Life in earth – the root microbiome to the rescue?

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Mauchline, T. H. and Malone, J. G. 2017. Life in earth – the root

microbiome to the rescue? Current Opinion in Microbiology. 37

(Supplement C), pp. 23-28.

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Life

in

earth

–

the

root

microbiome

to

the

rescue?

Tim

H

Mauchline

and

Jacob

G

Malone

Manipulationofthesoilmicrobiomeholdsgreatpromisefor contributingtomoreenvironmentallybenignagriculture,with soilmicrobessuchasPseudomonaspromotingplantgrowth andeffectivelysuppressingpathogenicmicroorganisms. Next-generationsequencinghasenabledanewgenerationof researchintosoilmicrobiomes,presentingtheopportunityto betterunderstandandexploitthesevaluableresources.Soil bacterialcommunitiesarebothhighlycomplexandvariable, andcontainvastinterspeciesandintraspeciesdiversity,both ofwhichrespondtoenvironmentalvariation.Therefore,we proposethatacombinationofwholemicrobiomeanalyseswith in-depthexaminationofkeymicrobialtaxawilllikelyprovethe mosteffectiveapproachtounderstandingrhizosphere microbialinteractions.Thisreviewhighlightsrecenteffortsin thisdirection,basedaroundtheimportantbiocontrolbacterium

Pseudomonasfluorescens.

Addresses

1

DepartmentofAgroEcology,RothamstedResearch,Harpenden,UK 2_{MolecularMicrobiologyDepartment,JohnInnesCentre,Norwich,UK} 3_{SchoolofBiologicalSciences,UniversityofEastAnglia,Norwich,UK}

Correspondingauthor:Malone,JacobG(jacob.malone@jic.ac.uk)

CurrentOpinioninMicrobiology2017,37:23–28

ThisreviewcomesfromathemedissueonEnvironmental microbiology

EditedbyMarcioCSilvaandJorgeVivanco

ForacompleteoverviewseetheIssueandtheEditorial

Availableonline21stApril2017

http://dx.doi.org/10.1016/j.mib.2017.03.005

1369-5274/ã2017TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

The Green Revolution boosted global agricultural production in the 20th century through innovations centredonthedevelopmentofhigh-yieldingdwarfcrop varieties that respond well to chemical fertilizers and other agrochemicals. It is estimated that this process savedbetween18–27millionhectaresoflandfrombeing convertedtoagriculture[1]andthattheassociatedyield gains prevented over one billion people from starving. However, thecontinued heavy use of agrochemicals is costly, ecologically damaging, and unsustainable in the medium to long term.Theuse of precision agriculture, involving the better use of external inputs alongside genetically modified cropswith more efficient nutrient-use characteristics is likely to be hugely important in

achievingfutureproductivitygains[2].Additionally,the soilmicrobiome holdsgreatpromise for contributingto more environmentally benign agriculture. Naturally occurring soil-dwellingmicrobes influenceplant health, resource-use efficiency and biocontrol [3,4]. However, theirpotentialhasbeenunder-exploitedtodate.Recent advances in nucleic acid sequencing technologies have enabledanew generationofresearchintosoilmicrobial communities,andoffertheopportunityto better under-stand,andhenceexploit,thisresource.

Advances

in

soil

microbiome

analysis

Soilmicrobiomesareintricate,highlydiverseecosystems containing thousands of interacting microorganisms—a recentanalysisofthemicrobiomeofdisease-suppressive soilsidentified over33000bacterialandarchaealOTUs inthesugarbeetrhizosphere[5].Recently,theabilityto rapidly sequenceand identifyDNAextracted fromsoil sampleshasenabledthedevelopmentofseveralpowerful metagenomicanalysistechniques[6].Forexample, inter-rogationofthegeneticsofwholemicrobialcommunities allows us to probe the physiological characteristics and potentialofplant-associatedmicroorganisms[7,8]. Ampli-con sequence analysis of marker genes, typically 16S rRNA in the caseof bacteria, enable usto characterize the relative abundance of different species in phyllosphere and rhizosphere communities [9], while metatranscriptomicapproachesmaybeusedto examine themetabolicactivitiesand regulatorymechanismsthat functionin differentenvironments[10–12].

reconstructionofadraftgenomefromanovelsoil metha-nogenindicatesthatthismaybecomemorecommonplace inthefuture[25].Nonetheless,inrealitythe reconstruc-tionof discretemicrobial genomeswill alwaysbe prob-lematic. Bacterial genomes are composed of multiple, often plastic genetic elements, leading to problems in assemblinggenomecomplements.Thisisespeciallythe casein complexcommunities where speciescomplexes arecommonplace.Therefore,advancesinsequence anal-ysiswill mostlikelygiverise to thecreationof‘species metagenomes’. The production of broader culturable metagenomes[26],coupledwith anincreasedability to sequenceindividualmicrobialisolateswillbeusefulfor verifyinggenomereconstructionfrommetagenomes,and alsoforuseinmanipulativeexperimentation.Wepropose thatacombinationoftotalcommunitystudies,withmore in-depth analysis of key culturable microbial taxa will furtherourunderstandingofrhizospheremicrobial inter-actions more effectively than either approach taken in isolation.

Biocontrol

pseudomonads

in

the

soil

microbiome

Astheharmfulenvironmental impactsof chemical pes-ticidesbecomemore apparent, manipulationof thesoil andplant-associatedmicrobiotaisgainingincreasing rec-ognitionasapotentialalternativetreatmentforarangeof crop diseases and pests. This may occur on a whole-microbiomelevel,forexamplethroughthedevelopment of suppressive soils or the control of potato scab by irrigation,or alternativelythroughthe stimulation/intro-ductionofkeybiocontrolmicroorganisms,suchasBacillus

orPseudomonasspp.Manyimportantfungalandbacterial diseases including fire blight (Erwinia amylovora, [27]), potatoscab(Streptomycesscabies,[28])andtake-all( Gaeu-mannomycesgraminisvar.tritici,[29])areeffectively sup-pressedbymembersofthePseudomonasfluorescensspecies group. These important, widespread soil-dwelling microbes have an established role in the development of take-all suppressive soils [29–33], where the fungal pathogenis maintained ata low levelin the soilbut is unabletocausedisease.Take-allisadestructivefungal cropdiseasethatcausessubstantiallossesincerealcrops [34,35],andisthereforeanattractivetargetforthe devel-opment of Pseudomonas biocontrol agents. However, to dateeffortsinthisdirectionhavebeenplaguedby incon-sistency[36],inlargepartduetothehugecomplexityof theplant/pathogen/soilecosystem.

Pseudomonas

fluorescens

P.fluorescensareadiversecladeofGramnegative,g -pro-teobacteria that non-specifically colonise a number of differentplantspecies.Theyrepresentamajor constitu-ent of the rhizosphere microbiome, and exploit root exudatesassourceofnutrientsandenergy.P.fluorescens

spp. are flexible, generalist bacteria that are able to colonisemanydifferentenvironmentalnichesandcarbon

sources. Their genomes are correspondingly complex, encodingaround6000genes,and withahighdegree of intraspecies diversity—the Pseudomonas core genome represents as little as 20% of an individual bacterial genome[19],withmuchoftheaccessorygenomegiven over to signal transduction, phenotypic output loci and secondary metabolism [15,19]. The high degree of genomicandmetabolicplasticityamongthesoil pseudo-monadsallowsbothindividualbacteria,andthemicrobial population as a whole, to effectively adapt to different plant–soil–microbiomeenvironments.

Pseudomonasplantcolonisationisacomplex,tightly con-trolled process that begins with chemotaxis into the rhizospherealong a gradient of root exudates, followed by surface association and migration on the rhizoplane [37],andultimately theformation ofabacterial biofilm [38].The earlystages of colonisationarefacilitated by flagellaandtypeIVpili,andtheproductionof biosurfac-tants,whichtogetherenablecoordinatedswarming motil-ity [37,39]. The later stages are characterised by the formation of micro-colonies on the plant surface, then establishmentofamaturebiofilm.Inadditiontobacterial cellsthisprotectivematrixiscomposedofproteinaceous adhesins[40],lipopolysaccharide[41]andvarious exopo-lysaccharidemolecules[38,42].Tosuccessfullycolonise the plant rhizosphere, many Pseudomonas spp. produce enzymesthatenablethemtomanipulateplants, encour-aginggrowthanddisruptingstressresponses.For exam-ple,enzymesthatsynthesise andcataboliseauxins[15] and plant growth-promoting volatiles such as 2-3-buta-nedioland acetoin[43] have beenidentified in several

Pseudomonasgenomes[15,19].Inaddition,many Pseu-domonas spp. produce ACC deaminase, which protects plants from environmental stresses by short-circuiting ethyleneproduction[44].

P.fluorescensintherhizosphereisundercontinuousattack fromothermembersof thesoilmicrobiome.Thistakes the form of competition and antagonism from other microorganisms,as wellaspredationbynematodes and insects. To counter this second threat, and to prevent insect predationof theirhost plants,many Pseudomonas

spp. produce insecticidal molecules such as the Mcf, IPD072AaandFittoxins[15,45,46].Meanwhile,tofight againsthostile bacteria, oomycetes and fungi,soil Pseu-domonasspp.secretebacteriocins[47,48],alongsidetoxins and other natural products using specialised protein secretion pathways. Type III and Type VI complexes inject toxins and effector proteins into eukaryotic and bacterialcells,andcontributetovariouscytotoxicityand virulence-associatedphenotypes[49].TypeIIsecretion systemsarediverse proteinexporters,and facilitatethe secretion ofbacteriocins, surfaceadhesinsand extracel-lular enzymes [40]. Pseudomonas secrete a number of these exoenzymes including plant tissue-degrading lyases, proteinases and chitinases that contribute to

biocontrolbyhydrolysingfungalcellwalls[50,51].Aswell asaffectingplantbehaviour,somePseudomonasspp.also disrupt signal transduction by other rhizosphere micro-organisms, for examplebyproducing AHL lactonaseto suppressquorumsensing[52].

Pseudomonasspp.alsoproduceadiversearrayofsecreted naturalproducts. Thesehavevaried functions,although manyserve tokillor suppressplant predatorsand com-peting microorganisms [15,53]. Even those molecules withawell-definedalternativefunctionoftenfunctionas antimicrobials. These include the metal ion-chelating siderophores, which also inhibit pathogenic fungi by inducing metal ion starvation in model rhizospheres [54]. Phenazines; flavin coenzyme analogues that func-tionas electronshuttlesin microoxicenvironments[55] alsoinhibitelectrontransportinplantpathogens[56,57], and are linked to ecologicalfitness in take-allinfected wheat rhizospheres [58]. Likewise, viscosin and other cyclic lipopeptides act both as surfactants to enable swarmingmotility[37],andantibioticsthatsolubilisecell membranes [59]. Soil Pseudomonas spp. also produce a host of dedicated antimicrobials,such as the antifungal compoundspyoluteorin andpyrrolnitrin [60,61], phloro-glucinolslike2-4-DAPG[62],andhydrogencyanide[63]. A recentlyconducted metabolic profilinganalysisbased onsoilisolatesfromRothamsted Research(Harpenden, U.K.)demonstratedaremarkablelevelofnaturalproduct diversitywithintherhizospherePseudomonaspopulation, withisolatesfromasinglewheatfieldproducinga com-parable natural product complement to an extensive library of global isolates from diverse environmental sources [64].

Dependingontheexactconditionsintheirenvironment,

P. fluorescenspopulations selectfromthehugepotential within the accessory genome to produce an optimal geneticandmetabolicresponse.Clearly,ifwecandefine thegeneticlociandphenotypiccharacteristicsthat con-tribute to rhizosphere colonisation and biocontrol, and determine how these change with different plant/soil environments,we willbemuchbetter placedto exploit the soil Pseudomonas population to develop better crop management strategiesandnovelbiocontrolagents.

Analyzing

genomic

diversity

in

plant

associated

Pseudomonas

populations

A recent two-year experiment at Rothamsted [65] pre-senteduswithanopportunitytoexaminetherelationship betweenthePseudomonasgenomeandtheenvironment, inthecontextofinfectionwithtake-all.Thisexperiment compared high (Hereward) and low (Cadenza) take-all inoculumbuilding(TAB)wheatvarieties,andtheimpact on crop yield in the second wheat [65]. We isolated hundreds of Pseudomonas CFUs from the rhizospheres of second year wheat plants, and subjected them to

extensive phenotypic, genotypic and genomic analysis, includingwholegenomesequencingof 19isolates.

AphylogenetictreeofallPseudomonasisolatesbasedon ERIC PCR profiles and housekeeping gene sequences showedthatthewheatvarietygrowninyearoneexerted considerable selective pressure on both the extentand natureofPseudomonasgenomicdiversity.Herewardplots showed increased take-all build-up and Pseudomonas

genomicrichness,alongsideyieldlossesof3t/ha( Fig-ure1)[66].However,whiledistinctclustersofgenotypes wereobservedwhenyearonewheatvarietywas consid-ered, no pattern was observed with cultivars from year two.Thesefindingsagreewitha16SrRNAgene ampli-consequenceanalysisoftherhizospheresoilineachplot, which showed that year one Hereward plots contained significantly larger Pseudomonas populations, alongside severaldifferentgeneraof saprophytes[21].

Wethentookastatisticalapproachtocombineourvarious datasets, conducting correlation coefficient analyses to identify the phenotypes and genes that were selected bydifferentcultivarcombinationsoverthecourseofthe field trial. This analysis identified several interesting correlations between phenotypes, genotypes, and the wheat varieties from which strains were isolated [21]. At least two distinct, mutually exclusive phenotypic/ genotypic groups emerged from our analysis. The first oftheseshowedincreasedlevelsofantimicrobialactivity towards Streptomyces spp., and contained operons for cyclic-lipopeptide andLPS biosynthesis,typeVI secre-tion and toxin production.The second group produced

Figure1

Low

Low

Low High

High High

Hereward Cadenza

Pseudomonas richness

Pseudomonas abundance

Disease incidence

First year variety

Second year outcome

Current Opinion in Microbiology

SoilPseudomonasgenotypicrichnessisassociatedwithmoresevere

diseaseincidenceinwheatroots.

highlevelsoffluorescentsiderophores,aphenotypethat stronglycorrelated withacetoincatabolismloci[21].

Excitingly,we alsosaw correlations betweenindividual

Pseudomonasgenesandthewheatvarietiesgrowninthe firstyear.TheoperonsassociatedwithyearoneHereward cultivation (high TAB) also positively correlated with

Streptomyces suppression, while loci that positively correlated with year one Cadenza (low TAB) strongly associatedwithincreasedpyoverdinproduction[21].In addition,Pseudomonasisolatesfromthisfieldexperiment were used to construct synthetic community fungal antagonism assays [1]. Increased Pseudomonas spp. richness positively correlated with in vitro pathogen growth. This supported the field observation that first yearHerewardplots,withahigherrhizospheregenotypic richnessthanfirstyearCadenza,developedmoresevere take-all disease, demonstrating a negative biodiversity effect(Figure 1).We proposethat the increasedlevels ofsenescentroottissueandsaprophyticmicroorganisms thataccompanyHerewardgrowthinyearonemayleadto an increased abundance of pseudomonads that are adapted to niche competition with other microbes, whereas the comparatively benign environment associatedwithCadenzarhizospheresfavours Pseudomo-nasgenotypesthatarebetteradaptedtoplant–host com-municationandincreasedproductionofmetalscavenging siderophores(Figure 2).

Clearly,itremainstobeestablishedwhetherthemodel weproposefortheinterplaybetweenwheat,take-alland

Pseudomonas is correct, or whether there is a different reasonforthepopulationshiftswesee.Nonethelessthe impactofthefirstyearwheatcultivarwasstilldetectable twoyearsafterthebeginningoftheexperiment, consis-tentwithsubstantial selectivepressureonthefirst-year rhizospherepopulation.Asecondlong-termwheat exper-iment that will capture the full disease epidemic is underway,asareseverallaboratoryexperimentsincluding root exudate metabolomic analysis, to strengthen and refine ourinitial conclusions. Ourexperiments indicate that first year wheat genotype affects both the overall

Pseudomonaspopulation,andalsothedistributionof indi-vidual genotypes in the second year rhizosphere [21,65,66].Inturn,theseexperimentssupportour con-tentionthatabetterunderstandingofthesoilmicrobiota, combined with smart manipulation of plant cropping systemsmaypresentareliablefutureroutetosustainable yieldimprovementandbiocontrol.

Acknowledgements

RothamstedResearchreceivesstrategicfundingfromBBSRC,andTM acknowledgessupportfromtheBBSRCISPG;Optimisationofnutrientsin soil–plantsystems(BBS/E/C/00005196).JMwassupportedinthisworkby theUniversityofEastAngliaandtheBBSRCISPG;BioticInteractionsfor CropProductivity(BB/J004553/1),totheJohnInnesCentre.

26 Environmentalmicrobiology

Figure2

T6SS

Saprophytic microbes

Siderophores

Plant hormones Toxins

Viscosin Pili

Hereward-selected

Pseudomonas

Cadenza-selected

Pseudomonas

Year 1 Hereward – High Take-all Year 1 Cadenza – Low Take-all

Current Opinion in Microbiology

Amodelforyear1wheatcultivarselectionofsoilPseudomonasgenotypes.

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