Trichoderma polysporum selectively inhibits white-nose syndrome fungal pathogen Pseudogymnoascus destructans amidst soil microbes


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Trichoderma polysporum selectively inhibits white-nose syndrome fungal pathogen Pseudogymnoascus destructans amidst soil microbes

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Title:
Trichoderma polysporum selectively inhibits white-nose syndrome fungal pathogen Pseudogymnoascus destructans amidst soil microbes
Series Title:
Microbiome
Creator:
Singh, Amanpreet
Lasek-Nesselquist, Erica
Chaturvedi, Vishnu
Chaturvedi, Sudha
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English

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Pseudogymnoascus ( local )
Nose Syndrome ( local )
Metagenomics ( local )
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serial ( sobekcm )

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Abstract:
Pseudogymnoascus destructans (Pd), the causative fungal agent of white-nose syndrome (WNS), has led to the deaths of millions of hibernating bats in the United States of America (USA) and Canada. Efficient strategies are needed to decontaminate Pd from the bat hibernacula to interrupt the disease transmission cycle without affecting the native microbes. Previously, we discovered a novel Trichoderma polysporum (Tp) strain (WPM 39143), which inhibited the growth of Pd in autoclaved soil samples. In the present investigation, we used culture-based approaches to determine Tp-induced killing of native and enriched Pd in the natural soil of two bat hibernacula. We also assessed the impact of Tp treatment on native microbial communities by metagenomics.
Original Version:
Microbiome, Vol. 6, no. 139 (2018-01-01).

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K26-00031 ( USFLDC: LOCAL DOI )
k26.31 ( USFLDC: LOCAL Handle )

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RESEARCHOpenAccess Trichodermapolysporumselectivelyinhibitswhite-nosesyndromefungalpathogenPseudogymnoascusdestructansamidstsoilmicrobesAmanpreetSingh1,EricaLasek-Nesselquist2,VishnuChaturvedi1,3andSudhaChaturvedi1,3*AbstractBackground:Pseudogymnoascusdestructans(Pd),thecausativefungalagentofwhite-nosesyndrome(WNS),hasledtothedeathsofmillionsofhibernatingbatsintheUnitedStatesofAmerica(USA)andCanada.EfficientstrategiesareneededtodecontaminatePdfromthebathibernaculatointerruptthediseasetransmissioncyclewithoutaffectingthenativemicrobes.Previously,wediscoveredanovelTrichodermapolysporum(Tp)strain(WPM39143),whichinhibitedthegrowthofPdinautoclavedsoilsamples.Inthepresentinvestigation,weusedculture-basedapproachestodetermineTp-inducedkillingofnativeandenrichedPdinthenaturalsoiloftwobathibernacula.WealsoassessedtheimpactofTptreatmentonnativemicrobialcommunitiesbymetagenomics.Results:OurresultsdemonstratedthatTpattheconcentrationof105conidia/gsoilcaused100%killingofnativePdinculturewithin5weeksofincubation.A10-foldhigherconcentrationofTp(106conidia/gsoil)killedanenrichedPdpopulation(105conidia/gsoil).The12,507fungaloperationaltaxonomicunits(OTUs,dominatedbyAscomycotaandBasidiomycota)and27,427bacterialOTUs(dominatedbyAcidobacteriaandProteobacteria)comprisedthenativesoilmicrobesofthetwobathibernacula.NosignificantdifferencesinfungalandbacterialrelativeabundanceswereobservedbetweenuntreatedandTp-treatedsoil(105Tpconidia/gsoil,p0.05).Conclusions:OurresultssuggestthatTp-inducedkillingofPdishighlyspecific,withminimaltonoimpactontheindigenousmicrobespresentinthesoilsamples.ThesefindingsprovidethescientificrationaleforthefieldtrialsofTpintheWNS-affectedhibernaculafortheeffectivedecontaminationofPdandthecontrolofWNS.Keywords:White-nosesyndrome,Pseudogymnoascusdestructans,Trichodermapolysporum,Biologicaldecontamination,Biocontrolagent,Selectiveinhibition,Bathibernacula,Nativesoilmicrobiota,MetagenomicsBackgroundPseudogymnoascusdestructans(Pd),theetiologicalagentofwhite-nosesyndrome(WNS),hascausedsignificantreductionsinhibernatingbatpopulationsacrosstheUSAandCanada[1–6].MortalitywasfirstobservedinhibernatingbatsinHowesCavenearAlbany,NewYork(NY),in2006,andhassincespreadextensivelyto32USstatesand7Canadianprovinces[7].RecentlyPdwasdetectedinalittlebrownbat(Myotislucifugus)asfarawayasWashingtonState[8].Severalspeciesofbatsarealreadythreatenedwithextinction,includingMyotislucifugus(littlebrownbat),Myotissodalis(Indianabat),andMyotisseptentrionalis(northernlong-earedbat)[3,9].Pseudogymnoascusdestructans,apsychrophilicfungus,iswelladaptedtogrowinthecoldconditionsprevailingincavesandmines[2,10].Ithasbeenshowntosecreteproteolyticenzymessimilartothefungithatcauseskininfections(dermatophytes);[2]andhasaclonalpopula-tionintheUSA[11–13].Lowbodytemperature,alongwithreducedimmunesysteminhibernatingbats,providestheoptimalgrowthenvironmentforPd[14–17]. *Correspondence:sudha.chaturvedi@health.ny.gov1MycologyLaboratory,WadsworthCenter,NewYorkStateDepartmentofHealth,120NewScotlandAvenue,Albany,NY12208,USA3DepartmentofBiomedicalSciences,SchoolofPublicHealth,UniversityatAlbany,Albany,NY,USAFulllistofauthorinformationisavailableattheendofthearticle ©TheAuthor(s).2018OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.0InternationalLicense(http://creativecommons.org/licenses/by/4.0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.TheCreativeCommonsPublicDomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle,unlessotherwisestated.Singhetal.Microbiome (2018) 6:139 https://doi.org/10.1186/s40168-018-0512-6

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Alarmingly,Pdhasbeenfoundtosurviveintheaffectedhibernacula,evenintheabsenceofbats[18–20].Thus,aninfectedhibernaculumcouldremaincontaminatedwithPdforprolongedperiodsoftimeandserveasthefocifornewinfections[21].MathematicalmodelshavepredictedthatreducingPdincavesandminesmaypreventWNS-associatedbatmortality[20,22].Currently,effortsarebe-ingdevotedtothedevelopmentandtestingofchemicalandbiologicalagentsfortheeffectiveeradicationofPdfrombathibernaculaandhibernatingbats.Althoughthesecontrolstrategiesappeartobepromising,theyarenotbeingusedforthelarge-scaledecontaminationofhiber-nacula,becauseofthelikelyoff-targeteffectsonthenativemicrobialcommunities[23–27].ConsideringthemassmortalityofbatscausedbyWNSandtheeconomiclossof22.9billiondollarstoagriculturalpestcontrolintheUSAannually[28],imminentstepsareneededtodecon-taminatePdfrombathibernaculaandbreakitstransmis-sioncycle.Previously,wecharacterizedanovel,psychrotolerantTrichodermapolysporum(Tp)strain(WPM39143)fromtheWilliamPreserveMine,UlsterCounty,NY,oneoftheminesattheepicenteroftheWNSzoonotic[29].Tpgrewwellatlowtemperaturesof6–15°CandinhibitedPdinlaboratorymediaandautoclavedsoilsamples[29].ThepresentstudyaimedatfurtherevaluationofTpasaneffectivebiocontrolagentagainstPd.Specifically,weexamined(a)ifTpcouldactasaneffectivebiocontrolagentagainstPdinthenaturalsoilfrombathibernaculaand(b)whetherTptreatmentimpactedthenativemicro-bialcommunitiesinthenaturalsoilfrombathibernacula.MethodsFungalstrainsandmediaPseudogymnoascusdestructans(Pd)strainM1379andTpstrainWPM39143wereusedasdescribedpreviously[29,30].AllfungalisolatesweremaintainedonSabour-auddextroseagar(SDA)slantsat4°Candstoredin20%glycerolat70°Cinsterilecryogenicvials.Sabour-auddextroseagarfortifiedwithanenhancedpanelofantibacterials(SDA-A;Additionalfile1),SDA-Awithcycloheximide(0.2g/L),androsebengalagarwithchlor-amphenicol(RBC;100g/ml)wereusedfortheisolationofPd,Tp,andotherfungifrombathibernaculaasdescribedpreviously[30].Potatodextroseagar(PDA)andwateragar(WA)wereusedtoinducesporeforma-tioninPdandTp.Milletseedsextractwasusedtofor-tifynutrientsinthesoilsamplesfrombathibernaculaforTpandPdinteractionstudies.Inbrief,50gofmilletseedswasaddedin250mlwater,wasautoclavedat121°Cfor20min,andwasmixedwithanadditional250mlsteril-izedwater.Theresultingextractwaspassedthroughmus-lincloth,autoclavedasabove,allowedtocool,andstoredat4°Cuntilused.Alltheexperimentswereperformedinthebiosafetycabinetinabiosafetylevel2laboratory.BiocontrolapplicationofTpinnaturalsoilTodetermineTp-inducedkillingofPdinanaturalsoil,onesoilsamplefromAeoluscave(AC),BenningtonCounty,VT(darkblackincolor;collectiondateof11/13/2015),andonesoilsamplefromBartonHillMine(BHM),EssexCounty,NY(coursesediments;collectiondateof12/28/2015),wereused.Soilsamples,approximately100g,fromeachsitewereweighed,transferredtoanautoclavedmortarandpestle,andmixedgentlytoobtainahomogeneousmixture.Themixturewasaliquotedintoeightvialsforeachsite,witheachvialcontaining5gofsoil.FourvialswereinoculatedwithPd(105conidia/gsoil),andtheremainingfourvialsreceivedsterilizedwater.Following1-weekpost-incubation,twoofthefourPd-containingvialsreceivedTp(105conidia/gsoil)toob-taina1:1ratioofPdtoTp.TheothertwoPdcontainingvialsreceivedsterilizedwater(Pdonly).OftheremainingfourvialsinwhichPdwasnotinoculated,tworeceivedTp(105conidia/gsoil),whichservedasTponlycontrols,whiletheremainingtwovialsreceivedsterilizedwaterandservedassoilonlycontrols(Additionalfile2).Allthevialswereincubatedat10°Cfor5weeks.TodeterminethenumberofTprequiredforkillingPdinsubsequentexperiments,10-fold(106conidia/gsoil)and100-fold(107conidia/gsoil)higherconcentra-tionsofTpconidiathanPdconidia(105conidia/gsoil)wereaddedandthesesoilsampleswereincubatedat10°Candthenprocessedat1,3,and5weekspost-incubation(Additionalfile3).CulturerecoveryofPd,Tp,andotherfungiFollowingincubationofsoilsamplesat10°Cfor1–5weeks,100mgofsoilsamplewasremovedfromeachvialindupli-cateandtransferredinto2-mlscrewcapvials.Thesoilsamplewassuspendedin500lofsterilizedwaterandvor-texedvigorously.Ten-folddilutionsofthesupernatantwereprepared,and50lofeachdilutionwasplatedontovariousmediaplates(150mmdiameter)induplicate.Theplateswereincubatedat10°Candcheckedperiodicallyforthere-coveryofPd,Tp,andotherfungiasdescribedpreviously[30].Inbrief,SDA-AmediumwasusedtodeterminetheTpcolony-formingunits(CFUs),SDA-Awithcyclohexi-midewasusedtodeterminePdCFU,andtheRBCmediumwasusedforthedeterminationofotherfungipresentinthecaveandminesoilsamples.ThepercentkillingofPdbyTpwascalculatedusingaformula1(PdCFUexperi-ment/PdCFUcontrol)×100.DualculturechallengestudiesFordualculturechallengestudies,approximately1×1mmoffreshlygrownTpfungalhyphalmatwasinoculatedonSinghetal.Microbiome (2018) 6:139 Page2of11

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onesideoftheSDAplate.Similarly,freshlygrownfungalhyphalmatfromvariousfungiobtainedfrombothACandBHMwereinoculatedontheoppositesideoftheplate.TheinteractionsbetweenTpandotherfungalisolateswereassessedbymeasuringcolonydiameterfollowing18dayspost-incubationat10°C.TheTpanddifferentfungaliso-lateculturesaloneservedasacontrol.Statisticalanalysisforculture-basedassays(CFUenumer-ationandcolonydiameter)wasperformedusingGraphPadPrismsoftware(GraphPad,SanDiego,CA,USA).Thecom-parisonoftwogroupswasperformedusingatwo-tailedun-pairedt-testwithapvalueof0.05acceptedassignificant.MetagenomicsofsoilsamplesDNAlibrariesForthemicrobialcommunityanalysis,DNAfrom100mgofuntreatedandTp-treated(105and106conidia/gsoilforACand105conidia/gsoilforBHM)soilsamples(indu-plicate)wereextractedwiththePowersoilDNAisolationkit(MOBIOLaboratories,Carlsbad,CA,USA)(Add-itionalfile4).DNAconcentrationsweremeasuredusingaNanodropspectrophotometerND2000(Nano-DropTechnologies,Wilmington,DE,USA).DNAlibrarieswerepreparedusingatwo-stepPCR.InthefirstPCR,thepri-mersetstargetedtheITS2regionoftheribosomalRNA(rRNA)geneoffungi[31]andthehypervariableregionV4ofthe16SrRNAgeneofbacteriaandarchaea[32].ThesameIlluminaadaptorsequenceswereaddedtobothfungalandbacterial/archaealprimers.Thus,theprimersetusedforthefirstPCRforfungihadthefollowingsequence:Forwardprimer5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGAACTTTYRRCAAYGGATCWCT-3(locusspecificsequenceinbold)andReverseprimer5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGCCTCCGCTTATTGATATGCTTAART-3(locus-specificsequenceinbold).TheprimersetusedforthefirstPCRforbacteria/archaeahadthefollowingse-quence:Forwardprimer5-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGYCAGCMGCCGCGGTAA-3(locus-specificsequenceinbold)andReverseprimer5-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACNVGGGTWTCTAAT-3(locus-specificse-quenceinbold).PCRreactionswerecarriedoutinatotalvolumeof25l.ThreemicrolitersofextractedDNA(5ng/l)wasaddedtothePCRreactioncontaining15.1lofster-ilewater,0.2lofbovineserumalbumin,2.5lofAccutaqLAbuffer,2lofdNTPs,0.2lofAccutaq™LADNApoly-merase(Sigma-Aldrich,St.Louis,MO,USA),and1leachoffungalandbacterial/archaealforwardandreverseprimers.AllthePCRreactionswerecarriedoutintriplicateonaC1000TouchThermalCycler(BioRad,Hercules,CA,USA).Thethermocyclingconditionsforfungal-specificPCRwereinitialdenaturationat95°Cfor1min,followedby27cyclesofdenaturationat94°Cfor30s,annealingat55°Cfor1min,extensionat68°Cfor1min,followedbyfinalexten-sionat68°Cfor5min.Forbacterial/archaeal-specificPCR,thermocyclingconditionswere95°Cfor1min,followedby27cyclesofdenaturationat94°Cfor30s,annealingat63°Cfor1min,extensionat68°Cfor1min,andafinalexten-sionat68°Cfor5min.Thefirst-stagePCRproductswererunontheagarosegeltoconfirmthelengthoftherespect-ivebands.TriplicatesampleswerepooledtolimitpossiblePCRartifacts.AnAgencourtAMPureXPPCRpurificationkit(BeckmanCoulter,Inc.,Indianapolis,IN,USA)wasusedforthepurificationofthePCRproducts.ThesecondPCRstepaddeddualindices(barcode)alongwithIlluminase-quencingadaptorsusingtheNexteraXTindexkit(Illumi-na-16SMetagenomicsprotocol).PCRreactionswerecarriedoutinatotalvolumeof50l,whichcontained5lofPCRproductfromthefirstPCRandthe2×KAPAHifiHotStartReadymix.Thereactionconditionswereinitialde-naturationat95°Cfor3min,followedbyeightcyclesofde-naturationat95°Cfor30s,annealingat55°Cfor30s,extensionat72°Cfor30s,andafinalextensionat72°Cfor5min.AllPCRreactionswereperformedintriplicate.ThePCRproductswerecleanedusinganAgencourtAMPureXPPCRpurificationkit.DNAconcentrationwasmeasuredusingaQubit2.0Fluorometer(Invitrogen,Carlsbad,CA,USA)andthesizewasmeasuredwithanAgilentTapesta-tionusingtheD1000HighSensitivitykit.TheresultingDNAlibrarieswerepooled,denatured,andsequencedonMiSequsingMiSeqReagentkitv3(600cycles)(IlluminaInc.,SanDiego,CA,USA)bytheWadsworthCenterAd-vancedSequencingCoreFacility.SequenceprocessingTheWadsworthSequencingCoredemultiplexedreadsandremovedprimerandlinkersequencesbeforeana-lysis.ThesubroutinesofBBToolsv36.38[33],BBmergeandBBduk,mergedallpairsofforwardandreversereads,qualitytrimmedmergedpairs(withtheparame-terstrimq=20,minq=20,minlength=150,minavgqual-ity=20,efilter=3,mininsert=250,mininsert0=250),andremovedanyremainingforwardandreverseprimersforfungalandbacterialsamples.TheITS2regionfortaxonomicidentificationwasextractedfromfungalse-quencesusingITSxv.1.0.11[34],whichremoved5.8Sand28Sregionsfrommergedsequences.ChimericsequenceswereremovedusingUSearch61withamodi-fiedUniteITS2-onlyŽreferencedataset[35](version7.2release28.06.2017)whichwasdesignedtoserveasachimera-freereferencedatabase.Chimeric16SsequenceswereremovedfrombacterialsamplesinQIIMEv.1.9.0[36]viaUsearch61[37,38]andthemostrecentversionoftheGreengenesdatabase(gg_13_8minorreleaseofgg_13_5from15.08.2013).Usingdefaultparameters,QIIMEfurtherprocessedquality-filteredsequences,whichwerethenclusteredintooperationaltaxonomicunitsSinghetal.Microbiome (2018) 6:139 Page3of11

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(OTUs)at97%similaritybytheUCLUSTalgorithm[37]andassignedtaxonomybymothurv.1.25[39,40]withthepickopenreferenceOTUoptionandadefaultconfidencelevelof0.5.ThelatestreleasesoftheGreengenes16s(gg_13_8)andUNITEdatasets(version7release28.06.2017)[41],bothclusteredat97%similarity,servedasthereferencedatabasesforclusteringandtaxonomicassignmentofbacterialandfungalsequences,respectively.SamplestatisticsForbacterialandfungaldatasets,Chao1,observedspe-cies,andSimpsonsindexestimatesofalphadiversitywerecalculatedinQIIMEwithrarefiedOTUtables.Sig-nificantdifferencesacrosstreatmentsandlocationswereassessedviaanon-parametrict-testwith999permuta-tionsofthepvalue.QIIMEalsocalculatedbetadiversitywithBrayCurtis,andEuclideanmetricsandsignificantdifferencesincommunitycompositionwereidentifiedwithananalysisofsimilarities(ANOSIM).PrincipalcomponentanalysisplotsweregeneratedinRv.3.3.0[42]withEuclideandistancematricestoconfirmconsistencyamongreplicatesandverifythatmostofthevariationindatasetswasderivedfromdifferencesintreatmentandlo-cation(datanotshown).Bacterialandfungalsampleswererarefiedtodepthsof190,000and92,226,respectively,andrelativeabundancesweresummarizedtoagenuslevel(ifpossible).AbundancecountatthephylumandgenuslevelswereexportedfromQIIMEandanalyzedinDESeq2v.3.5[43]inRtoidentifysignificantchangesintaxonomiccomposition.OnlytaxawithfourormorecountsacrosssampleswereincludedinDESeq2analysestoremovesparseOTUs.FungalidentificationAllfungirecoveredfromthesoilsampleswereidentifiedbymorphologicalandmolecularmethods[44].Formo-leculartesting,DNAfrompurefungalcolonieswasex-tractedusingMasterPureTMCompleteDNAandRNApurificationkits(Epicenter,Madison,WI,USA)aspermanufacturersinstructions.TheextractedDNAwasusedfortheamplificationofinternaltranscribedspacer(ITS)regions1and2(ITS1,5.8S,andITS2)oftheribo-somalgeneasdescribedpreviously[30].Insomein-stances,wheretheITSregionfailedtoprovidefungalidentification,theD1/D2regionofthelargesubunit(LSU)ofthe28SrDNAgenewasamplified[30].PCRwascarriedoutasdescribedinWhiteetal.[45].PCRproductswerecleanedwithExoSAP-IT(USBCorp.,Cleveland,OH,USA)andsequencedattheWadsworthCenterAdvancedGenomicsCore.ThesequenceswereassembledandeditedforaccuracyusingSequenchersoftware4.8(GenCodesCorp.,AnnArbor,MI,USA).AllunknownsequenceswerecomparedtotheNCBIGenBankdatabasewithblast[46]andtheWesterdijkFungalBiodiversityInstitutedatabase[47]forfungalidentifications;%identityof97wasusedforspeciesconfirmation.Incaseofdiscrepantresultsbetweenthetwodatabases,theWesterdijkFungalBiodiversityInsti-tutedatabasewaspreferredasithascuratedsequences.ResultsBiocontrolofPdinnaturalsoil–Aculture-basedapproachPdwasrecoveredfromnativeACsoilat~104CFU/gsoil(Fig.1).PdrecoverywasverylowfromBHM(~8CFU/gsoil),consistentwithourpreviousobservations[30].TreatmentofACsoilwithTp(105conidia/gsoil)resultedin100%inhibitionofthenativePdpopulationwithin5weekspost-incubationat10°C,confirminghighbiocon-trolpotentialofTp(Fig.1).However,whenACandBHMsoilsampleswereenrichedwithPd(105conidia/gsoil),theTp-inducedkillingofPdwasmarkedlyreducedtoapproximately40to43%(datanotshown).MaximumkillingofPdinenrichedsoilwasobserved,whenTpin-oculumwasincreasedfrom1-fold(105conidia/gsoil)to10-fold(106conidia/gsoil)tothatofPd(105conidia/gsoil).AlthoughTpkilledPdasearlyas1-weekpost-incubation,95%killingwasachievableat5-weekpost-incubation(Fig.2a).FurtherincreaseinTpto100-foldofPdwasnoteffectiveasthekillingofPdwasobservedtobeonly84%at5-weekpost-incubation(Fig.2a).EnrichingsoilnutrientsbyaddingmilletseedsextractdidnotimpactTp-inducedkillingofPdasonly87and72%Pdwaskilledwiththeadditionsof10-foldand100-foldmoreTp,respectively(Fig.2b).Thesere-sultsindicatedthattheratioofTptoPdof10:1 Fig.1InhibitionofnativePdbyTpinACsoilsamples.Untreatedsoil(soilalone)andsoiltreatedwithTp(105conidia/gsoil)wereincubatedat10°C.Fiveweekspost-incubation,twoaliquotsfromeachsamplewereprocessedfortherecoveryofPd.Tpinduced100%killingofnativePdintheACsoilsamples(p<0.05)Singhetal.Microbiome (2018) 6:139 Page4of11

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appearedtooptimallyrenderthemaximuminhibitoryeffect.ThesoilmicrobialcommunitiesdidnothamperTpgrowthinanyofthesoilsamplestested.TheTpgrowthwasnotonlysustainedbutalsoincreasedwithprolongedincubation(datanotshown).EffectofTptreatmentonmicrobialcommunitiesofcaveandminesoilsamples—ametagenomicsapproachSequenceanalysisAtotalof2,003,427fungalsequencespassedinitialqualityfilters.Ofthese,96%(1,921,639)passedQIIMEsqualitystandardsaswell,while0.07%(1347)wereflaggedaschimericandremoved(Table1).Thisre-sultedinanaverageof192,163sequencespersample(N=10),whichwereclusteredinto12,507OTUs.Forallbacterialsamples,atotalof3,295,648sequencespassedtheinitialqualityfiltersofBBtoolswith97%ofthesesequences(3,177,499)acceptedbyQIIMEand0.6%(21,503)identifiedaschimericandremoved(Table1).Onaverage,therewere317,749sequencespersample(N=10),whichclusteredinto27,427OTUs. Fig.2InhibitionofenrichedPdbyTpinACsoilsamples.aSoilsamplesfromACwereinoculatedwithPd(105conidia/gsoil)induplicate.Following7dayspost-incubationat10°C,Tpat10-fold(106conidia/gsoil)or100-fold(107conidia/gsoil)tothatofPd(105conidia/gsoil)wasadded.Untreatedsoil(soilalone)andsoilcontainingTpservedascontrols.TwoaliquotsfromeachsamplewereprocessedfortherecoveryofPdincultureatweek1,3,and5post-incubation.Approximately,50and67%killingofPdwasobservedwith10-foldand100-foldhigherTpatweek1post-incubation,whichgraduallyincreasedto57and72%atweek3post-incubationand95and84%atweek5post-incubation.bSoilsampleswereinoculatedwithPdandtreatedwithTpasexplainedina,exceptthatthesesamplesalsoreceived1%milletseedsextract.AdditionofmilletseedsextractdidnotenhanceTpkillingofPd. Table1FungalandbacterialcommunityanalysesbyhighthroughputsequencingSampleFungiBacteriaBBmergeandbbdukQIIMEandUsearch61BBmergeandbbdukQIIMEandUsearch61ACSoilalone196,99392,225247,465237,561ACSoilalone2161,501153,529321,874308,610ACSoil+TpA1214,333204,336393,209377,754ACSoil+TpA2166,355158,564344,568330,694ACSoil+TpB1323,672312,063444,201427,840ACSoil+TpB2116,711105,622207,965199,979BHSoilalone1156,003150,631275,597267,273BHSoilalone2233,462225,964306,924298,202BHSoil+TpA1205,092200,594304,892295,134BHSoil+TpA2329,305318,111448,953434,452Total2,003,4271,921,6393,295,6483,177,499SoilsamplesfromAeolusCave(AC)andBartonHillMine(BHM)weretreatedwithTpattheconcentrationof105conidia/gsoil(A1,A2)or106conidia/gsoil(B1,B2)followedbygDNAextraction,PCR,andhighthroughputsequencing.Untreatedsoilsamples(soilalone)wereincludedforcomparison.Note,duetosmallnumberofPdrecoveredfromBHMsoil,theTptreatmentwaslimitedto105conidia/gsoilSinghetal.Microbiome (2018) 6:139 Page5of11

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TptreatmentandlocationeffectsonalphaandbetadiversityofsoilsamplesNosignificantdifferencesinthealphadiversitywereob-servedacrosstreatmentsorgeographicallocationsforeitherbacterialorfungalcommunities(Additionalfile5).Fungalcommunitycompositionchanges,asassessedbythebetadiversityestimates,alsodidnotdiffersignifi-cantlyduetoTptreatmentofeitherACorBHMsoils.However,therewasasignificantdifferenceincommu-nitycompositionbetweenthetwolocations(ANOSIMteststatistic=1.0,pvalue0.003).Similarly,bacterialbetadiversitywasnotsignificantlyalteredbyTptreatmentwithinACorBHMbutvariedsignificantlybetweensites.TreatmenteffectontheabundanceoffungalandbacterialcommunitiesatthephylumandgenuslevelsForfungi,97%ofallsequencescouldbeassignedatthephylumlevel,withsevenphylaidentifiedfromACandfivephylaidentifiedfromBHM.BothACandBHMweredominatedbyOTUsfromAscomycotaandBasid-iomycota,whichcomprisedover80%ofthefungalcom-munities(Fig.3a).SoilsubjectedtoTptreatment(105conidia/gsoil)showednosignificantdifferencesinthephylumabundancesforfungiateitherACorBHM(Additionalfile6).However,therewasasignificantin-creaseinAscomycotaforACsoilsubjectedtoahigherTptreatment(106conidia/gsoil,Additionalfile6),whichreflectedthedramaticincreaseinTrichodermainthesesamples(Fig.3b).Similarly,analysisatthegenuslevelrevealedhighlysignificantincreasesinTrichodermaforbothACandBHMforbothtreatments,withfewothertaxabeingaffected(Fig.3b,Additionalfile7).Al-thoughnosignificantchangesinPseudogymnoascusabundancewereobserved(Additionalfile7),thegenusencompassesseveralspecies,whichmightnotbeinflu-encedbyTptreatment.Forbacteria/archaea,atotalof50phylawereidentifiedfromAC,whereasonly37wereidentifiedfromBHMwith0.27%ofthesequencesdesignatedasunclassified(Bacteria;Other).ThefivemostabundantphylainACwereAcidobacteria(28%),Proteobacteria(18%),Plancto-mycetes(13%),Chloroflexi(10%),andActinobacteria(9.5%)(Fig.3c).ThemostabundantphylaidentifiedfromBHMincludedPlanctomycetes(11%),Acidobac-teria(10%),Firmicutes(10%),andActinobacteria(8.8%),withProteobacteria(44%)comprisingalmosthalfoftheBHMbacterialcommunity(Fig.3c).Despiteapparentdifferencesinthecommunityrichnessandevennessbe-tweentheACandBHM,therewerenosignificantdiffer-encesinalphadiversity(Additionalfile5).Therewerealsonosignificantdifferencesinbacterialrelativeabun-dancesbetweenuntreatedandTp-treatedsoil(105Tpconidia/gsoil)forACatthephylumorgenuslevel(Additionalfiles8and9).ForACsoilsamplestreatedwith106Tpconidia/gsoil,12phylaand40generaweresignificantlyaffectedcomparedtountreatedsoilsamples(Additionalfiles8and9).ForBHMsoilsamplestreatedwith105Tpconidia/gsoil,nophylaandonly12generashowedsignificantchanges(Additionalfiles8and9).Tpinteractionwithothercave/minefungi—aculture-basedapproachAtotalof39fungalspecieswererecoveredfromAC,andofthese,85%belongedtoAscomycota,13%belongedtoearlydivergingfungallineage(EDFL),and2%belongedtoBasidiomycota(Fig.4a,Additionalfile10).Ofthe31fun-galspeciesrecoveredfromBHM,87%belongedtoAsco-mycota,10%belongedtoEDFL,and3%belongedtoBasidiomycota(Fig.4a,Additionalfile11).Thus,Ascomy-cotadominatedthefungalspeciesfollowedbyEDFLandBasidiomycotafrombothACandBHM.DualchallengestudyThedualinteractionstudiesindicatedthatTpishighlyspecificininhibitingPd.InadditiontoPd,onlyonefun-galisolatebelongingtogenusMicroascuswasinhibited.Therestofthe67fungalspeciescollectivelyidentifiedfrombothACandBHMwerenotinhibited,includingthecloselyrelatedspeciesPseudogymnoascuspannorum(Fig.4b,Additionalfiles10and11).DiscussionWedemonstratedthatthebiocontrolagentTpinhibitedPdinthepresenceofmicrobesthatarenativetothesoilfromtheaffectedhibernacula.Thisfindingfurtherex-pandedourearlierobservationsoftheefficacyofTpinkillingofPdinautoclavedsoilsamples[29].AnotherimportantfindingofthisstudywasthespecificityofTpforkillingPdwithminimaltonoimpactonthemicro-bialdiversityandcommunitystructuresofsoilsamplestestedfrombothACandBHM.Eventhoughthemicro-bialcommunitycompositionsofACandBHMweresignificantlydifferent,theywerenotaffectedbyTptreat-ment,suggestingthatthesoilcommunitiesarerelativelyrobustandindifferenttoTptreatment.Thecombinedculture-basedandmetagenomicsapproachesallowedustofollowthefateofthebiocontrolagentanditstargetinthetreatedsoil.Culture-basedmonitoringofPdandTpwasimportanttoestimatelossofviableorganismswhileDNA-basedapproachesprovidedbettercensusofmicrobialcommunities.SeveralDNAandculture-basedstudieshaverevealedwidedistributionandpersistenceofPdinbathibernac-ula[18,21,48,49].OtherpublishedcavefungalsurveysindicatethatPdcouldsurviveandpersistinbathiber-naculaforprolongedperiodsandcanhaveimpactonbothWNSdiseasemanagementandepidemiology[18].Singhetal.Microbiome (2018) 6:139 Page6of11

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Fig.3(Seelegendonnextpage.)Singhetal.Microbiome (2018) 6:139 Page7of11

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ToeradicatePdfrombathibernacula,weneedhighlycompetentbiocontrolagent,whichcangrow,sustain,andselectivelykillPdwithoutimpactingthehibernaculaecosystem.TheTpstrainusedinthisinvestigationful-fillsallthesecriteria,therebystrengtheningtheargu-mentforapplicationofTpasapotentialbiocontrolagentagainstPdincavesandmines.Inconcordancewithmetagenomicsanalysis,thedual-culturechallengestudiesofTpwithseveralfungirecoveredfromACandBHMrevealedthatexceptforoneisolateofMicroascusspecies,thegrowthofotherfungiwasnotaffectedbyTp.Microascusisasoilsapro-phyteandacommonagentofbio-deterioration[50].SinceotherspeciesinthegenusMicroascuswerenotinhibitedbyTp,wedonotanticipatedeleteriouseffectsoncave/mineecosystem.Conversely,Tpgrewwellinthepresenceofbothfast-andslow-growingfungi,aswellasinthepresenceofothermicrobialcommunitiesindigenoustothesoilsamplestested.ThehighsurvivalpotentialofTpinhibernaculasoilsuggestsitsabilitytosurviveunderunfavorableconditionsandhighrepro-ductivecapacity.Microbialcommunitiesplayseveralcriticalrolesinthesoil,includingorganicmatterdecompositionandcontrolofitscycle,regulationofmineralnutrientavailability,andnitrogenfixation[51].Thousandsofbacterial,archaeal,andeukaryoticorganismsarepresentinnaturalsoilandcollectivelycontributetomaintainingthemyriadoffunctionsofsoil.Microbialinoculationofabiocontrolagentcancausetremen-douschangesinthenumberandcompositionoftaxonomicgroups.Thesechangescanincreasethedi-versityofthesoilsampleswhilealsohavingtoxicef-fectsontheindigenousmicrobes[52].Thus,thepracticaluseofanymicrobialinoculationshouldberigorouslytestedinalaboratorysettingtoavoidanydeleteriouseffectstomicrobialdiversityinsoils.Tothisend,wehaverigorouslytestedtheuseofTpasabiocontrolagentfortheeradicationofPdincaveandminesoilsamplesandTptreatmentinlargehadnoimpactonthenativemicrobialcommunitiesotherthanPd.ConclusionsThepresentstudydemonstratestheremarkablespeci-ficityandhighpotencyofTpforkillingPdinthepresenceofindigenousmicrobialcommunitieswithminimaltonoimpactonthemicrobialcommunity (Seefigureonpreviouspage.)Fig.3RelativeabundanceoffungalandbacterialcommunitiesinsoilsamplesfrombathibernaculawithorwithoutTptreatment.ThesoilsamplesfromAeolusCave(AC)andBartonHillMine(BHM)weretreatedwithTpattheconcentrationof105conidia/gsoil(A1&A2)or106conidia/gsoil(B1&B2).Untreatedsoilsamples(soilalone)wereincludedforcomparison.gDNAwasextractedfollowedbyPCRandhighthroughputsequencing.aTherelativeabundanceoffungalphylaisshown.Ascomycotadominated,followedbyBasidiomycotaandearlydivergingfungallineages(Chytridiomycota,Entomophthoromycota,Mortierellomycota,Mucormycota,andRozellomycota)frombothACandBHM.bTherelativedistributionof25mostabundantfungalgeneraisshown.TheincreasesinTrichodermaasrepresentedby“T”inbothACandBHMwereduetotheexogenousadditionofTp.cTherelativeabundanceofbacterialphylaisshown.AcidobacteriadominatedACsoilandProtobacteriadominatedBHMsoil Fig.4FungalrecoveryfrombathibernaculaandtheirinteractionwithTp.aSoilsamplesfromAeolusCave(AC)andBartonHillMine(BHM)weresuspendedinsterilewaterandthenspreadonculturemediaplates.ColoniesrecoveredwereidentifiedbysequencingoftheITSandD1/D2regionsoftheribosomalgenefollowedbyBLASTsearch.Piechartsrepresenttherelativedistributionsoffungalphyla.AscomycotadominatedfollowedbyEarlyDivergingFungalLineage,andBasidiomycotainbothACandBHM.bInteractionofTpwithotherfungalspeciesisolatedfromACandBHMwascarriedoutonSDAplateandtheresultsoftheseinteractionswereassessed18dayspost-incubationat10°C.UpperpaneldenotesTp-inducedinhibitionofPdandMicroascusspecies(Ms).LowerpaneldenotesfungalspeciesnotinhibitedbyTp.TwosuchexamplesareMortierellaclonocystis(Mc)andPenicilliumsopii(Ps)Singhetal.Microbiome (2018) 6:139 Page8of11

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structureordiversitypresentinACandBHMsoilsamples.ThestudyrigorouslytestedtheapplicationofTpinnaturalsoilsamplesinalabsetting,withre-sultsthatstrengthenstheargumentforTpsapplica-tionasabiocontrolagentunderfieldconditions.AdditionalfilesAdditionalfile1:ConcentrationsofextendedpanelofantibioticsinSabourauddextroseagar.(DOCX13kb)Additionalfile2:FlowchartofPdandTp(1:1ratio)interactioninACandBHMsoilsamples.(TIF294kb)Additionalfile3:FlowchartofPdandTp(1:10and1:100ratio)interactioninACsoilsamples.(TIF324kb)Additionalfile4:FlowchartofPdandTpinteraction(1:1and1:10ratio)inACsoilandPdandTpinteraction(1:1ratio)inBHMsoilfollowedbyDNAextractionandmetagenomicsanalysis.(TIF184kb)Additionalfile5:Comparisonofalpha-diversityamongTptreatmentsandlocationsusinganon-parametrict-test.(XLSX55kb)Additionalfile6:DESeq2analysisofchangesintheabundanceoffungalphylainsoilfrombathibernaculawithorwithoutTptreatment.(XLSX55kb)Additionalfile7:DESeq2analysisofchangesintheabundanceoffungalgenerainsoilfrombathibernaculawithorwithoutTptreatment.(XLSX97kb)Additionalfile8:DESeq2analysisofchangesintheabundanceofbacterialphylainsoilfrombathibernaculawithorwithoutTptreatment.(XLSX19kb)Additionalfile9:DESeq2analysisofchangesinabundanceofbacterialgenerainsoilfrombathibernaculawithorwithoutTptreatment.(XLSX193kb)Additionalfile10:FungirecoveredfromACsoilandassessedforgrowthinhibitionbyTp.(DOCX23kb)Additionalfile11:FungirecoveredfromBHMsoilandassessedforgrowthinhibitionbyTp.(DOCX20kb)AbbreviationsAC:Aeoluscave;ANOSIM:Analysisofsimilarities;BHM:BartonHillMine;CFU:Colony-formingunit;EDFL:Earlydivergingfungallineage;ITS:Internaltranscribedspacer;LSU:Largesubunit;OTUs:Operationaltaxonomicunits;Pd:Pseudogymnoascusdestructans;PDA:Potatodextroseagar;RBC:Rosebengalagarwithchloramphenicol;rRNA:RibosomalRNA;SDA:Sabourauddextroseagar;Tp:Trichodermapolysporum;WA:Wateragar;WNS:White-nosesyndromeAcknowledgementsWearethankfultotheWadsworthCenterMedia&TissueCultureCoreforthepreparationofvariousmediaandreagentsandWadsworthCenterAdvancedGenomicsCoreforsequencingofDNAsamples.WealsothankMr.AlHicksforprovidingussoilsamplesforbothACandBHM.ThemembersofChaturvediLabareacknowledgedfortheirsupportandhelp.FundingThisstudywassupportedinpartwithfundsfromFishandWildlifeService(F15AS00188)toSCandVCandinpartwithfundsfromtheNationalScienceFoundation(1203528)toVCandSC.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.AvailabilityofdataandmaterialsThedatasetsgeneratedandanalyzedduringthecurrentstudyhavebeendepositedintheNCBIBioProjectdatabase(accessionnumberPRJNA433166)(https://www.ncbi.nlm.nih.gov/bioproject/).RawsequencesandmetadataareavailableintheSRAdatabasewiththefollowingaccessionnumbers:SRR6676481,AeolusCaveSoilAlone,fungaldata,replicate1;SRR6676487,AeolusCaveSoilAlone,fungaldata,replicate2;SRR6676483,AeolusCaveSoil+Tp(105conidia/gsoil),fungaldata,replicate1;SRR6676489,AeolusCaveSoil+Tp(105conidia/gsoil),fungaldata,replicate2;SRR6676477,AeolusCaveSoil+Tp(106conidia/gsoil),fungaldata,replicate1;SRR6676491,AeolusCaveSoil+Tp(106conidia/gsoil),fungaldata,replicate2;SRR6676479,BartonHillMineSoilAlone,fungaldata,replicate1;SRR6676493,BartonHillMineSoilAlone,fungaldata,replicate2;SRR6676475,BartonHillMineSoil+Tp(105conidia/gsoil),fungaldata,replicate1;SRR6676485,BartonHillMineSoil+Tp(106conidia/gsoil),fungaldata,replicate2;SRR6676480,AeolusCaveSoilAlone,bacterialdata,replicate1;SRR6676486,AeolusCaveSoilAlone,bacterialdata,replicate2;SRR6676482,AeolusCaveSoil+Tp(105conidia/gsoil),bacterialdata,replicate1;SRR6676488,AeolusCaveSoil+Tp(105conidia/gsoil)bacterialdata,replicate2;SRR6676476,AeolusCaveSoil+Tp(106conidia/gsoil)bacterialdata,replicate1;SRR6676490,AeolusCaveSoil+Tp(106conidia/gsoil),bacterialdata,replicate2;SRR6676478,BartonHillMineSoilAlone,bacterialdata,replicate1;SRR6676492,BartonHillMineSoilAlone,bacterialdata,replicate2;SRR6676474,BartonHillMineSoil+Tp(105conidia/gsoil),bacterialdata,replicate1;SRR6676484,BartonHillMineSoil+Tp(105conidia/gsoil)bacterialdata,replicate2.Apermanentlinktothedatacanbefoundathttps://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP132276.Authors’contributionsAPSandSCconceivedanddesignedtheexperiments.APSperformedtheexperiments.APS,ELN,andSCanalyzedthedata.APS,ELN,SC,andVCwrotethepaper.Allauthorsreadandapprovedthefinalmanuscript.EthicsapprovalandconsenttoparticipateThecollectionofsoilsamplesfromBartonHillMineinNewYorkState(NYS)wasdonewiththepermissionandcooperationofDEC,assupplementalactivitiesduringstandardwinterhibernaculasurveysconductedbytheNYSDepartmentofEnvironmentalConservation(NYSDEC).AeolusCavesoilsampleswerecollectedwiththepermissionandcooperationoftheVermontDepartmentofFishandWildlife(VDFW)asasupplementalactivityduringascheduledvisittothesite.Thesampleswerecollectedasmembersoftheagencyteamswerecountingthebats,withnoadditionaldisturbanceevents.Novisitationandsamplingpermitswererequiredineitherstate.ConsentforpublicationNotapplicable.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Publisher’sNoteSpringerNatureremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations.Authordetails1MycologyLaboratory,WadsworthCenter,NewYorkStateDepartmentofHealth,120NewScotlandAvenue,Albany,NY12208,USA.2BioinformaticsCore,WadsworthCenter,NewYorkStateDepartmentofHealth,Albany,NY,USA.3DepartmentofBiomedicalSciences,SchoolofPublicHealth,UniversityatAlbany,Albany,NY,USA.Received:18February2018Accepted:2July2018 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