Chapter 4

Microbial Interactionsin the Rhizosphere

Jose-Miguel Barea, Marıa-Jose Pozo, Rosario Azcon, andConcepcion Azcon-AguilarDepartamento de Microbiologıa del Suelo y Sistemas Simbioticos, EstacionExperimental del Zaidın, CSIC, Spain

4.1 INTRODUCTION

The diverse genetic and functional groups of the extensivesoil microbial populations are known to carry out activ-ities exerting a critical impact on soil functions (Bareaet al., 2005b; Avis et al., 2008). Among other activities,soil microorganisms propel the biogeochemical cyclingof nutrients and organic matter and improve plant perfor-mance and soil quality, key issues for agroecosystem self-sustainability (Gianinazzi et al., 2010; Jeffries and Barea,2012). These topics are relevant not only for optimizingthe stability and productivity of the agroecosystems butalso to prevent erosion and to minimize negative culturaland environmental stresses (Buscot, 2005).

Micobial activities are particularly relevant in theroot–soil interface microhabitat, known as the rhizo-sphere, a dynamic environment where microorganismsinteract with plant roots and soil constituents (Bowen andRovira, 1999).

Strategic and applied research has demonstrated thatmicrobial interactions in the rhizosphere can be man-aged, as a low input biotechnology, to help sustainableenvironmentally friendly agrotechnological practices(Ramos-Solano et al., 2009; Azcon and Barea, 2010).Thus, the formation, development, significance, function-ing, and managing of rhizosphere microbial populationsare topics of current research interest, which havebeen reviewed recently (Berg and Smalla, 2009; Joneset al., 2009; Lambers et al., 2009; Hartmann et al.,2009; Dessaux et al., 2010). Particularly, the molecular

Molecular Microbial Ecology of the Rhizosphere, Volume 1, First Edition. Edited by Frans J. de Bruijn. 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.

determinants involved in rhizosphere formation andfunctioning are receiving much attention (Matilla et al.,2007; Ferrer et al., 2008; Leitner et al., 2008; Faure et al.,2009; Mathesius, 2009; Tarkka et al., 2009; Lopez-Raezet al., 2012; Christensen and Kolomiets, 2011; Joussetet al., 2011; Santoro et al., 2011).

The soil microbiota is often separated into theso-called microorganisms and the larger “microfauna”(Bowen and Rovira, 1999). Although it is acknowledgedthat microfauna affect plant growth and above-groundfood webs (Scheu et al., 2005), this review will concen-trate on microorganisms and discuss the ecological andmolecular aspects of rhizosphere microbial interactions,analyzing (i) trophic and functional groups of microor-ganisms involved in rhizosphere interactions; (ii) directmicrobe–microbe interactions benefiting agroecosystems;and (iii) microbial interactions involving arbuscularmycorrhiza, the omnipresent fungal–plant (root) symbio-sis. The research trends of this thematic area are thusoutlined. As these topics were reviewed by us in Bareaet al. (2005b), most information concerns new advancesin rhizosphere interactions.

4.2 DIVERSITY OF TROPHICAND FUNCTIONAL GROUPS OFRHIZOSPHERE MICROORGANISMS

A variety of microbial forms, either culturable or not, canbe found in rhizosphere microhabitats as stimulated by

29

30 Chapter 4 Microbial Interactions in the Rhizosphere

root-released compounds and signal molecules (Roeschet al., 2007). A general view of the microbial typesinvolved and their cooperative activities are discussedhere.

4.2.1 Rhizosphere MicrobialPopulationsIt is commonly accepted that any microbial group candevelop important functions in the ecosystem (Giriet al. 2005). However, most studies on rhizospheremicrobiology, especially those describing cooperativeplant–microbial interactions, have focused only onbacteria and fungi. Accordingly, this review will focuson these two types of microbes.

Bacteria and fungi have different trophic/living habits,and a variety of saprophytic or symbiotic relationships,either detrimental (pathogens) or beneficial (mutualists),have been reported (Kobayashi and Crouch, 2009). Detri-mental microbes include major plant pathogens and minorparasitizing and nonparasitizing deleterious rhizosphereorganisms. Beneficial saprophytes bacteria and fungi canpromote plant growth and health. Beneficial plant mutu-alistic symbionts include the N2-fixing bacteria and thearbuscular mycorrhizal (AM) fungi (Barea et al., 2005b;see Section 6).

4.2.2 Nonsymbiotic BeneficialRhizosphere Bacteria and FungiParticular attention has been devoted to the so-calledplant-growth-promoting rhizobacteria, the PGPR (Kloep-per et al., 1991; see Chapter 53). The moleculardeterminants of root colonization by PGPR are beinginvestigated (Gamalero et al., 2004; Brotman et al., 2008;Barret et al., 2011; Liu et al., 2011; see Section 7). ThePGPR participate in many important ecosystem processes,such as the biological control of plant pathogens, nutrientcycling and/or seedling development, and bioremediationof contaminated soils (Barea et al., 2005b; Adesemoyeet al., 2009; Hayat et al., 2010). Figure 4.1 summarizesthe main ecosystem roles of PGPR.

Numerous PGPR have been identified as biocontrolagents of plant pathogens, Pseudomonas sp. being oneof the major groups involved (Haas and Defago, 2005;Mendes et al., 2011). Biological control of soil-bornediseases is known to result from (i) the reduction in thesaprophytic growth of the pathogens followed by reduc-tion in the frequency of the root infections through micro-bial antagonism; (ii) reduction of pathogen virulence;and (iii) the stimulation of “induced systemic resistance(ISR)” in the host plant (Zhang et al., 2004; Compantet al., 2005; see Chapters 54, 55). Microbial antagonismmay be achieved through the release of antibiotics by the

Plant growthpromotion

Biocontrol ofplant pathogens

Improvementof soil quality

Regulation ofsoil biodiversity

AntibiosisAntagonismAntioxidantsInducedresistance

Quorum sensing

Plant hormonesN2–fixationP–solubilizationACC deaminase Siderophores

Figure 4.1 Rhizobacteria functions in the soil-plant system: Maineffects and mechanisms responsible for plant growth and healthpromotion, and for the regulation of soil diversity and eco-dynamicsof microbial populations.

PGPR. Among the different antifungal factors producedby PGPR, acetylphloroglucinols (see Chapter 56) andphenazines (Schouten et al., 2004; Haas and Defago, 2005;Weller, 2007; Gross and Loper, 2009; Pierson and Pierson,2010; see Chapter 54) are receiving most attention. Theproduction of siderophores as pyoverdine and pyochelincontributes to pathogen control through competition foriron (Gross and Loper, 2009). Production of cell wallhydrolases with lytic activity against fungal pathogenshas been shown to also contribute to biological controlby PGPR (Chernin and Chet, 2002; Someya et al., 2007).The reduction of pathogen virulence, achieved throughdetoxification or degradation of virulence factors, includ-ing autoinducer signals (quenching thereby pathogenquorum-sensing capacity) is another relevant mechanismin biocontrol (Zhang and Birch, 1997; Dong et al., 2001,2004; Molina et al., 2003; see Chapters 75, 76).

Bacterial determinants triggering ISR and mech-anisms regulating resistance in the plant are underextensive scrutiny (Kloepper et al., 2004; Bakker et al.,2007, Ramos-Solano et al., 2008, 2010). Determinantsalready identified include lipopolysaccharides, antibiotics,diacetylphloroglucinol, 2,3-butanediol, and siderophores(Ryu et al., 2004; Hofte and Bakker, 2007; Bakker et al.,2007). The signaling pathways controlling the resistanceresponse in the plant are being elucidated (Pozo andAzcon-Aguilar, 2007; Pozo et al., 2008; van Wees et al.,2008; Weller et al., 2012; see Chapter 54).

Among rhizosphere fungi described as biocontrolagents Trichoderma spp. is a representative example.Trichoderma controls fungal pathogens by acting bothas antagonist, based on competition, antibiosis, andmycoparasitism, and by inducing localized and systemicdefensive responses in the plant (Shoresh et al., 2010;Druzhinina et al., 2011; see Chapter 54). Trichodermagrows toward the fungal pathogen and releases a largevariety of secondary metabolites such as the antibiotics

4.2 Diversity of Trophic And Functional Groups of Rhizosphere Microorganisms 31

gliotoxin, gliovirin, polyketides, pyrones, peptaibols(Mukherjee et al., 2012a), and a battery of lytic enzymes,mainly chitinases, glucanases, and proteases. Theseenzymes facilitate penetration of Trichoderma into thehost and the utilization of the host components fornutrition (Kubicek et al., 2011). Induction of plantdefense mechanisms in biocontrol by Trichoderma hasbeen highlighted (Harman et al., 2004; Vinale et al.,2008; Shoresh et al., 2010; Hermosa et al., 2012), andthe determinants and regulatory mechanisms involved arebeing investigated (Djonovic et al., 2006, 2007; Segarraet al., 2009; Mukherjee et al., 2012a, 2012b).

Some endophytes and nonpathogenic strains of fungalpathogens can also act as biocontrol agents (Waller et al.,2005; Vallance et al., 2009; Kaur et al., 2010).

With regard to the role of PGPR in nutrient cycling,the main processes refer primarily to nitrogen fixation andphosphate solubilization (Richardson et al., 2010). Certainbacterial taxa are the only organisms able to fix N2, thefirst step in cycling N from the atmosphere to the bio-sphere, a key N input to plant productivity (Arrese-Igor,2010; see Chapter 44). Many asymbiotic diazotrophic bac-teria have been described and tested as biofertilizers underfield conditions, but their effectiveness cannot be gener-alized (Ramos-Solano et al., 2009).

Diverse rhizobacterial (and rhizofungal) taxa areable to mineralize and/or solubilize sparingly availablephosphate sources (Khan et al., 2010) largely by releas-ing specific enzymes and/or chelating organic acids(Marschner, 2008; Richardson et al., 2010; see Chapter58). Phosphate-solubilizing bacteria (PSB) selectedfrom PGPR populations have been assayed under fieldconditions but their effectiveness may be limited becauserefixation of solubilized phosphate ions on their way tothe root (Barea et al., 2007; Zaidi et al., 2010).

Azospirillum species are also considered PGPR(Dobbelaere and Okon, 2007; Gutierrez-Manero andRamos-Solano, 2010; Hartmann and Bashan, 2009;Couillerot et al., 2010; Bashan et al., 2011). A keyaction mechanism of these bacteria is the productionof auxin-type phytohormones, which affect the rootingpatterns thereby benefiting plant nutrient uptake (Baudoinet al., 2010; see Chapters 27, 29, 30).

Recent advances on the role and mechanisms ofPGPR helping bioremediation of contaminated soils havebeen discussed (Zhuang et al., 2007; de Lorenzo, 2008;Wenzel, 2009; Dimkpa et al., 2009; Ruız-Lozano andAzcon, 2011; Wenzel et al., 2011; see Section 12).

4.2.3 The MutualisticPlant–Microbe SymbiontsBeneficial plant microbial symbionts include the N2-fixingbacteria and the multifunctional mycorrhizal fungi.

The bacteria able to fix N2 in symbiosis withlegumes belong to diverse genera, collectively termedas rhizobia (Willems, 2007; see Chapter 44). Howthese bacteria interact with legume roots leading to theformation of N2-fixing nodules, the signaling processesinvolved, the evolutionary history, and, particularly, themolecular aspect determinants of host specificity in therhizobial–legume symbiosis are described elsewhere inthis book (see Chapter 45). Other bacteria, belonging tothe genus Frankia (actinomycetes), also form N2-fixingnodules on the roots of the so-called actinorrhizal species(Normand et al., 2007a). The genome of Frankia sp. isbeing explored (Normand et al., 2007b). The genetic basesfor their endosymbiosis establishment have been found tohave common features with that of AM fungi and rhizobia(Gherbi et al., 2008; Markmann and Parniske, 2009).

The other major groups of mutualistic microbial sym-bionts are the fungi that establish (mycorrhizal) associ-ations with the roots of most plant species (Smith andRead, 2008; see Chapter 43). Mycorrhizal fungi engage insymbiotic, generally mutualistic, associations, establishedwith most vascular plants where both partners exchangenutrients and energy. The soil-borne mycorrhizal fungicolonize the root cortex biotrophically and then developan external mycelium, a bridge connecting the root withthe surrounding soil microhabitats (Smith and Read 2008).Mycorrhizal symbioses can be found in almost all ecosys-tems worldwide to improve plant fitness and soil qualitythrough key ecological processes (Azcon-Aguilar et al.,2009). Most of the major plant families form AM asso-ciations, the most common mycorrhizal type (Brundrett,2009). The AM fungi are obligate symbionts, which arenot able to complete their life cycle without colonizing ahost plant (Bago and Cano, 2005). They are ubiquitoussoil-borne microbial fungi, whose origin and divergencedate back to more than 450 million years (Redecker et al.2000; Bonfante and Genre, 2008; Dotzler et al., 2009;Honrubia, 2009; Smith et al., 2010; Schußler and Walker,2011). Molecular analyses and the fossil record indicatethat AM associations evolved as a symbiosis, facilitat-ing the adaptation of plants to the terrestrial environmentand suggest that AM fungi played a crucial role in landcolonization by plants (Schußler and Walker, 2011).

The AM fungi belong to the phylum Glomeromycota(Schußler et al., 2001; Rosendahl, 2008; Helgason andFitter 2009; Gamper et al. 2010; Schußler and Walker,2011).

Because of the microbial character of the AM fungiduring their entire life cycle, this review will focus onlyon AM symbiosis and fungi. However, the importance ofmicrobial interactions involving other mycorrhizal typesmust be recognized as well.

Earlier studies on the diversity of AM fungalcommunities were based largely on the morphological

32 Chapter 4 Microbial Interactions in the Rhizosphere

characterization of their large multinucleate spores. How-ever, molecular tools are now available for a challengingdissection of AM fungal population dynamics (Robinson-Boyer et al. 2009). For molecular identification, thePCR-amplified ribosomal DNA (rDNA) fragments of thespores and/or the mycelia from AM fungi are usuallysubjected to cloning, fingerprinting, and sequencing(Hempel et al., 2007; Opik et al., 2008, 2010; Toljanderet al., 2008; Alguacil et al. 2009; 2011; Rosendahl et al.,2009; Sonjak et al., 2009; Kruger et al., 2011). Alternativemolecular tools now exist to quantitatively analyze theeffect of environment, management, or inoculation ofsoils on more diverse AM fungal communities. Q-PCRcan be used for simultaneous specific and quantitativeinvestigations of particular taxa of AM fungi in rootsand soils colonized by several taxa (Gamper et al.,2008; Koenig et al., 2010). In addition, new techniquesof high throughput sequencing (e.g., pyrosequencing)are rapidly developing (Roesch et al., 2007) and arenow being used for AM fungi (Lumini et al., 2010; seeChapter 106). A lack of relationship between geneticand functional diversities has been shown (Munkvoldet al., 2004; Croll et al., 2008; Ehinger et al., 2009).Despite the advancement in molecular techniques, theidentification approaches employed for AM fungi basedon morphological characteristics are still valid andused, being considered complementary to the molecularmethods (Morton, 2009; Oehl et al., 2009).

Recent advances in the genetics and genomics ofthe AM fungi have been reviewed (Ferrol et al., 2004;Gianinazzi-Pearson et al., 2004, 2009; Pawlowska, 2005;Parniske, 2008; Sanders and Croll, 2010; see Chapter 43).The complete genome of the model AM fungus Glomusintraradices is being determined (Martin et al., 2008).

The AM fungi contribute to nutrient, particularlyphosphate, acquisition and supply to plants therebyaffecting rates and patterns of nutrient cycling in bothagricultural and natural ecosystems (Barea et al., 2008).The AM symbiosis also improves plant health throughincreased protection against environmental stressesincluding biotic, as derived from pathogen attack (Pozoet al., 2010; Lopez-Raez et al., 2011), or abiotic, ascaused by drought (Ruız-Lozano et al., 2008; Arocaet al., 2011), salinity (Porcel et al., 2012), heavy metals(HMs) (Turnau et al., 2006; Azcon et al., 2009b) ororganic pollutants (Leyval et al., 2002). Additionally,AM associations improve soil structure through aggre-gate formation, necessary for appropriate soil quality(Rillig and Mummey, 2006). The role of flavonoids andstrigolactones in root exudates as signals in mycorrhizalinteractions has been evidenced (Akiyama et al., 2005;Steinkellner et al., 2007; Lopez-Raez et al., 2012; seeChapters 33, 34, 35, 51).

4.3 MICROBE–MICROBEINTERACTIONS BENEFITINGAGROECOSYSTEM DEVELOPMENT

Direct interactions occurring between members ofdifferent microbial types often result in the promotionof key processes benefiting plant growth and health.It is obvious that all interactions taking place in therhizosphere are, at least indirectly, plant mediated.However, this section deals with direct microbe–microbeinteractions themselves, with the plant as a “supportingactor” in the rhizosphere. Three types of interactionshave been selected for discussion here because of theirrelevance to the development of sustainable agroecosys-tems. These are (i) the cooperation between rhizobiumand other PGPR for improving N2 fixation; (ii) microbialantagonism for the biocontrol of plant pathogens; and(iii) interactions between rhizosphere microbes and AMfungi to establish a functional mycorrhizosphere.

4.3.1 PGPR Impact to Improve N2Fixation by Rhizobia–LegumesAssociationsAs they share common microhabitats in the root–soilinterface, rhizobia and other PGPR can interact dur-ing their processes of root colonization. It has beenshown that some PGPR can improve nodulation andN2 fixation through mechanisms including produc-tion of plant hormones, flavonoids, Nod factors, orenzymes (Tilak et al., 2006; Remans et al., 2008;Dardanelli et al., 2008; Bansal, 2009; Mishra et al.,2009; Medeot et al., 2010; Ahmad et al., 2011;Fox et al., 2011; see Chapter 53).

Inoculation of PGPR that are able to solubilize phos-phate (PSB) enhances legume nodulation and N2 fixation(15N methods) (Barea et al., 2008; Azcon and Barea, 2010;Zaidi et al., 2010).

Azcon et al. (2009a) demonstrated that PGPR iso-lated from a HM-contaminated soil increased nodulationof legumes growing in these soils where control plantsdid not form nodules. The explanation may be relatedto the role of PGPR accumulating HM in soil and there-fore reducing HM concentrations and uptake by plants andrhizobia, thereby preventing toxicity and enabling nodu-lation. In addition, an increase in soil enzymatic activi-ties (phosphatase, β-glucosidase, dehydrogenase, etc.) andin auxin production around PGPR-inoculated roots couldalso be involved in the PGPR effect on nodulation.

Tolerant PGPR have been shown to improve nodu-lation, N2 fixation, and legume performance in stressedenvironments, including drought, salinity, nutrient defi-ciency, acidity, or alkalinity (Zahran, 2010; Ahmad et al.,2011).

4.3 Microbe–Microbe Interactions Benefiting Agroecosystem Development 33

4.3.2 Microbial Antagonism forthe Biological Control of PlantPathogensThe role of microbial populations in the rhizosphere asthe first barrier to pathogen infection was highlighted inthe 1970s, when the term “suppressive soils” becameestablished (Barea et al., 2005b). It is now widelyknown that some soils are naturally suppressive tosoil-borne plant pathogens such as Fusarium, Gaeuman-nomyces, Rhizoctonia, Pythium, and Phytophthora. As themicroorganisms with antagonistic properties toward plantpathogens are diverse (Heydari and Pessarakli, 2010),deciphering the metagenome of disease-suppressive soilsis a major challenge in identifying the microbes andmolecular mechanisms involved in such suppression(van Elsas et al., 2008; Mendes et al., 2011). The useof high-density 16S rDNA oligonucleotide microarrays(PhyloChip) has revealed that Proteobacteria, Firmicutes,and Actinobacteria are consistently associated withdisease suppression. In addition to Pseudomonadaceae,species of Agrobacterium, Bacillus, Streptomyces, andBurkholderia have been shown to be effective antagonistsof soil-borne pathogens (Whipps, 2001). Bacteriophagesare recognized nowadays as biocontrol agents for bacte-rial diseases (Jones et al., 2007, Chandanie et al., 2010;see Chapter 57). A variety of fungal species and isolateshave been applied in biocontrol, as nonpathogenicspecies of fungi as Pythium and Fusarium (Whipps2001; Vallance et al., 2009; Kaur et al., 2010) butthe ubiquitous Trichoderma species clearly dominate(Druzhinina et al., 2011; see Chapter 54). Trichodermaspp. efficiently controls a broad range of phytopathogenicfungi such as Rhizoctonia solani, Pythium ultimum,and Botrytis cinerea. Interactions between Trichodermaand mycotoxigenic Fusarium have been assayed forthe biocontrol of Fusarium head blight (Matareseet al., 2012). Pseudomonas and Trichoderma spp. areexamples of combination of multiple mechanisms foreffective pathogen suppression (Heydari and Pessarakli,2010).

The use of beneficial microorganisms for biocontrolstrategies should aim to produce a high level of plant pro-tection and wide range of effectiveness. To achieve thatgoal and reduce the variability in the results, biocontrolresearch has to face the challenges of finding appropri-ate screening procedures to select suitable microorgan-isms in diverse soil environments (Pliego et al., 2011).Understanding the impact of a changing environment onthe biocontrol agent performance, and predicting the out-put of multiple interactions in the rhizosphere, is funda-mental to develop effective combinations of antagonisticmicroorganisms (Xu et al., 2011).

4.3.3 Interactions Between AMFungi and Other RhizosphereMicrobes for Mycorrhiza/Mycorrhizosphere EstablishmentRhizosphere microorganisms can either interfere withAM fungi or benefit mycorrhiza establishment (Pivatoet al. 2009; Larsen et al., 2009). A typical beneficialeffect is that exerted by the so-called mycorrhiza-helper-bacteria (MHB), a term referring those bacteriathat enhance mycorrhiza formation (Frey-Klett et al.,2007; see Chapter 49). Soil microorganisms producecompounds that increase the rates of root exudation. This,in turn, stimulates AM fungal mycelia in the rhizosphereor facilitates root penetration by the fungus. Planthormones, as produced by soil microorganisms, affectAM establishment (Barea et al., 2005b). Rhizospheremicroorganisms influence the presymbiotic stages of AMdevelopment, such as spore germination rate and mycelialgrowth (Franco-Correa et al., 2010; Fernandez-Bidondoet al., 2011). Conversely, the establishment of PGPRinoculants in the rhizosphere can be benefited by AMfungal co-inoculation (Toljander et al., 2007).

AM colonization changes the chemical composi-tion of root exudates, whereas the AM soil myceliumintroduces physical modifications into the environmentsurrounding the roots thereby affecting microbial struc-ture and diversity (Barea et al., 2005b). In addition, thereare specific modifications in the environment surroundingthe AM mycelium itself, the mycorrhizosphere (Requenaet al., 1999; Hooker et al., 2007; Toljander et al.,2007; Finlay, 2008; Vestergard et al., 2008; Lioussanneet al., 2010).

A case of particular interest concerns the relationshipsbetween AM fungal structures and certain intimatelyassociated endobacteria (Bonfante and Anca, 2009). Thepresence of endobacteria inside AM fungal cytoplasmhas long been documented by electron microscopy,which distinguished two endobacterial morphotypes. Thefirst, restricted to the Gigasporaceae, is an unculturedtaxon, Candidatus Glomeribacter gigasporarum, relatedto Burkholderia (Lumini et al., 2007). The other bacterialtype that has been detected inside AM fungal spores andhyphae colonizing plant roots sampled in the field is calledbacterium-like organism (BLO) (MacDonald et al., 1982).

Recently, Naumann et al. (2010) analyzed 28 cul-tured AM fungi, from diverse evolutionary lineages andfour continents, showing that most of the AM fungalspecies investigated possess BLOs. Analyzing the 16SrDNA they found that BLO sequences from divergent lin-eages all clustered in a well-supported monophyletic cladethat was not closely related to any described bacterialgroup, but with the Mollicutes. The intracellular loca-tion of BLOs was revealed by confocal microscopy and

34 Chapter 4 Microbial Interactions in the Rhizosphere

fluorescent in situ hybridization (FISH), and confirmed bypyrosequencing. These bacteria diverged from their sistergroup more than 400 million years ago, colonizing theirfungal hosts before main AM fungal lineages separated.The BLO–AM fungal symbiosis can, therefore, be datedback at least to the time when AM fungi formed the ances-tral symbiosis with emergent land plants.

Research aimed to isolate and characterize bacteriadesignated as “probable endobacteria” from AM fungalspores (Gigaspora margarita) has been recently published(Cruz and Ishii, 2011). Bacterial isolates were charac-terized by both morphological and molecular methodsrevealing the presence of Bacillus spp. and Penibacillusspp. These bacteria showed phosphate-solubilizing capac-ity and possessed nitrogenase activity. Besides, they wereantagonistic to fungal plant pathogens but stimulated AMhyphal growth. The use of a new type of scanning elec-tron microscope demonstrated the presence of bacterialaggregates, apparently similar to biofilms, on the surfaceof hyphae and spores.

4.4 INTERACTIONS BETWEENSELECTED AM FUNGI AND PGPRFOR IMPROVING AGROECOSYSTEMSUSTAINABILITY

Managing microbial interactions involving selected AMfungi and PGPR (mycorrhizosphere tailoring) is recog-nized as a feasible biotechnological tool to improve plantgrowth and health, and soil quality, as summarized inFigure 4.2.

Many co-inoculation experiments using selected AMfungi and rhizosphere microorganisms have been reported.The main conclusions from key information will be sum-marized here with special emphasis on the ecological andmolecular aspects of interactions related to (i) symbioticN2 fixation; (ii) phosphate solubilization; (iii) phytoreme-diation of HM-contaminated soils; (iv) biological controlof root pathogens; and (v) improvement of soil quality.

4.4.1 Interactions with SymbioticN2-Fixing BacteriaThe widespread presence of the AM symbiosis in legumesand its role in improving nodulation and N2 fixationby legume–rhizobia associations are both universallyrecognized processes (Azcon and Barea, 2010). Actually,rhizobial bacteria and AM fungi are known to interactamong themselves and with their common legumehost roots, either at the colonization stages or at thesymbiotic functional level (Lesueur and Sarr, 2008).Particularly interesting are the studies (i) on the geneticand molecular relationships of AM fungi and rhizobia

Rhizosphere

Roots

* Plant establishment and development (phytostimulation)* Nutrient cycling and supply (biofertilization)* Plant protection, induced resistance (biocontrol)* Phytoremediation (bioremediation)* Soil conservation (agro ecosystems restoration)

Tailored mycorrhizosphere

Co inoculation withtarget bacteria

Microbe–microbeinteractions

Mycorrhiza

Mycorrhizosphere

Figure 4.2 Mycorrhizosphere tailoring: Microbial interactionsinvolving selected AM fungi and PGPR can be managed to improveplant fitness and soil quality through key ecological processes.

in establishing their endosymbioses; (ii) analyzing thephysiological interactions related to the formation andfunctioning of the tripartite symbiosis; and (iii) using 15Nto ascertain and quantify the AM role on N2 fixation bylegume–rhizobia associations.

Developmental genetics and evolution timing analysisof microbe–plant symbioses, including both mutualistic,either N2-fixing or mycorrhizal, and pathogenic associa-tions, have revealed a common developmental programfor all of these compatible microbe–plant associations(Markmann et al., 2008; Parniske, 2008; Provorovand Vorobyov, 2009b; den Camp et al., 2011). As therhizobia–legume symbiosis evolved much later than theAM symbiosis (Martınez-Romero, 2009; Provorov andVorobyov, 2009a; Zhukov et al. 2009), the cellular andmolecular events occurring during legume nodulationmay have evolved from those already established in theAM symbiosis. The legume–rhizobia symbiosis seemsto have evolved from a set of preadaptations duringcoevolution with AM fungi; thus some plant genescan modulate both legume symbioses (Parniske, 2004;Saito et al., 2007; Chen et al., 2009; Horvath et al.,2011; see Chapters 43, 45). A series of common plantgenes required for early developmental stages of bothAM and rhizobial symbioses have been identified byforward genetics approaches (Parniske, 2008). Theseinclude a receptor kinase, which is also required foractinorrhizal symbiosis, and has been implicated in theevolution of nodulation (Markmann et al., 2008). Theuse of mycorrhiza-defective legume mutants (Myc−)

4.4 Interactions Between Selected AM Fungi and PGPR 35

has provided relevant information allowing the commoncellular and genetic programs responsible for the legumeroot symbioses to be dissected (Gianinazzi-Pearson et al.,2009).

Both AM fungi and root-nodulating Frankia spp.coexist in the roots of some nonlegume (actinorrhizal)plant species and interact to facilitate establishment oftheir endosymbioses and to benefit plant–soil devel-opments (He and Critchley, 2008; Orfanoudakis et al.,2010). Genomic and transcriptomic studies have shownthat the actinorrhizal–Frankia symbioses also sharesome of the genes involved in the common SYMpathway described for AM fungi and legume–rhizobiumsymbioses (Gherbi et al., 2008; Hocher et al., 2011).

A great number of reports have focused on the phys-iological and biochemical basis of AM fungal–rhizobiainteractions. The AM fungi are known to exert a generalinfluence on plant nutrition, based on the supply of P bythe AM fungi to satisfy the high P demand of symbioticN2 fixation, but more localized effects of AM fungi occurat the root, nodule, or bacteroid levels (Azcon and Barea,2010).

The addition of a small amount of 15N-enriched inor-ganic fertilizer and an appropriate “nonfixing” referencecrop is the basis to ascertain and quantify the amount ofN, which is actually fixed by legume–rhizobia consortiain a particular situation and to measure the contributionof the AM symbiosis to the process. A lower 15N-to-14Nratio in the shoots of rhizobia-inoculated AM plants withrespect to those achieved by the same rhizobial strainin nonmycorrhizal plants has been found. This indicatesan enhancement of the N2 fixation rates (an increase in14N from the atmosphere), as induced by the AM activity(Barea et al., 2005a; Chalk et al. 2006).

4.4.2 Interactions withPhosphate-Solubilizing BacteriaThe interactions between AM fungi and phosphate-solubilizing microorganisms (PSM) are relevant to Pcycling and plant nutrition. Because the Pi made availableby PSM acting on sparingly soluble P source has limiteddiffusion in soil solution, and may not reach the root sur-face, AM fungi could tap the phosphate ions solubilizedby the PSM and translocate them to plant roots (Bareaet al., 2005a; Azcon and Barea, 2010). The microbialinteraction of AM fungi and PSB has been tested inexperiments using 32P-tracer methodologies (Barea et al.,2007). Upon adding a small amount of 32P to label theexchangeable soil P pool, the isotopic composition, or“specific activity” (SA = 32P/31P quotient), was deter-mined in plant tissues. It was found that dual inoculationreduced the SA of the host plant, indicating that theseplants acquired P from sources that were not directly

available to noninoculated or singly inoculated plants.Microbial inoculation improved biomass production andP accumulation in plants, demonstrating the interactiveeffects of PSB and AM fungi on P capture, cycling, andsupply in a tailored mycorrhizosphere (Barea et al., 2007).

Multi-microbial interactions, including those amonglocally isolated AM fungi, PSB, and Azospirillum, havealso been reported, which indicate that microorganismscan act synergistically when co-inoculated (Azcon andBarea; 2010).

4.4.3 Interaction forPhytoremediation of SoilContaminated with Heavy MetalsAM fungi improve phytoremediation of soils contami-nated with HMs, radionuclides, or polycyclic aromatichydrocarbons (Leyval et al., 2002). Most phytoreme-diation assays involving mycorrhizosphere interactionsconcern HMs and different strategies of phytoremediationhave been investigated (Turnau et al., 2006; Ruız-Lozanoand Azcon, 2011; see Section 12). These studies mostlyconcentrated on Zn, Cu, Cd, Pb, or Ni, but remediationof As-contaminated soils has also been attempted (Donget al., 2008).

Interactions between rhizobacteria and AM fungihave been investigated in diverse experiments toascertain whether they cooperate to benefit phytoreme-diation (Azcon et al., 2009a, 2009b, 2010). The mainachievements resulting from these experiments usingHM-multiple contaminated soils and Trifolium as testplants were: (i) a number of bacteria and the AM fungiwere isolated from an HM-contaminated soil and iden-tified by 16S rDNA or 18S rDNA, respectively; (ii) thetarget bacteria were able to accumulate large amountsof metals; (iii) co-inoculation with an HM-adaptedautochthonous bacteria and AM fungi increased biomass,N and P content as compared to noninoculated plants andalso enhanced the establishment of symbiotic structures(nodule number and AM colonization), which werenegatively affected as the level of HM in soil increased;(iv) dual inoculation lowered HM concentrations inTrifolium plants, inferring a phytostabilization-basedactivity; however, as the total HM content in plant shootswas higher in dually inoculated plants, due to the effect onbiomass accumulation, a possible phytoextraction activitywas suggested; and (v) inoculated HM-adapted bacteriaincreased dehydrogenase, phosphatase, and β-glucanaseactivities and auxin production in the mycorrhizosphere,indicating an enhancement of microbial activities relatedto plant development.

The physiological/biochemical mechanisms by whichthe tested bacterial isolates enhanced phytoremediationactivity in AM plants include (i) improved rooting, and

36 Chapter 4 Microbial Interactions in the Rhizosphere

AM formation and functioning; (ii) enhanced microbialactivity in the mycorrhizosphere; and (iii) accumulation ofmetals in the root–soil environment, thus avoiding theirtransfer to the trophic chain, or to aquifers (Ruız-Lozanoand Azcon, 2011).

Inoculation of autochthonous AM fungi and bacteria,together with the application of treated agrowaste residue,changed bacterial community structure and enhancedphytoextraction to remediate HM-contaminated soils(Azcon et al., 2009a). An enhancement of antioxidantactivities in plants inoculated with AM fungi and bacteriaand agrowaste residue was evidenced (Azcon et al.,2009b). Such a mycorrhizosphere effect seems to helpplants to limit oxidative damage to biomolecules inresponse to metal stress. The molecular mechanismsinvolved in HM tolerance in AM inoculated plantshave been recently discussed (Cornejo et al., 2008;Gonzalez-Guerrero et al., 2009; Zhang et al., 2009; Alouiet al., 2011).

4.4.4 Interactions for theBiological Control of PlantPathogensAM establishment has been shown to reduce damagecaused by soil-borne plant pathogens with an enhance-ment of plant resistance/tolerance in mycorrhizal plants(Barea et al., 2005b; Pozo and Azcon-Aguilar, 2007;Pozo et al., 2009, 2010). As specific microorganismsantagonistic to plant pathogens are being used asbiological control agents, a major aim in rhizospherebiotechnology is to exploit the prophylactic ability of AMfungi in association with these antagonists (Barea et al.,2005b). The mycorrhizosphere has been hypothesized toconstitute an environment conductive to microorganismsantagonistic to soil-borne pathogen proliferation. Indeed,various antagonistic bacteria have been identified withinAM extraradical structures or in the mycorrhizosphere ofseveral AM species, thus favoring biological control ofpathogens (Lioussanne, 2010).

A key point is to ascertain whether an antifungalbiocontrol agent would negatively affect beneficial AMfungi. This is fundamental to exploit the possibilitiesof dual (AM fungi and microbial antagonists) inocu-lation to aid plant defense against root pathogens. Invitro and in field experiments aiming to evaluate theimpact of Pseudomonas strains producing the antifungaldiacetylphloroglucinol on AM formation and functioningconfirmed the lack of effect of the bacteria on thenumbers or diversity of the AM fungal population andperformance. Moreover, the antifungal Pseudomonasimproved the plant nutritional benefits from the AMassociation (Barea et al., 1998). Although some invitro experiments show the capacity of Trichoderma to

parasitize AM fungi, no inhibition or even promotionof plant colonization by AM fungi has been reported(Martınez-Medina et al., 2009). In some cases, althoughco-inoculation with other organisms resulted in a reduc-tion in AM colonization, the effects on plant health weremaintained or improved (Gamalero et al., 2010).

Most co-inoculation studies involving AM fungi andbiocontrol agents have dealt with bioprotection againstsoil-borne pathogens such as Fusarium or Rhizoctonia(Saldajeno et al. 2008) and other fungi (Chandanie et al.,2009). However, neutral and even antagonistic effects ofsuch interactions on the bioprotection against both, soil-and air-borne pathogens have also been described (Salda-jeno et al., 2008).

The enhanced capacity of bioprotection achieved bydual inoculation usually results from the combinationof the mechanisms used by each organism individually,such as competition, altered root exudates, morphologicalchanges in the root system, antibiosis, and the activationplant defense responses (Saldajeno et al., 2008). This isillustrated in Figure 4.3.

As already mentioned, most antagonistic microorgan-isms trigger plant defense mechanisms that may differdepending on the signaling pathway activated. Trigger-ing simultaneously different defense signaling pathwaysin the plant may alter the spectrum of pathogens to whichthe protection achieved is effective, as cross talk betweenphytohormones is a key element in the regulation of plantresistance (van Wees et al., 2008; Pieterse et al., 2009).

In summary, experimental evidence supports theargument that mycorrhizosphere management addressedat enhancing plant resistance/tolerance to pathogenattack is a promising biotechnological tool. However, assynergism and interference in the biocontrol propertiesof different organisms have been described, research onthe optimal microbial combinations and conditions forsynergism is essential for the successful application ofmicrobial consortia in sustainable agricultural practices.

4.4.5 Interactions for ImprovingSoil QualityPhysical–chemical soil properties are fundamental for soilquality, with soil structure being one of the most influen-tial factors (Buscot, 2005). Soil structure depends on theaggregation status of soil particles and well-aggregatedsoil ensures appropriate soil tilth, soil–plant water rela-tions, aeration, root penetrability, and organic matter accu-mulation (Miller and Jastrow, 2000). The contributionof microbial interactions to the formation and stabiliza-tion of soil aggregates has been demonstrated (Miller andJastrow, 2000).

As a result of degradation/desertification processes,disturbance of natural plant communities is often

4.5 Conclusions 37

(A)

(C)

(D)

(E)

(B)

(A)Defense

Figure 4.3 Synergistic interactions among different antagonisticmicroorganisms and mycorrhizas for the biological control of plantpathogens: This is illustrated in this mycorrhizosphere modelincluding plant roots (represented in brown), mycorrhizal hyphae (inblue), bacteria, fungi, nematodes and other microbes. Antagonisticbacteria are living on the root surface (A) or associated to spores ormycelia (B) from mycorrhizal plants (C). Trichoderma spp, amycoparasitic fungus is also involved (D). These microorganismssynergistically interact and protect the plant against pathogens (E)through the combination of different mechanisms involvingcompetition for nutrients and colonization sites, production ofdifferent antimicrobial compounds or siderophores, parasitism and, insome cases, priming of plant defenses, resulting in Induced SystemicResistance.

accompanied, or preceded by, loss of physical–chemicaland biological soil properties, such as soil structure, plantnutrient availability, organic matter content, and micro-bial activity (Jeffries and Barea, 2012). Accordingly,management of AM fungi, together with rhizospherebacteria, was proposed for the integral restoration ofdegraded ecosystems. This has been investigated in adesertified semi-arid ecosystem with Anthyllis cytisoides,a drought-tolerant legume, as a test plant (Requena et al.,2001). Anthyllis seedlings inoculated with indigenousrhizobia and AM fungi were transplanted to field plots fora 5-year trial. The tailored mycorrhizosphere enhancedseedling survival and growth, P acquisition, N fixation,and N transfer from N fixing to associated nonfixing

species in the natural succession. The improvementin the physical–chemical properties in the soil aroundthe Anthyllis plants was shown by the increased levelsof N, organic matter, and number of hydrostable soilaggregates. Glomalin-related glycol proteins, producedby the external hyphae of AM fungi, are seen to beinvolved in the initiation and stabilization of water-stablesoil aggregates, due to its glue-like hydrophobic nature(Miller and Jastrow 2000; Rillig and Mummey, 2006;Bedini et al., 2009).

4.5 CONCLUSIONS

There is considerable experimental evidence that bacteriaand fungi living at the root–soil environments carryout a variety of interactive activities known to benefitplant growth and health, and also soil quality. In thepast years, research has attempted to improve theunderstanding of diversity, dynamics, and significance ofrhizosphere microbial populations, and to gain moreinformation on the molecular determinants of theircooperative interactions.

From the practical/ecological point of view, the aimswill be to improve sustainable plant productivity and foodquality while preserving the environment. However, toachieve this, we need first to carry out basic and strategicstudies to gain a better understanding of microbial inter-actions taking place in the rhizosphere. Only then can thecorresponding agrobiotechnology be applied successfully.Hence, future investigations in the field of microbialcooperation in the rhizosphere will include: (i) advancesin visualization technology; (ii) analysis of the molecularbasis of root colonization; (iii) signaling processes in therhizosphere; (iv) functional genomics; (v) mechanismsinvolved in beneficial cooperative microbial activi-ties; (vi) engineering of microorganisms for beneficialpurposes; and (vii) biotechnological developments forintegrated management.

Nondisruptive in situ visualization techniques arealready being used for detailed studies on the interactionsof microorganisms within the rhizosphere, both betweenthemselves and with the root. Improving these techniques,based on the use of confocal laser scanning microscopyand fluorescent proteins, will allow not only the simulta-neous imaging of different populations of microbes in therhizosphere but also the temporal–spatial visualizationof gene expression. Novel research is needed to improveimmunofluorescence techniques to assess gene transfer inrhizosphere environments without the need of cultivatingthe microorganisms.

Many traits of root colonization by rhizo-microbeshave already been identified, but novel molecularapproaches are being used to screen for new traits. These

38 Chapter 4 Microbial Interactions in the Rhizosphere

are important to decipher the genes encoding proteinsinvolved in transport or signal transduction pathways incolonization. An increase in our knowledge on quorum-sensing systems will be important for understanding theecodynamics of microbial populations in the rhizosphere,and the cellular and molecular aspects of signalingprocesses in microbe–microbe interactions.

Future functional genomics (including transcrip-tomics, proteomics, and metabolomics) developmentswill be useful to identify the genes expressed in therhizosphere that play key roles in processes such asnutrient mobilization or suppression of plant diseases.The use of promoters to drive gene expression specificallyat the root–soil interfaces will allow the engineering ofmicroorganisms for beneficial purposes.

The specific management of mycorrhizal fungi/bacte-ria interactions, through the design of appropriate myc-orrhizospheres, should be one of the main objectivesof applied studies in the future. The use of microbialinoculants must take into account the importance ofretaining microbial diversity in the rhizosphere, andin achieving realistic and effective biotechnologicalapplications (“rhizosphere technology”). The improve-ment of molecular-biology-based approaches will befundamental for analyzing microbial diversity and com-munity structure, and to predict responses to microbialinoculation/processes in the environment (“ecologicalengineering”). Further studies must address the con-sequences of the cooperation between microbes inthe rhizosphere under field conditions to assess theirecological impacts and biotechnological applications.

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