BA SE Biotechnol. Agron. Soc. Environ. 2011 15(2), 327-337 Focus on: Beneficial effect of the rhizosphere microbial community for plant growth and health Venant Nihorimbere (1, 2 *) , Marc Ongena (3 *) , Maïté Smargiassi (3) , Philippe Thonart (3, 4) (1) University of Burundi. Faculty of Agricultural Sciences. PO Box 1550. Bujumbura (Burundi). E-mail: venant. [email protected](2) University of Burundi. Institut Supérieur d’Agriculture. BP 7268. Bujumbura (Burundi). (3) Univ. Liège - GemblouxAgro-Bio Tech. Centre Wallon de Biologie Industrielle. Passage des Déportés, 2. B-5030 Gembloux (Belgique). (4) Univ. Liège. Service de Technologie microbienne. Boulevard du Rectorat, 29. B-4000 Liège (Belgique). * Both authors equally contributed to this article. Received on May 5, 2010; accepted on August 24, 2010. Plant rhizosphere is the soil nearest to the plant root system where roots release large quantity of metabolites from living root hairs or fibrous root systems. These metabolites act as chemical signals for motile bacteria to move to the root surface but also represent the main nutrient sources available to support growth and persistence in the rhizosphere. Some of the microbes that inhabit this area are bacteria that are able to colonize very efficiently the roots or the rhizosphere soil of crop plants. These bacteria are referred to as plant growth promoting rhizobacteria (PGPR). They fulfil important functions for plant growth and health by various manners. Direct plant growth promotion may result either from improved nutrient acquisition and/or from hormonal stimulation. Diverse mechanisms are involved in the suppression of plant pathogens, which is often indirectly connected with plant growth. This paper describes the different mechanisms commonly used by most PGPR in their natural habitats to influence plant-growth and health. Keywords. Rhizosphere, PGPR, root exudation, plant-microbe interaction. Effet bénéfique de la communauté microbienne de la rhizosphère sur la croissance et la santé des plantes. La rhizosphère est le volume du sol situé au voisinage immédiat des racines des plantes et qui se caractérise par la présence d’exsudats racinaires (rhizodépôts). Ces exsudats sont utilisés par la microflore endémique en tant que signaux chimiques en plus d’être un substrat nutritif disponible pour la croissance et le développement de ces microorganismes dans la rhizosphère. Certaines de ces bactéries du sol, appelées PGPRs (Plant Growth Promoting Rhizobacteria), sont capables de coloniser les racines ou bien encore la rhizosphère, mais à la différence des autres bactéries rhizosphériques elles ont, en retour, un effet bénéfique sur la plante. Cet effet bénéfique peut être direct, ou indirect. La promotion directe de la croissance est le résultat du pouvoir d’acquisition des nutriments ou de la stimulation des hormones de la plante. D’autres mécanismes indirects, mais le plus souvent liés à la croissance des plantes, sont impliqués dans la réduction/suppression des pathogènes des plantes. Cet article décrit les différents mécanismes mis en jeu par les PGPRs dans leur environnement naturel pour influencer favorablement la croissance et la santé des plantes. Mots-clés. Rhizosphère, PGPR, exsudation racinaire, interaction plantes/micro-organismes. 1. IntroductIon According to a general view, the rhizosphere includes plant roots and the surrounding soil. This is a wide and wise definition, already coined more than hundred years ago by Hiltner (1904). In that particular environment, very important and intensive interactions take place between the plant, soil, and microfauna. Biochemical interactions and exchanges of signal molecules between plants and soil microbes have been described and reviewed (Pinton et al., 2007). The rhizosphere inhabiting microorganisms compete for water, nutrients and space and sometimes improve their competitiveness by developing an intimate association with plant (Hartmann et al., 2009). These microorganisms play important roles in the growth and ecological fitness of their host. An understanding of the basic principles of rhizosphere microbial ecology,
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BASE Biotechnol. Agron. Soc. Environ.201115(2),327-337 Focus on:
Plantrhizosphereisthesoilnearesttotheplantrootsystemwhererootsreleaselargequantityofmetabolitesfromlivingroothairsorfibrousrootsystems.Thesemetabolitesactaschemicalsignalsformotilebacteriatomovetotherootsurfacebutalsorepresentthemainnutrientsourcesavailabletosupportgrowthandpersistenceintherhizosphere.Someofthemicrobesthatinhabitthisareaarebacteriathatareabletocolonizeveryefficientlytherootsortherhizospheresoilofcropplants.Thesebacteriaare referred toasplantgrowthpromoting rhizobacteria (PGPR).They fulfil important functions forplantgrowthandhealthbyvariousmanners.Directplantgrowthpromotionmayresulteitherfromimprovednutrientacquisitionand/orfromhormonalstimulation.Diversemechanismsareinvolvedinthesuppressionofplantpathogens,whichisoftenindirectlyconnectedwithplantgrowth.ThispaperdescribesthedifferentmechanismscommonlyusedbymostPGPRintheirnaturalhabitatstoinfluenceplant-growthandhealth.Keywords.Rhizosphere,PGPR,rootexudation,plant-microbeinteraction.
Effet bénéfique de la communauté microbienne de la rhizosphère sur la croissance et la santé des plantes.Larhizosphèreest le volumedu sol situé auvoisinage immédiat des racinesdesplantes et qui se caractérisepar la présenced’exsudatsracinaires(rhizodépôts).Cesexsudatssontutilisésparlamicrofloreendémiqueentantquesignauxchimiquesenplusd’êtreunsubstratnutritifdisponiblepourlacroissanceetledéveloppementdecesmicroorganismesdanslarhizosphère.Certainesdecesbactériesdusol,appeléesPGPRs(PlantGrowthPromotingRhizobacteria),sontcapablesdecoloniserlesracinesoubienencorelarhizosphère,maisàladifférencedesautresbactériesrhizosphériquesellesont,enretour,uneffetbénéfiquesurlaplante.Ceteffetbénéfiquepeutêtredirect,ouindirect.Lapromotiondirectedelacroissanceestlerésultatdupouvoird’acquisitiondesnutrimentsoude la stimulationdeshormonesde laplante.D’autresmécanismes indirects,mais leplussouventliésàlacroissancedesplantes,sontimpliquésdanslaréduction/suppressiondespathogènesdesplantes.CetarticledécritlesdifférentsmécanismesmisenjeuparlesPGPRsdansleurenvironnementnaturelpourinfluencerfavorablementlacroissanceetlasantédesplantes.Mots-clés.Rhizosphère,PGPR,exsudationracinaire,interactionplantes/micro-organismes.
1. IntroductIon
According to a general view, the rhizosphereincludesplant rootsand thesurroundingsoil.This isawideandwisedefinition,alreadycoinedmorethanhundredyearsagobyHiltner(1904).Inthatparticularenvironment,veryimportantandintensiveinteractionstake place between the plant, soil, and microfauna.Biochemical interactions and exchanges of signal
molecules between plants and soil microbes havebeen described and reviewed (Pinton et al., 2007).The rhizosphere inhabiting microorganisms competeforwater,nutrientsandspaceandsometimesimprovetheir competitiveness by developing an intimateassociationwithplant(Hartmannetal.,2009).Thesemicroorganismsplayimportantrolesinthegrowthandecological fitness of their host.An understanding ofthebasicprinciplesofrhizospheremicrobialecology,
includingthefunctionanddiversityofmicroorganismsthat reside there, is necessary before soil microbialtechnology can be applied in the rhizosphere. Herewe review differentmechanisms commonly used bythebeneficial rhizospherebacteria to influenceplant-growthandhealthinthenaturalenvironment.
2. thE rhIzosphErE EFFEct
During seed germination and seedling growth,the developing plant interacts with a range ofmicroorganisms present in the surrounding soil. Asseedsgerminateand rootsgrow through the soil, thereleaseoforganicmaterialprovidesthedrivingforceforthedevelopmentofactivemicrobialpopulationsinazonethatincludesplantrootandsurroundingsoilinafewmmofthickness.Thisphenomenonisreferredastherhizosphereeffect(Morganetal.,2001).
Broadly, there are three distinct componentsrecognized in the rhizosphere; the rhizosphereper se (soil), the rhizoplane, and the root itself. Therhizosphereisthusthezoneofsoilinfluencedbyrootsthroughthereleaseofsubstratesthataffectmicrobialactivity.Therhizoplane is the rootsurface, includingthe strongly adhering root particles. The root itselfis a part of the system, because certain endophyticmicroorganismsareabletocolonizeinnerroottissues(Bowenetal.,1999).Therhizosphereeffectcanthusbeviewedas thecreationof adynamicenvironmentwheremicrobescandevelopandinteract.
3. MaIn charactErIstIcs oF thE root ExudatIon procEss
Root exudation is the release of organic compoundsfrom living plant roots into the surrounding soil; itis an ubiquitous phenomenon (Jones et al., 1995).Roots release compounds via at least two potentialmechanisms, and the rates of exudation sensu strictovary widely among species and environmentalconditions (Kochian et al., 2005). Exudates aretransportedacrossthecellularmembraneandsecretedinto the surrounding rhizosphere. Plant products arealsoreleasedfromrootsbordercellsandrootborder-like cells which separate from border as they grow(Baisetal.,2006).However,itisimportanttonotethatitisverydifficulttoidentifyrootexudateswithrespecttothechemicalcompositionandtheconcentrationinthesoilbecauseofmethodologicaldifficulties(Stolp,1988).Atthemomentofexudationandthereafter,theorganicmaterialsaresubject tomicrobialattack,andthuscannotbeenrichedandseparatedfromtherootsin the natural environments. Data on the nature andquantity of root exudates have been obtained from
sterile hydroponic cultures; but the results, however,are difficult to extrapolate to the natural conditions(Stolp,1988).Inthiscontext,rootexudationhasbeenquantified by measuring the production of labelledCO2 in the rhizosphere of
14C-labelled plants, and ithasbeenestimatedthat12-40%ofthetotalamountofcarbohydratesproducedbyphotosynthesisisreleasedinto the soil surrounding roots (Brimecombe et al.,2007).Root exudates aremainly composed ofwatersoluble sugars, organic acids, and amino acids, butalso contain hormones, vitamins, amino compounds,phenolicsandsugarphosphateesters(Uren,2001).
Releaseoftheselowmolecularweightcompoundsis a passive process along the steep concentrationgradientwhichusually exists between the cytoplasmofintactrootcells(millimolarrange)andtheexternal(soil) solution (micromolar range).Direct or passivediffusion through the lipid bilayer of the plasmamembrane is determined bymembrane permeability,whichdependson thephysiological state of the rootcellandonthepolarityofthecompounds,facilitatingthepermeationoflipophilicexudates(Rudrappanetal.,2007). The efficiency of the exudation process maythusbeenhancedbystressfactorsaffectingmembraneintegrity such as nutrient deficiency, temperatureextremes,orexudationstress(Ratnayaleetal.,1978).
Theproportionof carbon released from rootshasbeenestimatedtoasmuchas50%intheyoungplants(Whipps,1990)butlessinplantsgrowntomaturityinthefield (Jensen,1993).Thenatureofexudatesmayalsovaryaccordingtothegrowthstageoftheplant.Forinstance,therearemorecarboxylatesandrootmucilageat the six leaf stage than earlier.On the other hand,nitrogenisalsoofconsiderableimportancetonutrientcycling,usuallyasNH4
+,NO3-(Wacquantetal.,1989),
aminoacids(Boulteretal.,1966),celllysates,sloughedroots,andotherroot-deriveddebris.ItisestimatedatthematuritythattherhizodepositionofNamountedto20%of the total plant nitrogen (Jensen, 1996).Rootexudationisalsolargelydependentonthenutritionalstatus of the plant regarding oligoelements. LowconcentrationsofsomenutrientssuchasK+,Na+andMg++readilystimulatetheactivityofmajorenzymesofthe glycolytic pathway, namely phosphofructokinaseand pyruvate kinase, which together regulateglycolysis in plant cells (Plaxton, 1996). Individualmicronutrientsaresimilarlyimportantcomponentsofmajorenzymes,whichregulateallbiologicalprocesses
in plants. It is clear from these considerations thatlow nutrient availability can constraint plant growthin many environments of the world, especially thetropics where soils are extremely deficient in theseoligoelement nutrients (Pinton et al., 2007). SomespeciestypicallyexudeorganicacidanionsinresponsetoPandFedeficiencyorphytosiderophoresduetoFeandZndeficiency(Haynes,1990).
4. thE rhIzosphErE-InhabItIng MIcroFlora
4.1. diversity
The rhizosphere microflora include bacteria, fungi,nematodes, protozoa, algae and microarthrops(Raaijmakers et al., 2001). Of the soil microbes,98% cannot be cultured. Theiridentification, characterizationand the description of their roleare therefore particularly difficult.Recently, nucleic acid basedtechniques including analysis ofDNAandrRNAmoleculesfromsoilsamples have revealed enormousdiversity in the rhizosphereinhabiting microbial flora (Suzukietal.,2006).ThemolecularmethodsusedforsoilmicrobialdiversityarecoveredinthereviewbyNannipieriand collaborators (2003). Thenumberofmicrobialspeciespresentin soil may vary from thousandsto millions. Many studies indeedsuggest that the Proteobacteriaand the Actinobacteria form themost common of the dominantpopulations (>1%, usually muchmore) found in the rhizosphere ofmanydifferentplantspecies(Singhet al., 2007).These groups containmany“cultured”members.Theyarethemoststudiedoftherhizobacteria,andassuch,containthemajorityoftheorganisms investigated,bothasbeneficialmicrobial inoculants andaspathogens.
The specific content of rootexudates may create a niche thatinfluences which microorganismsare to colonize the rhizosphere,thereby altering the compositionand diversity of microorganismscolonizingtherhizosphereinaplantspecific manner (Grayston et al.,
1998). Plant species, plant developmental stage andsoil type have thus been indicated as major factorsdeterminingthecompositionofrhizospheremicrobialcommunities (Broecklingetal.,2008).Thatsaid, theextent towhich the above-cited factors contribute tomicrobial communities is not fully understood andthere are several contrasting reports in the literatureindicatingeitherplantorsoiltypeasdominantfactor(Nunanetal.,2005).Owingtotheabovestatement,itcanbegeneralizedthatthediversityandpredominanceof rhizosphere microbial population depend on anumberofabioticandbioticfactorsprevailinginthatparticularecologicalniche(Figure 1).
4.2. population level
Studies based on the use of growth media steadilyshowed that bacterial populations residing in the
Figure 1.Ecologicalfactorsinfluencingtherootexudationprocessandtherebyrhizosphere colonization by beneficial rhizobacteria— Facteurs écologiques influençant le processus d’exsudation racinaire et par conséquent, la colonisation de la rhizosphère par les rhizobactéries du sol.
rhizosphere are several orders of magnitude largerthanthoseresidinginbulksoils.Rhizospherebacteriaconcentrationcanreachbetween1010and1012cellspergram of soil (Foster, 1988), and they are transferredto various associated environments including plants,foods,animals,marineandfreshwaterhabitats(Buéeet al., 2009). Only few groups of these bacteria areconsidered to be soilborne, probably because non-sporeformingbacteriacannotsurvivewellinsoilforlongperiods.
Theeffectofrootexudatesdependsonthedistancethat they can diffuse away from rhizoplane (Guptaetal.,2002).Bacterialcommunitiesarenotuniformlydistributed along root axes, and differ between rootzones. Distinct bacterial community compositionsare obtained by molecular fingerprints in differentrootzones,likethoseofemergingrootsandroottips,elongating roots, sites of emergence of lateral roots,andolderroots(Yangetal.,2000).Ithasbeenproposedthat populations residing in the rhizosphere oscillatealongrootaxesinawave-likefashion(Semenovetal.,1999).Accordingly,bacterialcommunitiestemporarilyprofitfromthenutrientsreleasedbyyoungerrootsintheroothairzones,andwave-likefluctuationsinbacterialcell numbers can be explained by death and lysis ofbacterialcellsuponstarvationwhennutrientsbecomedepleted, followedbycelldivisions in survivingandthusviablepopulationsaspromotedbythereleaseofnutrientsfromdeadanddecayingcells(Semenovetal.,1999).Bacterialcommunitiesinrhizospheresoilsarethusnotstatic,butwillfluctuateovertimeindifferentrootzones.
4.3. the rhizosphere as a battle field
The number and diversity of microorganisms arerelated to the quantity and quality of the exudatesbut also to theoutcomeof themicrobial interactionsthat occur in the rhizosphere (Somers et al., 2004).Soilbiota (bacteria, fungi,micro-faunaand theplantroot) are themselves embedded in food webs andthus interactions with consumers or predators in themicrobial as well as macro- and mesofaunal worldare important to understand rhizosphere processes.A high number of soil microbes attained propertiesenabling them to interactmore efficientlywith rootsand withstand the quite challenging conditions ofrhizospherelife.
The rhizosphere inhabiting microorganismscompete each other for water, nutrients and spaceand sometimes improve their competitiveness bydeveloping an intimate association with plant. Thisprocess can be regarded as an ongoing process ofmicro-evolution in low-nutrient environments,whichare quite common in natural ecosystems (Schloteretal.,2000).
5. plant-bactErIa IntEractIons In thE rhIzosphErE
Microorganisms present in the rhizosphere playimportantrolesinecologicalfitnessoftheirplanthost.Important microbial processes that are expected tooccur in the rhizosphere includepathogenesisand itscounterpart,plantprotection/growthpromotion,aswellas theproductionof antibiotics, geochemical cyclingofmineralsandplantcolonization(Kentetal.,2002).Plant-microbe interactions may thus be consideredbeneficial,neutral,orharmfultotheplant,dependingonthespecificmicroorganismsandplantsinvolvedandontheprevailingenvironmentalconditions(Baisetal.,2006).Exploringthesemicroorganismsbyunravellingtheir possible relationships with plants has launcheda new and fascinating area of investigations in therhizosphereresearch.
5.1. pathogenic interactions
Roots exudates can attract beneficial organisms (seebelow), but they can also be equally attractive topathogenic populations (Schroth et al., 1964), thatmanyexpressvirulenceononly a limitednumberofhostspecies.Manypathogenicorganisms,bacteriaaswell as fungi, have coevolvedwith plants and showa high degree of host specificity (Raaijmakers et al.,2009).Innaturehowever,plantdiseaseistheexceptionrather than the rule because the conditions that areoptimizedfortheplantgrowthmaynotbefavourableforpathogens(Paulitzetal.,2001).
Plants are not defenceless. In fact, it is estimatedthatonlyabout2%oftheknownfungalspeciesareabletocolonizeplantsandcausedisease(Buchananetal.,2000). Even though plants are in permanent contactwith potential pathogens such as fungi, bacteria orviruses,successfulinfectionisrarelyestablished.Suchageneral resistanceagainstmostpathogenshasbeennamed“horizontalresistance”or“non-host-resistance”(Heath,1981).Thisreflectsthefactthattheplantisnotasuitabletargetforinfectionbyaspecificpathogenduetopreformed,passiveresistancemechanismsresultingin“basicincompatibility”.Theseresistancemechanismscomprisestructuralbarriersandtoxiccompoundsthatare present in the unaffected, healthy plant and limitsuccessfulinfectiontospecializedpathogensthathavethe ability to overcome these factors and thereforeexhibit“basiccompatibility”.Ifcontactisneverthelessestablishedwith theplant tissue,pathogensareoftenconfronted with preformed chemical componentsnamed phytoanticipins (van Etten et al., 1994). Thisterm comprises a variety of compounds producedby different biosynthetic pathways which possessantimicrobialproperties.Theselowmolecularweightsecondary metabolites are mainly stored in inactive
However, in some instances, pathogens canovercomethepre-formedbarriersanddevelopvirulentinfection processes leading to plant disease. Plantdiseasesplayadirectroleinthedestructionofnaturalresources in agriculture. In particular, soil-bornepathogenscauseimportantlosses,fungibeingthemostaggressive.Theextentoftheirharmfuleffectsrangesfrommildsymptomstocatastropheswherelargefieldsplanted with agricultural crops are destroyed. Thus,theyaremajorandchronicthreatstofoodproductionandecosystemstabilityworldwide.Commonandwellinvestigated bacterial agents include Gram- bacteriaErwinia carotovora, Pseudomonas, Ralstonia spp.and the Gram+ bacterium Streptomyces scabies.The fungal and oomycete phytopathogens includemembers of Fusarium, Phytophthora, Pythium,Rhizopus, Rhizoctonia and Verticillium (Tournas etal.,2005).Fromtheforestpathogens,amongthemostimportantarethefilamentousfungiHeterobasidionandArmillariella(Asiegbuetal.,2005),andPhytophthoraspp.(Rizzoetal.,2005).
5.2. beneficial microorganisms and modes of action
Plant-beneficial microbial interactions can beroughly divided into three categories. First, thosemicroorganisms that, in association with plants, areresponsibleforitsnutrition(i.e.,microorganismsthatcanincreasethesupplyofmineralnutrientstotheplant).Inthiscase,whilemostmaynotdirectlyinteractwiththeplant,theireffectsonsoilbioticandabioticparameterscertainly have an impact on plant growth. Second,thereisagroupofmicroorganismsthatstimulateplantgrowthindirectlybypreventingthegrowthoractivityofpathogens.Suchmicroorganismsarereferredtoasbiocontrolagents,andtheyhavebeenwelldocumented.A third group involves those microorganismsresponsiblefordirectgrowthpromotion,forexample,byproductionofphytohormones.Therehasbeenalargebody of literature describing potential uses of plantassociatedbacteriaasagentsstimulatingplantgrowthandmanaging soil andplantfitness (Welbaumet al.,2004).Onanotherhand,apparentlyneutralinteractionsare found extensively in the rhizosphere of all cropplants. Saprophytic microorganisms are responsibleformanyvital soil processes, such asdecompositionoforganicresiduesinsoilandassociatedsoilnutrientmineralization or turnover processes.Whereas theseorganismsdonotappear tobenefitorharm theplantdirectly (hence the term neutral), their presence isobviously vital for soil dynamic, and their absence
Rhizosphere-living bacteria that exert a globalbeneficialeffectonplantgrowtharereferredasplantgrowth promoting rhizobacteria (PGPR) (Kloepperetal., 1978). The number of bacterial speciesidentified as PGPR increased recently as a result ofthenumerousstudiescoveringawiderrangeofplantspeciesandbecauseoftheadvancesmadeinbacterialtaxonomyandtheprogressinourunderstandingofthedifferent mechanisms of action of PGPR. Presently,PGPR include representatives from very diversebacterialtaxa(Lucyetal.,2004)andinthefollowingsectionswe are not giving a thorough description ofall thegeneraandspeciesofPGPR,but rathera fewexamplestoillustratethediversityandmodesofactionof these beneficial bacteria. Diverse PGPR strainshave been used successfully for crop inoculationsThese comprise members of the bacterial generaAzospirillum(Cassánetal.,2008),Bacillus(Jacobsenet al., 2004), Pseudomonas (Loper et al., 2007),Rhizobium (Long, 2001),Serratia (DeVleeschauweret al., 2007), Stenotrophomonas (Ryan et al., 2009),and Streptomyces (Schrey et al., 2008). Some fungibelongingtothegeneraAmpelomyces,Coniothyrium,and Trichoderma have also been described to bebeneficialforthehostplant(Harmanetal.,2004).ThemodesofactionofPGPRinvolvecomplexmechanismstopromoteplantgrowth,developmentandprotection.Importantamongthemarebiofertilization(increasingtheavailabilityofnutrientstoplant),phytostimulation(plantgrowthpromoting,usuallybytheproductionofphytohormones) andbiocontrol (controllingdiseases,mainlybytheproductionofantibioticsandantifungalmetabolites, lytic enzymes and induction of plantdefenseresponses).PseudomonasandBacillusgeneraarethemostcommonlyinvestigatedPGPR,andoftenthe dominating bacterial groups in the rhizosphere(Morgan et al., 2005). One has to mention that, inmany cases of individual beneficial plant-microbeinteractions,severalmechanismsareinvolved(Mülleret al., 2009). Ad planta, direct mechanisms of plantgrowth promotion are difficult to differentiate fromdisease suppression and the relative importanceon a specific mechanism can vary within differentpathosystems(Chetetal.,2002).
colonization. In all successful plant-microbeinteractions,thecompetencetocolonizeplanthabitatsisimportant(Lugtenbergetal.,2002;Kamilovaetal.,2005). Single bacterial cells can attach to surfacesand, after cell division and proliferation, form denseaggregates commonly referred to as macrocoloniesor biofilms. Steps of colonization include attraction,recognition, adherence, invasion (only endophytesandpathogens),colonizationandgrowth,andseveral
strategies toestablish interactions.Plantroots initiatecrosstalkwithsoilmicrobesbyproducingsignalsthatarerecognizedbythemicrobes,whichinturnproducesignals that initiate colonization (Berg, 2009). PGPRreach root surfaces by active motility facilitated byflagellaandareguidedbychemotacticresponses(Pintonetal.,2007).ThisimpliesthatPGPRcompetencehighlydepends eitheron their abilities to take advantageofaspecificenvironmentorontheirabilitiestoadapttochangingconditionsorplantspecies.Asanexample,when strain S499 ofBacillus subtiliswas applied toplant seedlings, it showedmoredistinctbuteffectivecolonizationof the rootsystemof twodistinctplants(Figure 2). In most cases, the population of manyPGPR inoculants actually declines progressively intimeafterinoculationfrom107-109cellspergramdrysoilto105-106cellspergramdrysoilafter2-3weeks(DeFlaun et al., 1993). Nevertheless this populationthresholdisoftensufficienttoprovidebeneficialeffects(Raaijmakers et al., 2002). Rhizosphere competenceof biocontrol agents thus involves effective rootcolonizationcombinedwiththeabilitytosurviveandproliferatealonggrowingplantrootsoveralargetimeperiod, in the presence of the indigenous microflora(Weller,1988;Lugtenbergetal.,1999).
pathogen inhibition.Bacteria and fungi live aroundroots and feed on root exudates and dead root cells.Competitionbetweenmicrobialspeciesinthisareaisstiff.Inthebattleforestablishmentandpersistenceintheniche,bacteriauseseveralstrategies.
Antagonism. Root colonization not only results inhighPGPRpopulationdensitiesontherootsystem,it
also functions as the delivery system of antagonisticmetabolitesthatareinvolvedindirectinhibitionofplantpathogens (Shoda, 2000; Raaijmakers et al., 2002).It includes antibiosis i.e. the inhibition of microbialgrowth by diffusible antibiotics and volatile organiccompounds, toxins,andbiosurfactants,andparasitismthat may involve production of extracellular cellwall-degradingenzymessuchaschitinasesandβ-1,3-glucanase(Compantetal.,2005;Haasetal.,2005).Thedegradation of pathogenicity factors of the pathogensuchastoxinsbythebeneficialorganismhasalsobeenreportedasprotectivemechanism (Haaset al., 2005).To demonstrate the role of antibiotics in biocontrol,mutants impaired in biosynthesis or over-producingmutantshavebeenused togetherwith, insomecases,the use of reporter genes or probes to show efficientproduction of the compound in the rhizosphere. Asexample,Bacillus subtilis strainsproduceavarietyofpowerful antifungalmetabolites, e.g., zwittermicin-A,kanosamineandlipopeptidesfromthesurfactin,iturinand fengycin families (Emmert et al., 1999; Ongenaetal., 2006).Dunne and collaborators (2000) showedthat overproduction of extracellular protease in themutant strains ofStenotrophomonasmaltophiliaW81resulted in improved biocontrol ofPythium ultimum.Excretion of chitinases and glucanases by speciesof Trichoderma and Streptomyces has also beenshowntoplayanimportantroleinmycoparasitismofphytopathogenicfungi(Whipps,2001).
Competition. Competition for resources such asnutrientsandoxygenoccursgenerallyinsoilbetweensoil-inhabiting organisms. For biocontrol purpose,it occurs when the antagonist directly competes
Figure 2.RootcolonizationbystrainS499ofBacillus subitilis — Colonisation racinaire par la souche de Bacillussubtilis S499.
Microscopyvisualizationofrootof — Visualisation par microscopie des racines de:a:treatedsalad — salade traitée;b:untreatedsalad — saladenon traitée;c:treatedtomato — tomate traitée;d:untreatedtomato — tomate non traitée;Thearrowsinaandcindicatethebiofilmformation — Les flèches en a et c indiquent la formation de biofilm;Thecrosses-surroundedinbanddindicateabsenceofbiofilmformationbythestrainontherootsystem — Les croix entourées en b et d indiquent l’absence de formation de biofilm sur le système racinaire par la souche;Surface-sterilizedtomatoandsaladseedsweresuspendedinbacterialsuspensionof108cells.ml-1andgerminatedfortwoweeksintheconditionedchamberandingelifiedsterileplantnutrientmediumasdefinedbyMurashigeandSkoog(1962) — Les graines de tomate et de salade stérilisées sont suspendues dans une solution bactérienne de 108 cellules.ml-1 et sont mises à germer pendant deux semaines dans une chambre d’air conditionnée sur un milieu nutritif pour plantes mis au point par Murashige et Skoog (1962).
with pathogens for these resources. Root inhabitingmicroorganisms compete for suitable sites at the rootsurfaces. Competition for nutrients, especially forcarbon,isassumedtoberesponsibleforthewell-knownphenomenonoffungistasischaracterizingtheinhibitionoffungalsporegerminationinsoil(Alabouvetteetal.,2006). Given the relative abundance of substrates inthe rhizosphere, the efficiency of nutrient uptake andcatabolismbybacteriaisakeyfactorincompetitiveness(Chin-A-Woeng et al., 2003). The capacity for rapidgrowthwhensubstratesareencounteredisnottheonlyfactoraffectingrhizospherecompetence,asrhizobacteriadeploymany othermetabolic strategies. For example,the capacity for extracellular conversionof glucose togluconic acid and 2-ketogluconic acid enables somebacteria, including several species ofPseudomonas tosequester glucose effectively and gives a competitiveadvantageovermicroorganismsthat lacktheability toutilisethesecompounds(Gottschalk,1986).
Competitionfortraceelements,suchasiron,copper,zinc,manganese,etc.alsooccursinsoils.Forexample,ironisanessentialgrowthelementforalllivingorganismsandthescarcityofitsbio-availableforminsoilhabitatsresults in a furious competition (Loper et al., 1997).Siderophores, low molecular weight compounds withhighironaffinity,areproducedbysomemicroorganisms(also by most biocontrol agents) to solubilize andcompetitively acquire ferric ion under iron-limitingconditions, thereby making iron unavailable to othersoilmicroorganismswhich cannot grow for lack of it(Loperetal.,1997;Haasetal.,2005).Thebacteriumthatoriginallysynthesizedthesiderophorestakesuptheironsiderophorecomplexbyusingareceptorthatisspecifictothecomplexandislocatedintheoutercellmembrane.Suppressionofsoilborneplantpathogensbysiderophoreproducing Pseudomonads has been reported in someinstances(Loper,1988;Wegeretal.,1988;Buysensetal.,1996).
Induced resistance.Plant-associatedbacteriacanreducethe activity of pathogenic microorganisms not onlythroughmicrobial antagonisms, but also by activatingtheplant tobetterdefenditself,aphenomenontermed“inducedsystemicresistance”,“ISR”(Shoda,2000;VanLoon,2007).Sometimes,themechanismofISRelicitedbyPGPRoverlapspartlywiththatofpathogen-inducedsystemicacquiredresistance(SAR).BothISRandSARrepresentastateofenhancedbasalresistanceoftheplantthatdependsonsignallingcompoundssuchasjasmonicacid, ethylene and salicylic acid (Van Loon, 2007).Expressionofnaturaldefense reactionagainst stressesfrombioticorabioticorigin isexhibitedbyallplants,suchas:- physicalstresses(heatorfrost);- inoculation by pathogenic or non-pathogenic organisms;
Early recognition of the aggressor by the plantis one of the mechanisms involved in elicitation ofplant defense reactions (Lugtenberg et al., 2002).Recognition of the aggressor immediately initiates acascadeofmolecular signalsand the transcriptionofmanygenes,whicheventuallyresultsintheproductionof defence molecules by the host plant (van Loon,2000).Suchdefencemolecules includephytoalexins,pathogenesis-related(PR)proteins(suchaschitinases,β-1,3-glucanases,proteinaseinhibitors,etc.)andligninfor reinforcement of cellwalls (vanLoon, 2000). Infact, cell wall thickenings, wall appositions or rapiddeathoftheinjuredplantcellsresultinginnecrosisofthe immediateadjacent tissuesarebarrierswhichcutthepathogenoffitsnutrientsandcontributetoslowingdownofthefungusprogressiveinvasion(Lugtenbergetal.,2002;Alabouvetteetal.,2006).
plant growth promotionPhytostimulation. Phytostimulation enhances plantgrowthinadirectway.Intheprocessesofplantgrowth,phytohormones [e.g., production of indole-3-aceticacid(IAA),auxins,cytokinins,andgibberellins]playanimportantrole.Thesehormonescanbesynthesizedby the plant themselves but also by their associatedmicroorganisms such as Azospirillum spp., besideshavingnitrogen-fixingability(Steenhoudtetal.,2000).SpeciesofPseudomonasandBacilluscanproduceasyet notwell characterized phytohormones or growthregulators that cause crops to have greater amountsof fine rootswhich have the effect of increasing theabsorptive surface of plant roots for uptake ofwaterand nutrients. The phytohormones they produceinclude indole-acetic acid, cytokinins, gibberellinsandinhibitorsofethyleneproduction.Indole-3-aceticacidisaphytohormonewhichisknowntobeinvolvedin root initiation, cell division, and cell enlargement(Salisbury, 1994). This hormone is very commonlyproduced by PGPRs (Barazani et al., 2001).Auxinsare quantitatively themost abundant phytohormonessecreted by Azospirillum, and it is generally agreedthat their production, rather than nitrogen-fixation,is themajor factor responsible for the stimulationofrootingand,hence,enhancedplantgrowth(Bloembergetal., 2001). Furthermore, plant-associated bacteriacan influence the hormonal balance of the plant.Ethylene is an important example to show that thebalanceismostimportantfortheeffectofhormones:at low levels, it canpromoteplantgrowth in severalplantspeciesincludingArabidopsis thaliana,whileitisnormallyconsideredasaninhibitorofplantgrowthand known as a senescence hormone (Pierik et al.,2006).
The general effect on the plant can be direct,that is throughplantgrowthpromotion,or indirect,that is through improving plant nutrition via thebetter development of the roots, and it is difficultto distinguish between them. The elevation of rootIAAlevelinlodgepolepineplantlets,inoculatedwithPaenibacillus polymyxa, and, of dihydroxyzeatinriboside root concentration in plants inoculatedwith Pseudomonas fluorescens (Fuentes-Ramirezetal.,2005),mightbeattributedtotheinductionofplant hormone synthesis by the bacteria. However,the uptake of bacterial synthesized phytohormonescan not be excluded, since both P. polymyxa andPseudomonas produce IAA and cytokinins in vitro(Fuentes-Ramirezetal.,2005).
Biofertilization. The mechanisms by which PGPRincreases crop performance is notwell understood.There are several PGPR inoculants currentlycommercialized that seem to promote growththrough at least one mechanism; suppression ofplantdisease(termedbioprotectants),phytohormoneproduction (termed biostimulants), or improvednutrient acquisition (termed biofertilizers). ThemodeofactionofPGPRbybiofertilizersacteither,directly by helping to provide nutrient to the hostplant, or indirectly by positively influencing rootgrowthandmorphologyorbyaidingotherbeneficialsymbiotic relationships (Vessey, 2003). The mostprominent example is bacterial nitrogen fixation.Thesymbiosisbetweenrhizobiaanditslegumehostplants is an important example for plant growth-promoting rhizobacteria (PGPR). Bacteria of thisgroupmetabolizerootexudates(carbohydrates)andin turnprovidenitrogen to theplant foraminoacidsynthesis.The ability to fix nitrogen also occurs infree-livingbacteria likeAzospirillum,Burkholderia,and Stenotrophomonas (Dobbelare et al., 2003).Biofertilization accounts for approximately 65% ofthenitrogensupply tocropsworldwide(Bloemberget al., 2001).Anothernutrient is sulfate,which canbe provided to the plant via oxidation by bacteria(Banerjee et al., 2002). Bacteria may contributeto plant nutrition by liberating phosphorous fromorganic compounds such as phytates and thusindirectlypromoteplantgrowth(Unnoetal.,2005).Azospirillum treatment resulted in enhancement ofroot growth and activities (e.g., acidification of theroot surroundings) that increases phosphorous andother macroelements and microelements uptake(Dobbelaere et al., 2007). Recently, De Werraand collaborators (2009) showed that the abilityof Pseudomonas fluorescens CHA0 to acidify itsenvironment and to solubilizemineral phosphate isstronglydependentonitsabilitytoproducegluconicacid.
6. conclusIon
Therhizosphereisthezoneofsoilsurroundingaplantrootwhere the biology and chemistry of the soil areinfluenced by the root.As plant roots grow throughsoil they mostly release water soluble compoundssuch as amino acids, sugars and organic acids thatsupply food for the microorganisms. High levelsof exudates in the rhizosphere attract a plethora ofmicroorganisms to a larger extend than elsewhere inthesoil.Thecompositionandpatternofrootexudatesaffect microbial activity and population numbers.Plantspecies,plantdevelopmentalstageandsoiltypehavebeen indicatedasmajor factorsdetermining thecomposition of rhizosphere microbial communities(Broecklinget al., 2008).As shown inmany studies,there isnogeneraldecisionabout thekeyplayer: thediversity and predominance of rhizospheremicrobialpopulationdependon a numberof abiotic andbioticfactorsofaparticularecologicalniche.
A better understanding of the basic principles ofthe rhizosphere ecology, including the function anddiversityof inhabitingmicroorganisms ison thewaybut further knowledge is necessary to optimize soilmicrobial technology to the benefit of plant-growthand health in the natural environment. In sum, thiscan constitute overwhelming evidence indicatingthat an ever exploitation of plant growth promotingrhizobateria (PGPR) can be a true success story insustainable agriculture. As a consequence, currentproductionmethods in agriculture,e.g., the improperuseofchemicalpesticidesandfertilizerscreatingalonglistofenvironmentalandhealthproblems,willreduce.
acknowledgements
VenantNihorimbereisrecipientofagrantfromtheBelgianTechnical Cooperation (BTC/CTB). Marc Ongena isResearchAssociateattheF.R.S.-FNRS(NationalFundsforScientificResearch,Belgium).
bibliography
AlabouvetteC.,OlivainC.&SteinbergC.,2006.Biologicalcontrolofplantdiseases:theEuropeansituation.Eur. J. Plant Pathol.,114,329-341.
AsiegbuF.O. & NahalkovaJ.L.G., 2005. Pathogen-inducible cDNAs from the interaction of the root rotfungusHeterobasidion annosumwithScotspine(Pinus sylvestrisL.).Plant Sci.,168,365-372.
BowenG. & RoviraA., 1999. The rhizosphere and itsmanagementtoimproveplantgrowth.Adv.Agron.,66,1-102.
BrimecombeM.J., De LeijF.A.A.M. & LynchJ.M.,2007. Rhizodeposition and microbial populations.In: PintonR., VeraniniZ. & NannipieriP., eds. The rhizosphere biochemistry and organic substances at the soil-plant interface.NewYork,USA:Taylor&FrancisGroup.
BroecklingC.D. et al., 2008. Root exudates regulate soilfungal community composition and diversity. Appl. Environ. Microbiol.,74,738-744.
BuchananR.,GruissemW.&JonesR.L.,2000.Biochemistry and molecular biology of plants.Rockville,MD,USA:AmericanSocietyofPlantBiologists.
BuéeM. et al., 2009. The rhizosphere zoo: an overviewof plant-associated communities of microorganisms,including phages, bacteria, archaea, and fungi, and ofsomeoftheirstructuringfactors.Plant Soil,321,189-212.
CassánF. & García SalamoneI., 2008. Azospirillum sp.: cell physiology, plant response, agronomic and environmental research in Argentina. BuenosAires:AsociacionArgentinadeMicrobiologia.
ChetI.&CherninL.,2002.Biocontrol,microbialagentsinsoil. In: BittonG., ed.Encyclopedia of environmental microbiology.NewYork,USA:Willey,450-465.
Chin-A-WoengT.F.C.,BloembergG.V.&LugtenbergB.J.,2003. Phenazines and their role in biocontrol byPseudomonasbacteria.New Phytol.,157,503-523.
CompantS. et al., 2005. Use of plant growth-promotingbacteria for biocontrol of plant diseases: principles,mechanisms of action, and future prospects. Appl. Environ. Microbiol.,71,4951-4959.
De VleeschauwerD. & HöfteM., 2007. Using Serratia plymuthica tocontrol fungalpathogensofplants.CAB Rev.,2,46.
DeFlaunM.F.&GerbaC.P.,1993.MonitoringrecombinantDNA microorganisms and viruses in soil. In:MettingF.B.J.,eds.Soil microbial ecology: application in agricultural and environmental management.Washington:MarcelDekkerInc.,131-150.
DobbelareS., VanderleydenJ. & OkonY., 2003. Plant-growth promoting effects of diazotrophs in therhizosphere.Crit. Rev. Plant Sci.,22,107-149.
DobbelaereS. & OkonY., 2007. The plant growthpromotingeffectsandplant responses. In:ElmerichC.& NewtonW.E., eds. Associative and endophytic nitrogen-fixing bacteria and cyanobacterial associations (Nitrogen fixation: origins, applications and research progress).Heidelberg,Germany:Springer,145-170.
DunneC.,Moenne-LoccozY., deBruijnF.J.&O’GaraF.,2000. Overproduction of an inducible extracellularserineproteaseimprovesbiologicalcontrolofPythiumultimumbyStenotrophomonas maltophilia strainW81.Microbiology,146,2069-2078.
GraystonS.J.,WangS.Q.,CampbellC.D.&EdwardsA.C.,1998.Selectiveinfluenceofplantspeciesonmicrobialdiversity in the rhizosphere. Soil Biol. Biochem., 30,369-378.
HaynesR.J., 1990.Active ion uptake andmaintenance ofcationanion balance: a critical examination of theirroleinregulatingrhizospherepH.Plant Soil,126,247-264.
HeathM.C., 1981.A generalized concept of host-parasitespecificity.Phytopathology,71,1121-1123.
HiltnerL.,1904.ÜberneuereErfahrungenundProblemeaufdemGebiete der Bodenbakteriologie unter besondererBerücksichtigung der Gründüngung und Brache. Arb. Dtsch. Landwirtsch. Ges.,98,59-78
JacobsenB.J., ZidackN.K. & LarsonB.J., 2004. Therole of Bacillus-based biological control agents inintegrated pest management systems: plant diseases.Phytopathology,94,1272-1275.
JensenB., 1993. Rhizodeposition by 14CO2-pulse-labelledspringbarleygrowninsmallfieldplotsonsandyloam.Soil Biol. Biochem.,25,1553-1559.
JonesD.L.&DarrahP.R.,1995.Influxandeffluxoforganicacidsacross thesoil-root interfaceofZeamaysL.andits implications in rhizosphereCflow.Plant Soil,173,103-109.
KamilovaF. et al., 2005. Enrichment for enhancedcompetitive plant root tip colonizers selects for a newclass of biocontrol bacteria. Environ. Microbiol., 7,1809-1817.
KloepperJ.W. & SchrothM.N., 1978. Plant growthpromoting rhizobacteria on radish. In: Station depathologie végétale et phyto-bacteriologie, ed.Proceedings of the 4th Conference plant pathogenic bacteria, Angers, INRA,879-882.
KochianL., PiñerosM. & HoekengaO., 2005. Thephysiology, genetics and molecular biology of plantaluminumresistanceandtoxicity.Plant Soil,274,175-195.
LongS.R., 2001. Genes and signals in the Rhizobium-legumesymbiosis.Plant Physiol.,125,69-72.
LoperJ.E., 1988. Role of fluorescent siderophoreproductioninbiologicalcontrolofPythium ultirnumbyaPseudomonas fluorescensstrain.Phytopathology,78,166-172.
LoperJ.E. & HenkelsM.D., 1997.Availability of iron toPseudomonas fluorescens in rhizosphere and bulk soilevaluated with an ice nucleation reporter gene. Appl. Environ. Microbiol.,63,99-105.
LoperJ.E.&GrossH.,2007.GenomicanalysisofantifungalmetaboliteproductionbyPseudomonasfluorescensPf-5.Eur. J. Plant Pathol.,119,265-278.
LucyM.,ReedE.&GlickB.R.,2004.Applicationsoffreeliving plant growth promoting rhizobacteria. Antonie Van Leeuwenhoek,86,1-25.
LugtenbergB.J.J. & DekkersL.C., 1999. What makesPseudomonasbacteriarhizospherecompetent.Environ. Microbiol.,1,9-13.
LugtenbergB.J.J.,Chin-A-WoengT.F.C.&BloembergG.V.,2002. Microbe-plant interactions: principles andmechanisms.Antonie Van Leeuwenhoek,81,373-383.
MorganJ.A.W. & WhippsJ.M., 2001. Methodologicalapproaches to the study of rhizosphere carbon flowand microbial population dynamics. In: PintonA.,VaraniniZ. & NannipieriP., eds. The rhizosphere. Biochemistry and organic substances at the soil-plant interface. New York, USA: Marcel Dekker, 373-409.
MorganJ.A.,BendingG.D.&WhiteP.J.,2005.Biologicalcosts and benefits to plant-microbe interactions in therhizosphere.J. Exp. Bot.,56,1729-1739.
MüllerH. et al., 2009. Quorum-sensing effects in theantagonisticrhizospherebacteriumSerratiaplymuthicaHRO-C48.FEMS Microbiol. Ecol.,67,468-478.
MurashigeT.&SkoogF.,1962.Arevisedmediumforrapidgrowth and bioassays with tobacco cultures. Physiol. Plant,15,473-497.
NannipieriP. et al., 2003. Microbial diversity and soilfunctions.Eur. J. Soil Sci.,54,655-670.
NunanN.etal.,2005.Linksbetweenplantandrhizoplanebacterial communities in grassland soils, characterizedusingmolecular techniques.Appl. Environ. Microbiol.,71,6784-6792.
OngenaM. & ThonartP., 2006. Resistance induced inplants by non-pathogenic microorganisms: elicitationand defense responses. In: Teixeira da SilvaJ.A., ed.Floriculture, ornamental and plant biotechnology: advances and topical issues. London: Global ScienceBooks,447-463.
PierikR. et al., 2006.The Janus face of ethylene: growthinhibition and stimulation.Trends Plant Sci., 11, 176-183.
PintonR., VeraniniZ. & NannipieriP., 2007. The rhizosphere. Biochemistry and organic substances at the soil-plant interface.NewYork,USA:Taylor&FrancisGroup,LLC.
PlaxtonW.C., 1996. The organization and regulation ofplant glycolysis.Annu. Rev. Plant Physiol.Plant Mol. Biol.,47,185-214.
RaaijmakersJ.M. & WellerD.M., 2001. Exploitinggenotypic diversity of 2,4-diacetylphloroglucinol-producing Pseudomonas spp.: characterization ofsuperior root-colonizing P. fluorescens strain Q8r1-96.Appl. Environ. Microbiol.,67,2545-2554.
RaaijmakersJ.M., VlamiM. & de SouzaJ.T., 2002.Antibiotic production by bacterial biocontrol agents.Antonie Van Leeuwenhoek,81,537-547.
RaaijmakersJ.M.etal.,2009.Therhizosphere:aplaygroundand battlefield for soilborne pathogens and beneficialmicroorganisms.Plant Soil,321,341-361.
RatnayaleM., LeonardR.T. & MengeA., 1978. Rootexudation in relation to supply of phosphorus and itspossiblerelevancetomycorrhizalinfection.New Phytol.,81,543-552.
RizzoD.M., GarbelottoM. & HansenE.A., 2005.Phytophthora ramorum: integrative research andmanagement of an emerging pathogen in CaliforniaandOregon forests.Annu. Rev. Phytopathol.,43, 309-335.
RudrappanT.,QuinnW.J.,Stanley-WallN.R.&BaisH.P.,2007.Adegradationproductofthesalicylicacidpathwaytriggers oxidative stress resulting in down-regulationof Bacillus subtilis biofilm formation on Arabidopsis thalianaroots.Planta,226,283-297.
SuzukiM. et al., 2006. Biosynthesis and secretion ofmugineicacidfamilyphytosiderophoresinzinc-deficientbarley.Plant J.,48,85-97.
TournasV.H.&KatsoudasE.,2005.Mouldandyeastflorain fresh berries, grapes and citrus fruits. Int. J. Food Microbiol.,105,11-17.
UnnoY.etal.,2005.Plantgrowthpromotionabilitiesandmicroscale bacterial dynamics in the rhizosphere oflupin analysed by phytate utilization ability. Environ. Microbiol.,7,396-404.
UrenN.C.,2001.Types,amountsandpossiblefunctionsofcompoundsreleasedintotherhizospherebysoil-grownplants. In: PintonR.,VaraniniZ.&NannipieriP., eds.The rhizosphere. Biochemistry and organic substances at the soil-plant interface. New York, USA: MarcelDekker,19-40.
vanEttenH.D.,MansfieldJ.W.,BaileyJ.A.&FarmerE.E.,1994. Two classes of plant antibiotics: phytoalexinsversusphytoanticipins.Plant Cell,6,1191-1192.
vanLoonJ.C.,2000.Inducedresistance.In:SlusarenkoA.J.,FraserR.S.S. & Van LoonJ.C., eds. Mechanisms of resistance to plant diseases.Dordrecht,TheNetherlands:KluwerAcademicPublishers,521-574.
VanLoonL.C., 2007. Plant responses to plant growthpromotingbacteria.Eur. J. Plant Pathol.,119,243-254.
WacquantJ.P.,OukniderM.&JacquardP.,1989.Evidencefor a periodic excretion of nitrogen by roots of grass-legumeassociations.Plant Soil,116,57-68.
WegerL.A. et al., 1988. Siderophore-mediated uptake ofFe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizospheremicroorganisms.J. Bacteriol.,170,4693-4698.
