Microbiota Characterization of Compost Using Omics Approaches Opens New Perspectives for ...

19 Pages·2016·2.89 MB·English

RESEARCHARTICLE
Microbiota Characterization of Compost
Using Omics Approaches Opens New
Perspectives for Phytophthora Root Rot
Control
JosefaBlaya1*,FrutosC.Marhuenda2,JoseA.Pascual1,MargaritaRos1
1 DepartmentofSoilandWaterConservationandOrganicWastesManagement,CentrodeEdafologíay
BiologíaAplicadadelSegura(CEBAS-CSIC),Murcia,Spain,2 DepartmentofAgrochemistryand
Biochemistry.UniversityofAlicante,Alicante,Spain
*[email protected]
a11111
Abstract
PhytophthorarootrotcausedbyPhytophthoranicotianaeisaneconomicallyimportantdis-
easeinpeppercrops.Theuseofsuppressivecompostsisalowenvironmentalimpact
methodforitscontrol.Althoughattemptshavebeenmadetorevealtherelationship
OPENACCESS betweenmicrobiotaandcompostsuppressiveness,littleisknownaboutthemicroorgan-
ismsassociatedwithdiseasesuppression.Here,anIonTorrentplatformwasusedto
Citation:BlayaJ,MarhuendaFC,PascualJA,Ros
M(2016)MicrobiotaCharacterizationofCompost assessthemicrobialcompositionofcompostsmadeofdifferentagro-industrialwasteand
UsingOmicsApproachesOpensNewPerspectives withdifferentlevelsofsuppressivenessagainstP.nicotianae.Bothbacterialandfungal
forPhytophthoraRootRotControl.PLoSONE11(8):
populationsrespondeddifferentlydependingonthechemicalheterogeneityofmaterials
e0158048.doi:10.1371/journal.pone.0158048
usedduringthecompostingprocess.Highproportions(67–75%)ofvineyardpruningwaste
Editor:SabrinaSarrocco,UniversitadegliStudidi
wereusedinthemostsuppressivecomposts,COM-AandCOM-B.Thismaterialmayhave
Pisa,ITALY
promotedthepresenceofhigherrelativeabundanceofAscomycotaaswellashigher
Received:February15,2016
microbialactivity,whichhaveprovedtobeessentialforcontrollingthedisease.Althoughno
Accepted:June9,2016 uniquefungiorbacteriahavebeendetectedinneithersuppressivenorconducivecom-
Published:August4,2016 posts,relativelyhighabundanceofFusariumandZopfiellawerefoundincompostCOM-B
andCOM-A,respectively.Tothebestofourknowledge,thisisthefirstworkthatstudies
Copyright:©2016Blayaetal.Thisisanopen
accessarticledistributedunderthetermsofthe compostmetabolome.Surprisingly,compostsandpeatclusteredtogetherinprincipalcom-
CreativeCommonsAttributionLicense,whichpermits ponentanalysisofthemetabolicdataaccordingtotheirlevelsofsuppressiveness
unrestricteduse,distribution,andreproductioninany
achieved.Thisstudydemonstratedtheneedforcombiningtheinformationprovidedbydif-
medium,providedtheoriginalauthorandsourceare
ferenttechniques,includingmetagenomicsandmetametabolomics,tobetterunderstand
credited.
theabilityofcomposttocontrolplantdiseases.
DataAvailabilityStatement:Thesequencesare
availablefromBioSampledatabasewiththethe
Biosampleaccessionnumber:SAMN03609932.
Funding:ThisworkwassupportedbyLIFEproject
AGROWASTE(LIFE10/ENV/ES/469)http://ec.
Introduction
europa.eu/environment/life/.
PhytophthoranicotianaevanBredadeHaan(=PhytophthoraparasiticaDastur(1896))stands
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. outamongplantpathogenssinceitisathreattoplantproductivityonaglobalscaleforabroad
PLOSONE|DOI:10.1371/journal.pone.0158048 August4,2016 1/19
OmicsApproachesandPhytophthoraRootRotControlbyComposts
rangeofhosts[1].ThehostrangeofP.nicotianaeincludes255plantgenerain90families[2].
InSpainandTunisia,P.nicotianaecausesroot-andcollar-rotinpepperplants(Capsicum
annuumL.)andhasbecomeamajordiseaseduringthelastyears[3–5].Managementofthis
diseaseisbasedonsoilfumigationusingcompoundssuchasmethylbromide,1,3-dichloropro-
pene,chloropicrin,metalaxylandmefenoxam[6].Thebanneduseofmostoftheseproducts
andtheirinabilitytototallycontrolthedisease,havepromptedtheexplorationandidentifica-
tionofnewapproaches.
Theuseofcompostmadeofagro-industrialwasteandby-productsisapromisingalterna-
tive.Thesecompostsarenotonlyfreeofxenobioticsandexcessivecontentofheavymetalsbut
also,theyhavebeenproventosuppressawidevarietyofsoil-borneplantpathogens[7–9].
Suppressivecompostsareexamplesofnaturalbiologicalcontrolofdiseasesastheresultofa
three-wayinteractionbetweenthemicroorganismsinthecompost(composition,diversityand
function),plantpathogen,andplant[10].Theextantmicrobiotaofcompostshasshowntobe
themainfactorresponsibleforsuppressiveness[11,12].Inparticular,suppressiveeffects
againstPhytophthoraspp.havebeenrelatedtotheabilityofcompoststomaintainahigh
microbialactivity[7].Nevertheless,themechanismsofsuppressionoftheseoomyceteshave
notbeenunraveled.
Compostmicrobiotahighlydependsonthematerialsusedduringthecompostingprocess.
Thequalityofcompostorganicmatterisimportantwithrespecttotheefficacyofthesuppres-
sionandtheregulationandmaintenanceofmicrobialcommunitiesincomposts[13].Shiftsin
thechemicalcompositionofcompostorganicmatterhavebeenpreviouslycharacterizedby
13C-NuclearMagneticResonance(13C-NMR)spectroscopy[14].However,itsecologicalsig-
nificanceshouldbeelucidatedinrelationtothemicrobialcommunitiesinhabitingtheorganic
matter[15],whichcanbestudiedthroughmolecularanalysis—suchasfingerprintingtech-
niquesandsequencingmethods[16–18].High-throughputsequencing(e.g.IlluminaorIon
Torrent)isapowerfulalternativefortheidentificationatagreaterdepthofthemicrobialcom-
munitycompositionanddiversity.Amongthestudiesregardingthemetagenomicsofcompost
[19–22],onlyYuetal.[22]investigatedtheconnectionsbetweenthemicrobialcommunities
andthediseasesuppressionabilityofcompostagainstPythiumultimum.
Itshouldbenotedthatgenomicinformationitselfisnotenoughforunderstandingthebio-
logicalprocessesthattakeplacewithincompost.Intheattempttofullydescribethecompost
microbiome,metabolomicshaveemergedasafunctionalapproachthatprovidesinsightsinto
themetabolicactivitiesengagedbywholecommunitiesofmicroorganisms[23].Severalstudies
pointedoutthecentralroleplayedbymetabolitesincellularactivitiesandthemileagethat
couldbegainedbymonitoringatthelevelofthemetabolome[24].Metabolites(low-molecu-
lar-weightcompoundssuchasaminoacids,sugars,andlipids)playsignificantrolesinthe
microbialregulationofthecentralandsecondarymetabolism.Notonlycantheycontributeto
externalsignalsasindicatorsoftheenvironmentalconditionsorbysensingsuchsignals,but
alsotheyvaryinresponsetoavarietyofstimuli(e.g.nutritionaldeficit,externalstressors,or
disease)[25].Currently,theimplementationofmetabolomicsforenvironmentalmonitoringis
stillatanearlystage,mostlyappliedasascreeningtooltoassessthepotentialtoxiceffectof
pollutants[26].
Wehypothesizethatsuppressivecompostswillcontainmoresimilarmicrobialcommuni-
tiesamongthemcomparedtonon-suppressivecomposts,andwilldependonthequantityand
stateoftheorganicmatterineachcompost.Basedonthis,thespecificobjectivesofthisstudy
were(i)tostudytheinvivoabilityoffouragro-industrialwaste-basedcompoststocontrolP.
nicotianaeinpepperplants,(ii)tocomparethechemicalheterogeneityoforganicmatterpres-
entinthedifferentcomposts,and(iii)tocharacterizeandcomparethemicrobialcomposition
andmetametabolomeofsuppressiveandnon-suppressivecomposts.
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
Methods
Thecompostsassayedandtheiranalysis
Fouragro-industrialcompostsweremadefromdifferentwastes(expressedasdryweight)as
follows:CompostA(COM-A):peppersludge(125gkg-1),pepperwastes(125gkg-1),and
vineyardpruningwastes(750gkg-1);CompostB(COM-B):pepperwastes(160gkg-1),arti-
chokewastes(160gkg-1),andvineyardpruningwastes(680gkg-1);CompostC(COM-C):
peppersludge(190gkg-1),pepperwastes(20gkg-1),garlicwastes(20gkg-1),carrotwastes
(350gkg-1),almondshells(40gkg-1),andvineyardpruningwastes(380gkg-1);CompostD
(COM-D):artichokesludge(150gkg-1),artichokewastes(264gkg-1),vineyardpruningwastes
(500gkg-1),andcompost(86gkg-1).
Thecompostswereproducedinopen-airpilesof200kg,thebio-oxidativephaselasting75
daysandmaturation42days.Themoisturecontentwasinitiallysetat40–50%andwasmain-
tainedbywatering.Thepileswereturnedperiodicallytoensureaeration,andthetemperature
evolutionwasmonitoredperiodically(datanotshown).Oncethecompostingprocesswasfin-
ished,thecompostsweremilledandpassedthrougha1-cmsieve.Threesamplesofeachcom-
postpileweretakenbymixingninesub-samplesfromrandomsiteswithineachpile.The
sampleswerestoredat-20°Cand4°Cforsubsequentanalysis.
Differentphysical-chemical,chemical,andbiologicalcharacteristicsofthecompostsand
peatweremeasured.ThepHandelectricalconductivity(EC)ofthecompostsandpeatwere
measuredina1:10(w/v)water-solubleextract,inaconductivitymeterandpHmeter,respec-
tively.Thetotalorganiccarbon(TOC)andnitrogenweremeasuredwithanElementalAna-
lyzer(LECOTruSpecC/N)andnutrientsbyICP-OES(ICAP6500DUO).Dehydrogenase
activitywasmeasuredbythemethodofGarciaetal.[27].
Organicmatteranalysisby13C-NMR
Theorganicmattercompositionofthefourcompostsandonepeatwasestimatedbyspectral
intensityintegrationoverregionswithchemicalshiftcharacteristicsofdifferentorganiccarbon
functionalgroups.TheCPMAS13C-NMRexperimentswereperformedinaBrukerAdvance
DRX500,operatingat125.75MHzfor13C.Thesampleswerepackedintoa4–mm-diameter
cylindricalzirconiarotorwithKel-Fend-capsandspunat10000±100Hz.Aconventional
CPMASpulsesequence[28]wasused,witha1.0-mscontacttime.Between2000and5000
scanswereaccumulated,withapulsedelayof1.5s.Thelinebroadeningwasadjustedto50Hz.
Spectraldistributions(thedistributionoftotalsignalintensityamongvariouschemicalshift
ranges)werecalculatedbyintegratingthesignalintensity,expressedasapercentage,infive
chemicalshiftregions:0–45(aliphaticstructures),45–60(methoxygroups),60–110(polysac-
charidesstructuresregion),110–160(aromaticstructures),and160–210(carboxyl,carbonyl,
amideC)[29].Thealkyl/O-alkylratiowasalsocalculated[14].
DNAextraction,sequencing,andanalysis
1
TotalDNAwasextractedusingtheFastDNA SpinKitforsoil(Q-Biogene,Carlsbad,CA,
USA),followingthemanufacturer´sinstructions.TheDNAconcentrationsofthesamples
1
weredeterminedusingaNanoDrop ND-1000Spectrophotometer(ThermoFisherScientific
Inc.,DE,USA);thesampleswerethenstoredat-20°Cuntilrequired.Forthemolecularanaly-
sisofbacterialcommunities,the16SrRNAgenewasamplifiedusingprimerpairs8F/120R,
F388/R534,F968/R1073and8F/R361[30,31]andforthefungalcommunity,theITS1and
ITS2regionsofthefungalrRNAgenewereamplifiedusingtheITS5/ITS2andITS3/ITS4
primerpairs[32].Eachsamplewasamplifiedintriplicate;theampliconswerepurifiedusing
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
theQIAquickPCRPurificationKit(Qiagen,Hilden,Germany)andcompositedtogetherat
equimolarconcentrationpriortosequencing.ForPCRamplification,each25-μLPCRmix
containedthefollowingreagents:1XKAPA2GFastHotStartReadyMix2(2X)(KapaBiosys-
tems,Boston,MA,USA),1.5mMMgCl 0.5μMofeachprimer,and5μLofDNA.
2,
ThePCRwithprimers8F/120R,F968/R1073and8F/R361wasperformedusingthefol-
lowingconditionsasfollows:15cyclesofdenaturationat90°Cfor30s,amplificationwitha
temperaturegradientof70°C−50°Cfor30s,andafinalextensionof72°Cfor30s.Addition-
ally,sampleswereheldfor30cyclesofdenaturationat94°Cfor45s,amplificationat50°C
for45s,andafinalextensionof72°Cfor45s.ThePCRsforprimerpairF388/R534andthe
ITSregionhadaninitialdenaturationstepat95°Cfor3min,followedby25–40cyclesof
denaturationat95°Cfor15s,amplificationat60°Cfor15s,extensionat72°Cfor15s,anda
finalextensionof72°Cfor1min.
AlibrarywascreatedusingtheIonPlusFragmentLibraryKit,andbarcodeswereaddedby
theIonXpress™BarcodeAdapters1–96Kit.Thetemplatepreparationwasperformedwiththe
IonOneTouch™2SystemandtheIonPGM™TemplateKitOT2400.Finally,theplatform
sequencedthesamplesusingIonTorrentPGM(LifeTechnologies,Carlsbad,CA,USA)with
theSequencingKitIonPGM400,inchipsIon318ChipkitandIon314Chipkit.
ThedataanalysiswasperformedusingthesoftwarepackagesQIIMEv1.8.0.andUSEARCH
v7.0.1090.Sequencesshorterthan60bpand/orQmeanqualityscoresbelow25wereremoved.
Primersandbarcodeswereremovedandachimerafilterwasused.Theremaininghighquality
sequencesweregroupedinoperationaltaxonomicunits(OTUs),followingtheOpenReference
method:sequenceswereclusteredagainsttheGreenGenesv13_8,forthebacterialcommunity,
andagainstUNITE/QIIME12_11ITS,forthefungalcommunity,usingtheunclustmethod
with97%similarity.Sequencesnotmatchingthedatabaseweresubsequentlyclusteredde
novo.ArepresentativesetofOTUswasgeneratedandthenthetaxonomyofeachoftheOTUs
wasassignedusingthesamedatabase.ThesequenceshavebeendepositedinNCBIunderBio-
ProjectPRJNA283180.
Shannondiversitywasusedtoestimatediversityofbacterialandfungalcommunitiesas
indicatedbyNeheretal.[19].ShannondiversitywascalculatedasH´=-S(pilnpi)wherep
representstheproportionoftaxoniinthecommunity.
Metaboliteextractionandanalysis
Metaboliteextractionwasperformedbywaterextraction(1:10w/v,composttodeionized
water)ofsixreplicatesofthecompostsandpeat.Themixtureswereshakenfortwohours,
afterwhichthesupernatantwaspassedthrougha0.2-μmfilter.Thesupernatantwasanalyzed
usinganAgilent1290InfinityUPLCsystemcoupledtoa6550AccurateMassquadrupole
TOFmassspectrometer(AgilentTechnologies,Waldbronn,Germany)usinganelectrospray
interfacewithjetstreamtechnology.SeparationwasachievedonareversephasePoroshell
120EC-C18column(3X100mm,2.7μm:Agilent)operatingat30°C.Themobilephaseswere
water:formicacid(99.9:0.1v/v;phaseA)andacetonitrile:formicacid(99.9:0.1v/v;phaseB).
Anisocraticflowof95%phaseAand5%phaseBwasmaintainedfor3min.Theflowrate
wassetconstantat0.4mL/minandtheinjectionvolumewas3μL.Theoptimalconditionsof
theelectrosprayinterfacewereasfollows:gastemperature280°C,dryinggas9L/min,neb-
ulizer45psi,sheathgastemperature400°C,sheathgasflow12L/min.Spectrawereacquired
insingleMSmodewithanm/zrangeof100–1100,negativepolarity,andanacquisitionrate
of1.5spectra/s.Internalmasscalibration,bysimultaneousacquisitionofreferenceionsand
massdriftcompensation,wasusedtoobtainlowmasserrors.Datawereprocessedusingthe
MassHunterQualitativeAnalysisSoftware(versionB.06.00,AgilentTechnologies).After
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
analysisofthedataobtainedfromthemetabolic,apeakgroupingwascarriedout,followinga
script,byRsoftware.
Suppressivenessbioassay
ThepathogenicstrainCC2ofP.nicotianae(accessionnumberKJ000327)previouslyisolated
frompepperplantswithdiseasesymptomswasusedinthisstudy[4].TheinoculumofP.nico-
tianaewasproducedbytransferringoneagarplug(5mm)of7-day-oldmyceliaonpeaagar
medium(100gL-1groundpeas,100mgL-1β-sitosterol,and20gL-1technicalagar,adjusted
topH5.5),autoclavedat121°Cfor20minandamendedwith100mgL-1sterilizedstreptomy-
cin.Theculturewasmaintainedat28°Cfor7days.Themyceliawererecoveredfromthecon-
tentoftwoPetridishesandmixedwith100mLofsteriledistilledwater,usingablender.
Compostsweremixedwithacommercialpeat(50/50v/v)toobtaindifferenttreatments:
TCOM-A,TCOM-B,TCOM-C,TCOM-D,andTPeat(100%peat,asacontrol).Seedsofpep-
per(Capsicumannuumcv.Lamuyo)weresownintraysof150pots,withoneseedperpotand
acoveringofvermiculite.Sixreplicatesofeachtreatmentwereestablishedrandomly,eachrep-
licateconsistingof10seeds.Germinationwascarriedoutinagerminationchamberat
28±1°C.Oncetheseedshadgerminated,thetrayswereplacedinagrowthchamberunder
daylightconditions.Fourreplicatesofeachtreatmentwereinoculatedwith2mLofP.nicotia-
nae(~103cfug-1substrate)afterthefirsttrueleafappeared.Thesuppressiveeffectofthediffer-
enttreatmentswasdeterminedbymeasuringthediseaseincidence(numberofdiseasedplants)
23daysafterinoculation.
Statisticalanalysis
Thephysical,physical-chemical,andbiologicalcharacteristicsofthecompostsandpeat,as
wellastheresultsfromthesuppressivenessbioassay,weresubjectedtoone-wayanalysisofvar-
iance(ANOVA).WhentheF-statisticwassignificant,Tukey’sposthoctest(p(cid:1)0.05)wasused
toseparatemeans.Pearsoncorrelationsweremadebetweenalldata.Thestatisticalanalyses
wereperformedusingSPSS19.0software(SPSSInc.,Chicago,IL,USA).
StatisticalanalysisofmetabolitedatawascarriedoutwithMetaboanalysis2.5software.A
multi-variantanalysisofmasscompoundsbyprincipalcomponentanalysiswasused.Forheat-
mapclusteringofsamplesandmasscompounds,thesquaredEuclideandistanceandward
linkagewereutilized.
Results
Physical,physical-chemical,andbiologicalanalyses
Themainphysical-chemicalandbiologicalcharacteristicsofthecompostsandpeatareshown
inTable1.BothpHandECshowedsignificantdifferencesdependingonthesubstrate
(F=745;p<0.001;F=236;p<0.001,respectively).
PeathadthelowestpHvalueswhereas,COM-B,COM-C,showedthehighestpHvalues.
CompostCOM-A,COM-ChadthelowestECvalueandCOM-Dthehighest.
TheTOCofcompostsandpeatrangedfrom273to480gkg-1,withthehighestvaluesfor
peatfollowedbyCOM-A,COM-C,COM-BandCOM-D(F=3118;p<0.001).
Aswasexpected,peatshowedthesignificantlylowestlevelofvaluesforN,PandKcom-
paredwithcomposts.ThecontentofNwasintherange22–28.5gkg-1forcomposts,Prange
was3.7–5.6gkg-1andKbetween16.6–28gkg-1.
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
Table1. Physical-chemicalandbiologicalpropertiesofcompostsandpeat.
COM-A COM-B COM-C COM-D Peat
pH 8.5c 8.9d 8.8d 6.2b 5.5a
ECa(mScm-1) 1.9ab 2.6c 1.8a 3.8d 2.0b
TotalorganicC(gkg-1) 433d 316b 374c 273a 480e
TotalN(gkg-1) 25d 22b 29e 24c 13a
P(gkg-1) 3.7b 4.0c 4.0c 5.6d 0.3a
K(gkg-1) 24d 28e 18c 16.6b 0.6a
Dehydrogenaseactivity(mgINTg-1) 27.2d 37.0e 9.4c 3.2b 0.32a
aEC,electricalconductivity.
Dataaremeanofthreereplicates.Foreachparameter,datafollowedbythesameletterarenotsignificantlydifferentaccordingtoTukey’sposthoctest
(p(cid:1)0.05).
doi:10.1371/journal.pone.0158048.t001
Dehydrogenaseactivitydifferedsignificantlyamongdifferentcompostsandpeat(F=139;
p<0.001),compostsCOM-AandCOM-Bshowingthehighestlevelsandpeatthelowest
(Table1).
Thesuppressiveeffectofdifferentgrowingmedia
TheincidenceofPhytophthorarootrotsymptomsofP.nicotianaeonpepper23daysafter
inoculationdifferedsignificantlyamongtreatments(F=10.039;p=0.001),indicatingthat
TPEATtreatment(100%peat)wasthemostconducivegrowingmedium,followedby
TCOM-DandTCOM-C,whichonlyreducedthediseaseincidenceby13%and23%,respec-
tively,comparedtoTPEAT(Fig1).TCOM-Atreatmentwasthemostsuppressiveorganic
Fig1.DiseaseincidenceinpepperseedlingsartificiallyinoculatedwithP.nicotianae.
doi:10.1371/journal.pone.0158048.g001
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
Fig2.Chemicalcompositionofthecompostsandpeatrevealedby13CNMR.(A)Distributionoforganiccarbon
functionalgroups:0–45ppm(aliphaticgroups);45–60ppm(methoxygroups),60–110ppm(carbohydrategroups);
110–160ppm(aromaticCstructures),and160–210ppm(carboxylandestergroup).(B)Alkyl/O-alkylratiovalues.
doi:10.1371/journal.pone.0158048.g002
mediumagainstP.nicotianae,withareductionof60%comparedtoTPEAT,followedby
TCOM-B,withareductionof50%(Fig1).Pepperplantsinnon-infestedgrowthmediadidnot
showanysymptomsofPhytophthorarootrot.
Compositionoftheorganicfractionsinthecompostsandpeat
Therelativeintegrationvaluesforthefivespecificorganiccarbonregionsfromthecomposts
andpeatareshowninFig2A.Significantdifferenceswereobservedamongfractions
(F=265.22;p<0.05).Thefraction0–45ppm,correspondingtothealiphaticfractionascribed
tolipids,waxes,terpenoids,cutins,andsuberins,andthefraction60–110ppm,corresponding
tothecarbohydrateregion(polysaccharides,aminoacids,aminosugars,ligninsubstitutes,and
others)(18)showedhigherrelativeabundancesthantherestofthefractions,namely45–
60ppm(methoxygroups),110–160ppm(aromaticCstructures),and160–210ppm(carboxyl
andestergroup)(Fig2A).
CompostsCOM-AandCOM-Bshowedlowerrelativeabundancesinthealiphaticstructure
regionscomparedtocompostsCOM-C,COM-Dandpeat(P)(Fig2A).Forcarbohydrate
structureregion,COM-Cshowedthelowestrelativeabundance.Peatshowedthehighestrela-
tiveabundanceinbothregions(Fig2A).TherelativeabundanceinthearomaticCstructure
regionfollowedthetrend:COM-B>COM-D>P>COM-A>COM-C,whileforcarboxyland
estergroupsitwasCOM-B>COM-A>COM-C>COM-D>P(Fig2A).Thealkyl/O-alkylratio
followedthetrendCOM-C>P>COM-A=COM-D>COM-B(F=320;p<0.05)(Fig2B).
Metabolomesofthedifferentcompostsandpeat
Tofinddifferencesamongthemetabolomesofcompostsandpeat,aprincipalcomponent
analysis(PCA)wasappliedtoconstructandvalidateastatisticalmodel.Thetworelevantaxes
explained88.6%ofthevariance(PC160.7%andPC227.9%)(Fig3).AccordingtoFactor1,
multivariateanalysisshowedthreedifferentclusters—peatbeingseparatedfromonecluster
consistingofcompostsCOM-AandCOM-Bandfromanothercomposedofcomposts
COM-CandCOM-D(Fig3).Theheatmapgeneratedwiththemassofthemostfrequently
metabolitesfoundacrosstheprofilesshowedthatmostofthemwerefoundinlowerrelative
abundanceinsuppressivecomposts(Fig4).Severalmasscompoundsreceivedahighloading
scoreinFactor1andcontributedthemosttotheseparationofpeatfromthecomposts(175;
97;247.8;278.9;179.9;216.9;374.8;232.9;330.8;194.9;336.9;352.8;218.9;164.9)(Fig4).
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
Fig3.PrincipalcomponentanalysisaccordingtothemetabolomeobtainedfromthecompostsCOM-A(greenballs),COM-B(pinkballs),
COM-C(redballs),COM-D(yellowballs),andpeat(blueballs),n=6.
doi:10.1371/journal.pone.0158048.g003
Othermasscompounds(184.9;260.9;300.9;238.9;254.9;316.9;262.9;310.9;186.9;278.9;
234.9;312.9)contributedtotheseparationofsuppressivefromconductivecomposts.
Fungalandbacterialcommunitiesofthedifferentcompostsandpeat
Of1904691reads,weobtainedatotalof679687readsafterqualityfilteringandchimeras,
508043from16SrRNAand171644fromfungalITSgenesequencesacrossallsamples.The
OTUclusteringandtaxonomicassignment,performedusingthesesequences,yielded25071
and3600individualOTUsfrom16SrRNAandfungalITSgenes,respectively.
Fungalcommunitycomposition. Theclassifiedsequencesforthecompostsandpeat
wereaffiliatedtothreefungalphyla.ThemostabundantphylumwasAscomycota,accounting
for54%ofallsequencereads,followedbyBasidiomycota(2.34%)andZygomycota(0.06%).
Thepercentageofsequencesclassifiedasotherfungiwas6.8%,whereas36.57%wasassigned
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Fig4.Aheatmapillustratingthe54metabolitesthatdifferamongthecompostsandpeat.Colorsindicaterelativequantityofeach
metabolite.
doi:10.1371/journal.pone.0158048.g004
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OmicsApproachesandPhytophthoraRootRotControlbyComposts
tounidentifiedfungi.Examinationofthetaxonomicstructureattheorderlevel(Fig5)showed
that,withinthephylumAscomycota,themostabundantorderswereSordariales,Hypocreales,
andMicroascales.CompostsCOM-AandCOM-BshowedhigherrelativeabundancesofAsco-
mycota(63.14and67.38%,respectively),inparticular,COM-Ahadthehighestrelativeabun-
danceofSordarialesandCOM-BthehighestabundanceofHypocreales(Fig5).Ontheother
hand,compostCOM-CshowedahighabundanceofSaccharomycetalesandcompostCOM-D
ofMicroascales,whiletheseorderswerealmostinexistentinpeat(Fig5).Peatshowedahigh
relativeabundanceofAscomycota,followedbyBasidiomycota(Fig5).Fungaldiversitywas
observedtobebetween2.38–3.75incompostsand2.64inpeat.
Atthegenuslevel,themostabundantclassifiedgenera(>1%)foreachcompostandpeat
areshowninTable2.Thegenerawiththehighestrelativeabundancesinthecompostswere
Zopfiella,Fusarium,Haematonectria,Galactomyces,Doratomyces,Geomyces,Coprinellus,and
Thermomyces.
Bacterialcommunitycomposition. Theclassifiedsequenceswereaffiliatedwith19bacte-
rialphyla,andtheremainingoneswereunassigned.Thedominantphyla,foundinallcom-
postsandpeat,weretheProteobacteria(39.89%oftotalsequencereads),Actinobacteria
(30.53%),Bacteroidetes(12.97%),Chloroflexi(6.25%),and—toalesserextent—Firmicutes
(4.87%),Gemmatimonadetes(1.97%),Acidobacteria(1.07%),andTM7andTM6(<0.41%).
CompostsCOM-AshowedhigherrelativeabundanceofProteobacteria,mainlyduetothe
highabundanceofAlphaproteobacteriaandGammaproteobacteria,aswellasthelowerabun-
danceofActinobacteria;whileCOM-BshowedhigherrelativeabundanceofChloroflexi(Fig
6).HigherrelativeabundanceofBacteriodeteswerefoundincompostCOM-AandCOM-B
comparedtotherestofcompostaswasfoundforGemmatinomidetesincompostCOM-D
(Fig6).Bacterialdiversitywasobservedtobebetween6.40–6.70incompostsand5.5inpeat.
Themostabundantclassifiedgenera(>1%)foreachcompostandpeatareshownin
Table3.Themostfrequentgenerainclude:Microbacterium,Mycobacterium,Streptomyces,
Devosia,andRhodoplanes.
Fig5.Relativeabundancesofthefungalordersidentifiedinthecompostsandpeat.
doi:10.1371/journal.pone.0158048.g005
PLOSONE|DOI:10.1371/journal.pone.0158048 August4,2016 10/19