Plant microbial diversity is suggested as the

key to future biocontrol and health trends

Gabriele Berg1,2, Martina Köberl1, Daria Rybakova1, Henry Müller3,

Rita Grosch4 and Kornelia Smalla5
1
Graz University of Technology, Institute of Environmental Biotechnology, Petersgasse 12,
8010 Graz, Austria
2
Austrian Centre of Industrial Biotechnology (ACIB GmbH), Petersgasse 14, 8010 Graz,
Austria
3
BioTenzz, Plüddemanngasse 39, 8010 Graz, Austria
4
Leibniz-Institute of Vegetable and Ornamental Crops (IGZ), Theodor-Echtermeyer-Weg 1,
14979 Großbeeren, Germany
5
Julius Kühn-Institut (JKI), Messeweg 11-12, 38104 Braunschweig, Germany

Correspondence: Gabriele Berg, Graz University of Technology, Institute of Environmental

Biotechnology, Petersgasse 12, 8010 Graz, Austria, Tel.:+43 316 8738310; fax: +43 316
8738819; email: Gabriele Berg@tugraz.at

Submitted to: FEMS Microbiology Ecology

MINI REVIEW

1
Abstract
The microbiome of plants plays a crucial role in both plant and ecosystem health. Rapid
advances in multi-omics tools are dramatically increasing access to the plant microbiome and
consequently to the identification of its links with diseases and to the control of those
diseases. Recent insights reveal a close, often symbiotic relationship between microorganisms
and plants. Microorganisms can stimulate germination and plant growth, prevent diseases, and
promote stress resistance and general fitness. Plants and their associated microorganisms form
a holo-biont and have to be considered as co-evolved species assemblages consisting of
bacterial, archaeal, and diverse eukaryotic species. The beneficial interplay of the host and its
microbiome is responsible for maintaining the health of the holo-biont, while diseases are
often correlated with microbial dysbioses. Microbial diversity was identified as a key factor in
preventing diseases and can be implemented as a biomarker in plant protection strategies.
Targeted and predictive biocontrol approaches are possible by developing microbiome-based
solutions. Moreover, combined breeding and biocontrol strategies maintaining diversity and
ecosystem health are required. The analysis of plant microbiome data has brought about a
paradigm shift in our understanding of its role in health and disease and has substantial
consequences for biocontrol and health issues.

Key words
Plant-associated diversity, plant microbiome, plant pathogens, biocontrol, plant protection,
breeding

2
Introduction

The plant microbiome has been known to be one of the key determinants of plant health and
productivity for more than a century. Lorenz Hiltner began intensive research on this topic as
early as 1901 (Hartmann et al., 2008). Important discoveries relating to plant-associated
microorganisms followed, for example the rhizosphere effect and the importance of
rhizosphere microorganisms in the protection of roots against soil-borne pathogens was
discovered (Cook et al., 1995; Weller et al., 2002) as well as the communication of
microorganisms with their plant host (Ryu et al., 2003; Hartmann & Schikora, 2012). Despite
a taxonomic and functional overlap within the plant microbiota (Bai et al., 2015), distinct
microbiomes were identified for each organ (Vorholt, 2012; Philippot et al., 2013; Hardoim et
al., 2015) and plant species (Berg & Smalla, 2009). The enrichment of microorganisms by the
plant root is not a random, but rather a targeted process. The current model shows the
involvement of seed- and soil-borne microorganisms (Adam et al., 2016a; Johnston-Monje et
al., 2016) and the attraction of microbes to roots by nutrients such as carbohydrates and
amino acids in combination with plant-specific secondary metabolites (Moe, 2013; Weston &
Mathesius, 2013). Differences in plant root exudates play an important role in the functioning
of both chemo-attractants and repellents (Badri & Vivanco, 2009). Plant defense signaling
plays an additional role in these processes (Doornbos et al., 2012). Although the structure of
plant microbiomes is well-studied, there are many knowledge gaps due to the plant species-
specific component and because most of the studies were performed on crops and model
plants like Arabidopsis (Bulgarelli et al., 2012). The gaps are especially related to plants in
natural ecosystems and their relationship to plant health.
Multi-omics technologies allow much deeper insights into the structure of plant-
associated microbial communities, which support and often extend the current body of
knowledge (Berg et al., 2016; Jansson & Baker, 2016). In addition, the tools revealed new
functions of the plant microbiome and interactions within the ecosystem. Altogether the plant
microbiota has profound effects on soil, plant, and agro-ecosystem health (Sessitsch & Mitter,
2015). Moreover, the One Health and EcoHealth concepts, which emphasize a holistic and
interdisciplinary understanding of health, needs plant health involvement and strategies,
which are just in the beginning stages of investigation (Zinsstag et al., 2011). This overview
of the state-of-the-art in current literature in addition to own research insights shows that the
analysis of plant microbiome data has brought about a paradigm shift in our understanding of
its role in health and disease and has substantial consequences for biocontrol and beyond.
3
Novel insights into plant microbiomes

Plant-associated microbial communities and their functions for plant hosts

Microbial diversity and balance is a key for healthy plants (Yan et al., 2017). Traditional
knowledge and recent insights form a clearer picture of the functioning of the plant holo-biont
(Fig. 1). Microbial species have the ability to contribute multiple aspects to the system,
including essential functions such as (i) seed germination and growth support through the
provision of hormones, (ii) nutrient supply, e.g. by fixation of nitrogen, and the mobilization
of phosphorus and minerals such as iron, (iii) resistance against biotic stress factors (pathogen
and parasite defense), (iv) resistance against abiotic factors, and (v) physiology and
production of bioactive metabolites. In detail, already in the first step of a plants’ life cycle,
the plant and especially the seed microbiome are crucial for the host (Truyens et al., 2015).
Seed-associated microorganisms can be essential for the germination procedure in different
plant phyla (Hornschuh et al., 2006; Alavi et al., 2013; Jacquemyn et al., 2015; van der
Heijden et al., 2016). The underlying mechanisms comprise the production of
phytohormones, fixation of nitrogen, and the mobilization of phosphorus and minerals (Tkacz
& Poole, 2015). In these cases of germination support, microorganisms are essential, and this
may be one reason that these keystone microorganisms are vertically transmitted (Bragina et
al., 2012; Truyens et al., 2015). The plant microbiome is involved in resistance against biotic
and abiotic stress factors (Bragina et al., 2013). While the mechanisms involved in pathogen
defense are well-studied (Weller et al., 2002; Berg, 2009; Lugtenberg & Kamilova, 2009); the
phenomenon of abiotic stress protection, for example against high salinities and drought, is
only at the beginning of the investigations (Yang et al., 2009; Zolla et al., 2013; Rolli et al.,
2015). The plant microbiota also has an influence on the physiology and production of
bioactive metabolites (Zabetakis et al., 1999; Verginer et al., 2010). This was also shown for
medicinal plants and their active ingredients (Köberl et al., 2013a; Schmidt et al., 2014).
Interestingly, the rhizosphere microbiome was able to be linked to insect feeding behavior,
which was most probably a result of microbiome-driven changes in the leaf metabolome of
Arabidopsis (Badri et al., 2013). Peñuelas et al. (2014) were able to show that the removal of
the floral microbiome of Sambucus nigra resulted in a reduced floral terpene emission which
plays a key role in pollination and consequently in fruit and seed production. Recent studies
also revealed the direct impact of the root microbiome on plant phenology (Wagner et al.,
2014; Panke-Buisse et al., 2015). Additional essential roles of the plant microbiome for
phenotypic and epigenetic plasticity as well as the evolution of plants were suggested by
4
Partida-Martínez & Heil (2011) and recently evidenced for mycorrhizal fungi (van der
Heijden et al., 2016).

Inter-kingdom signaling and inter-connections of the plant microbiome

The cooperation between plants and millions of microbes as well as in between them requires
an intense communication (Venturi & Keel, 2016). A high number of signatures encoding
interaction via quorum sensing and other signaling molecules were found in plant-associated
microbial metagenomes (Bragina et al., 2015); however the mechanism by which
microorganisms interact as a community to confer beneficial traits to plants is still poorly
understood. Volatile organic compounds are responsible for “microbial small talk” but can
also act as long-distance messengers for interaction with the plant host (Schmidt et al., 2016).
A recent review by Rosier et al. (2016) highlights the importance of chemical signaling, and
biochemical and genetic events which determine the efficacy of benign microbes in
promoting the development of beneficial traits in plants.
Beyond the plant host, the plant microbiome is interconnected with the ecosystem.
Although the plant microbiome is divided into specific compartments linked with specific
microorganisms, it is also connected with the surrounding environment. The rhizosphere
represents the plant-soil interface, from which organic material and signaling compounds
migrate into subsoil zones facilitating long-term mineralization processes as part of the
biogeochemical and ecological disequilibrium (Berendsen et al., 2012; Gocke et al., 2017).
The phyllosphere and all above-ground organs are connected with the atmosphere and are at
the interface for permanent exchange with the air microbiome (Lindow & Brandl, 2003). Both
ectophytic microhabitats – the phyllosphere and rhizosphere – present a higher microbial
diversity than their endophytic counterparts, the endorhiza and endosphere, because resident
and transient microorganisms are co-occurring. Endosphere, carposphere and spermosphere
are colonized by specifically adapted microorganisms representing an intimate interaction;
they present the internal phytomicrobiome (Fig. 2). The sporosphere included the spores of
mosses as well as pollen grains of higher plants, which are morphologically homolog.

The seed microbiome plays a crucial role in plant’s development

The seed microbiome is crucial because it ensures the dissemination and preservation of a co-
evolved and specific microbiome to the next generation. In seeds, most of the microorganisms
are in a dormant stage and are difficult to isolate (Truyens et al., 2015). Through the
application of DNA-based techniques, highly diverse seed microbiomes were shown to exist

5
in many plant species (Adam et al., 2016a; Johnston-Monje et al., 2016; Klaedtke et al.,
2016). There are still only a few plant species being analyzed but it seems that seeds transfer a
core microbiome to the new plantlets similar to all other organisms including mosses (Bragina
et al., 2012) and humans (Blaser, 2014). However, in textbooks and international trade rules
there is evidence that the use of pathogen-free seeds is very important in the production of
healthy crops. A large amount of physical and chemical treatments have been developed to
obtain pathogen-free seeds. These measures can also lead to the general reduction of
microbial diversity and could potentially cause diseases in seedlings due to fact that beneficial
microorganisms supporting germination and growth were also killed. Therefore, we
recommend the evaluation of existing methods by omics technologies. We also believe that a
combination with biologicals (biopesticides, biostimulants, biofertilizers) can improve the
effects of common seed disinfection techniques as suggested by Adam et al. (2016b).

The plant microbiome and agriculture

Nearly half of the world's land is used for agriculture according to FAO (Food and
Agriculture Organization) statistics. All our crops are “domesticated” by breeding and man-
made evolution. Co-evolution between plants and associated microbial communities, known
for wild type species, has already been hypothesized based on culture-dependent results
obtained for the rhizosphere of ancient and modern wheat cultivars (Germida & Siciliano,
2001). Co-evolution was recently shown to be prevalent amongst other plants, e.g. maize,
sugar beet, wheat, pumpkin and lettuce, by application of deep sequencing techniques (Peiffer
et al., 2013; Bulgarelli et al., 2015; Cardinale et al., 2015; Adam et al., 2016a). Crop breeding
was identified as strong driver of natural evolution (Berg et al., 2013). In some cases, the
breeding strategy was targeted at combating pathogens, increasing yield and various growth
characteristics, but historically it was a mainly random selection process for plant phenotypes,
and changes in the plant microbiome were neglected.
In general terms, most of the management strategies and treatments in agriculture have
an impact on microbial diversity and community composition of soils. The concept of the soil
memory effect as a potential mechanism for facilitating sustainable plant growth and
productivity was recently suggested by Lapsansky et al. (2016). Evidence was found in field
studies that organically managed crops enrich their own biocontrol potential (Cooper et al.,
2006; Fließbach et al., 2007; Crowder et al., 2010, Schmid et al., 2011). For example,
statistically significant differences in the structure and function of above-ground grapevine-
associated microorganisms from organically and conventionally managed vineyards were

6
found in a study by Schmid et al. (2011). Aureobasidium pullulans, a copper-detoxifying
fungus and biocontrol agent, played a key role in explaining these differences.

Plant pathogens, disease outbreaks and suppressiveness

What is a plant pathogen?

The presence of microbes in plants became initially noticeable when diseases appeared.
However, the vast majority of microbes in plants are not causal agents of damage in plants
(Mendes et al., 2013). Microbes that are in general able to incite diseases on plant are
collectively called plant pathogens. The most common plant diseases are caused by fungi,
oomycetes, bacteria, viruses, viroids, virus-like organisms, phytoplasmas, nematodes and
parasitic higher plants. In the context of this review, we discuss fungi and bacteria, the largest
groups of plant pathogens, as causal agents of plant diseases. A classical definition of plant
pathogens assumes that in order to fulfill Koch’s postulates, the pathogenic microorganisms
must be characterized by pathogenicity/virulence factors. Pathogenicity is defined as the
ability of a pathogen to infect and cause disease on a particular host plant, and virulence as a
quantitative property is defined as the degree of damage caused to the host (Sacristán &
García-Arenal, 2008). The degree of a disease is assumed to be negatively correlated with the
host fitness (D’Arcy et al., 2001). In addition, plant-fungal pathogen interaction can be race-
specific and race-non-specific. Race-specific interaction is known for a large number of leaf
pathogens such as rust or powdery mildew fungi (Hovmøller et al., 2008), while race-non-
specific interaction is described for various soil-borne pathogens. Fungi enter plants through
natural openings (e.g. stomata), wounds or by direct penetration of plant tissue. In contrast,
bacteria can enter plants only through wounds or natural openings. Diverse pathogens thrive
on plant surfaces, while other pathogens inhabit internal plant tissues. Our present knowledge
on the factors driving the ecology of plant pathogens is still rather limited as plant pathogens
are typically studied mainly on diseased plants. However, there is increasing evidence that
members of the microbiome have an important role in the establishment and development of
plant diseases (Trivedi et al., 2012; Erlacher et al., 2014).
Many plant pathogens are either cultivable or diagnostic tools exist for known, not
cultivable obligate pathogens. However, despite the substantial progress made in cultivation-
dependent methodology (Bai et al., 2015), not all microbes retrieved from natural
environments are cultivable. Hence, aside from obligate parasites, a lot of pathogens are not
cultivable or they might have entered a state called viable but non-cultivable in response to
7
different stresses such as low temperature, salt or UV (Podolich et al., 2015). Here, molecular
markers and cultivation-independent methods become important for their detection. In
addition, there are diseases for which, up until now, no pathogen could be identified or
diseases, for which a microbiome imbalance (dysbiosis) of the soil microbiota is likely to play
a role, e.g. replant diseases (Mazzola & Manici, 2012; Yim et al., 2013).
Pathogens can interact with a wide variety of plants independent of the mode of life
(biotroph, necrotroph; obligate or facultative). The hosts may range from a few to several
hundred plant species. In addition to an endophytic pathogenic lifestyle in a host plant, fungal
pathogens can adopt to a contrasting non-pathogenic lifestyle when growing on various other
non-host plant species (Freeman et al., 2001; Berg et al., 2005; Vilhelmsson et al., 2016). The
fungal plant pathogen Verticillium dahliae was identified as a frequent inhabitant of the
endosphere in many plants (Götz et al., 2006) and used for biological control (Tyvaert et al.,
2014; Postma & Goossen-van de Geijn, 2016). Rhizoctonia is another interesting example; it
can cause serious symptoms in many crops and has a certain degree of host specificity
(Wibberg et al., 2015).

Microbial diversity and disease outbreaks

The ubiquitous occurrence of potential pathogens on plants raises the question as to whether
the outbreak of certain “microbiome diseases” is not always dependent on microbial diversity.
We know that the resistance of the microbial community to the invasion of pathogens is
linked to its level of diversity (Jousset et al., 2011; van Elsas et al., 2012; Mallon et al., 2015)
and “missing microbes” can support disease outbreaks (Blaser, 2014). The microbiome of
each plant forms a network; these networks can be visualized by co-occurrence patterns
(Cardinale et al., 2015). The strength and connection of the network is crucial for the invasion
success of pathogens but also for the establishment of biocontrol agents. Microbial invasions
can cause microbiome shifts, and endophytes can be involved in controlling pathogens in that
same manner (Trivedi et al., 2012; Erlacher et al., 2014; Mercado-Blanco & Lugtenberg,
2014; Podolich et al., 2015). Mendes et al. (2007) observed the suppression of Fusarium
moniliforme on sugarcane by endophytic isolates of the Burkholderia cepacia complex.
Microbes can also inhibit the induction of pathogenic genes by impairing them with quorum
sensing or otherwise the pathogens have to compete with microbes for plant resources
(Friesen et al., 2011). In addition, microbes can directly influence plant defense response by
producing antagonistic molecules within plant tissue or on the surface of the plants (Fravel,
1988). An indirect influence on a plant host’s defensive response level is evidenced either as

8
an alteration in defense pathways or as an improvement of plant vigor (e.g. via the production
of hormones). On the one hand we know that microorganisms can support the pathogens
(Erlacher et al., 2014). These are often members of the Enterobacteriacea family, known for
their degrading capacity of plant tissues. Both responder groups, those who support and
antagonize the pathogen, have to be considered as interesting targets for biocontrol
approaches in the development of plant protection methods.
Interestingly, soil microbiota was identified as causal agent of suppressive soils.
Historically, crop rotation is one of the oldest plant protection methods as cited in biblical
teachings. In recent decades, crop rotations became shorter and in the US there is a long
tradition of monocultures. There is no doubt that crop rotation is a highly efficient method of
controlling many diseases and this can be explained by the great extent of specificity of the
plant microbiome, which enhances the overall microbial diversity in soil. A multitude of
pathogens existing as normal members of these communities have a low outbreak potential. In
monocultures, an increase of pathogen density resulted in severe disease followed by an
increase of antagonistic microorganisms which in a best case scenario cause suppressiveness
(Mazzola, 2004). This phenomenon was intensively studied in wheat monocultures in the
Pacific Northwest (Weller et al., 2002) and was recently defined as soil immune response to
pathogens (Raaijmakers & Mazzola, 2016).
There are important future perspectives for linking plant microbiome and diseases by
monitoring. Microbiome-wide association studies in human medicine revealed complex
interactions among the microbiome and the environment as first keys for developing precision
diagnostics and therapies that are based on the microbiome (Gilbert et al., 2016). This can be
endorsed as a worthy objective not only for the promotion of plant health but also for the
promotion of soil health, which are closely connected.

Microbiome-based solutions for plant protection

Microbiome and biocontrol strategies

Plant diseases are the cause of major economic losses for farmers worldwide. The FAO
estimated that pests and diseases are responsible for about 25% of crop loss (Martinelli et al.,
2014). There are regional differences reported: it is estimated that diseases typically reduce
crop yields by 10% every year in more developed settings, but yield loss due to diseases often
exceeds 20% in less developed areas. Disease control is reasonably successful for most crops
and is achieved by use of plants that have been bred for appropriate resistance against many
9
diseases, by plant cultivation approaches such as crop rotation, use of pathogen-free seeds,
appropriate planting date and plant density, but also by chemical and biological control. Most
of these plant protection methods can influence the plant microbiome and therefore new
insights should be taken into consideration for the improvement of plant protection strategies.
In many cases, diseases are associated with microbiome imbalances or shifts which makes
exploitation of the entire microbiome a desirable objective. Analyzing the plant microbiome
as well as the metabolic interplay with the host plant opens new doors for advanced
biocontrol technologies (Berg, 2015).
New insights reveal an impressive microbial diversity amongst all plants as well as
novel antagonistic microorganisms towards phytopathogens. Despite the fact that plant
microbial diversity depends on many factors, there were some facts reported which should be
considered in future screening strategies. Mosses, the oldest land plants on Earth, are
characterized by a unique microbial diversity (Bragina et al., 2012, 2015) and they harbor an
extraordinary potential of antagonists due to their ecology (Opelt et al., 2007). Furthermore,
medicinal plants have been evaluated as being an interesting source for biodiversity (Köberl
et al., 2013b). This originates from their rich secondary metabolism, which triggers the plant
microbiome composition. Endemic plants were also identified as being unique sources for
biodiversity and antagonists (Zachow et al., 2014). Due to novel insights, we expect that
endophytes, and in particular seed endophytes, can serve as sources for novel biocontrol
agents. Up to now, mainly bacteria and fungi have been exploited for biocontrol purposes.
Archaea were recently identified as plant microbiome members (Müller et al., 2015); their
functions on plants and their potential for biocontrol are completely unknown.
As mentioned above, all microorganisms associated with plants form a network which
can be influenced by microbial invasion. These network models of soil and plant microbiomes
provide new opportunities for enhancing disease management and can be deciphered for
biocontrol. Poudel et al. (2016) suggested a framework for interpreting microbiome networks,
illustrating how observed network structures can be used to generate testable hypotheses
about candidate microbes affecting plant health. They presented four different types of
network analyses, i) general network analysis to identify candidate taxa for maintaining an
existing microbial community, ii) host-focused analysis to include plant responses, iii)
pathogen-focused analysis to identify taxa with direct or indirect associations with taxa
known a priori as pathogens, and iv) disease-focused analysis to identify taxa associated with
disease. Interestingly, there are a lot of parallels to manage invasive plants by symbiotic
microorganisms (Kowalski et al., 2015).

10
 Most of the potential biocontrol agents were screened by in vitro antagonisms against
the pathogen. Despite of the fact that many of these screenings yielded in successful
biocontrol agents, there is a long and intense debate about this screening assay. One reason is
that many of the efficient plant growth promoting and biocontrol organisms in vivo show no
or only modest in vitro antagonisms (Adesina et al., 2009; Schreiter et al., 2014a). High
throughput ad planta assays were developed, but they also showed disadvantages because of
their artificial character. For example, treatment of oilseed rape seeds with Paenibacillus
showed a negative effect on the plant growth under gnotobiotic conditions, no effect in sterile
soil, but plant growth promotion in natural soil (Rybakova et al., 2016a). Thus, testing of the
potential plant growth promoting and biocontrol strains needs to be done in soils and the
effects of plant species and soil types need to be tested. Schreiter et al. (2014b) could show
that similar genera, among them Pseudomonas, were enriched in the rhizosphere of lettuce
grown under field conditions in three different soil types. This finding might explain that the
soil types did neither influence the rhizo-competence nor the biocontrol activity. Recently,
microbiome shifts were discussed as potential novel mechanisms for biocontrol (Schmid et
al., 2011; Grosch et al., 2012; Erlacher et al., 2014; Schreiter et al., 2014a). The potential
mechanisms of microbiome shifts are not well-studied but it is assumed that they might
include several direct interactions with plant pathogens as well as indirect interactions via the
plant by stimulation of the plant immune system (Berg, 2009; Lugtenberg & Kamilova,
2009). Bacillus amyloliquiefaciens FZB42, which is a commercialized and efficient plant
strengthener is able to enhance overall microbial diversity (Erlacher et al., 2014). Targeting
enhanced diversity as biomarker for screening methods, will be an interesting objective.
The development and application of biological control products is an increasingly
attractive objective world-wide. While single organisms were mainly used in the past, often
correlated with inconsistent effects, it is now possible to develop microbiome-based
biocontrol strategies (Berg et al., 2013). Emmert & Handelsman (1999) recommended Gram-
positive bacteria as a good potential candidate for biocontrol applications. And indeed,
looking on the registered products and those which are in the pipeline, most of the products
are based on Bacillus (e.g. EU pesticides database). There are mainly technological reasons
for the prevalent use of Bacillus as a base product: in comparison to Gram-negative bacteria,
bacilli are able to develop spores to survive unfavorable conditions. These spores are easy to
formulate and have a high shelf life. When we examined the plant-associated microorganisms
which comprise thousands of bacterial species, we came to the realization that only a small
proportion of their taxonomic diversity is currently being exploited for biocontrol purposes.

11
Moreover, Firmicutes (e.g. Bacillus) normally presents a small proportion of the plant
microbiome; Janssen (2006) reported them as being contributors to a mean of only 2% (range
0-8%) in the total bacterial soil community. Exceptions were published by Köberl et al.
(2011) who found up to 37% of Firmicutes (Bacillus and Paenibacillus) in soils of arid
environments. There is still an ongoing debate regarding the mode of action of Bacillus on
plants as well as towards plant pathogens, and although induced resistance and plant growth
hormones are reported to be involved in plant-microbe interaction, it is clear that all Bacillus
and Paenibacillus strains are able to produce a long list of bioactive substances and antibiotics
(Cawoy et al., 2015; Rybakova et al., 2016b). Due to the fact that Bacillus and Paenibacillus
strains and spores are highly persistent in the environment, a reduction of plant-associated
microbial diversity may be an outcome. Therefore, we would again like to emphasize our
demand for more diversity amongst and within biocontrol products. New cultivation strategies
should be developed and applied to exploit the entire antagonistic diversity associated with
plants (Zachow et al., 2013).
Antibiotic resistances are spreading globally, threatening our ability to treat common
infectious diseases, resulting in prolonged illness, disability, and death. Unfortunately,
agricultural practices such as the use of antibiotics in the livestock industry lead to increased
levels of resistance (Ferber, 2002; Jechalke et al., 2014). The spreading of manure can
transfer resistances into plant production systems; due to their bioactive secondary
metabolites plants often enriched resistant bacteria in their rhizosphere (Berg et al., 2005).
Although antibiotic resistance is ancient and widespread in environmental bacteria, risk
assessment studies to minimize the emergence and impact of resistance for the whole cycles
are needed. Although many biocontrol agents showed a low persistence on plants and in soil
(Scherwinski et al., 2008), the massive application of spore-forming bacteria could change
this picture. Moreover, as most soil bacteria, bacterial antagonists in general, and
Paenibacillus in particular, contain not only antibiotic production genes but show also high
numbers of antibiotic resistance genes resulting from their ecological background and their
antagonistic function within the microbiome. The potential mobilization of these resistance
genes via different routes of horizontal gene transfer needs further investigation.

Future health trends

Targeted microbiome engineering for crops is a future trend. Biodiversity should be a
biomarker for these microbiome modulations. Higher plant-associated diversity can be
achieved not only through the implementation of biological control agents which shifts the

12
microbiome (Erlacher et al., 2014), but also by the application of microbial consortia. In this
context, crop-specific biological consortia can be taken together from a pool of selected
biocontrol agents. Unfortunately, the application of several microbial strains for biocontrol
reasons is currently limited in the European Union by the necessity of registering each of the
strains, which is a very long and expensive process.
David et al. (2014) showed that food-borne microbes from diet are able to survive and
transiently colonized the gut. The plant-associated microbial diversity can be transferred to
the gut microbiome because fruits and vegetables are the major component of a healthy diet.
However, outbreaks are due to alterations of the gut microbial diversity and are associated
with chronic disease, e.g. allergies, obesity, mental diseases (Gilbert et al., 2016). Increasing
chronic diseases in children can be explained by the “missing microbe theory” published by
Blaser (2014). Microbial diversity is already established as a key factor in preventing human
diseases and should be implemented in plant protection measures.

Concluding remarks
Continued advances in phytopathology and microbial ecology are needed to improve disease
control, and to keep pace with changes in disease pressure caused by the ongoing evolution
and movement of plant pathogens and changes in agricultural practices. In order to effectively
address this issue, all crop plants have to be recognized as holo-bionts. Plant-associated
microbial diversity is a crucial factor for plant health. Although the task remains for us to
clarify the question of what specifically constitutes a healthy microbiome, microbial diversity
can be used as a biomarker for healthy plant microbiomes, e.g. in breeding and biocontrol
strategies. Targeted and predictive biocontrol approaches are possible by taking a holistic
approach and integrating microbiome-based solutions. Moreover, combined breeding and
biocontrol strategies maintaining diversity and ecosystem health are required. These systemic
approaches are required in order to avoid further losses of our biodiversity and to ensure
sustainable agricultural practices.

Funding
This work was supported by the EU-Egypt Innovation Fund [RDI ENPI/2014/342-707] and
by the European Union in frame of FP7-KBBE-2013-7-single-stage [BIOCOMES; No.
612713]. The cooperation of G.B. was funded by a project in the Austrian Centre of Industrial
Biotechnology, which has been supported by the Austrian BMWFW, BMVIT, SFG,

13
Standortagentur Tirol and ZIT through the Austrian FFG-COMET-Funding Program. M.K.
acknowledges support by the Austrian Science Fund FWF [J 3638, T 847].

Acknowledgements
We would like to thank Timothy Mark (Graz) and Elisabeth Jungkurth (Braunschweig) for
English revision.
Conflict of interest. None declared.

References
Adam E, Bernhart M, Müller H, et al. The Cucurbita pepo seed microbiome: genotype-
specific composition and implications for breeding. Plant Soil 2016a, DOI:
10.1007/s11104-016-3113-9.
Adam E, Groenenboom AE, Kurm V, et al. Controlling the microbiome: microhabitat
adjustments for successful biocontrol strategies in soil and human gut. Front Microbiol
2016b;7:1079.
Adesina MF, Grosch R, Lembke A, et al. In vitro antagonists of Rhizoctonia solani tested on
lettuce: rhizosphere competence, biocontrol efficiency and rhizosphere microbial
community response. FEMS Microbiol Ecol 2009;69:62–74.
Alavi P, Starcher MR, Zachow C, et al. Root-microbe systems: the effect and mode of
interaction of Stress Protecting Agent (SPA) Stenotrophomonas rhizophila
DSM14405(T.). Front Plant Sci 2013;4:141.
Badri DV, Vivanco JM. Regulation and function of root exudates. Plant Cell Environ
2009;32:666–81.
Badri DV, Zolla G, Bakker MG, et al. Potential impact of soil microbiomes on the leaf
metabolome and on herbivore feeding behavior. New Phytol 2013;198:264–73.
Bai Y, Müller DB, Srinivas G, et al. Functional overlap of the Arabidopsis leaf and root
microbiota. Nature 2015;528:364–9.
Berendsen RL, Pieterse CM, Bakker PA. The rhizosphere microbiome and plant health.
Trends Plant Sci 2012;17:478–86.
Berg G. Plant-microbe interactions promoting plant growth and health: perspectives for
controlled use of microorganisms in agriculture. Appl Microbiol Biotechnol
2009;84:11–8.
Berg G. Beyond borders: investigating microbiome interactivity and diversity for advanced
biocontrol technologies. Microb Biotechnol 2015;8:5–7.
14
Berg G, Smalla K. Plant species and soil type cooperatively shape the structure and function
of microbial communities in the rhizosphere. FEMS Microbiol Ecol 2009;68:1–13.
Berg G, Eberl L, Hartmann A. The rhizosphere as a reservoir for opportunistic human
pathogenic bacteria. Environ Microbiol 2005;7:1673–85.
Berg G, Zachow C, Müller H, et al. Next-generation bio-products sowing the seeds of success
for sustainable agriculture. Agronomy 2013;3:648–56.
Berg G, Rybakova D, Grube M, et al. The plant microbiome explored: implications for
experimental botany. J Exp Bot 2016;67:995–1002.
Blaser M. Missing Microbes. London: Oneworld Publications, 2014.
Bragina A, Berg C, Cardinale M, et al. Sphagnum mosses harbour highly specific bacterial
diversity during their whole lifecycle. ISME J 2012;6:802–13.
Bragina A, Cardinale M, Berg C, et al. Vertical transmission explains the specific
Burkholderia pattern in Sphagnum mosses at multi-geographic scale. Front Microbiol
2013;4:394.
Bragina A, Berg C, Berg G. The core microbiome bonds the Alpine bog vegetation to a
transkingdom metacommunity. Mol Ecol 2015;24:4795–807.
Bulgarelli D, Rott M, Schlaeppi K, et al. Revealing structure and assembly cues for
Arabidopsis root-inhabiting bacterial microbiota. Nature 2012;488:91–5.
Bulgarelli D, Garrido-Oter R, Münch PC, et al. Structure and function of the bacterial root
microbiota in wild and domesticated barley. Cell Host Microbe 2015;17:392–403.
Cardinale M, Grube M, Erlacher A, et al. Bacterial networks and co-occurrence relationships
in the lettuce root microbiota. Environ Microbiol 2015;17:239–52.
Cawoy H, Debois D, Franzil L, et al. Lipopeptides as main ingredients for inhibition of fungal
phytopathogens by Bacillus subtilis/amyloliquefaciens. Microb Biotechnol 2015;8:281–
95.
Cook RJ, Thomashow LS, Weller DM, et al. Molecular mechanisms of defense by
rhizobacteria against root disease. Proc Natl Acad Sci USA 1995;92:4197–201.
Cooper JM, Schmidt CS, Wilkinson A, et al. Effect of organic, 'low-input' and conventional
production systems on disease incidence and severity in winter wheat. Asp Appl Biol
2006;80:121–6.
Crowder DW, Northfield TD, Strand MR, et al. Organic agriculture promotes evenness and
natural pest control. Nature 2010;466:109–13.
D’Arcy CJ, Eastburn DM, Schumann GL. Illustrated glossary of plant pathology. Plant
Health Instr 2001, DOI: 10.1094/phi-i-2001-0219-01.
15
David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human
gut microbiome. Nature 2014;505:559–63.
Doornbos RF, Van Loon LC, Bakker PA. Impact of root exudates and plant defense signaling
on bacterial communities in the rhizosphere. A review. Agron Sustain Dev
2012;32:227–43.
Emmert EA, Handelsman J. Biocontrol of plant disease: a (Gram-)positive perspective. FEMS
Microbiol Lett 1999;171:1–9.
Erlacher A, Cardinale M, Grosch R, et al. The impact of the pathogen Rhizoctonia solani and
its beneficial counterpart Bacillus amyloliquefaciens on the indigenous lettuce
microbiome. Front Microbiol 2014;5:175.
Ferber D. Antibiotic resistance. Livestock feed ban preserves drugs' power. Science
2002;295:27–8.
Fließbach A, Oberholzer HR, Gunst L, et al. Soil organic matter and biological soil quality
indicators after 21 years of organic and conventional farming. Agric Ecosyst Environ
2007;118:273–84.
Fravel DR. Role of antibiosis in the biocontrol of plant diseases. Annu Rev Phytopathol
1988;26:75–91.
Freeman S, Horowitz S, Sharon A. Pathogenic and nonpathogenic lifestyles in Colletotrichum
acutatum from strawberry and other plants. Phytopathology 2001;91:986–92.
Friesen ML, Porter SS, Stark SC, et al. Microbially mediated plant functional traits. Annu Rev
Ecol Evol Syst 2011;42:23–46.
Germida J, Siciliano S. Taxonomic diversity of bacteria associated with the roots of modern,
recent and ancient wheat cultivars. Biol Fertil Soils 2001;33:410–5.
Gilbert JA, Quinn RA, Debelius J, et al. Microbiome-wide association studies link dynamic
microbial consortia to disease. Nature 2016;535:94–103.
Gocke MI, Huguet A, Derenne S, et al. Disentangling interactions between microbial
communities and roots in deep subsoil. Sci Total Environ 2017;575:135–45.
Götz M, Nirenberg H, Krause S, et al. Fungal endophytes in potato roots studied by
traditional isolation and cultivation-independent DNA-based methods. FEMS Microbiol
Ecol 2006;58:404–13.
Grosch R, Dealtry S, Schreiter S, et al. Biocontrol of Rhizoctonia solani: complex interaction
of biocontrol strains, pathogen and indigenous microbial community in the rhizosphere
of lettuce shown by molecular methods. Plant Soil 2012;361:343–357.

16
Hardoim PR, van Overbeek LS, Berg G, et al. The hidden world within plants: ecological and
evolutionary considerations for defining functioning of microbial endophytes. Microbiol
Mol Biol Rev 2015;79:293–320.
Hartmann A, Schikora A. Quorum sensing of bacteria and trans-kingdom interactions of N-
acyl homoserine lactones with eukaryotes. J Chem Ecol 2012;38:704–13.
Hartmann A, Rothballer M, Schmid M. Lorenz Hiltner, a pioneer in rhizosphere microbial
ecology and soil bacteriology research. Plant Soil 2008;312:7–14.
Hornschuh M, Grotha R, Kutschera U. Moss-associated methylobacteria as phytosymbionts:
an experimental study. Naturwissenschaften 2006;93:480–6.
Hovmøller MS, Yahyaoui AH, Milus EA, Justesen AF. Rapid global spread of two aggressive
strains of a wheat rust fungus. Mol Ecol 2008;17:3818–26.
Jacquemyn H, Waud M, Merckx VS, et al. Mycorrhizal diversity, seed germination and long-
term changes in population size across nine populations of the terrestrial orchid Neottia
ovata. Mol Ecol 2015;24:3269–80.
Janssen PH. Identifying the dominant soil bacterial taxa in libraries of 16S rRNA and 16S
rRNA genes. Appl Environ Microbiol 2006;72:1719–28.
Jansson JK, Baker ES. A multi-omic future for microbiome studies. Nat Microbiol
2016;26:16049.
Jechalke S, Heuer H, Siemens J, et al. Fate and effects of veterinary antibiotics in soil. Trends
Microbiol 2014;22:536–45.
Johnston-Monje D, Lundberg DS, Lazarovits G, et al. Bacterial populations in juvenile maize
rhizospheres originate from both seed and soil. Plant Soil 2016;405:337–55.
Jousset A, Schulz W, Scheu S, et al. Intraspecific genotypic richness and relatedness predict
the invasibility of microbial communities. ISME J 2011;5:1108–14.
Klaedtke S, Jacques MA, Raggi L, et al. Terroir is a key driver of seed-associated microbial
assemblages. Environ Microbiol 2016;18:1792–804.
Köberl M, Müller H, Ramadan EM, et al. Desert farming benefits from microbial potential in
arid soils and promotes diversity and plant health. PLoS One 2011;6:e24452.
Köberl M, Schmidt R, Ramadan EM, et al. The microbiome of medicinal plants: diversity and
importance for plant growth, quality and health. Front Microbiol 2013a;4:400.
Köberl M, Ramadan EM, Adam M, et al. Bacillus and Streptomyces were selected as broad-
spectrum antagonists against soilborne pathogens from arid areas in Egypt. FEMS
Microbiol Lett 2013b;342:168–78.

17
Kowalski KP, Bacon C, Bickford W, et al. Advancing the science of microbial symbiosis to
support invasive species management: a case study on Phragmites in the Great Lakes.
Front Microbiol 2015;6:95.
Lapsansky ER, Milroy AM, Andales MJ, et al. Soil memory as a potential mechanism for
encouraging sustainable plant health and productivity. Curr Opin Biotechnol
2016;38:137–42.
Lindow SE, Brandl MT. Microbiology of the phyllosphere. Appl Environ Microbiol
2003;69:1875–83.
Lugtenberg B, Kamilova F. Plant-growth-promoting rhizobacteria. Annu Rev Microbiol
2009;63:541–56.
Mallon CA, Elsas JD, Salles JF. Microbial invasions: the process, patterns, and mechanisms.
Trends Microbiol 2015;23:719–29.
Martinelli F, Scalenghe R, Davino S, et al. Advanced methods of plant disease detection. A
review. Agron Sustain Dev 2014;35:1–25.
Mazzola M. Assessment and management of soil microbial community structure for disease
suppression. Annu Rev Phytopathol 2004;42:35–59.
Mazzola M, Manici LM. Apple replant disease: role of microbial ecology in cause and
control. Annu Rev Phytopathol 2012;50:45–65.
Mendes R, Pizzirani-Kleiner AA, Araujo WL, et al. Diversity of cultivated endophytic
bacteria from sugarcane: genetic and biochemical characterization of Burkholderia
cepacia complex isolates. Appl Environ Microbiol 2007;73:7259–67.
Mendes R, Garbeva P, Raaijmakers JM. The rhizosphere microbiome: significance of plant
beneficial, plant pathogenic, and human pathogenic microorganisms. FEMS Microbiol
Rev 2013;37:634–63.
Mercado-Blanco J, Lugtenberg B. Biotechnological applications of bacterial endophytes.
Curr Biotechnol 2014;3:60–75.
Moe LA. Amino acids in the rhizosphere: from plants to microbes. Am J Bot 2013;100:1692–
705.
Müller H, Berg C, Landa BB, et al. Plant genotype-specific archaeal and bacterial endophytes
but similar Bacillus antagonists colonize Mediterranean olive trees. Front Microbiol
2015;6:138.
Opelt K, Chobot V, Hadacek F, et al. Investigations of the structure and function of bacterial
communities associated with Sphagnum mosses. Environ Microbiol 2007;9:2795–809.

18
Panke-Buisse K, Poole AC, Goodrich JK, et al. Selection on soil microbiomes reveals
reproducible impacts on plant function. ISME J 2015;9:980–9.
Partida-Martínez LP, Heil M. The microbe-free plant: fact or artifact? Front Plant Sci
2011;2:100.
Peiffer JA, Spor A, Koren O, et al. Diversity and heritability of the maize rhizosphere
microbiome under field conditions. Proc Natl Acad Sci USA 2013;110:6548–53.
Peñuelas J, Farré-Armengol G, Llusia J, et al. Removal of floral microbiota reduces floral
terpene emissions. Sci Rep 2014;4:6727.
Philippot L, Raaijmakers JM, Lemanceau P, et al. Going back to the roots: the microbial
ecology of the rhizosphere. Nat Rev Microbiol 2013;11:789–99.
Podolich O, Ardanov P, Zaets I, et al. Reviving of the endophytic bacterial community as a
putative mechanism of plant resistance. Plant Soil 2015;388:367–77.
Postma J, Goossen-van de Geijn H. Twenty-four years of Dutch Trig® application to control
Dutch elm disease. BioControl 2016;61: 305–12.
Poudel R, Jumpponen A, Schlatter DC, et al. Microbiome networks: a systems framework for
identifying candidate microbial assemblages for disease management. Phytopathology
2016;106:1083–96.
Raaijmakers JM, Mazzola M. Ecology. Soil immune responses. Science 2016;352:1392–3.
Rolli E, Marasco R, Vigani G, et al. Improved plant resistance to drought is promoted by the
root-associated microbiome as a water stress-dependent trait. Environ Microbiol
2015;17:316–31.
Rosier A, Bishnoi U, Lakshmanan V, et al. A perspective on inter-kingdom signaling in plant-
beneficial microbe interactions. Plant Mol Biol 2016;90:537–48.
Rybakova D, Schmuck M, Wetzlinger U, et al. Kill or cure? The interaction between
endophytic Paenibacillus and Serratia strains and the host plant is shaped by plant
growth conditions. Plant Soil 2016a;405:65–79.
Rybakova D, Cernava T, Köberl M, et al. Endophytes-assisted biocontrol: novel insights in
ecology and the mode of action of Paenibacillus. Plant Soil 2016b;405:125–40.
Ryu CM, Farag MA, Hu CH, et al. Bacterial volatiles promote growth in Arabidopsis. Proc
Natl Acad Sci USA 2003;100:4927–32.
Sacristán S, García-Arenal F. The evolution of virulence and pathogenicity in plant pathogen
populations. Mol Plant Pathol 2008;9:369–84.

19
Scherwinski K, Grosch R, Berg G. Effect of bacterial antagonists on lettuce: active biocontrol
of Rhizoctonia solani and negligible, short-term effects on nontarget microorganisms.
FEMS Microbiol Ecol 2008;64:106–16.
Schmid F, Moser G, Müller H, et al. Functional and structural microbial diversity in organic
and conventional viticulture: organic farming benefits natural biocontrol agents. Appl
Environ Microbiol 2011;77:2188–91.
Schmidt R, Köberl M, Mostafa A, et al. Effects of bacterial inoculants on the indigenous
microbiome and secondary metabolites of chamomile plants. Front Microbiol
2014;5:64.
Schmidt R, Etalo DW, de Jager V, et al. Microbial small talk: volatiles in fungal-bacterial
interactions. Front Microbiol 2016;6:1495.
Schreiter S, Ding GC, Grosch R, et at. Soil type-dependent effects of a potential biocontrol
inoculant on indigenous bacterial communities in the rhizosphere of field-grown lettuce.
FEMS Microbiol Ecol 2014a;90:718–30.
Schreiter S, Ding GC, Heuer H, et al. Effect of the soil type on the microbiome in the
rhizosphere of field-grown lettuce. Front Microbiol 2014b;5:144.
Sessitsch A, Mitter B. 21st century agriculture: integration of plant microbiomes for improved
crop production and food security. Microb Biotechnol 2015;8:32–3.
Tkacz A, Poole P. Role of root microbiota in plant productivity. J Exp Bot 2015;66:2167–75.
Trivedi P, He Z, Van Nostrand JD, et al. Huanglongbing alters the structure and functional
diversity of microbial communities associated with citrus rhizosphere. ISME J
2012;6:363–83.
Truyens S, Weyens N, Cuypers A, et al. Bacterial seed endophytes: genera, vertical
transmission and interaction with plants. Environ Microbiol Rep 2015;7:40–50.
Tyvaert L, França SC, Debode J, et al. The endophyte Verticillium Vt305 protects cauliflower
against Verticillium wilt. J Appl Microbiol 2014;116:1563–71.
Van der Heijden MG, de Bruin S, Luckerhoff L, et al. A widespread plant-fungal-bacterial
symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment. ISME J
2016;10:389–99.
Van Elsas JD, Chiurazzi M, Mallon CA, et al. Microbial diversity determines the invasion of
soil by a bacterial pathogen. Proc Natl Acad Sci USA 2012;109:1159–64.
Venturi V, Keel C. Signaling in the rhizosphere. Trends Plant Sci 2016;21:187–98.

20
Verginer M, Siegmund B, Cardinale M, et al. Monitoring the plant epiphyte
Methylobacterium extorquens DSM 21961 by real-time PCR and its influence on the
strawberry flavor. FEMS Microbiol Ecol 2010;74:136–45.
Vilhelmsson O, Sigurbjörnsdóttir MA, Grube M, Höfte M. Are lichens potential natural
reservoirs for plant pathogens? Mol Plant Pathol 2016;17:143–5.
Vorholt JA. Microbial life in the phyllosphere. Nat Rev Microbiol 2012;10:828–40.
Wagner MR, Lundberg DS, Coleman-Derr D, et al. Natural soil microbes alter flowering
phenology and the intensity of selection on flowering time in a wild Arabidopsis
relative. Ecol Lett 2014;17:717–26.
Weller DM, Raaijmakers JM, Gardener BB, et al. Microbial populations responsible for
specific soil suppressiveness to plant pathogens. Annu Rev Phytopathol 2002;40:309–
48.
Weston LA, Mathesius U. Flavonoids: their structure, biosynthesis and role in the
rhizosphere, including allelopathy. J Chem Ecol 2013;39:283–97.
Wibberg D, Rupp O, Blom J, et al. Development of a Rhizoctonia solani AG1-IB specific
gene model enables comparative genome analyses between phytopathogenic R. solani
AG1-IA, AG1-IB, AG3 and AG8 isolates. PLoS One 2015;10:e0144769.
Yan Y, Kuramae EE, de Hollander M, et al. Functional traits dominate the diversity-related
selection of bacterial communities in the rhizosphere. ISME J 2017;11:56–66.
Yang J, Kloepper JW, Ryu CM. Rhizosphere bacteria help plants tolerate abiotic stress.
Trends Plant Sci 2009;14:1–4.
Yim B, Smalla K, Winkelmann T. Evaluation of apple replant problems based on different
soil disinfection treatments – links to soil microbial community structure? Plant Soil
2013;366:617–31.
Zabetakis I, Moutevelis-Minakakis P, Gramshaw JW. The role of 2-hydroxypropanal in the
biosynthesis of 2,5-dimethyl-4-hydroxy-2H-furan-3-one in strawberry (Fragaria ×
ananassa, cv. Elsanta) callus cultures. Food Chem 1999;64:311–4.
Zachow C, Müller H, Tilcher R, et al. Catch the best: novel screening strategy to select stress
protecting agents for crop plants. Agronomy 2013;3:794–815.
Zachow C, Müller H, Tilcher R, et al. Differences between the rhizosphere microbiome of
Beta vulgaris ssp. maritima – ancestor of all beet crops – and modern sugar beets. Front
Microbiol 2014;5:415.
Zinsstag J, Schelling E, Waltner-Toews D, et al. From "one medicine" to "one health" and
systemic approaches to health and well-being. Prev Vet Med 2011;101:148–56.

21
Zolla G, Badri DV, Bakker MG, et al. Soil microbiomes vary in their ability to confer drought
tolerance to Arabidopsis. Appl Soil Ecol 2013;68:1–9.

22
Fig. 1. The interplay between the plant and its microbiome in health and disease. Healthy
plants are associated with their microorganisms by metabolic co-operation and exchange of
signals, hormons and nutrients. Diseases are characterized by a microbial dysbioses and a
response of specific microbes, which can act as antagonists or synergists towards pathogens.

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Fig. 2. The relationship between plant habitats and its interconnection with the
ecosystem.

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