes (SARC-1
and Pioneer-32F10) from study-1 were grown at five P levels in three different textured
soils. Plants were harvested when they reached the flowering stage and chemical and
growth parameters were measured. Carboxylates were collected at harvesting and
analyzed on HPLC. Lower levels of P application increased carboxylates exudation.
Carboxylate release was the highest in sandy clay loam soil. Malate was the highest
carboxylates released by both wheat and maize plant. Maize plant roots released more
carboxylates than a wheat plant in the rhizosphere. The release of carboxylates also
decreased the pH of the rhizosphere by 1.02 to 4.27 units in wheat and 3.70 to 5.39 units
in maize in all soils. An increase in carboxylate also increased rhizosphere soil available
P (8.5 mg kg-1 for wheat and 11.2 mg kg-1 for maize). Decrease in P application levels
increased P acquisition parameters and the highest values for P uptake (9.21 mg plant-1
for wheat, 53.51 mg plant-1 for maize), PAE (0.047 mg P cm-1 RL for wheat, 0.036 mg P
cm-1 RL for maize), shoot P (1.34 mg g-1for wheat, 1.70 mg g-1 for maize), the lowest R/S
ratio (0.12 for wheat and 0.12 for maize), and minimum RMF (0.106 g g-1for wheat,
0.110 g g-1 for maize) were obtained at P application lower (33.75 mg P application kg-1
for wheat and 45 mg P application kg-1 for maize) than recommended dose. Root
parameters and PUE (5.69 g2 SDM mg-1 P) in wheat and (18.5 g2 SDM mg-1 P) in maize
improved under low P application (0 mg P application kg-1 sandy loam soil). PSF was the
lowest (-11.99% for wheat) at 33.75 mg P application kg-1 in wheat and (-21.68% for
maize) at 45 mg P application kg-1 in maize. With the increase in P application, an
increase in SL, SFW, SDW and root P (0.18 mg g-1 for wheat, 0.91 mg g-1 for maize) was
observed. Plant physiological and root morphological parameters were improved at
decreasing P application.
This research revealed that P application @ 33.75 mg P application kg-1 for wheat and 45
mg P application kg-1 for maize can be better in giving optimized growth and improving P
acquisition characteristics and carboxylate release. The ability of plants to release
carboxylate at lower P can be helpfull in improving the availability of adsorbed P.
172
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CARBOXYLATE RELEASE AND PHOSPHORUS

ACQUISITION IN WHEAT AND MAIZE

INSTITUTE OF SOIL & ENVIRONMENTAL SCIENCES

The Greatest Social Reformer and a Model for Mankind

I am very thankful to Higher Education Commission of Pakistan for providing all

I feel proud to mention the feelings of commitments towards my family and

3.3.1. Pre-sowing soil Analysis 30

3.3.2. Experimental procedure for P adsorption 33

3.3.3. Experimental procedure for carboxylate effects on P adsorption 34

3.3.4. Analysis of adsorption data 34

3.3.5. Statistical Analysis 35

Study 3: Influence of soil types and deficient levels of

3.4.1. Soil preparation 36

3.4.2. Growth conditions 36

3.4.3 Treatment plan for wheat 37

3.4.4. Treatment plan for maize 37

3.4.6. Plant measurement 38

3.4.7. Total chlorophyll content (SPAD value) 38

3.4.8. Physiological parameters 38

Analysis of carboxylates, pH measurement, Plant analysis, and P

3.4.11. P acquisition parameters measurement 38

3.4.12. Root Analysis 39

3.4.13. Statistical analysis 40

Chapter 4 Results and Discussion 41

4.1.1.4. Effects of P on Phosphorus use efficiency (PUE) in various 49

4.1.2.1. Growth responses of maize genotypes 59

4.1.2.2. Root P in various maize genotypes 64

4.1.2.3. Shoot P in various maize genotypes 64

Effects of P on Phosphorus use efficiency (PUE) in various

4.1.2.7 Effects of P on citrate release in various maize genotypes 70

4.1.2.8. Effects of P on oxalate release in various maize genotypes 73

4.1.2.9. Effects of P on malate release in various maize genotypes 73

Study 2: Effect of carboxylates (organic acids) on P adsorption

4.2.1.3.2. Langmuir Equation 87

4.2.2. Effect of carboxylates (Organic acids) on phosphorus adsorption. 92

4.3. Study 3: Influence of soil types and deficient levels of P on the

4.3.1.1. Growth responses 113

2.1. Rhizosphere acidification in different plant species 12

3.1. Physico-chemical properties of soils used in the experiment 32

4.3. Classification of wheat genotypes on the basis of % increase in total 58

4.6. Classification of maize genotypes on the basis of % increase in total 76

4.7. Amount of P in soil solution and P adsorbed 86

4.13. Freundlich adsorption isotherm parameters for P adsorption affected by 98

4.14. Langmuir adsorption isotherm parameters for P adsorption affected by 99

2.2. Mechanism of carboxylate secretion from the root 20

Phosphorus availability due to mobilization by carboxylates and induction of carboxylates

➢ To check the influence of carboxylate on P availability in different types of soils

➢ To study different levels of P application on P acquisition characteristics, plant

Problem statement and thesis outline

2.1. Problem of P availability in Pakistani soils

2.2. Strategies to improve P availability

2.3. Plant adaptation to low P availability

2.3.1. Morphological adaptations of plants

2.3.1.1. Root architecture

Morphological traits of roots are helpful in P acquisition because of the movement of P in

2.3.1.2. Root biomass

In P deprivation, plants adapt to a wide array of physiological strategies to ‘chemically

2.3.2.1. Proton release

An interesting aspect of cluster roots is their capacity to produce immense quantities of