Annals of Applied Biology ISSN 0003-4746

REVIEW ARTICLE

Microbiology is the basis of sustainable agriculture: an opinion

I.A. Tikhonovich & N.A. Provorov

Department of Biotechnology, All-Russia Research Institute for Agricultural Microbiology, St.-Petersburg, Russia

Keywords Abstract
Agricultural microbiology;
plant-growth-promoting rhizobacteria; Agricultural microbiology is presented as a synthetic research field responsible
plant–microbe interactions; sustainable for knowledge transfer from general microbiology and microbial ecology to
agriculture; symbiotic N2 fixation; vesicular the agricultural biotechnologies. The major goal of agricultural microbiology
arbuscular mycorrhiza.
is a comprehensive analysis of symbiotic micro-organisms (bacteria, fungi)
interacting with agriculturally important plants and animals: here we have
Correspondence
Dr N.A. Provorov, All-Russia Research Institute focussed on plants. In plants, interactions with micro-organisms are diverse,
for Agricultural Microbiology, St.-Petersburg, ranging from two-partite symbioses (e.g. legume–rhizobia N2 -fixing nodular
Russia. symbioses or arbuscular mycorrhiza) to multipartite endophytic and epiphytic
Email: [email protected] (root-associated, phyllosphere) communities. Two-partite symbioses provide
the clearest models for addressing genetic cooperation between partners,
Received: 21 October 2010; revised version resulting in the formation of super-organism genetic systems, which are
accepted: 2 May 2011.
responsible for host productivity. Analysis of these systems has now been
doi:10.1111/j.1744-7348.2011.00489.x extended considerably by using the approaches of metagenomics, which allow
the dissection of taxonomic/population structures and the metabolic/ecological
functions of microbial communities, which have resulted from the adaptation
of free-living, soil microflora in the endosymbiotic niches. Both beneficial
(nutritional, defensive, regulatory) and antagonistic (biocontrol) functions
expressed by symbiotic microbes towards their hosts are the potential subjects of
effective agronomic use. A fundamental knowledge of the genetics, molecular
biology, ecology and evolution of symbiotic interactions could enable the
development of microbe-based sustainable agriculture. This could achieve:
(a) an improvement of major adaptive functions and productivity in crop plants
by manipulating their microbial cohabitants; (b) partial or even full substitution
of ecologically hazardous agrochemicals (mineral fertilizers, pesticides) by
microbial preparations; (c) a decrease in the cost and an improvement of the
quality of agricultural products.

Introduction Ruaysoongnern, 2010). It is our view that the most

promising strategy to reach this goal is to substitute haz-
The intensive agricultural technologies, which ensured a ardous agrochemicals (mineral fertilizers, pesticides) with
‘green revolution’ in the middle of 20th century, had an environment-friendly preparations of symbiotic microbes
unexpectedly high ecological cost, contributing to global which could improve the nutrition of crops and livestock,
pollution, unfavourable climate change and loss of biodi- as well as their protection from biotic (pathogens, pests)
versity (Vance, 1998). In response to this, an increasing and abiotic (including pollution and climatic change)
attention has been paid to the development of sustain- stresses (Higa & Parr, 1994; Yang et al., 2009).
able agriculture in which the high productivities of plants Here agricultural microbiology is presented as a
and animals are ensured using their natural adaptive synthetic research field responsible for the transfer of
potentials, with a minimal disturbance of the envi- knowledge from general microbiology and microbial
ronment (Reganold et al., 1990; Vance, 2001; Noble & ecology to the agricultural biotechnologies. We have

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Agricultural microbiology I.A. Tikhonovich & N.A. Provorov

focussed on plants, but should emphasise the importance combined with the systemic regulation of nodule devel-
of micro-organisms in relation to livestock nutrition and opment, which involves the elements common to defence
health (Wang et al., 2009) and to the biocontrol of from pathogens (e.g. salicylic and jasmonic acids) and to
phytophagans (Romeis et al., 2006; Mohammed et al., the regulation of non-symbiotic development (CLAVATA-
2008). Analysis of the regular circulation of micro- like genes responsible for meristem persistence; HAR1 for
organisms between plant-, animal- and soil-borne niches development of lateral roots; Schnabel et al., 2005; Fergu-
is required to reconstruct the arrangement of the son et al., 2010). Importantly, in legumes the systemic sig-
microbiota in natural and agricultural ecosystems (Fig. 1) nal released by emerging nodular primordia is enhanced
and to address the beneficial microbial functions in the by external nitrate supply, leading to the restriction of
terms of nutritional and defensive symbioses (Table 1). nodulation by N fertilizer (Novak, 2010). This restriction
is implemented by signal(s) from leaves (Kinkema et al.,
2006), reflecting a dependency of symbiosis develop-
Microbial nutrition of plants
ment on the rate of photosynthesis. A symbiosis-specific
Micro-organisms form multiple symbioses with plants, catabolism of the photosynthetic products in legume nod-
which cannot obtain a balanced nutrient supply by ules involves a tight interaction of rhizobial bacteroids
themselves: they are subjected to a general metabolic with mitochondria and plastids (Fig. 2), the symbiotically
misbalance obtaining an excess of C but suffer a defi- derived cellular organelles (Margulis & Sagan, 2002).
ciency of other macro-elements, mainly of N and P. The As plants provide on their surfaces, in tissues and
best studied genetic models for nutritional symbioses sometimes in cells, the niches for accommodating their
are two-partite plant–microbe associations, such as the microbial partners, host genes controlling the develop-
N2 -fixing legume–rhizobia nodular symbioses (Franche ment of the symbioses may be considered as the major
et al., 2009). determinants for the beneficial performance of agricultur-
Mutualistic interactions are most effective if based ally important microbes (Fig. 3). The developmental pro-
on positive (mutually stimulatory) metabolic feedback grammes responsible for generating these niches are the
between partners: the intensive flow of symbiotrophi- products of more than 60 million years of co-evolution
cally acquired N or P from nodules or mycorrhizal roots of legumes with rhizobia (Sprent, 2007). This resulted in
stimulate plant photosynthesis, supporting the symbioses the co-evolution of genetic systems between complemen-
development and microbial proliferation (Smith & Read, tary partners encoding for the symbiotic subprogrammes
2008; Novak, 2010). In legumes, these feedbacks are responsible for: (a) infection; (b) nodule histogenesis and
(c) endocytosis of bacteria by the plant cells where the
stable symbiosomes are developed (Oldroyd & Downie,
2008; Den Herder & Parniske, 2009). The first subpro-
gramme was made available by an ancestral vesicular
arbuscular mycorrhiza (VAM) symbiosis, with which the
nodular infection shares many regulatory Sym genes
(Parniske, 2008). The nodule histogenesis programme
was originated de novo in legumes, possibly based
on root storage outgrowth formation (Provorov &
Vorobyov, 2010a).
From the rhizobia side, symbiosis development is acti-
vated by host-specific signals (lipochitooligosaccharide
Nod factors) encoded by several dozens of nod/nol/noe
genes (Franche et al., 2009). Originally, these genes were
considered to be present in all rhizobia species; however,
Figure 1 Circulation of agriculturally important micro-organisms in the it has been recently recognised that there is a diver-
ecosystem. Wide arrows represent the major pathways of microbial sity of infection mechanisms, including the evolutionary
migration (open arrows – transformations of different types of multipartite ‘primitive’ epidermal crack entry of rhizobia and their
microbial communities; solid arrows – specialization of the communities
active uptake by the specially deformed root hairs (Sprent,
into the two-partite symbioses). Dotted arrows represent the hypothetical
pathways of microbial migration and curved arrows represent release
2007). The taxonomic diversity of rhizobia (over 20 dis-
of microbial communities from animals into the soil. The endophytic tinct lineages of α- and β-proteobacteria), which diverged
microflora have been separated into the root/stem (1) and leaf (2) from a common ancestor much earlier than the legume
parts having divergent structures and sources of origin (Rosenblueth hosts originated (Balachandar et al., 2007), suggests that
& Martinez-Romero, 2006; Ryan et al., 2008). some rhizobia may elicit the nodular symbiosis bypassing

156 Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors
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 Table 1 Biodiversity of beneficial plant–microbe symbioses

Microsymbionts Functions Forms of symbiosis Taxonomic groups of microsymbionts Plant hosts Basic mechanisms of interaction

Fungi Nutritional Vesicular arbuscular Glomeromycota (formerly Glomales, 80–90% of land plants Transfer of nutrients (mainly phosphates)
mycorrhiza Zygomycetes), 150–170 species to host via arbuscules – the intrusions
(endomycorrhiza) of hyphae penetrating the root cortical
I.A. Tikhonovich & N.A. Provorov

cells
Ectomycorrhiza Basidiomycota (5000–6000 species) and About 5000 species of dicots and Transfer of nutrients (mainly
Ascomycota (400–500 species) gymnosperms (mainly trees and N-compounds) to host via a system of
shrubs) hyphae colonising the intercellular

Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors

spaces in roots (Hartig net)
Orchid mycorrhiza Different fungi including saprotrophic, Orchidaceae Transfer of nutrients (mainly
ectomycorrhizal and pathogenic ones C-compounds) to host via the hyphae

Annals of Applied Biology © 2011 Association of Applied Biologists

colonising the root cells; induction of
embryogenesis
Defensive Endophytic systems Ascomycota, Clavicipitaceae (Claviceps, Monocots, mainly cereals Syntheses of toxins inhibiting
Epichloë, Neotyphodium) phytophagans (invertebrates or
vertebrates)
Ascomycota (Phialophora, Gliocladium) Synthesis of antibiotics inhibiting the
pathogenic fungi
Epiphytic associations Ascomycota, Clavicipitaceae: Cordyceps Various plants Lethal infections in phytophagous insects
Ascomycota (Trichoderma) Inhibition of pathogenic fungi
Bacteria Nutritional (N2 Root (sometimes stem) Rhizobia (multiple genera of Dicots from 10 Eurosid I families: about Infection of plants via root hairs or
fixation) nodules gram-negative bacteria including 90% genera of Fabaceae and epidermal cracks; development of
Azorhizobium, Bradyrhizobium, Parasponia, Ulmaceae (the hosts for nodules from primordia induced de
Mesorhizobium, Rhizobium, rhizobia); some genera from novo in cortex or in pericycle;
Sinorhizobium) or actinobacteria Betulaceae, Casuarinaceae, penetration of plant cells and
(Frankia) Coriariaceae, Datiscaceae, differentiation of bacteria into N2 -fixing
Elaegnaceae, Myricaceae, non-reproducible cellular forms:
Rhamnaceae, Rosaceae (the hosts for bacteroids in rhizobia, vesicles in
Frankia) Frankia surrounded by the host-made
membranes (formation of
symbiosomes)
Syncyanoses Cyanobacteria (Nostoc, Anabaena)a Angiosperm Gunnera Infection of glands at the bases of leaf
petioles; penetration of the plant cells
Gymnosperms (Cycadales) Colonisation of intercellular spaces in the
cortex of coralloid roots

157
Agricultural microbiology
 158
Agricultural microbiology

Table 1 Continued

Microsymbionts Functions Forms of symbiosis Taxonomic groups of microsymbionts Plant hosts Basic mechanisms of interaction

Aquatic fern Azolla Colonisation of cavities inside the water

leaves; bacteria are transmitted
through the plant generations (via
megaspores)
Endophytic systems Gram-negative bacteria (Azoarcus, Mostly cereals Colonisation of intercellular spaces and
Gluconacetobacter, Herbaspirillum) vascular bundles; N2 fixation may be
implemented by non-reproducible
cellular forms (Azoarcus)
Rhizosphere associations Gram-negative bacteria (Azospirillum, Various plants Colonisation of rhizosphere and
Flavobacterium, Enterobacter) rhizoplane supported by root exudates;
N2 fixation combined with bacterial
synthesis of phytohormones and other
growth stimulatory substances
Defensive Endophytic systems Gram-positive coryneform bacteria, Various plants Synthesis of toxins against phytophagans
Clavibacter (Rathayibacter) toxicus (infection via nematode vectors
Anguina spp.)
Different bacteria (gram-positive and Synthesis of antibiotics against
gram-negative) pathogenic fungi and bacteria
Rhizosphere associations Different bacteria (Agrobacterium Various plants Synthesis of antibiotics against
radiobacter, Bacillus cereus, pathogenic fungi and bacteria;
Pseudomonas chlororaphis, induction of plant defence reactions
P. corrugata, P. fluorescens, P. putida,
Serratia marcescens)

a In Nostoc and Anabaena, N2 fixation is implemented by irreversibly differentiated cellular forms (heterocysts), plant infection – by motile filaments (hormogonia).
I.A. Tikhonovich & N.A. Provorov

Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors

Annals of Applied Biology © 2011 Association of Applied Biologists
I.A. Tikhonovich & N.A. Provorov Agricultural microbiology

formation of super-organism genetic systems (‘sym-

biogenomes’), which could improve the productivity
and adaptive potentials of the partners (Tikhonovich &
Provorov, 2009). Importantly, in plant–microbe sym-
bioses no structural integration of symbiont genes into
the host genome was detected (Raven, 2010), suggest-
ing an adaptive advantage of temporary hosting of the
beneficial microbes over collecting the potentially useful
microbial genes into the plant genome.
An ecologically very important type of nutritional
symbiosis is represented by VAM formed by land plants
with Glomeromycotan fungi (Smith & Read, 2008).
Through this symbiosis, 80–90% of terrestrial plants
obtain a supply of nutrients, mainly of phosphates.
The defence against various root pathogens also has
many similarities and common evolutionary routes with
VAM (Provorov et al., 2002; Parniske, 2008). The genes
Figure 2 Metabolic co-operation of partners’ cells at the late stages encoding for leucine-rich repeat (LRR) protein kinases,
of legume–rhizobia symbiosis (amide nodule type). TCA – tricar- which control either local (within roots) or systemic
boxylic acid cycle; PEP – phosphoenolpyruvate; OA – oxaloacetate; (root↔shoot) signal transduction, are involved in the
PEPC – phosphoenolpyruvate carboxylase; MDH – malate dehydroge- regulation of both nodule and VAM development and
nase; GS – glutamine synthetase; GOGAT – NADH-dependent glutamate
have been the best studied determinants of plant–microbe
synthase; AS – asparagine synthetase; AAT – aspartate aminotransferase.
interactions (Dardick & Ronald, 2006; de Lorenzo et al.,
2009; Lin et al., 2010).
the Nod factor-dependent pathway (Masson-Boivin et al.,
2009). The crack entry of some Bradyrhizobium strains
Multipartite symbiotic communities
into Aeschynomene stems may be induced by cytokinin-
like bacterial signals, whilst Nod factor synthesis is absent Nearly all plants support in their tissues and on
in these bacteria (Giraud et al., 2007). surfaces, multipartite microbial communities utilising
Analysis of the intranodule population dynamics of a broad range of adaptive functions (Clay & Poole,
rhizobia during the symbiosis development suggests 2010). These communities have been the subject of
that co-adaptation of legumes and rhizobia is based molecular research implemented by 16S rDNA-based
on signalling and metabolic feedbacks between partners techniques such as terminal restriction fragment length
(Provorov & Vorobyov, 2009). At the early symbiotic polymorphism (t-RFLP) analysis, denaturing gradient gel
stages (nodule infection and histogenesis) these feedbacks electrophoresis, rDNA cloning and sequencing (Ryan
are mostly negative, as they are dependent upon defence- et al., 2008). In addition, Raman microspectroscopy has
like reactions (Jones et al., 2007). However, at the late been used to link the structure and functions of microbial
stages (endocytosis, metabolic exchange of partners), the communities at the single-cell level (Wagner, 2009).
defence-like responses are superseded by the mutualism- This research has revealed the great taxonomic and
specific mechanisms for bacteria population control functional diversity of endophytic microflora, which is
(Schumpp & Deakin, 2010). They are based on the composed of hundreds of species and may be nearly
feedbacks from the host stimulating the beneficial as diverse as the soil microflora (although they have
activities in positive microbial partners (Provorov & quite different compositions). Only some representatives
Vorobyov, 2009). of endophytic communities have been genetically and
Collectively, these feedbacks elicit symbiosis-stipulated functionally characterised, e.g. the N2 fixer Azoarcus sp.
natural selection pressures, which may be simulated (Krause et al., 2006) and defensive symbiont Clavibacter
using mathematical composition approaches, demon- (Rathayibacter) toxicus (Schmitz et al., 2007), while for the
strating that an increased symbiotic efficiency may result majority of endophytes, adaptive impacts remain obscure.
in improving the integrity of the partners (Provorov & The major functions of endophytic microbes (reviewed
Vorobyov, 2010b). Analysis of the molecular background in Ryan et al., 2008) are biocontrol of pathogens/pests,
for this integrity demonstrates that two-component sym- detoxification of environmental contaminants and per-
bioses such as legume–rhizobia can provide models haps separation of plants from their own metabolic
for investigating genetic interactions resulting in the wastes. N2 fixation as a major host-beneficial function is

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Agricultural microbiology I.A. Tikhonovich & N.A. Provorov

Figure 3 Genetic dissection of the legume developmental programme used for hosting the rhizobia inside cells and tissues in the N2 -fixing nodules.
The central part of arrow represents the infection developmental subprogramme (Pisum sativum – Rhizobium leguminosarum bv. viceae symbiosis)
dissected into the steps: Ngi – nod gene induction; Hac – root hair curling; Iti – infection thread (IT) initiation; Ith – IT growth in the root hair; Itr –
IT development in the root; Itn – IT growth in the juvenile nodule; Idd – infection droplet differentiation; Bad – bacteroid development; Nop – nodule
persistence. Another developmental subprogramme is represented by Nodule tissue development (Ntd) subjected to Autoregulation of nodulation
(AON); the detailed description of these programmes is in Borisov et al. (2003) and Shtark et al. (2010). The plant genes involved in symbiosis are: RH42,
44 – genes encoding for acid proteins synthesised in the curled root hairs; Sym – genes, the mutations of which arrest the respective developmental
steps/subprogrammes; ENOD – genes encoding for early nodulins expressed during development of nodular structures. The late nodulins expressed
together with the nitrogen fixation onset are: Lb – leghaemoglobin; GS – glutamine synthetase; GOGAT – NADH-dependent glutamate synthase; AAT –
aspartate aminotransferase; PEPC – phosphoenolpyruvate carboxylase; MDH – malate dehydrogenase (their operation is presented in Fig. 2). Bacterial
genes expressed at different developmental stages encode for: nodD – transcriptional regulation of the nod/nol/noe genes under impacts of the plant
flavonoids; nodABC – synthesis of core part of Nod factor; hsn – host-specific nodulation (dependent on the chemical modifications of Nod factors); exo,
ndv – synthesis of surface components (exopolysaccharides, cyclic β-glucans) required for the bacterial dialogue with the host defence systems and for
osmotic adaptations; nif, fix – synthesis of nitrogenase and associated enzymes; bacA – bacteroid differentiation.

specific for only some endophytic bacteria species because (b) modulation of their synthesis in plants. The first is
nif genes have only been found in a small fraction of these implemented by Azospirillum spp. when they are resi-
species (Martı́nez et al., 2003; Pedrosa & Elmerich, 2007). dent in the plant rhizosphere or in outer root tissues.
However, the frequency of N2 fixers may be increased Importantly, when these bacteria occupied the para-
markedly in endophytic communities following N star- nodules induced on cereal (wheat, maize) roots by the
vation of the host (Mano & Morisaki, 2008). Several auxin analogue 2,4-D, increases in nitrogenase activ-
other trophic functions of endophytes may be adaptively ity, photosynthetic rate and biomass yield were detected
important, including an uptake of soil N, P and Fe as well (Saikia et al., 2007). The second strategy is represented
as the in planta interconversion and allocation of different by the bacterial synthesis of 1-aminocyclopropane-1-
metabolites (Rosenblueth & Martinez-Romero, 2006). carboxylate (ACC) deaminase catalysing the metabolism
The nutritional and defensive outputs of plant– of ACC, the precursor of plant ethylene which down-
microbial interactions may be improved because of regulates different types of symbiotic interactions (Guinel
plant growth regulation, implemented via modulation & Geil, 2002; Belimov et al., 2009). The abilities to syn-
of the host hormonal status. These impacts are espe- thesise the major phytohormones and to modulate the
cially important under environmental stresses, e.g. soil internal host hormonal synthesis were found in some
drought or acidity (Belimov et al., 2009; Dimkpa et al., plant-associated microbes which probably do not display
2009). Two major strategies may be involved in this any specific nutritional or defensive functions (Dodd et al.,
regulation: (a) bacterial production of phytohormones; 2010). These data may suggest a background to define a

160 Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors
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I.A. Tikhonovich & N.A. Provorov Agricultural microbiology

‘regulatory’ type of plant–microbe symbioses in addition categories – localised and systemic. With a localised type,
to the previously recognised nutritional and defensive plants display a potential to regulate the performance
types (Table 1). of the endosymbiotic cohabitants, via positive and neg-
The mechanisms of formation of the microbial com- ative feedbacks established between the infected and
munities residing in plant tissues are variable. The major non-infected organs. For example, an effective supply
pathway of plant occupation by external microflora is of N and/or P to the shoots leads to a reverse flow of
via epidermal cracks induced by the emergence of lat- C directed to the root portions (nodules; mycorrhizal
eral roots (Yrjälä et al., 2010). It is the underground sections of the cortex) occupied by the microsymbionts
plant organs that are most accessible to the endophytic (Provorov & Vorobyov, 2009). With the multicomponent
microbes, while only some may be able to spread sys- endophytic communities, the regulatory mechanisms are
temically into the shoots (Mano & Morisaki, 2008). For less clear; perhaps they differ greatly from the regula-
bacteria, this spread (which occurs mostly via the vascu- tion of localised symbionts. In managing the systemically
lar bundles) is carried out by various cellular components distributed microbes, an important role may be imple-
including the pili, different types of protein secretion sys- mented by quorum sensing circuits, because a range of
tems and the surface components, mostly exo- and lipo- host-beneficial microbial functions are auto-induced or
polysaccharides (Rosenblueth & Martinez-Romero, 2006; auto-repressed by homoserine lactones. Plants can inter-
Ryan et al., 2008). However, above-ground organs have fere with this regulation by signals imitating the bacterial
their own sources of colonisation, e.g. leaves are often regulators (Sanchez-Contreras et al., 2007).
infected via stomata by epiphytic (phyllosphere) microbes The differences in regulation of local and systemic
which may be soil-, air- or insect-borne (van der Lelie communities are possibly related to the alternative
et al., 2009; Newton et al., 2010). The differences in micro- strategies of their evolution. The phylogenetic studies of
bial composition between below- and above-ground parts Claviceps and Epichloë/Neotyphodium defensive symbionts
are because of: (a) difference in the infection sources, e.g. (Schardl et al., 2004) suggest that an enhancement of
phyllosphere microbes are subjected to stresses induced pathogenic effects (which are more pronounced in the
by UV, drought, lack of nutrients and therefore are less evolutionary advanced Claviceps spp.; White et al., 2002)
diverse than the rhizosphere microbes; (b) differential is correlated with the transitions from systemic to a
multiplication in various organs and tissues; (c) migration localised type of symbiosis. This evolutionary pathway
barriers between different parts of the plant. was supported by the data of Redman et al. (1999), who
Additional mechanisms for establishing endophytic obtained mutations of the root pathogen Colletotrichum
microflora may include: (a) ‘vertical’ (via seeds) trans- magna, which resulted in mutualistic defensive symbionts,
mission of microsymbionts; (b) their introduction via with a relaxation of host control, thus allowing the
soil-born eukaryotic vector organisms (Mano & Morisaki, fungus to colonise the upper parts of stems. Endophytic
2008). The latter is exemplified by the introduction of strains of Fusarium oxysporum in banana plants ensure the
defensive C. (Rathayibacter) toxicus strains via Anguina effective biocontrol of sedentary endoparasitic nematodes
nematodes (Riley & Reardon, 1995; Schmitz et al., 2007) by direct suppression using mycotoxins and indirect
or of N2 -fixing Azoarcus strains via endophytic fungi (Karg suppression via systemic defence reactions (Sikora et al.,
& Reinhold-Hurek, 1996; Hurek et al., 2002; Krause et al., 2008). These examples suggest that some severe and
2006). The availability of mechanisms for a regular re- widespread pathogens of crop plants might have evolved
establishment of plant–microbial systems enables us to from the regular components of endophytic microflora,
address their genetic and adaptive potentials in terms of which operate as beneficial (defensive) symbionts in wild
the microbiome concept, which was initially introduced plants.
to define the integral gene pool of microbiota residing in In contrast to defensive symbioses, for nutritional
the human colon (Lederberg & McCray, 2001). A heuris- symbioses, the localised types are more beneficial for
tic potential of this concept is to represent the symbiotic the host plants. The nodular N2 -fixing symbioses in
microbial communities as the products of genetic inte- dicots (Eurosid I clade) are generally more effective
gration of plant hosts with their microbial allies, which (in terms of plant biomass and N accumulation) than
underlie the structural/functional unity of the partners the endophytic and rhizospheric associations widespread
(Tikhonovich & Provorov, 2009). This integration is most in cereals (Franche et al., 2009). In legumes, the
clearly expressed during the co-evolution of the partners, nodular N2 -fixing symbioses are more effective than
which can be assessed using population genetics and anodular (root endophytic) symbioses, thus steering
molecular phylogeny approaches (Arnold et al., 2010). plant evolution towards complicated nodule structures
Summarising the present knowledge, plant- (Bryan et al., 1996). Moreover, the evolution of legumes
microbial symbioses may be classified into two broad with the more restrictive penetration type, requiring

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Agricultural microbiology I.A. Tikhonovich & N.A. Provorov

transition from the crack entry of rhizobia to their 2008; Hartmann & Bashan, 2009; Dadnia et al., 2010;
active uptake by root hairs induced by the rhizobia- Hungria et al., 2010). Importantly, under stress condi-
made Nod factors (Sprent, 2007), may be correlated tions, rhizosphere communities may undergo a regular
with an improved efficiency of symbiotic N2 fixation. change in spatial structure and functional patterns. For
Specifically, root hair infections may be implemented by instance, roots attacked by fungal pathogens excrete
single cells or microcolonies providing clonal structures increased amounts of exudates, which have concentra-
in the intranodule rhizobia populations. These structures tions of organic acids that are favourable for the formation
can ensure an effective allocation of C-compounds of a defensive Pseudomonas population (Kamilova et al.,
to highly active N2 fixers, resulting in the selection 2006). Analysis of root-associated bacterial communities
of intensive in planta N2 fixation, within rhizobia in barley and oats exposed to the toxic heavy metal Cd
populations (Provorov & Vorobyov, 2010b). suggested that this stress enhances the release of nutrients
It is our view that the well-studied two-partite from roots. This release stimulated bacterial multiplica-
plant–microbe symbioses represent the evolutionary tion in the rhizoplane and subsequent migration to the
descendants of much more complicated multipartite rhizosphere, where a protective barrier against Cd was
communities. For instance, multipartite interactions are formed (Pishchik et al., 2002, 2009). At lower subtoxic
involved in the widely distributed VAM symbiosis, Cd concentrations, this migration may result in increased
because the Glomeromycotan fungi (as well as other plant productivity and bacterial population density.
groups of mycorrhizal fungi) usually colonize the plants
in cooperation with satellite (helper) bacteria. Some of
Ecological, agronomic and biotechnological impacts
the bacteria are located inside hyphae, whilst others are
on their surfaces (Artursson et al., 2006). Some rhizo- The broad application of microbes in sustainable
bia species were perhaps recruited from these satellites, agriculture may be because of the genetically based
suggesting that the nodular symbioses may represent dependency of plants on the beneficial functions provided
evolutionary derivatives of the more ancient multicompo- by symbiotic cohabitants (Douglas, 1994; Seckbach, 2002;
nent (plants + fungi + bacteria) communities (Provorov Noble & Ruaysoongnern, 2010). The agronomic potential
et al., 2010). of plant–microbial symbioses proceeds from the analysis
The epiphytic microbial communities are most numer- of their ecological impacts which have been best studied
ous and functionally diverse in the rhizosphere and for N2 fixing (Franche et al., 2009) and mycorrhizal
rhizoplane. The plant–microbe interactions within the (Smith & Read, 2008) symbioses. This analysis has been
rhizosphere have been widely reviewed (Lugtenberg based on ‘applied co-evolutionary research’ (Thompson,
et al., 2001; Lugtenberg & Kamilova, 2009) and we shall 2005; Arnold et al., 2010), addressing the ecological
not address them in much detail. However, it is necessary and molecular mechanisms for mutual adaptation and
to emphasise that up to now, attention has only been paid parallel speciation of plant and microbial partners. For
to a few microbes, e.g. to N2 -fixing Azospirillum (Franche plant–fungal interactions, it has been demonstrated
et al., 2009; Isawa et al., 2010) and defensive Pseudomonas that the host genotype represents the leading factor in
(Lugtenberg & Kamilova, 2009) species. At present, a the biogeographic distribution of mycobionts and for
more holistic approach to root-associated organisms is their evolution within the mutualist↔antagonist and
being developed assuming that these microbes are the specialist↔generalist continua (Peay et al., 2010). In
components of taxonomically diverse, but functionally mycorrhizal symbioses, this evolution may be elicited
integrated communities, supported not only by the root by an enormous diversity of beneficial interactions, in
exudates but also by interactions with mycorrhizal fungi addition to major nutritional effects. These involve the
(Johansson et al., 2004; Richardson et al., 2009). Because increased resistance of plants to soil acidity (Arriagada
of these mycorrhiza–rhizosphere interactions, bacteria et al., 2007), salinity (Evelin et al., 2009), natural and
are able to withdraw nutritional elements from soil anthropogenic contaminants (Giller et al., 2009; Weyens
organic compounds and primary minerals, thus ensuring et al., 2009), drought, pathogen attacks and other stresses
a high trophic potential of plant-associated communities (Yang et al., 2009; Feddermann et al., 2010).
(Uroz et al., 2009). The multifunctional aspects of mycorrhizal symbioses
A combination of nutrition, defensive and regula- play a key role in the invasions of plants into novel
tory functions may be widespread in the rhizosphere ecological zones including an artificial introduction into
communities, e.g. Azospirillum spp. combine N2 fixa- cropping areas (Pringle et al., 2009). The key adaptive
tion with phytohormone [Indole-3-acetic acid (IAA) (trophic) impacts of mycorrhizae may be related to
synthesis], which provides better root growth and nutri- a more economical use of photosynthesis products
ent assimilation (Kravchenko et al., 1994; Remans et al., for nutrient uptake via mycorrhizal fungal hyphae, as

162 Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors
Annals of Applied Biology © 2011 Association of Applied Biologists
I.A. Tikhonovich & N.A. Provorov Agricultural microbiology

compared to root hair uptake (Rooney et al., 2009). symbioses where the strong correlations between the
Nevertheless, an increased respiration rate in mycorrhizal ecological efficiency of mutualism and its genotypic
roots, as compared to sterile roots, has been demonstrated specificity are evident (Provorov & Vorobyov, 2010b). For
(Hughes et al., 2008). Promising genetic models to study the associations between non-legumes and rhizosphere
the ecological impacts of mycorrhizae are available in bacteria, the ecological efficiency and genotypic specificity
the VAM-defective mutants of leguminous and non- of interactions between partners are poorly correlated
leguminous plants (reviewed in Provorov et al., 2010), (Kozhemyakov et al., 2004). Therefore, improvement of
which may be out-competed by parental genotypes these associations may be achieved by selecting microbial
because of repressed P uptake in the mutants (Facelli strains for inoculation of a wide range of plant genotypes.
et al., 2010). ‘Symbiotic’ selection could be based on a broad
The major impact of agricultural microbiology on sus- involvement of wild plant germplasm, which has
tainable agriculture would be to substitute agrochemicals retained the high adaptive potential of different types
(mineral fertilizers, pesticides) with microbial prepara- of plant–microbial interactions: root nodular symbioses
tions. However, this substitution is usually partial and (Provorov & Tikhonovich, 2003), rhizosphere associations
only sometimes may be complete, e.g. in recently domes- (Kravchenko et al., 1993) and VAM (Jakobi et al., 2000).
ticated leguminous crops, which retain a high potential for Selection for rhizosphere-related traits (Wissuwa et al.,
symbiotrophic N nutrition typical for many wild legumes 2009), e.g. for the amount and composition of root
(Provorov & Tikhonovich, 2003). The application of nutri- exudates (Kamilova et al., 2006), could be implemented
tional symbionts could be based on plant mixotrophy, using a quantitative trait loci (QTL) approach. In Phaseolus
e.g. on a simultaneous symbiotrophic and combined N vulgaris, several QTLs linked with high responsiveness to
nutrition. This is why the maximal productivity of the auxin and Azospirillum applications have been identified
majority of crops is reached using an optimal (species- and (Remans et al., 2008). Breeding legumes for improved
genotype-specific) combination of both nutritional types photosynthetic activity may be useful to utilize the N2 -
because of which a high sustainability of legume produc- fixing potential in super-nodulating genotypes, which
tion may be achieved (Provorov et al., 1998). Moreover, display a decreased productivity because of energy over-
the energy costs for N2 fixation and for assimilation of expenditure for excessive nodulation and nitrogenase
combined N differ by less than 10% (Andrews et al., activities (Novak, 2010).
2009). The balance between symbiotrophic and com- At present, a wide spectrum of preparations of diverse
bined N nutrition may be improved by a rapid removal of microbial species may be used to enhance crop production
N-compounds from the actively N2 -fixing symbioses, as (Smith, 2004; Humphry et al., 2007; Andrews et al.,
has been suggested for tropical forest ecosystems (Hedin 2010). However, different approaches for improving the
et al., 2009). nutritional and defensive types of microbial mutualists
Unfortunately, domestication and selection of plants need to be developed. For the nutritional types, an
have resulted in a loss of their symbiotic activities, effective colonisation of plants in a host-specific manner
e.g. the balance between symbiotrophic and combined is optimal and the impacts of beneficial symbionts are
N nutrition has decreased in many legumes because increased in parallel with their host specificity (Provorov
of their cultivation under high N supply (Provorov & Vorobyov, 2009).
& Tikhonovich, 2003). Moreover, some rhizobia have For improving the defensive symbiotic types, direct
evolved from beneficial (N2 -fixing) symbionts of legumes and indirect suppression of plant pathogens and pests
into non-N2 -fixing ‘cheaters’ (Provorov & Vorobyov, could be combined. This combination could be especially
2009). A loss of rhizosphere-related traits involving important with severe plant infections such as the take-
microbe-facilitated uptake of nutrients has been suggested all disease in wheat (caused by Gaeumannomyces graminis
for a range of non-legumes (Kravchenko et al., 1993; var. tritici), which is suppressed by a multicomponent
Wissuwa et al., 2009). Some defensive symbionts may community dominated by acidobacteria, Planctomycetes,
evolve into severe plant pathogens, e.g. the deleterious Nitrospira, Chloroflexi, Azospirillum and Thermoanaerobacter
F. oxysporum races represent the descendants of defensive (Sanguin et al., 2009). The specificity of defensive
endophytes (Sikora et al., 2008). microbial symbionts with respect to host plant genotypes
To overcome the shortage of symbiotic activities in may be less pronounced than with nutritional symbionts,
cultivated plants, a coordinated breeding of plants and while the specificity towards pest genotypes is important.
micro-organisms could be implemented with the aim to In some crops, indirect suppression of pathogen infections
create optimal combinations of genotypes of the different may be implemented by microbe-derived secondary
partners (Rengel, 2002; Provorov & Tikhonovich, 2003). metabolites, e.g. by chitin oligomers produced in
This approach is most promising in legume–rhizobia Escherichia coli engineered with rhizobial genes for

Ann Appl Biol 159 (2011) 155–168 © 2011 The Authors 163
Annals of Applied Biology © 2011 Association of Applied Biologists
Agricultural microbiology I.A. Tikhonovich & N.A. Provorov

Nod factor synthesis (Leppyanen et al., 2008). The 2006). Nevertheless, a careful monitoring of endophytic
application of microbial symbiotic signals or their microflora for the absence of pathogens is required, espe-
derivatives for remodelling plant developmental or cially in crops, which are consumed without cooking.
defensive functions may represent a promising field for In summary, it is our view that an increased knowl-
agricultural biotechnology. edge of microbe-based symbioses in plants could provide
The prospects for a future development of agricultural effective ways of developing sustainable agriculture in
microbiology may involve the construction of novel mul- order to ensure human and animal food production with
tipartite endo- and ecto-symbiotic communities based on a minimal disturbance of the environment. The effective
extended genetic and molecular (metagenomic) analy- management of symbiotic microbial communities is pos-
ses. The primary approach for such construction is to sible using molecular approaches based on the continuity
create composite inoculants, which simulate the natural of microbial pools which are circulating regularly between
plant-associated microbial communities. For balancing soil-, plant- and animal-provided niches in natural and
the host plant metabolism, a combination of N- and agricultural ecosystems (Popova et al., 2001; Kupriyanov
P-providing symbionts would appear promising, includ- et al., 2010; Fig. 1). Analysis of this circulation could
ing the endosymbiotic rhizobia + VAM-fungi (Shtark enable the creation of highly productive microbe-based
et al., 2010), or rhizosphere N2 -fixer Phyllobacterium + sustainable agricultural system, whilst addressing the eco-
P-solubilizer Bacillus (Rojas et al., 2001). logical and genetic consequences of the broad application
Special plant genotypes are required to support the of microbes in agricultural practice.
high simultaneous activities in different types of energy-
consuming microbes. The pea cultivar Triumph has been
Acknowledgements
created via the ‘inbred-backcross line’ strategy with wild
genotypes used as the donors of high symbiotic activity Supported by grants from Russian Foundation for
traits. The cultivar is capable of completing its N and P Basic Research, RFBR (09-04-00907a, 09-04-91007-
nutrition after the application of composite inoculants ANF_a, 09-04-92865-NISI_a, 10-04-01146-a), RFBR-
(rhizobia + VAM-fungi + rhizosphere bacteria) and NWO (047.018.001); Scientific School 3440.2010.4 and
represents an example of a plant genotype that has the by the State Contracts (02.740.11.0276, 02.740.11.0698).
potential for sustainable, microbe-based crop production
(Borisov et al., 2008). As the majority of legume Sym genes
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