Nutritional Capability of and Substrate Suitability for Pseudogymnoascus destructans, the Causal Agent of Bat White-Nose Syndrome


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Nutritional Capability of and Substrate Suitability for Pseudogymnoascus destructans, the Causal Agent of Bat White-Nose Syndrome

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Nutritional Capability of and Substrate Suitability for Pseudogymnoascus destructans, the Causal Agent of Bat White-Nose Syndrome
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PLOS One
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Raudabaugh, Daniel B.
Miller, Andrew N.
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Wns ( local )
Pseudogymnoascus Destructans ( local )
Substrate Suitability ( local )
P. Destructans ( local )
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serial ( sobekcm )

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Pseudogymnoascus destructans, the causal agent of bat white-nose syndrome, has caused nearly six million deaths in North American bats since its introduction into the United States in 2006. Current research has shown that caves can harbor P. destructans even after the infected bats are removed and bats no longer visit or inhabit previously infected caves. Our research focuses on elucidating reservoir requirements by investigating the nutritional capabilities of and substrate suitability requirements for six different P. destructans isolates from various localities including Illinois, Indiana, New York (Type specimen), and Pennsylvania. Enzyme assays implicate that both urease and b-glucosidase appear to be constitutive, lipase and esterase activity were more rapid than proteinase activity on 6% gelatin, gelatin degradation was accompanied by medium alkalinization, the reduction of thiosulfate generated hydrogen sulfide gas, chitinase and manganese dependent peroxidase activity were not visually demonstrated within eight weeks, and keratinase activity was not evident at pH 8 within eight weeks. We demonstrate that all P. destructans isolates are capable of growth and sporulation on dead fish, insect, and mushroom tissues. Sole nitrogen source assays demonstrated that all P. destructans isolates exhibit Class 2 nitrogen utilization and that growth-dependent interactions occur among different pH and nitrogen sources. Substrate suitability assays demonstrated that all isolates could grow and sporulate on media ranging from pH 5–11 and tolerated media supplemented with 2000 mg/L of calcium and 700 mg/L of three separated sulfur compounds: thiosulfate L-cysteine, and sulfite. All isolates were intolerant to PEG-induced matric potential with delayed germination and growth at −2.5 MPa with no visible germination at −5 MPa. Interestingly, decreasing the surface tension with Tween 80 permitted germination and growth of P. destructans in −5 MPa PEG medium within 14 days suggesting a link between substrate suitability and aq

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NutritionalCapabilityofandSubstrateSuitabilityfor Pseudogymnoascusdestructans ,theCausalAgentofBat White-NoseSyndrome DanielB.Raudabaugh 1,2 * ,AndrewN.Miller 2 1 DepartmentofPlantBiology,UniversityofIllinois,Urbana,Illinois,UnitedStatesofAmerica, 2 IllinoisNaturalHistorySurvey,UniversityofIllinois,Champaign,Illinois, UnitedStatesofAmerica Abstract Pseudogymnoascusdestructans ,thecausalagentofbatwhite-nosesyndrome,hascausednearlysixmilliondeathsinNorth AmericanbatssinceitsintroductionintotheUnitedStatesin2006.Currentresearchhasshownthatcavescanharbor P. destructans evenaftertheinfectedbatsareremovedandbatsnolongervisitorinhabitpreviouslyinfectedcaves.Our researchfocusesonelucidatingreservoirrequirementsbyinvestigatingthenutritionalcapabilitiesofandsubstrate suitabilityrequirementsforsixdifferent P.destructans isolatesfromvariouslocalitiesincludingIllinois,Indiana,NewYork (Typespecimen),andPennsylvania.Enzymeassaysimplicatethatbothureaseandb-glucosidaseappeartobeconstitutive, lipaseandesteraseactivityweremorerapidthanproteinaseactivityon6%gelatin,gelatindegradationwasaccompanied bymediumalkalinization,thereductionofthiosulfategeneratedhydrogensulfidegas,chitinaseandmanganesedependent peroxidaseactivitywerenotvisuallydemonstratedwithineightweeks,andkeratinaseactivitywasnotevidentatpH8 withineightweeks.Wedemonstratethatall P.destructans isolatesarecapableofgrowthandsporulationondeadfish, insect,andmushroomtissues.Solenitrogensourceassaysdemonstratedthatall P.destructans isolatesexhibitClass2 nitrogenutilizationandthatgrowth-dependentinteractionsoccuramongdifferentpHandnitrogensources.Substrate suitabilityassaysdemonstratedthatallisolatescouldgrowandsporulateonmediarangingfrompH5–11andtolerated mediasupplementedwith2000mg/Lofcalciumand700mg/Lofthreeseparatedsulfurcompounds:thiosulfateL-cysteine, andsulfite.AllisolateswereintoleranttoPEG-inducedmatricpotentialwithdelayedgerminationandgrowthat 2 2.5MPa withnovisiblegerminationat 2 5MPa.Interestingly,decreasingthesurfacetensionwithTween80permittedgermination andgrowthof P.destructans in 2 5MPaPEGmediumwithin14dayssuggestingalinkbetweensubstratesuitabilityand aqueoussurfacetensionalteringsubstances. Citation: RaudabaughDB,MillerAN(2013)NutritionalCapabilityofandSubstrateSuitabilityfor Pseudogymnoascusdestructans ,theCausalAgentofBatWhiteNoseSyndrome.PLoSONE8(10):e78300.doi:10.1371/journal.pone.0078300 Editor: VishnuChaturvedi,CaliforniaDepartmentofPublicHealth,UnitedStatesofAmerica Received June25,2013; Accepted September18,2013; Published October21,2013 Copyright: 2013Raudabaugh,Miller.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,which permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisstudywasfundedthroughawardsgivenbytheIllinoisDepartmentofNaturalResourcesStateWildlifeGrantsProgram(ProjectNumberT-78-R-1) andtheSection6EndangeredandThreatenedSpeciesProgram(ProjectNumberE-54-R-1)totheIllinoisNaturalHistorySurvey.Thefundershadnorole instudy design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:raudaba2@illinois.edu Introduction Thepsychrophilicfungus Pseudogymnoascus ( Geomyces ) destructans (Blehert&Gargas)Minnis&D.L.Lindner(Pds)hasbeen identifiedasthecausalagentofbatWhiteNoseSyndrome[1],[2] whichhasbeenimplicatedinalmostsixmillionNorthAmerican batdeathssinceitsinitialoutbreakin2006[3–5].Current scientificdatasuggeststhatPdsoriginatedfromEuropeandsince itsintroductiontoNewYorkStatein2006,hasspreadto22states and5Canadianprovinces[2]. Pseudogymnoascusdestructans isknown tocausesevereskinlesionsandcutaneousnecrosisinbatsduring hibernation,whichhasbeensuggestedtodisruptwingcirculation, cutaneousrespiration,andpromotedehydration[6],[7]. Pseudogymnoascusdestructans caninfecthibernatingbatsbecausethehost’s internalbodytemperatureisreducedtoslightlyaboveambient temperatures(ca.2to10 u C)andthebatimmunesystemis reportedtobesuppressed[3],[4].Ifaninfectedbatsurvives hibernation,thebat’sbodytemperatureandimmunesystem returntonormalandPdsiseradicatedfromthehost’sepidermal layers[8].RecentresearchhasshownthatPdsiscapableof survivingincavesnolongerpopulatedbyinfectedhibernatingbats [9],whichpointstothepotentialforinfectedcavestoactasPds reservoirs.ManyaspectsofPds(geographicdistribution,temperaturedependentgrowth,andgenomics)havebeenthoroughly researched,butnocomprehensiveresearchthusfarhasbeen conductedonthebasicbiologyofPds. LimitedstudieshaveshownPdstobetemperaturesensitive, producenumerousenzymesunderlaboratoryconditions,and containseveraldualvirulencefactors.Temperatureeffectsonthe growthandmorphologyofPdsdemonstratedthatPdshasactive growthfrom0toabout20 u C,withoptimalgrowthfrom12.5to 15.8 u C,andanincreasedoccurrenceofhyphalstress-related structures(hyphalthickening,presenceofarthrosporesand chlamydospore-likestructures)aboveca.15 u C[10]. Invitro ,Pds hasbeenshowntoproduce b -glucosidase,N-acetylb -glucosaminidase,acidandalkalinephosphatases,esterase/esteraselipase/ lipase,leucineandvailinearylamidase,naphthol-AS-B1-phophohydrolase,variousproteinases(albumin/casein/gelatin),and PLOSONE|www.plosone.org1October2013|Volume8|Issue10|e78300

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urease,whilenoenzymaticactivitywasindicatedforcystine arylamidase, a -chymotrypsin,alpha/betagalactosidase,trypsin, b glucoronidase, a -fucosidase,and a -mannosidase[11].Notably, someoftheseenzymespresentinPds(urease,proteinase(aspartyl), andsuperoxidedismutase)arefoundinotherpathogenicfungi [12],[13]andareconsidereddualvirulencefactors[14]. Ourresearchgoalsaretoobtainamorecomprehensive understandingofthereservoirrequirementsforPdsbyfocusing onthreekeyquestions:1)WhatisthenutritionalcapabilityofPds? 2)WhichcavesubstratesaresuitableforPdsgrowth?and,3)Does theincreaseinwatersurfacetensionatcoldtemperatureseffect substratesuitability?Toanswerourfirsttwoquestionswe categorizedcarboncontainingcaveresourcesintofourgroupsof substrates:keratinaceous,chitinaceous,cellulosic,andprotein/ lipidrichsubstrates.Inadditiontocommoncarboncontaining caveresources,weinvestigatedotherimportantenvironmental factorsincludingnitrogenutilization,pHtolerance,andtolerance tovariousenvironmentallevelsofcalciumandsulfurcompounds. Toelucidatehowtheincreaseinwatersurfacetensionatcold temperatureseffectsubstratesuitabilityweexaminedthegrowthof Pdsunderknownlevelsofmatric-inducedwaterstressand comparedthoseresultstogrowthunderidenticalconditions containingasurfacetensionreductionagent.Lastly,wepreformed anadditionalassaytoinvestigateifPdscouldproduceitsown biologicalsurfactant.MethodsAutomatedsystemsforcharacterizingfungalmetabolicfunctionsarebecomingmorecommoninresearch.Onesuchsystem, theBiologmicroplateiswidelyknownforitsbacterialplatesbut alsooffersafungalcarbonsourcespecificplate(FFGENIIplate). EachtestwellintheBiologFFGENIIplatecontainsonecarbon sourcewherecolordevelopmentandturbidityaremeasured.For ourcarbonassays,wedecidedtouseamoretraditionalapproach inordertomonitorsecondaryreactions,whichareequally importantforunderstandinghowsubstratesaredegraded.We determinedthattraditionalculturetechniqueswouldalsoallowfor greaterflexibilitybyquicklyadding,combining,oraltering environmentalresources,whichcaninfluencemetabolicfunction.CultureandassayconditionsSixPdscultureswereexamined;threefromIllinois(ILLS69284, ILLS69285,ILLS69286),andoneeachfromIndiana (GdINMSC7),NewYork(MYA-4855,Typeculture)and Pennsylvania(ILLS69283).Allcultureswereobtainedfrom publiclyavailablemycologicalculturecollectionsattheUniversity ofIllinois,WesternIllinoisUniversity,ATCC,andLockHaven University.Allisolatesweremaintainedin24hourdarknesson DifcoSabouraudÂ’sDextroseAgar(SDA)at7 u C.Topromote conidiation,200mg/LMnSO4(SigmaAldrich(SA))wasaddedto thestockculturemedium.Unlessnoted,allmediaweresterilized at121 u Cunder15PSIfor15minutes,wrappedwithParafilm (FisherScientific(FS))afterinoculation,andallplateswere invertedtwodaysafterinoculation.Allassayswereinoculatedwith activelygrowingmyceliumandreplicatedtwiceatdifferenttimes usingtworeplicateseachtime.Allassayswereincubatedin 24hourdarkness;carbon,sulfur,pH,calcium,andnitrogenassays wereconductedat9 u C,whilesurfacetension,matricpotential, andthebiosurfactantassayswereconductedat10 u C.AllPetri plateassayscontainedca.20mlofmediaunlessotherwisenoted. Glasswarewascleanedbyimmersionin30%glacialaceticacidor 6MHClforatleast6hours,rinsedwithdistilledwater,and autoclavedtwicepriortouse.AnalysesMatricpotentialwasadjustedusingpolyethyleneglycol(PEG) 8000(FS)followingtheequation; y =1.29[PEG]2T-140[PEG]24.0[PEG]where[PEG]=gramofPEG8000pergramofwater andT=temperature( u C)[15].Surfacetensionwasmeasuredwith astalagmometerusingfungalfree(centrifugedat14000rpmfor 2min)10 u Cmediafollowingtheequation L=(mL/mWater) waterwheremLandmwateraretheaverageweightof50dropsand water=74.23 6 102 3N/m.Sporecountwasconductedusingan improvedNeubauerhemocytometer,pHwasmeasuredwitha MilwaukeeMW102pH/temperaturemeter,microscopicevaluationswereconductedusinganOlympusSZX12stereomicroscope,cavesoilmatricpotentialwasdeterminedfollowingtwo equations;Wf , 45.3%:Log(h)=5.327-0.0779WforWf . 45.3%: Log(h)=2.412-0.0135WfwhereWfisfilterpaperwatercontent (%)andh=matricpotential(KPa)[16].One-wayANOVAwith TukeyÂ’sposttestwasperformedusingGraphPadPrismversion 5.03forWindowstodeterminestatisticalsignificanceforthe toleranceandbiosurfactantassays.WholecarbonsourceassaysFreeze-dried Locustamigratoria L.,fresh Poecilia sp.,anddried Lentinulaedodes (Berk.)PeglerwereplacedintoglassPetriplates, autoclaved,andinoculatedwithanautoclavedcottonswabthat waspre-moistenedina1%NaClsolutionandrubbedoverca. 5mm 6 5mmsporulatingPdsculture.Freshexoskeletonsof Pleoticusmuelleri Bate(50g)weresubjectedtoademineralization step[17],whichconsistedoftwo,300mlicecold0.25MHCl treatments(onefor5minutesandthesecondfor35minutes) followedbyneutralizationwithdistilledwater.Additionalfresh exoskeletonsweresubjectedtotheabovedemineralizationstep andadeproteinationstep[17],whichincorporatedthreeseparate 100ml(1.0MNaOH)treatmentsat95 u C;thefirstandsecond treatmentswere2hours,andthethirdwasfor1hourfollowedby neutralizationwithdistilledwater.Thedemineralizedand deproteinatedexoskeletonswereplacedinglassPetriplates, autoclaved,andinoculatedasabove.Allplateswerevisually monitoredtwiceaweekforthepresenceofmycelialgrowthand conidiation.CarbonsourceenzymaticassaysProteinaseactivity.Gelatindegradationandalkalinization assaysmediumconsistedofdistilledwatercontaining3%or6% gelatin(Knoxoriginalunflavored)and0.002%phenolred(SA)as thepHindicator[18].The3%gelatinmediumwasadjustedto pH6.0 6 0.1andthe6%gelatinmediumwasadjustedto pH7.0 6 0.1with3%KOHpriortosterilization.Eachsterilized gelatinmedium(150ml)waspipettedintoaseparateUV-sterilized 96-wellstandardmicroplateandeachtestwellwasinoculatedwith aBBsizepelletofscrapedmycelium.The96-wellplateswere coveredwithmicrotiterplatesealingfilm,incubated,and monitoreddailyforgelatindegradation(liquification)andpH change(colorchangefromyellowbelowpH6.8tored/pinkabove pH8.2).Ourgelatinmediumdifferedfrom[18]sincetheonly carbonandnitrogensourcewasgelatin.Wedidnotincludeother carbonornitrogensourcesbecausedermatophytesarereportedto useproteinsfortheirsolesourceofcarbonandnitrogen[19].We used3%gelatinforthedetectionoflowgelatinaseactivityand alkalinization,and6%gelatinmediumforgelatinaseactivityand toascertainiftherewasmediumacidificationpriortomedium alkalinization.Keratinaseactivity.Alpha-keratinbasalmedium(ca.pH8) consistedof0.1%glucose(SA),0.2%yeastextract(Difco),0.1% KH2PO4(FS),0.02%MgSO4N 7H2O(FS),1%Na2CO3(SA,Physiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org2October2013|Volume8|Issue10|e78300

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autoclavedseparatelyandaddedtoachievepH8),and1.5%agar (SA)L-1distilledwater[20].Keratinazure(SA)wascutintosmall ca.3mmlengthsections,autoclavedseparatelyinadrybeaker andaportionofthecooled(55 u C)basalmediumwasaddedtothe keratinazuretoproducea1%keratinazureoverlaymedium. Usingsteriletechniques,5mlofbasalmediumwasaddedto 10mlautoclavedglasstesttubes.Afterthebasalmedium solidified,1mlofthe1%azurekeratinoverlaymediumwas pipettedintotheglasstesttubes.Eachtesttubewasinoculated withaBBsizepelletofscrapedmycelium.Testtubesweresealed withParafilmandvisuallymonitoredfordyereleaseintothebasal mediumatweek6andweek8.Wedidnotincludecarboxymethyl celluloseinourmediumasameanstoeliminateanextraneous carbonsource,whichwouldnotbefoundonnaturalkeratin substrates.Keratindegradationcanbeinfluencedbyenvironmentalreducingagents[21],sotwoadditionaliterationsofthe alpha-keratinaseexperimentwereconductedusingthekeratin basalmedium,butsupplementingoneiterationwith0.5mg/L Na2S2O3? 5H2Oandsupplementingtheotherwith0.5mg/L Na2SO3.BothiterationswereconductedatpH7.5. Beta-keratinderivedassaymediumconsistedofsolublekeratin; 10gofwhitechickenfeatherswhererefluxedat100 u Cin500ml dimethylsulphoxidefor2hoursfollowedbyprecipitationusing 1000mlof-80 u Cacetone[22].Theprecipitatedkeratinwas filteredusingsterileWhatman # 1filterpaperandwashedfour times(50mleach)withsteriledistilledwater.Theprecipitatewas thenaddedtofiltersterilized0.1MphosphatebufferatpH7to obtaina0.06%solublekeratinsolution.Tothissolution,0.5g MgSO4N 7H2O,0.5gFeSO4(SA)and5mgZnSO4(SA)L-1was added,autoclaved,andportionsoftheresultingwetkeratinmat wereplacedinsterileglassPetridishes.Thekeratinmatswere inoculatedinthesamemannerasthewholecarbonsourceassays. Allplatesweremonitoredtwiceaweekformycelialgrowthand conidiation.Thismethoddiffersfrom[22]inthatwedidnot precipitateourkeratinin 2 90 u Cacetonebecausethisextreme precipitationtemperaturewouldonlyaffecttheresultingkeratin yield,andwebufferedourkeratintopH7toavoidkeratin degradationduringautoclaving[21].Ourgrowthmediumis similartotheliquidculturetechniquefoundin[22],exceptwe usedakeratinmatinsteadofkeratinparticlessuspendedinliquid, whichavoidspotentialaerationissuesassociatedwithsomeliquid culturetechniques[23].Chitinaseactivity.Colloidalchitinwasobtainedasfollows: 20gofcrabshellsweregroundintoapowder,aciddigestionat roomtemperaturewith150mlof12MHClfor1hourunder constantstirring,precipitationinicecoldwater,vacuumfiltration (coffeefilter:twolayers),neutralizationbywrappingtheprecipitateinmultiplelayersofcoffeefiltersandfloatingthewrapped precipitateintwochangesof500mlofdistilledwateratroom temperatureforatotalof24hours,unwrapped,autoclaved,and storedinasealedcontainerat10 u Cuntilneeded[24].Colloidal chitinmediaconsistedof2%moistcolloidalchitin(preparedfrom crabshellsasabove),0.7gK2HPO4(SA),0.3gKH2PO4,0.5g MgSO4N 7H2O,0.5gNaCl(FS),0.5gFeSO4,0.2mgZnSO4, 0.1mgMnSO4(SA),5mgthiaminechloride(SA),and100mg Biotin(SA),whichwasfiltersterilized(0.20mm)andaddedto 55 u Cmediaand2%agarL-1distilledwater.Abasalversionofthis medium(containingnochitin)waspouredintothePetriplateand allowedtosolidifyafterwhich2mlofcolloidalchitinmediumwas pipettedontop,inoculatedwitha5mmagarplug,andexamined twiceperweekforthepresenceofaclearingzonearoundthe colony.Ourextractiondifferedfrom[24]onlyintheneutralizing step;wewrappedthechitinprecipitateincoffeefiltersandfloated theprecipitateinwatertomaximizetheresultingyieldand prolongtheneutralizationstep.Toachieveclearresults,ourassay utilizedachitinfree-basalmediuminconjunctionwiththe2% chitinmediumasanoverlay.Cellulaseandligninaseactivity.Beta-glucosidaseactivity wasdeterminedusingesculinplusironagar:5gL-tartaricacid diammoniumsalt(SA),0.5gMgSO4N 7H2O,1gK2HPO4,0.1g yeastextract,0.001gCaCl2(SA),2.5gEsculinsesquihydrate (AlfaAesar),and8gagarL-1distilledwater[25].Themedium wasadjustedtopH7.0 6 0.1with3%KOHpriortosterilization. Aqueousferricsulphate(2%)wasaddedto55 u Cmediumataratio of1mlper100mlofmedia.Themediumwasthenpouredinto Petriplatesandinoculatedwitha5mmdiameteragarplugand evaluatedtwiceperweekfortheappearanceofabrownishblack precipitate.Manganese-dependentperoxidasewasassayedby supplementing39gDifcoPotatoDextroseAgar(PDA)with 100mg/Land200mg/LMnSO4L-1distilledwater[26].The mediumwassterilize,inoculatedasabove,andexaminedtwice perweekforablackprecipitate.Lipaseandesteraseactivity.RhodamineBagarmedium consistedof8gtryptone(Difco),4gNaCl,and10gagarL-1distilledwater[27].ThemediumwasadjustedtopH7.0 6 0.1 with3%KOHpriortosterilization.Themediumwascooledto 55 u Cpriortotheadditionof31.25mlofsterilelipid(eitherlardor oliveoil)and0.001%wt./vol.rhodamineB(Acros).Themedium wasshakenvigorouslyfor1minutetoemulsifythelipid,poured intoPetriplates,inoculatedwitha5mmdiameteragarplug,and monitoredtwiceaweekfortheappearanceoforangefluorescence whenexposedtoUVlightat365nm[28].TheVictoriaBlueB andTween80mediumconsistedof10gtryptone,5gNaCl,0.1g CaCl2(SA),0.01%wt./vol.VictoriablueB(Acros),10gTween 80(Acros,autoclavedseparately),and15gagarL-1distilledwater [29].ThemediumwasadjustedtopH7.0 6 0.1with3%KOH priortosterilization.Themediumwascooledto55 u Cpriortothe additionofTween80,shakenfor1minutetoemulsifytheTween 80,pouredintoPetriplates,inoculatedwitha5mmdiameteragar plug,andexaminedtwiceperweekforthepresenceofcalcium soap(fattyacid)crystals.Theoriginalbasemediafrom[27,28] usednutrientbroth,whichcontainspeptone(proteindigest),beef extractandNaCl.Wesubstitutednutrientbrothwithtryptone (proteindigest)sinceitisacomparablenutritionalsourceforPds andtheassayreactionwasnotcompromisedwhencomparedto thepositivecontrol( Serratiamarcescens Bizio).NitrogensourceassaysUreaseactivity.StuartÂ’sureabrothbase(0.1gyeastextract, 9.1gKH2PO4,9.5gNa2HPO4,0.012gphenolredL-1distilled water(pH6.8 6 0.2))andmodifiedChristensenÂ’sureabrothbase (1.0Tryptone,2gKH2PO4,1gdextrose(SA),5gNaCl,0.012g phenolredL-1distilledwater(pH6.8 6 0.2))withoutureawereused ascontrolmedia.Filtersterilized(0.22mm)urea(FS,20gL-1)was addedtoaportionofeachcooledmediumabovetotestforurease activity.Analiquotofeachmedia(150ml)withandwithouturea waspipettedintoaseparateUV-sterilized96-wellstandard microplatewell.Forcontrolpurposes,onlyhalfofeachofthe mediumtypeswereinoculatedwithaBBsizepelletofscraped mycelium.The96-wellplatewascoveredwithmicrotiterplate sealingfilmandwasmonitoreddaily(visually)forachangeincolor (yellowtopink)inboththeinoculatedandcontrolwells. ChristensenÂ’sureabrothbasewasmodifiedbythesubstitutionof tryptoneinplaceofpeptonesinceitisacomparablenutritional sourceforPds.Nitrate,nitrite,ammonium,L-asparagine,anduricacid assays.Thebasalmediaconsistedof20gD-glucose(SA),2g KH2PO4,0.5gMgSO4N 7H2O,0.5gFeSO4,0.2mgZnSO4,Physiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org3October2013|Volume8|Issue10|e78300

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0.1mgMnSO4,5mgthiaminechloride,and100mgBiotin: 0.20mmfiltersterilizedandaddedto55 u CmediaL-1distilled water[30]andoneofthefollowingfivenitrogensources:1)3.07g KNO3(FS),2)2.0g(NH4)2SO4(FS),3)2.58gKNO2(SA),4) 2.28gL-asparaginemonohydrate(FS),or5)1.275gC5H4N4O3(SA).Aftersterilizationandadditionofthemicronutrientsolution, eachmediumpHwasadjustedusingasaturatedsolutionof NaOHand12MHCl.AssayswereconductedatpH5,6,7,and 8forbasalmediacontainingnitrate,nitrite,L-asparagine, ammonium,ornoaddednitrogensource(control)andatpH8 foruricacid.Analiquotofeachmedium(150ml)waspipettedinto aseparateUV-sterilized96-wellstandardmicroplatewell.Each wellwasinoculatedwithaBBsizepelletofscrapedmycelium.The 96-wellplatewascoveredwithmicrotiterplatesealingfilmand monitoreddailyformycelialgrowth.SubstratesuitabilityassaysSincenumerousfungalresearchersusePDAasabasemedium forchemicaltoleranceassays[31],[32],weutilizedPDAforboth calciumandsulfurtoleranceassays.Tolerancewasdetermined25 daysaftermediainoculationbymeasuringtheradialdiameterof eachcolonytwiceat90 u anglesandaveragingthetwovalues.All replicatedatawerecompiledandanalyzedforstatisticalsignificanceaspreviouslydescribed.Calciumtoleranceassay.Assaymediaconsistedof39g DifcoPDAmediumsupplementedwith500mg/L,1000mg/L, and2000mg/Lcalciumchloride.Plateswereinoculatedwitha 5mmdiameteragarplug,incubated,andassayedfortoleranceas describedabove.Environmentalsulfurtoleranceandhydrogensulfide productionassays.Assaymediaconsistedof39gDifcoPDA mediumsupplementedwith0.2gFeSO4andoneofthefollowing: Na2S2O3? 5H2O(Baker),Na2SO3(Mallinckrodt),or97%Lcysteine(SA)at100mg,300mg,500mg,and700mgL-1distilledwater.Aftersterilization,Petriplateswereinoculatedwith one5mmdiameteragarplug,examinedtwiceperweekfora blackprecipitate,andassayedfortoleranceasdescribedabove.MediumpHassay.Ourbasalmediumconsistedof9gMalt broth(Difco),2.25gtryptone,and18gagarper900mLof distilledwater[33].Oneofthefollowingbuffersolutions(100ml) wasaddedaftersterilizationtoachievethefollowingpHlevels: pH5(6.9gNaH2PO4N H2O(FS)),pH7(3.904gNaH2PO4N H2O (FS)and3.105gNa2HPO4(Macron)),pH9(0.318gNa2CO3and3.948gNaHCO3(FS)),andpH11(5.3gNa2CO3).Plates wereinoculatedwitha5mmdiameteragarplugandtolerance wasevaluatedasdescribedabove.Theonlymodificationof[33] wasthesubstitutionoftryptoneinplaceofpeptonesinceitisa comparablenutritionalsourceforPds.Matricpotential,surfacetension,andsurfactant productionassays.Thematricpotentialmediumconsisted of0.2gsucrose,0.2gD-glucose,1gKNO3,1gKH2PO4,0.5g MgSO4N 7H2O,0.5gNaClL-1distilledwaterandadjustedto pH7withNaOH[34].ThemediumwasamendedwithPEG 8000asdescribedabovetogeneratethefollowingmatric potentials: 2 0.07MPa, 2 1MPa, 2 2.5MPa,and 2 5MPa.After sterilization,40mlofeachmediumwasinoculatedwith500mLof sporessuspendedinwater(averagesporecount =3.1 6 1076 1.1 6 106per500ml),sealedwithparafilm,andplaced onshakecultureat100rpmfor25days.Theassaywasvisually inspectedweeklyforgerminationandsubsequentgrowth. Tolerancewasqualitativelyassayedaseithervisiblegrowthor novisiblegrowthatday25.Thecurrentassayincreasedthespeed oftheshakeculturefrom[34]toaccountforthehighsurface tensionofwateratlowtemperatures[35]andincreasedthe mediumpHtoreducemycelialaggregation[36].Thesurface tensionassaywasidenticaltothematricpotentialassayexceptthat the500mlsporesuspensioncontained1%Tween80(average sporecount=4.1 6 1066 9.2 6 104per500ml)whichwasdemonstratedinasimilarstudy[36]toreducewatersurfacetension.The assaywasvisuallyinspectedweeklyforgerminationandsubsequentgrowth.TodetermineifPdsproducedextracellular biosurfactants,werepeatedthematricpotentialexperimentusing twoisolates(MYA-4855,GdINMSC7)inthe 2 1MPaconditions. Aftersterilization,40mlofthemediumwasinoculatedwitheither 500mLofsporessuspendedinwater(averagesporecount =3.1 6 1076 1.1 6 106per500ml)or500uLofsterilewater (control),sealedwithparafilm,andplacedonshakecultureat 100rpm.Onday15,fungalfreefiltratewasobtainedandthe surfacetensionwasmeasuredwithastalagmometeraspreviously describedabove.Results CarbonAllPdsisolateswerecapableofgerminationandsubsequent growthonautoclaved Locustamigratoria (insect), Poecilia sp.(fish), and Lentinulaedodes (mushroom)within30days(Figure1A–C). Germinationandsubsequentgrowthwasvisuallyobservedon demineralized Pleoticusmuelleri Bate(shrimp)exoskeletons,while minimalgrowthwasobservedonthedemineralizedanddeproteinatedexoskeletonsunder10 6 microscopicexamination (Figure1D).Gelatinliquefactionandalkalinizationoccurredon both3%and6%gelatin,andnodecreaseinpHwasobserved priortoalkalinizationonthe6%gelatinmedium.AllPdsisolates grewwellon a -keratinazuremedium,butnokeratinolyticactivity (dyerelease)wasvisuallypresent(Figure1E)andnogrowthwas evidentonthe b -keratinmat(Figure1F).Theadditionofsodium thiosulfateandsodiumsulfitetothekeratinazuremediumdidnot resultintheabilityofPdstodegradekeratinatpH7.5.Betaglucosidaseactivitywaspositivewithin1–2days,whileMndependentperoxidaseandchitinaseactivitywerenegative (Figure1G).Lipaseactivitywaspositiveonbotholiveoiland lard,andlipase/esteraseactivitywasobservedthroughthe presenceofcalciumfattyacidcrystals(Figure1H).NitrogenNitrogenassaysdemonstratedgrowthonmultiplenitrogen sources(nitrate,nitrite,ammonium,andL-asparagine)indicating aClass2nitrogenprofileforallPdsisolates.However,mycelial growthwasinfluencedbybothpHandsolenitrogensource (Figure1K).GrowthonnitratewasbetteratlowerpHvalues(5– 7),whilegrowthonL-asparagineandammoniumwasgreatestat higherpHvalues(7–8).Growthoccurredonnitritebyday20,but onlyatpH7andpH8.Growthwasminimalonuricacid.The ureaseassaywasonlypositiveonmediathatcontainedurea,with positiveresultstypicallywithin1–2daysonChristianson’smedia andwithin3–4daysonStewart’sbufferedmedium.Calcium,pH,andsulfurtoleranceAllisolateswereidenticalintheirtolerancetopH(Figure1I), calcium(Figure1J),sodiumthiosulfate,L-cysteine,andsodium sulfite.UsingANOVAanalysiswithTukey’sposttest,the combinedresultsofallsixPdsisolatesdemonstratednosignificant difference(p=0.87)incolonyradialdiameteroverthecalcium rangeofcontrol(20mg)to2000mg/Landnosignificant difference(p=0.69)incolonyradialdiameterwasevidentovera widepH(5–11)range(Figure2A–B). Pseudogymnoascusdestructans isolatesdemonstratedtolerancetoallthreesulfurcompoundswithPhysiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org4October2013|Volume8|Issue10|e78300

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nosignificantdifferenceinthecombinedcolonyradialdiameter overtherangeof100mg/Lto700mg/Lforsodiumthiosulfate (p=0.98),L-cysteine(p=0.75),andsulfite(p=0.83,Figure2C). Inaddition,Pdswasequallytolerant(p=0.13)toallthreesulfur compounds.HydrogensulfideproductionwasevidentonPDA amendedwithsodiumthiosulfate,whilenohydrogensulfidewas evidentonL-cysteineorsodiumsulfiteamendedPDA.MatricpotentialandsurfacetensionVisiblegrowthoccurredinitiallywherewaterwasunder minimalmatricforce( 2 0.07MPa),whilegreatestbiomassby day25occurredinthe 2 1MPamedium.Delayedgermination andsubsequentgrowthoccurredat 2 2.5MPabyday25,withno visiblegrowthoccurringat 2 5MPa.Incontrasttothematric potentialassay,growthoccurredinallsurfacetensionexperiments ( 2 0.07to 2 5MPa)(Figure1L–M).ANOVA-analysiswith Tukey’sposttestindicatednostatisticaldifferenceinsurface tension(p=0.6)betweenthe 2 1MPacontrolmediumand 2 1MPamediaexposedtotwodifferentPdsisolates.Discussion KeratinaceoussubstratesKeratinaceoussubstratescontainingcarbonfoundincaves includebirdfeathersandmammalhairandskin.Goodindicators infavorofPdshavingsomekeratinolyticactivityincludetheability togrowonasoleproteinsource[19],tolerantofhighpHandhigh environmentallevelsofL-cysteineandsulfite[37],abilitytodegrade gelatininconjunctionwithenvironmentalalkalinization[21],and Figure1.Pseudogymnoascusdestructansgrowthandmorphology. A) growthonPoecilia species.B)growthon Locustamigratoria .C)growth on Lentinulaedodes .D)growthondemineralizedshrimpexoskeletons(left)anddemineralized/deproteinatedexoskeletons(right).E)growthon keratinazuremedium(nodyereleased).F)nogrowthonbeta-keratinderivedmatassay.G)growthoncarbonmedia,clockwisefrombottom:PDA, Mn-amendedPDA, b -glucosidase( + Rx),collidialchitin.H)growthonlipidandestermedia,clockwisefrombottom:SDA,Oliveoil( + Rx),Lard( + Rx), Tween80.I)growthoncalciummedia,clockwisefrombottom:PDA,500mg/Lcalcium,1000mg/Lcalcium,2000mg/Lcalcium.J)growthonpH media,clockwisefrombottom:pH5,pH7,pH9,pH11.K)growthonnitrogenmedia,lefttoright:columns1–2=nitrate,columns3–4=nitrite, columns5–6=ammonium,columns7–8=L-asparagine,columns9–10=controlwithnonitrogensource,columns11–12=uricacid.RowsA–D (MYA-4855),RowsE-H(ILLS69283),rowsA,E(pH5),rowsB,F(pH6),rowsC,G(pH7),rowsD,H(ph8).L)matricpotentialassay,lefttoright:growth at 2 0.07MPa, 2 1Mpa,and 2 2.5Mpa;nogrowthat 2 5MPaand 2 7.5Mpa.M)surfacetensionreductionassaywiththeadditionof0.0125%Tween 80,lefttoright:growthat 2 1MPa, 2 2.5MPa,and 2 5MPa. doi:10.1371/journal.pone.0078300.g001 Physiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org5October2013|Volume8|Issue10|e78300

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theabilitytoconvertthiosulfatetohydrogensulfide(areducing agentofkeratin)[38].However,no invitro demonstrationof keratinolyticactivitywasseenonkeratinazure(withandwithoutan environmentalreducingagent)orthesoluble b -keratinderived substrate(Figure1E–F).Unfortunatelytheseresultsdonotresolve whetherPdsiskeratinophilicoractuallykeratinolytic.Keratinolytic abilitymaybedifficulttodemonstrate invitro becausekeratinaceous materialscontainsignificantamountsofnon-keratinproteinsand lipids[39],[40]andfungitendtousemorereadilyaccessiblenonkeratincarbonsourcespriortorevertingtomoreenergetically intensiveresources[21].Ourresultssuggestthatkeratinaceous substrates(suchasbatskin)mayshowsignsofstructuraldegradation withoutexplicitkeratinolyticactivity.RegardlessofwhetherPdsis keratinophilicorkeratinolytic,itiscapableofgeneratingamicroenvironmentinwhichkeratinaceoussubstratesfoundincavesoils aremoresusceptibletodegradationandcanserveasanimportant resourceforPds.ChitinaceoussubstratesArthropod(insects,crustaceans,arachnids,andmyriapods) remains,undigestedinsectpartsinguano,andfungalcellwall fragmentsofferanothercomplexandvariablecarbon-containing substratefoundincaveenvironments.Majorcomponentsfoundin arthropodexoskeletonsareproteinsandchitin,withsmall amountsoflipids[41],[42],whilefungalcellwallscontaingreater than90%polysaccharidesconsistingofglucans,mannans,and chitin[43]andfungalbasidiocarpscancontainbetween7.8to 21%lipids[44].ToelucidatewhichstructuralcomponentsPds isolatescouldutilizeinchitin-containingsubstrates,wepreformed threeseparateassaysusingshrimpexoskeletons,autoclavedfungal basidiocarps,andcolloidalchitin.Theshrimpexoskeletonswere demineralizedtoobtainasubstratethatwouldbesimilartobat digestedinsectexoskeletalremainsfoundinguano.Thedemineralizationprocesswasconsistentwithfoodpassagetimesfor severalbatspeciesincluding Myotislucifugus [45].Additional Figure2.Pseudogymnoascusdestructanstolerancetocalcium,pH,andthreesulfurcompounds. A)one-wayANOVAanalysisindicatedno significantdifference(p=0.87)inradialcolonydiameteronPDAamendedupto2000mg/Lofcalciumchloride.B)one-wayANOVAanalysis indicatednosignificantdifference(p=0.69)intheradialcolonydiameteroverthepHrangeof4–11.C)one-wayANOVAanalysisindicatedno significantdifferenceinradialcolonydiameteronPDAamendedwith200–700mg/Lofsodiumsulfite(p=0.83),L-cysteine(p=0.75),orsodium thiosulfate(p=0.98).PanelAandBdottedlinerefersto95%confidenceinterval,panelsA–Csolidlinereferstobestfitlineofmeanvalues. doi:10.1371/journal.pone.0078300.g002 Physiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org6October2013|Volume8|Issue10|e78300

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shrimpexoskeletonsreceivedasubsequentdeproteinationstepto obtainahighchitin/lowproteinsubstrate.AllPdsisolatesgrew wellondemineralizedshrimpexoskeletons,butmicroscopic examinationwasneededtovisualizegrowthondeproteinated shrimpexoskeletonremains(Figure1D).Similarly,Pdscouldgrow onautoclaved L.edodes basidiocarps(Figure1C),butoncolloidal chitinPdsdidnotdemonstrateaclearingzone(chitinaseactivity) andgrowthwassparse(Figure1G).Theseresultsstronglysuggest thatPdscannotdegradechitin,butratherPdsutilizesother nutritionalcomponentsfoundinchitinaceoussubstrates.Similar tokeratinaceoussubstrates,chitinaceoussubstratesareimportant resourcesforPdsduetothepresencesofothernutritional component(proteinsandlipids).CellulosicsubstratesThegenus Pseudogymnoascus iscurrentlyincludedinthefungal family Pseudoeurotiaceae Malloch&Cainofwhichmanymembers growsaprotrophicallyonwoodytissue[20].Researchhasshown thatmanyconfoundingvariables(temperature,substratestructure andcompositions,andphysiologicalconditions)existwhen assayingfungiforcellulolyticactivity[37].Becausewood degradationhasmanyconfoundingvariables,wefocusedour effortsondetecting b -glucosidaseandMn-dependentperoxidase activity.AllPdsisolatesdemonstratedastrongpositivereactionfor b -glucosidaseactivitywithin24–48hoursoftransfertoesculin ironmediasuggestingthat b -glucosidaseisconstitutive(Figure1G). Manganese-dependentperoxidasewasnegativeandnonoticeable increaseinhyphalormediumpigmentationoccurred(Figure1G). Sincemostmemberswithinthefamily Pseudoeurotiaceae grow saprotrophicallyonwoodytissuesandrottingvegetationandPds produces b -glucosidase,itishighlyprobablethatPdscandegrade cellulosicsubstrates.Recentresearchsupportsthisconclusionby demonstratingthatPdscouldpenetratedeadmosscellwalls[14]. Weconcludethatcellulosicsubstratescouldbepotentialsubstrates forPds,butcellulosicmaterialsmaynotprovetobeadequatefor long-termsubstratecolonization.Basedonourmatric-induced waterstressresultsandthefungalcellwallattributesof psychrophilicfungi[46],inorderforcellulosicsubstratetobe utilizedbyPds,thesubstratewouldneedtohavesufficient moisturetoreducethewaterpotentialofcellulosicdebris[47]. Consequently,cellulosicsubstrateswouldbeunsuitableforlong termcolonizationbyPdsincavesorportionsofcavesthathave frequentmoisturefluctuations.Protein/lipidrichsubstratesManycomplexsubstrates(batskin,arthropodexoskeletaland fishremains)containproteins,lipidsandestersaspreviously discussed.Fisharegenerallycomposedof15–30%protein,0–25% fat,and50–80%water[48],whilefishscalecompositioncanvary. AllPdsisolatesproducedamplemycelialgrowthandconidiation onthewholefishsubstrate(Figure1A),aswellaswholeinsect (Figure1B)andfungalbasidiocarps(Figure1C).Pdsisolatescould degradeoliveoil,lard,andTween80mediawithpositive reactionsoccurringwithinoneweek(Figure1H).Ourpositive lipaseandesteraseresultsareconsistentwithpreviouslypublished results[11].Inaddition,Pdsclearlywascapableofutilizinggelatin asasolesourceofnutrition.Takentogether,thelipidandprotein resultsindicatethatPdsiscapableofutilizingmanylipidand proteinsourcesfoundincomplexcarbonsubstrates.Unfortunately forhibernatingbats,theirskinandglandularsecretionscontain manyproteinsandlipids[49]makingthemgoodsubstratesfor Pds.NitrogenutilizationGuano,ammonium,urea,andbacterialfixationaccountfor someofthenitrogenresourcesavailablewithincaves,butmost cavenitrogenentersinadissolvedstateinrun-offwateror leechinggroundwater[50]. Pseudogymnoascusdestructans isolates demonstratedarapidpositiveureaseresultusuallywithin24– 48hours,whichsuggeststhatureaseisconstitutive. Pseudogymnoascusdestructans canutilizemanydifferentsourcesofnitrogen (Figure1K);howevertheuricacidassaywasweaklypositive indicatingthatitmaybeapoornitrogenresourceduetolow solubility[51].Overall,ourresultsindicatethatinneutralto alkalineenvironments,nitrate,nitrite,ammonium,andamino acidssourcesareallsufficientforgoodgrowth,whileuricacidisa potentialnitrogenresourceunderalkalineconditions.Ourresults areinagreementwithsimilarnitrogenstudiesusingbasidiomycetes[30], Penicillium , Aspergillus [52]and Monascus species[53]and reflecttheimportanceofhowpHinteractswithnitrogenuptake. Importantly,Pdshasdemonstratedureaseactivitywhichhasbeen proposedasadualusevirulencefactorinthepathogenesisof Cryptococcusneoformans (SanFelice)Vuill.andotherpathogenic fungi[12],[54].Calcium,pH,andsulfurtoleranceEnvironmentalchemistrycaninfluencefungalgrowthandits abilitytosuccessfullycolonizesubstrates[55].Carbonandnutrient availabilityandthesolubilityofmetalsareinfluencedbysoilpH [56]withhighlevelsofenvironmentalsulfurcompoundscapable ofinhibitingfungalgrowth[57],whilecalciumhasbeenshown in vitro toenhancethegrowthandreproductionofmanyfungi[18]. ThegrowthandsporulationofPdswasnotaffectedbythe additionofcalcium(2000mg/L),whichsuggeststhatenvironmentalcalciumlevelsdonotsignificantlyimpactsubstrate suitability(Figure2A).Similarly,substratepHassaysshowedthat Pdsisalkali-tolerantandcapableofuniformgrowthfrompH5to pH11(Figure2B),whichisalsopartiallysupportedbythe nitrogenassays(ph5–8).ThegrowthandsporulationofPds isolateswerenotinhibitedby700mg/Lofsodiumthiosulfate, sodiumsulfite,orL-cysteine(Figure2C)andhydrogensulfide productionwasonlydetectedinthiosulfateamendedmedium. TheresultsofthepHandsulfurcompoundassayssuggestthatPds hasthepotentialtoinhabitmostcavesandcavesubstratessince neitherpHnorinhibitorysulfurcompoundsfoundinsoils,urine, andbatskinappeartolimitsubstratesuitability.WaterinteractionsThepropertiesofwateratlowtemperatures(i.e.increased viscosityandsurfacetension)andwateravailabilityareimportant abioticfactorsthatinfluencesubstrate/fungalinteractionsandcan representmajorchallengesforhabitatcolonization[35],[58].To investigatePdstolerancetosubstratematricpotential,weutilized PEG8000sinceitisnon-toxicandnotmetabolizedbyfungi[59] andisanalogoustosoilcolloids[60].Matricpotentialistheability ofthesubstratetoholdontowaterthroughthecombinationof capillaryandparticleadsorptiveforces[61]andispredominantin unsaturatedsoils[62].CavesoilsamplesfromfourIllinoisPds positivecaveswereanalyzedformatricpotentialandhydroscopic moisturecontentinordertorelateour invitro waterinteraction experimentstoknownPdspositivecaves.TheaverageIllinoisPds positivecavesoilhydroscopicmoisturecontentwas 12.41 6 7.572%indicatinganunsaturatedenvironmentwitha matricpotentialrangeof 2 0.005to 2 1.26MPa,althoughspatial andtemporalheterogeneityistobeexpected.Whenexposedto PEG-inducedwaterstress(matricpotential),allPdsisolates conformedtotheTypeIIresponse[63](greatergrowthunderPhysiologyof Pseudogymnoascusdestructans PLOSONE|www.plosone.org7October2013|Volume8|Issue10|e78300

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minimalmatricstresswithareductionofgrowthasmatricstress increases),whichistypicalofmostsoilfungi[46](Figure1L).The matricpotentialresultsaresupportedbytheIllinoiscavesoil matricpotentialanalysissincePdssporegerminationandactive growthoccurredwithinasimilarrange.Inaddition,ourmatric potentialresultsaresupportedbythecellwallphysiologyof psychrophilicfungi;psychrophilicfungihaveagreaterdegreeof unsaturatedlipidsintheircellwallwhichisreportedtoreducethe abilityoffungitotoleratewaterstress[46].Interestingly,Pds isolateswerecapableofgrowthandsporulationuptoand including 2 5MPamediumbytheadditionof0.0125%Tween80 (Figure1M).Thisresultisconsistentwiththefactthatareduction insurfacetensionreducesthecapillaryforces[62]and, consequently,reducestheoverallmatricforceofthemedium. ThecombinationofthesetworesultssuggeststhatPdsissensitive tomatric-inducedwaterstress.Thesurfacetensioncomparisonof mediawithandwithoutexposuretoPdsindicatedthatPdswas notcapableofproducinganyextracellularbiosurfactantsor surfacetensionreducingcompounds.Collectively,thesethree assayssuggestthatsubstratescontainingsurfacetensionreducing agentswouldbebeneficialforthegrowthofPds.Inthecave environment,surfacetensionreducingsubstrateswouldcontain freefattyacidsorlipidsthatcouldbehydrolyzedtofattyacidsby Pds.Theskinofbatscontainsassociatedlipidsanditsglandular secretionscontainfreefattyacids[49]makingthesurfaceofbat skinexcellenthabitatsforPds.ConclusionsTheexaminationofsixisolatesfromfourEasternand MidwesternstatesdemonstratedthatPdspossessesClass2 nitrogenutilization,aTypeIIresponsetomatricstress,isalkalitolerant,andiscapableofsaprobicallyutilizingmanycomplex carbon-containingcavesubstrates.Basedoncurrentbiological information,temperatureandwateravailabilityaretheonly environmentallimitationsofacave,mine,orsimilarenvironment toactasareservoirforPds.Ourstudyofbioticandabioticfactors suggeststhatPdswouldbeabletoremaininpreviouslyinfected cavesasasaprobeusingsmallcomplexorganiccarbon-containing substratesthatactasresourceislandsbylimitingbioticcompetition.DuetotheslowgrowthcharacteristicsofPds,itislikelythat rapidandcopiousreproductionviaconidiationoccursasawayto islandhop,orbridgethegapbetweensuitableresourceislands. Althoughcavebioticandabioticfactorsmaylimitaccessibilityor assist/hindersubstratedegradationthatcannotbereproduced in vitro ,moreresearchisneededonthebasicbiologyandecologyof Pdssinceitwilllikelybecomeapermanentresidentinthemajority ofNorthAmericancaveecosystems.AcknowledgmentsTheauthorswishtothankthefollowingforprovidingisolatesusedinthis study:EdHeske,JoeKath,DanLindner,JoeMerritt,BarrieOverton, AndreaPorres-Alfaro,andSteveTaylor.AuthorContributionsConceivedanddesignedtheexperiments:DBR.Performedtheexperiments:DBR.Analyzedthedata:DBR.Contributedreagents/materials/ analysistools:ANM.Wrotethepaper:DBRANM.References1.LorchJM,MeteyerCU,BehrMJ,BoylesJG,CryanPM,etal.(2011) Experimentalinfectionofbatswith Geomycesdestructans causeswhite-nose syndrome.Nature480:376–378. 2.WarneckeL,TurneraJM,BollingerbTK,LorchJM,MisraV,etal.(2012) InoculationofbatswithEuropean Geomycesdestructans supportsthenovel pathogenhypothesisfortheoriginofwhite-nosesyndrome.ProcNatlAcad SciUSA.Accessed2013Apr09. 3.BlehertDS,HicksAC,BehrMJ,MeteyerCU,Berlowski-ZierBM,etal.(2009) Batwhite-nosesyndrome:anemergingfungalpathogen?Science323:227. 4.HayesMA(2012)TheGeomycesfungi:ecologyanddistribution.Bioscience62: 819–823. 5.GargasA,TrestMT,ChristensenM,VolkTJ,BlehertDS(2009)Geomyces destructanssp.nov.associatedwithbatwhite-nosesyndrome.Mycotaxon108: 147–154.doi:10.5248/108.147 6.CryanP,MeteyerCU,BoylesJG,BlehertDS(2010)Wingpathologyofwhitenosesyndromeinbatssuggestslife-threateningdisruptionofphysiology.BMC Biol8:135. 7.WibbeltG,KurthA,HellmannD,WeishaarM,BarlowA,etal.(2010)Whitenosesyndromefungus( Geomycesdestructans )inbats,Europe.EmergInfectDis16: 1237–1242. 8.MeteyerCU,ValentM,KashmerJ,BucklesEL,LorchJM,etal.(2011) Recoveryoflittlebrownbats(Myotislucifugus)fromnaturalinfectionwith Geomycesdestructans,white-nosesyndrome.JWildlDis47:618–626. 9.LorchJM,MullerLK,RusselRE,O’ConnorM,LindnerDL,etal.(2013) Distributionandenvironmentalpersistenceofthecausativeagentofwhite-nose syndrome,Geomycesdestructans,inbathibernaculaoftheEasternUnited States.ApplEnvironMicrobiol74:1293–1301. 10.VerantML,BoylesJG,WaldrepWJr,WibbeltG,BlehertDS(2012) Temperature-DependentGrowthof Geomycesdestructans ,theFungusThatCauses BatWhite-NoseSyndrome.PLoSONE7(9):e46280.doi:10.1371/journal.pone. 0046280 11.ChaturvediV,SpringerDJ,BehrMJ,RamaniR,LiX,etal.(2010) MorphologicalandMolecularCharacterizationsofPsychrophilicFungus Geomycesdestructans fromNewYorkBatswithWhiteNoseSyndrome(WNS). 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