Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field ...

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Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field experiments
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Front Microbiol. 2013; 4: 104
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Zhalnina, Kateryna
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Agricultural land management, such as fertilization, liming, and tillage affects soil properties, including pH, organic matter content, nitrification rates, and the microbial community. Three different study sites were used to identify microorganisms that correlate with agricultural land use and to determine which factors regulate the relative abundance of the microbial signatures of the agricultural land-use. The three sites included in this study are the Broadbalk Experiment at Rothamsted Research, UK, the Everglades Agricultural Area, Florida, USA, and the Kellogg Biological Station, Michigan, USA. The effects of agricultural management on the abundance and diversity of bacteria and archaea were determined using high throughput, barcoded 16S rRNA sequencing. In addition, the relative abundance of these organisms was correlated with soil features. Two groups of microorganisms involved in nitrogen cycle were highly correlated with land use at all three sites. The ammonia oxidizing-archaea, dominated by Ca. Nitrososphaera, were positively correlated with agriculture while a ubiquitous group of soil bacteria closely related to the diazotrophic symbiont, Bradyrhizobium, was negatively correlated with agricultural management. Analysis of successional plots showed that the abundance of ammonia oxidizing-archaea declined and the abundance of bradyrhizobia increased with time away from agriculture. This observation suggests that the effect of agriculture on the relative abundance of these genera is reversible. Soil pH and NH3 concentrations were positively correlated with archaeal abundance but negatively correlated with the abundance of Bradyrhizobium. The high correlations of Ca. Nitrososphaera and Bradyrhizobium abundances with agricultural management at three long-term experiments with different edaphoclimatic conditions allowed us to suggest these two genera as signature microorganisms for agricultural land use.
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Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Kateryna Zhalnina.
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ORIGINALRESEARCHARTICLE published:01May2013 doi:10.3389/fmicb.2013.00104 Ca .Nitrososphaeraand Bradyrhizobium areinversely correlatedandrelatedtoagri culturalpracticesinlong-term eldexperiments KaterynaZhalnina 1† PatriciaD.deQuadros 1,2† ,KelseyA.Gano 1† ,AustinDavis-Richardson 1 JennieR.Fagen 1 ,ChristopherT.Brown 1 ,AdrianaGiongo 1 ,JenniferC.Drew 1 LuisA.Sayavedra-Soto 3 ,DanJ.Arp 3 ,FlavioA.O.Camargo 2 ,SamiraH.Daroub 4 IanM.Clark 5 SteveP.McGrath 5 PennyR.Hirsch 5 and EricW.Triplett 1 1 DepartmentofMicrobiologyandCellScience,InstituteofFoodandAgriculturalSciences,UniversityofFlorida,Gainesville,FL,USA 2 DepartmentofSoilScience,FederalUniversityofRioGrandedoSul,PortoAlegre,Brazil 3 DepartmentofBotanyandPlantPathology,Or egonStateUniversity,Corvallis,OR,USA 4 EvergladesResearchandEducationCenter,UniversityofFlorida,BelleGlade,FL,USA 5 RothamstedResearch,Harpenden,Hertfordshire,UK Editedby: RichBoden,UniversityofPlymouth, UK Reviewedby: JoanaF.Salles,Universityof Groningen,Netherlands TrevorC.Charles,Universityof Waterloo,Canada *Correspondence: EricW.Triplett,Departmentof MicrobiologyandCellScience, InstituteofFoodandAgricultural Sciences,UniversityofFlorida, 1355MuseumRoad,Gainesville, FL32611-0700,USA. e-mail: ewt@u.edu   Theseauthorshavecontributed equallytothiswork. Agriculturallandmanagement,suchasfertilization,liming,andtillageaffectssoil properties,includingpH,organicmattercontent,nitricationrates,andthemicrobial community.Threedifferentstudysiteswer eusedtoidentifymicroorganismsthat correlatewithagriculturallanduseandtodeterminewhichfactorsregulatetherelative abundanceofthemicrobialsignaturesoftheagriculturalland-use.Thethreesitesincluded inthisstudyaretheBroadbalkExperimentatRothamstedResearch,UK,theEverglades AgriculturalArea,Florida,USA,andtheKelloggBiologicalStation,Michigan,USA.The effectsofagriculturalmanagementontheabundanceanddiversityofbacteriaandarchaea weredeterminedusinghighthroughput,barcoded16SrRNAsequencing.Inaddition,the relativeabundanceoftheseorganismswascorrelatedwithsoilfeatures.Twogroups ofmicroorganismsinvolvedinnitrogencyclewerehighlycorrelatedwithlanduseat allthreesites.Theammoniaoxidizing-archaea,dominatedby Ca .Nitrososphaera,were positivelycorrelatedwithagriculturewhileaubiquitousgroupofsoilbacteriaclosely relatedtothediazotrophicsymbiont, Bradyrhizobium ,wasnegativelycorrelatedwith agriculturalmanagement.Analysisofsuccessionalplotsshowedthattheabundanceof ammoniaoxidizing-archaeadeclinedandtheabundanceofbradyrhizobiaincreasedwith timeawayfromagriculture.Thisobservationsuggeststhattheeffectofagricultureon therelativeabundanceofthesegeneraisreversible.SoilpHandNH 3 concentrations werepositivelycorrelatedwitharchaealabundancebutnegativelycorrelatedwith theabundanceof Bradyrhizobium .Thehighcorrelationsof Ca. Nitrososphaeraand Bradyrhizobium abundanceswithagriculturalmanagementatthreelong-termexperiments withdifferentedaphoclimaticconditionsallowedustosuggestthesetwogeneraas signaturemicroorganismsforagriculturallanduse. Keywords:agriculturallanduse,ammonia-oxidizingarchaea,diazotr ophs, Ca .Nitrososphaera, Bradyrhizobium soilproperties INTRODUCTION Thetransformationofsoiltoagriculturalusecausessignicantchangesinitschemical,physical,andbiologicalfeatures, includingchangesinthemicrobialcommunitycomposition ( Kibblewhiteetal.,2008 ).Anumberofstudieshaveexamined theimpactofagricultureonmicrobialcommunitystructure.The effectofinorganicandorganicfertilizers,tillage,anddifferent croprotationswasanalyzed.Itwasfoundthatnitrogen(N)depositionincreasestheabundanceofcertainmicrobialphyla,such asActinobacteria,Proteobacteria,Bacteroidetes,andFirmicutes, butitalsodecreasestheabundanceofotherbacterialphyla, suchasAcidobacteriaandVerrucomicrobia( Ramirezetal.,2010, 2012;Fiereretal.,2012 ).Anincreaseinmicrobialbiomasswas detectedafterfarmyardmanureaddition( Kandeleretal.,1999 ), whilepreviousstudieshaveshownthatreducingtillageincreased microbialbiomass( BuckleyandSchmidt,2001;Plassartetal., 2008 ).Otherstudieshavefocusedonthechangesofspecic microbialtaxainvolvedinnutrientcyclinginsoil,suchasdiazotrophs( Mengetal.,2012 ),nitriers( Chuetal.,2008 ),and denitriers( Clarketal.,2012 )underspecicagriculturalmanagement.Therefore,allstudiesoftheagriculturalimpactonthe microbialcommunityarelimitedeithertothephylumlevel, whichrepresentsalargeanddiversesetoffunctionalmicrobial groups,ortothespecicphysiologicalgroup.Wehypothesize thattherearecommonmicrobialtaxastronglyassociatedwith agriculturallanduse,andthesetaxacanbeusedasindicators www.frontiersin.org May2013|Volume4|Article104 | 1

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Zhalninaetal. Keysignaturesforthelanduse tomonitorthelevelofthelanddisturbanceduringagricultural management. Oneoftheabundantgroupsfoundinmanysoilsunderagriculturaluseisammonia-oxidizingarchaea(AOA)( Heetal., 2007;Shenetal.,2008;Gubry-Ranginetal.,2010;Taketaniand Tsai,2010;WessŽnetal.,2010;Pratscheretal.,2011;Xiaetal., 2011 ).InsoilssuchasAmazonianAnthrosol,agriculturalplots haveahigherarchaealammoniamonooxygenasegene( amoA ) copynumberthaninadjacentsoils( TaketaniandTsai,2010 ). WessŽnetal.(2011) showedthathighnumbersofarchaeaare positivelycorrelatedwithnitrateleachingfrombothintegrated andorganicfarmingsystems.TwoagriculturalsoilsinGermany, OrthicLuvisolandGleyicCambisol,harboredhighabundancesof archaeal,butnotbacterial amoA genes( Schaussetal.,2009 ).AOA areimportantparticipantsinsoilnitricationbecausetheyare abletoperformtherststepofnitricationthroughtheammonia monooxygenaseenzyme(AMO)( Kšnnekeetal.,2005;Treusch etal.,2005 ).ThemostrepresentedsoilAOAwasfoundwithinwas Candidatus Nitrososphaera( TaketaniandTsai,2010;Xiaetal., 2011;Pesteretal.,2012 ). Agriculturalmanagementintensiesallprocessesrelatedto theNcycleincludinganincreaseinnitricationrates( Luetal., 2011 ).Increasednitricationcanresultinnitrateleachinginto thesurfaceandgroundwater,aswellastheemissionofnitrous oxidetotheatmosphere( Kimetal.,2012 ).Recently,thecontributionofAOAtonitricationwasshownbyassimilationof13C-CO2byAOAduringsoilnitrication( Zhangetal.,2010, 2012;Xiaetal.,2011 ).Inadditionahighcorrelationbetween AOAandnitricationactivitywasobserved( Offreetal.,2009 ) aswellashighabundanceofarchaeal amoA transcriptsinsoil ( Treuschetal.,2005;Nicoletal.,2008 ).Hence,AOAmaybe responsibleforalltheseconsequencesofintensiednitrication.Knowledgeofthefactorsthatmaydrivetheabundanceof thisgroupisnecessarytopreventagriculturallandmanagement fromnegativelyimpactingtheenvironment.Previousstudies haveinvestigatedtemperature,soiltype,andelevationasdrivers ofarchaealabundanceinsoils( Zhangetal.,2009;Taketaniand Tsai,2010 ).OtherstudieshaveexaminedpH,fertilization,carbontonitrogenratio,andtillageasanthropogenicdriversthat arearesultoflandmanagement( Kandeleretal.,1999;Fierer andJackson,2006;Enwalletal.,2007;Nicoletal.,2008;Taketani andTsai,2010;Batesetal.,2011;HermanssonandLindgren, 2001 ). Gubry-Ranginetal.(2011) founddifferentphylogenetic lineagesofAOAthatwereacidophilic,acido-neutrophilic,and alkalinophilic,andthesewerepositivelycorrelatedwithsoilpH levels. Bruetal.(2011) foundapositivecorrelationbetweenAOA abundanceandpH. PereiraeSilvaetal.(2012) haverecently shownthatAOAabundancewaspositivelycorrelatedwithpH inatemporalstudybasedoneightagriculturalsoils.However, Nicoletal.(2008) reportedresultswherearchaeal amoA gene andtranscriptabundancedecreasedwithhigherpHinacidic soils.ThefactorsresponsibleforthechangesinAOAabundance varydependingontheecosystem,thegeography,andsoiltype.It remainsunclearwhatfactorsaremainlyresponsiblefortriggering theabundanceofarchaeainsoil. Threelong-termexperimentsthatstudycropproduction, nutrientcycling,andenvironmentalimpactofagriculturewere includedinthiswork.Therstexperiment,theBroadbalk RothamstedExperiment,wasdesignedmorethan170yearsago totesttheeffectsofvariouscombinationsofinorganicfertilizers andfarmyardmanureontheyieldofthewheat.Thesecondstudy site,theKelloggBiologicalStationlong-termecologicalresearch project,wasinitiatedtoexaminethebasicecologicalrelationships ineld-cropecosystemstypicaloftheMidwesternU.S.Earlyin thelastcentury,280,000haofprimaryrichwithorganicmatter histosolsinSouthFloridaweredrainedtocreatetheEverglades AgriculturalArea,whichwasthethirdexperimentforourstudy. About25%ofUSwintervegetablesandsugarcanearecultured intheEAA.DespitethemanyyearsofstudyattheBroadbalk RothamstedExperiment,theEvergladesAgriculturalArea,and theKelloggBiologicalStation,therearefewstudiesdescribing howagriculturalmanagementpracticesaffectmicrobialtaxaat theselong-termsites( Castroetal.,2005;Ogilvieetal.,2008; Ramirezetal.,2010;Delmontetal.,2011;Clarketal.,2012; Fiereretal.,2012 ).Noneoftheseshowthedetaileddifferences intaxonomicgroupsthatoccurwithagriculturallanduseand noneofthemshowthisacrossmultiplesites.Also,noneofthese studiesprovideevidenceforbiomarkersoflanduseorshowhow microbialtaxachangewithlandusesuccession.Thegoalsofthis studyweretoexaminechangesinarchaealandbacterialcommunitycompositioninresponsetoland-usewithaparticular emphasisonammoniaoxidizers.Microbialcommunitiesinthe soilsofthreelong-termagriculturalsiteswereexaminedusing 16SrRNAbarcodedIlluminasequencing.Siteswerealsochosen sothattheeffectofsuccessiononmicrobialtaxacouldbeexamined.Soilcommunitycompositionwasalsocomparedtoseveral soilpropertiestoidentifythedriversofmicrobialdiversityand abundance.MATERIALSANDMETHODSSTUDYSITESSoilsamplesfromagriculturalandnon-agriculturalareasofthree differentlong-termexperimentalsiteswerecollectedforthis work: (a) BroadbalkRothamstedResearch(BRR) Locatedat RothamstedResearch,Harpenden,UK,theBRRsoilis anAlsolinty-siltyclayloam.BRRistheoldestlong-term agronomicexperimentintheworld.Apartfromoccasional fallowing,thearablemanagementplotshavebeenincontinuouswinterwheatfor168years.Nitrogenisaddedas farmyardmanure(FYM)at35t/hainautumnand/oras ammoniumnitrateinspringrangingfrom0to288kghaŠ 1peryear.TheBRRexperimentincludedtenagricultural treatments:sixNtreatments(0,48,96,144,192,288kg haŠ 1peryear);threeFYMwithorwithoutadditionalN(N: 0,96,192kghaŠ 1peryear);andoneisthenilapplication treatmentofnofertilizersororganicamendments.Two non-agriculturaltreatmentsincludeunfertilizedgrassland orwoodland.Alltreatmentshadthreepseudoreplicates andweresampledmonthlyover5-monthperiod(Mayto September)( Gouldingetal.,2000;Poulton,2012 ). (b) MichiganKelloggBiologicalStation(KBS) Locatedatthe MichiganKelloggBiologicalStation(KBS),Kalamazoo, FrontiersinMicrobiology |TerrestrialMicrobiologyMay2013|Volume4|Article104|2

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Zhalninaetal. Keysignaturesforthelanduse Michigan,USA,theKBSAlsolsoilsitehasplotsplanted toacorn-soybean-wheatrotationsunderconventionaltill systemsince1980.Ammoniumnitrateisaddedthreetimes duringthegrowingseason,beginninginAprilandendingin November.RatesofNapplicationrangefrom153to165kg haŠ 1peryearforcornandfrom56to90kghaŠ 1peryearfor wheat.AtKBS,15sampleswerecollectedfromagricultural plots(vebiologicalreplicateswiththreepseudoreplicatesof eachwerecollectedfromT1agriculturalplotsunderconventionaltillagewitharotationofcorn,soybean,andwheat), andninesampleswerecollectedfromnon-agriculturalplots (SF1,SF2,SF3plotswiththreepseudoreplicatesofeach).The non-agriculturalplotsSF2(LoudenField)andSF3(Turner Field)aresuccessionaloldeldsabandonedfromcroppingin 1951and1963,respectively.PlotSF1,CantonField,waslast managedasanagriculturalsystemin1971( Robertsonetal., 1993 ). (c) EvergladesAgriculturalArea(EAA) LocatednearBelle Glade,Florida,USA,theEAAHistosolcontainsrichorganic soilsoverlyinglimestone.Intheearly1900s,theEverglades regionbegantobedrainedforagriculturalpurposesforwintervegetablesandsugarcaneproduction.Drainageincreased thelevelofoxygeninsoilsandcreatedconditionsfavorable foraerobicmicroorganismsthatdecomposesoilorganicmatter(SOM).Sincethedraining,decompositionofSOMin EAAsoilsexceedtheiraccumulationresultinginsubsidence ofEvergladessoilsattheannualrateofabout1525mm ( Snyder,2005 ).Mineralizationoforganicnitrogenoccursat higherlevelsthanisrequiredforcrops,resultingindrainage watercontamination( BottcherandIzuno,1994 ).Three replicateseachoftheagriculturalsugarcaneplots,SR1,SR2, andSR3,andthenon-agriculturalvirginplots,VR1,VR2, andVR3,weresampledattheEAAmonthlyovera1-year period.SOILSAMPLINGAtallsites,soilsamplingwascollectedinthreereplicates:3cm diametercorers,pre-washedwith70%ethanol,wereinsertedinto thesoiltoadepthof10cm.Foreachreplicate,10coreswere pooledandsampleswerethensievedthrougha2mmsieveand thoroughlymixed.Eachreplicatewasthenfrozenat Š 80Cfor subsequentDNAextraction.ANALYSISOFSOILPARAMETERSForallsites,soilparametersincludingpH,percentmoisture, totalN,NO3-NandNH+ 4-Nweremeasured( WalkleyandBlack, 1934;SchoeldandTaylor,1955;Black,1965;Mulvaney,1996; RonghongandLawrence,2010 ).NH3levelswerecalculatedfrom thesoilNH+ 4-NconcentrationsusingthepKaofNH3(9.23) andsoilpH.Threemeasurementsweremadeforeachsampling plotandthenaveragedtogivearepresentativevalue.SoilpH wasmeasuredusingaglasselectrodein1:2suspensionofsoil indH2O( SchoeldandTaylor,1955 ).Gravimetricwatercontent(soilmoisture)wasdeterminedasgravimetricwatercontent bydrying10gsoilat105Cfor24h( Black,1965 ).AtBRR,total NandCweredeterminedusingthecombustionmethod(LECO CNS2000).NOŠ 3-NandNH+ 4-Nwereextractedwith2MKCl for2h.Afterextraction,thesupernatantwaslteredthrough WhatmanNo.1lterpaperandthesupernatantwasanalyzed forNH+ 4andNOŠ 3-Nbyanautomatedcolorimetricassay(Skalar SANPLUSSystem;Skalar,Breda,TheNetherlands).AtEAAand KBS,totalN,NOŠ 3-NandNH+ 4-Nweremeasuredaccordingto previouslydescribedprotocols( Mulvaney,1996;Ronghongand Lawrence,2010 ).AtBRRandKBSthe%soilorganiccarbon wasdeterminedbytheWalkley-Blackchromicacidwetoxidation method( WalkleyandBlack,1934 ).Soilorganicmatter(SOM) wascalculatedbymultiplying%organiccarbonbyafactor of1.72.DNAEXTRACTIONForeachsample,DNAwasisolatedfrom0.25gofsoilusing theMoBioPowerSoilDNAIsolationKit(Carlsbad,CA,USA). Extractionswereperformedaccordingtothemanufacturer'sprotocolforsamplescollectedfromEAAandKBS.Samplesfrom Broadbalkwereextractedasdescribedbythemanufacturerexcept fortheuseoftheMPBiomedicalsFastPrep-24machinefor30sat 5.5m/s,insteadofvortexagitation.AllgenomicDNAconcentrationandpuritywasdeterminedbyNanoDropspectrophotometry (ThermoScientic,Wilmington,DE,USA).ILLUMINAHIGH-THROUGHPUTSEQUENCINGOF16SrRNAGENESAND TAXONOMICCLASSIFICATIONOFSEQUENCEREADSBacterialandarchaeal16SrRNAgeneswereampliedusing barcodeduniversalprokaryoticprimers515F(5-GTGC CAGCAGCCGCGGTAA-3)and806R(5-GGACTACVSGG GTATCTAAT-3)( Caporasoetal.,2010 )andsequencedusing Illuminatechnologyasdescribedpreviously( Fagenetal., 2012 ).Classicationofreadswasdoneusingpreviousmethods ( Giongoetal.,2010a,b )modiedtothepaired-endIllumina platform( Fagenetal.,2012 ).Readsweretrimmedtoremove lowqualitybasesandtoremovetherst11basescorresponding totheprimerregionbyascriptbasedonTrim2( Huangetal., 2003 ,sourceavailableat:https://gist.github.com/1006830), andthenthereadswereseparatedbybarcode(sourceavailableat:https://gist.github.com/1006983).Thisresultedin 11,390,227,1,307,720,and1,739,319trimmedreadsfromBRR, KBS,andEAA,respectively,withanaveragereadlengthof 158bases.PairedreadswereassembledusingCLCAssembly Cellv3.0.2btothereferenceRibosomalDatabaseProject (RDP)( Coleetal.,2009 )16SSSUrRNAdatabase.FulltaxonomicdescriptionsbasedontheNCBItaxonomydatabase (http://www.ncbi.nlm.nih.gov)weregeneratedfortheentries intheRDPdatabaseusingTaxCollector( Giongoetal.,2010b ). Matcheswerelteredat80%lengthfractionandclassied atthe80%identitylevelfordomainandphylum,90%identifylevelforclass,orderandfamily,95%identitylevelfor genus,and99%identitylevelforspecies.Thetotalnumber ofpairsmatching16SrRNAsequencesinthedatabaseat eachlevelofsimilaritycreatedanOTUabundancematrix foreachleveloftaxonomyacrosssamples.Pairsthatdidnot matchtothesamesequenceintheRDPdatabasewereannotatedaccordingtotheirLastCommonAncestor(LCA),and pairsthatdidnothaveanLCA,oranymatchintheRDP database,wereconsideredtobeunclassied.Tonormalizefor www.frontiersin.orgMay2013|Volume4|Article104|3

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Zhalninaetal. Keysignaturesforthelanduse varyingsequencingdepths,theOTUabundancematricesfor eachsampleweredividedbythetotalnumberofpairsafter trimming.STATISTICALANALYSISStatisticalanalysiswasperformedusingtheRstatisticalpackage( RDevelopmentCoreTeam,2011 )andXLSTAT-Pro2011. Spearmancorrelations(using p 0 001)fornon-normallydistributeddatawereusedtoindependentlyevaluatethecorrelation ofeachmeasuredsoilparameterwiththerelativeabundanceof taxa.ForANOVA,therelativeabundanceoftaxawastransformed tothearcsinesquareroottosatisfythenormalityassumption. One-WayANOVAswereusedtocomparerelativeabundance oftaxainagriculturalandnon-agriculturalplotsatallstudy sites.ATwo-WayANOVAwasusedtodetermineifagriculture andstudysitehadasignicanteffectontherelativeabundance oftaxa.RESULTSSEQUENCEANALYSISOF16SrRNAGENESANDMICROBIAL COMMUNITYATAGRICULTURALANDNON-AGRICULTURALSITESThetotalnumberofbarcodedreadsobtainedfromsequencing rangedbetween1.3and11.4millionreadswithanaveragelength of158bp( Table1 ).94.3%,95.5%,and91.1%sequenceswere classiedas Bacteria ;3%,1.3%,and3.7%sequencesas Archaea atBRR,KBS,andEAA,respectively.Thetaxonomicclassicationofthetenmostabundantgenerafromeachofthethree studysitesincludes19differentgeneraintotal( Table2 ).Eight generawerefromtheProteobacteria(vealpha,twogamma, andonebeta),fourfromtheActinobacteria,threefromthe Bacteroidetes,twofromtheFirmicutes,andonegenuseach fromtheAcidobacteriaandThaumarchaeota. Ca .Nitrososphaera and Pseudomonas werethemostabundantgeneraatBroadbalk. FortheEverglades,themostabundantgenerawere Ca Nitrososphaeraand Rhodoplanes ,andattheKelloggBiological Table1|ResultsofIlluminasequencing. SiteTotalnumberofreadsAverageofpairedreadsNumberofoperationaltaxonomicunits persample PhylumClassOrderFamilyGenus BRR11,390,22764,7172440922321021 KBS1,307,72031,136253984201741 EAA1,739,31923,191254190217860 NumberofIlluminasequencingreadsatvetaxonomiclevelsfromRothamstedR esearch(BRR),t heEvergladesAgriculturalArea(EAA)andtheKelloggB iological Station(KBS). Table2|The10mostabundantgenerafoundinsoilsatthreeexperimentalsitesatagriculturalandnon-agriculturalplots. GenusBRRKBSEAA AgNon-agAverageStdevAgNon-agAverageStdevAgNon-agAverageStdev Ca. Nitrososphaera3.430.532.92(1943)1.451.410.411.04(323)0.883.561.682.62(608)1.23 Pseudomonas 1.480.441.39(926)2.610.20.230.22(70)0.200.110.030.06(14)0.08 Bradyrhizobium 0.781.940.97(647)0.540.651.330.89(276)0.440.090.630.35(80)0.32 Sphingomonas 0.970.640.92(609)0.622.430.261.79(555)1.210.470.410.44(101)0.20 Flavobacterium 0.780.720.77(512)0.721.180.250.20(63)0.180.350.060.22(50)0.30 Nocardioides 0.760.470.72(476)0.370.370.130.29(90)0.170.310.10.21(48)0.14 Rhodoplanes 0.491.360.63(417)0.400.530.50.53(165)0.120.571.370.96(223)0.46 Steroidobacter 0.600.710.62(410)0.150.310.090.24(74)0.160.170.240.19(45)0.13 Bacillus 0.461.210.59(394)0.590.721.310.92(285)0.710.60.450.55(127)0.46 Nitrospira 0.520.490.52(347)0.170.40.230.35(107)0.150.820.70.77(177)0.34 Paenibacillus 0.340.990.45(297)0.290.310.610.42(129)0.250.171.450.78(180)0.75 Streptomyces 0.440.530.46(304)0.190.630.240.52(162)0.300.050.310.19(45)0.16 Mycobacterium 0.311.000.42(282)0.300.430.710.54(166)0.250.020.180.1(23)0.11 Arthrobacter 0.440.100.38(255)0.260.250.080.20(62)0.110.660.010.33(77)0.36 Hyphomicrobium 0.360.390.36(242)0.120.220.140.19(61)0.080.380.130.27(62)0.15 Terrimonas 0.320.080.28(189)0.200.330.150.27(83)0.190.390.270.31(71)0.13 Flavisolibacter 0.150.020.13(85)0.130.770.120.54(168)0.430.210.130.17(39)0.11 Burkholderia 0.040.120.06(37)0.040.840.570.78(243)0.450.090.260.17(38)0.14 Ca. Koribacter0.020.00.02(12)0.010.620.570.62(191)0.390.00.910.42(98)0.53 Relativeabundancerepresentedasaproportionof16SrRNAgenereadsofthetotalnumberofreadsfromeachsite(%)atagricultural(Ag),non-agricul tural(Non-ag) plots,andaverageproportionof16SrRNAreadsateachexperiment.Num bersinthebracketsrepresentaveragenumberofreadsforeachtaxon. FrontiersinMicrobiology |TerrestrialMicrobiologyMay2013|Volume4|Article104|4

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Zhalninaetal. Keysignaturesforthelanduse Station,themostabundantgenerawere Ca .Nitrososphaeraand Sphingomonas Thegenerawitharelativeabundanceofatleast0.05%ofall total16SrRNAreadsfromeachsitewereanalyzedforallthree sitesandrepresentedinaVenndiagram( Figure1A ).Twenty-ve genera(13.2%)werecommontoallthreesites. Correlationsbetweentherelativeabundanceofarchaealand bacterialtaxaandagriculturalmanagementwerecalculated acrossallsites.Onlythosegenerathatrepresentedatleast 0.05%oftotalreadsacrossanysitewereexamined.Twentysevengeneraweresignicantlypositivelycorrelated(rho 0.5, p 0 001)withagriculturalusewhile23generawerenegatively correlatedwithagriculture(rho Š 0.5, p 0 001, Table3 ). Onlytwogenera, Ca .Nitrososphaeraand Bradyrhizobium, showedsignicantcorrelationswithagriculturalmanagement atallthreesites( Figure1B Table3 ).Inaddition,ninegeneraweresignicantlycorrelatedwithagricultureatBRRand EAA. Marmoricola Blastococcus Ramlibacter ,and Lysobacter were positivelycorrelatedwithBRRandEAAagriculturalmanagement( Table3 ),while Rhodoplanes Mycobacterium Paenibacillus, and Burkholderia abundancesweresignicantlyhigheratnonagriculturalplotsandnegativelycorrelatedwithagricultural land-use( Tables2 3 ). Therelativeabundancesof Ca .Nitrososphaeraand Bradyrhizobium wereplottedtogethertodepicttherelationshipbetweentherelativeabundanceofthesegeneraand agriculturalmanagement( Figure2 ).Thesetwogenerawere inverselycorrelatedwitheachother(rho =Š 0.26, p 0 001). Non-agriculturalplotsexhibitedthelowerabundanceof Ca. Nitrososphaeraandthehigherabundanceof Bradyrhizobium Conversely,agriculturalplotswererepresentedbythehigher abundanceof Ca .Nitrososphaeraandthelowerabundanceof Bradyrhizobium Inaddition,therelativeabundancesofthe16SrRNA from Ca.Nitrososphaeraand Bradyrhizobium wereevaluated atKBSsuccessionalplots( Figure3 ).Theproportionof Ca. Nitrososphaeradeclinedsteadilywithtimeawayfromagriculture. Incontrast,theproportionof Bradyrhizobium increasedduring FIGURE1|Venndiagramofthemostabundantgenera. Venndiagram showingoverlapofthemostabundantgenera(thosewith 0.05%ofall 16SrRNAreads)betweensoilsfromthreeexperimentalsitesatthe BroadbalkExperimentatRothamstedResearch(BRR),theEverglades AgriculturalArea(EAA)andtheKelloggBiologicalStation(KBS) (A) .Venn diagramshowingoverlapofthemostabundantgeneracorrelatedwith agriculturalmanagementatthethreeexperimentalsites(rho 0.5, p 0 001) (B) therst38yearswithoutagriculturebutremainedconstant thereafter.AMMONIA-OXIDIZINGARCHAEAINAGRICULTURALAND NON-AGRICULTURALSITESThediversityofammonia-oxidizingarchaea(AOA)wasexaminedateachsite.Thaumarchaeotaand Ca. Nitrososphaerawere themostprevalentarchaealphylumandgenus,respectively,at allsitesandinagriculturalandnon-agriculturalplots( Figure4 ), showingaconsistentpatternatallthreesites.AtBRR, Ca. Nitrososphaeracomprisedanaverageof96%(agriculturalplots) and94%(non-agriculturalplots)oftotalarchaealreads,representingthehighestrelativeabundanceofThaumarchaeotaamong allthreesites. Ca. Nitrososphaeracomprised76%(agricultural plots)and72%(non-agriculturalplots)oftotalArchaeafor EAAand77%(agriculturalplots)and75%(non-agricultural plots)oftotalArchaeaforKBS.OtherAOA, Nitrosopumilus and Ca. Nitrosocalduswerefoundinagriculturalsoilsinverylow abundance. Therelativeabundancesof Ca .Nitrososphaera16SrRNA geneswerecomparedinagriculturalandnon-agriculturalplots atthethreestudysites( Figure5A ).Atallsitestherelativeabundancesof Ca .Nitrososphaeraweresignicantlyhigherinagriculturalthaninnon-agriculturalplots( Figure5A ).Therelative abundanceofthisgenushasincreasedtwofoldwithagricultureatEAAandthreefoldatKBS.TheBRRagriculturalplots hadthehighestsevenfoldincreaseintherelativeabundance ofthe Ca .Nitrososphaera( p 0 001)comparedtothenonagriculturalunfertilizedgrasslandandwoodlandplots.Priorto thestartoftheBRRexperimentin1843,thissitehadbeen incultivationforatleast200years( Powlsonetal.,1986 )and probablyevenlonger(theRothamstedestatemapfrom1623 showsthesiteasarable).Therefore,thedecreaseintherelativeabundanceofarchaeainthenon-agriculturalplotshas probablyoccurredwithinthelast125yearssincecultivation ceased.Bradyrhizobium INAGRICULTURALANDNON-AGRICULTURALSITESTheproportionof Bradyrhizobium 16SrRNAgenereads washigheratallthreesitesonnon-agriculturalcompared toagriculturalplots( Figure5B ).Thehighestabundanceof Bradyrhizobium wasfoundatBRRnon-agriculturalplotsfollowedbythenon-agriculturalEAAplots.Therelativeabundance ofthisgenusloweredwithagriculturebytwoandthreefoldat BRRandKBS,respectively,andsevenfoldatEAAcomparedto non-agriculturalplots.SOILPROPERTIESAtallthreelocations,soilpHwassignicantlyhigherinagriculturalplots,whichwereslightlybasicatBRRandEAA.pH waslowerinnon-agriculturalplots,whichwereslightlyacidic ( Table4 ).Thesoilorganicmatter(SOM)wassignicantlyhigher inallnon-agriculturalplotsascomparedtotheagricultural plots( Table4 ).TheaveragemoisturecontentatBRRwashigher innon-agriculturalplots,butatEAA,themoisturelevelwas higherinsoilsundercultivation.Thetotalsoilnitrogenwas higherinnon-agriculturalplotsatBRR,whereasKBShadmore www.frontiersin.orgMay2013|Volume4|Article104|5

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Zhalninaetal. Keysignaturesforthelanduse Table3|Generahighlycorrelatedwithagricultureinbold(16SrRNA 0.05%,rho 0.5, p 0 001). GenusPhylumSpearmancorrelation(rhovalue) AgriculturalpHNH3 BRREAAKBSBRREAAKBSBRREAAKBS Ignavibacterium Chlorobi Š 0.87 ŠŠ 0.85 ŠŠ 0.67 Š Marmoricola Actinobacteria 0.510.85 Š 0.09 0.76 Š 0.03 0.61 Š Arthrobacter Actinobacteria 0.580.85 Š 0.19 0.74 Š 0.20 0.65 Š Ca. EntotheonellaProteobacteria Š 0.84 ŠŠ 0.83 ŠŠ 0.62 Š Adhaeribacter Bacteoidetes Š 0.83 ŠŠ 0.76 ŠŠ 0.55 Š Blastococcus Actinobacteria 0.620.83 Š 0.480.74 Š 0.370.66 Š Hyphomicrobium Proteobacteria Š 0.80 ŠŠ 0.71 ŠŠ 0.59 Š Ramlibacter Proteobacteria 0.570.80 Š 0.20 0.79 ŠŠ 0.06 0.58 Š Ca. Nitrososphaera Thaumarchaeota 0.630.780.700.440.73 0.50 0.370.56 Š 0.54 Nocardioides Actinobacteria Š 0.78 ŠŠ 0.66 ŠŠ 0.48 Š Prosthecomicrobium Proteobacteria Š 0.76 ŠŠ 0.70 ŠŠ 0.62 Š LysobacterProteobacteria 0.600.73 Š 0.510.71 Š 0.18 0.47 Š Woodsholea Proteobacteria Š 0.71 ŠŠ 0.60 ŠŠ 0.68 Š Pseudomonas Proteobacteria Š 0.57 ŠŠ 0.42 ŠŠ 0.36 Š Flavobacterium Bacteoidetes Š 0.52 ŠŠ 0.54 ŠŠ 0.35 Š Antarcticicola Proteobacteria 0.50 ŠŠ 0.56 ŠŠ 0.42 ŠŠ Azospirillum Proteobacteria 0.55 ŠŠ 0.35 ŠŠ 0.15 ŠŠ Cystobacter Proteobacteria 0.55 ŠŠ 0.41 ŠŠ 0.26 ŠŠ Dechloromonas Proteobacteria 0.51 ŠŠ 0.10 ŠŠŠ 0.12 ŠŠ Desulfuromonas Proteobacteria 0.55 ŠŠ 0.24 ŠŠ 0.13 ŠŠ Flavisolibacter Bacteroidetes ŠŠ 0.84 ŠŠ 0.66 ŠŠŠ 0.07 Luteimonas Proteobacteria 0.56 ŠŠ 0.32 ŠŠ 0.01 ŠŠ Methylobacterium Proteobacteria 0.57 ŠŠ 0.59 ŠŠ 0.39 ŠŠ Nostoc Cyanobacteria0.59 ŠŠ 0.40 ŠŠ 0.17 ŠŠ Skermanella Proteobacteria 0.60 ŠŠ 0.47 ŠŠ 0.31 ŠŠ Sphingomonas Proteobacteria ŠŠ 0.84 ŠŠ 0.61 ŠŠŠ 0.06 Terrimonas Bacteroidetes 0.56 ŠŠ 0.42 ŠŠ 0.07 ŠŠ Actinoallomurus Actinobacteria Š Š 0.87 ŠŠ Š 0.83 ŠŠ Š 0.71 Š Ca. KoribacterAcidobacteria Š Š 0.87 ŠŠ Š 0.80 ŠŠ Š 0.69 Š Dokdonella Proteobacteria Š Š 0.85 ŠŠ Š 0.80 ŠŠ Š 0.69 Š Rhodoplanes Proteobacteria Š 0.64 Š 0.85 Š Š 0.41 Š 0.82 Š Š 0.25 Š 0.70 Š Actinomadura Actinobacteria Š Š 0.84 ŠŠ Š 0.82 ŠŠ Š 0.66 Š Acidiphilium Proteobacteria Š Š 0.84 ŠŠŠ 0.81 ŠŠ Š 0.71 Š Ca. SolibacterAcidobacteria Š Š 0.84 ŠŠ Š 0.75 ŠŠ Š 0.64 Š Rhodocista Proteobacteria Š Š 0.84 ŠŠ Š 0.80 ŠŠ Š 0.61 Š Cupriavidus Proteobacteria Š Š 0.84 ŠŠ Š 0.76 ŠŠ Š 0.69 Š Mycobacterium Actinobacteria Š 0.59 Š 0.83 Š Š 0.44 Š 0.82 Š Š 0.29 Š 0.66 Š Paucimonas Proteobacteria Š Š 0.83 ŠŠ Š 0.72 ŠŠ Š 0.69 Š Streptomyces Actinobacteria Š Š 0.82 ŠŠ Š 0.75 ŠŠ Š 0.65 Š Paenibacillus Firmicutes Š 0.64 Š 0.82 Š Š 0.46 Š 0.78 Š Š 0.29 Š 0.68 Š BradyrhizobiumProteobacteria Š 0.63 Š 0.82 Š 0.80 Š 0.74 Š 0.83 Š 0.81 Š 0.54 Š 0.69 Š 0.07 Rhodopseudomonas Proteobacteria Š Š 0.74 ŠŠ Š 0.66 ŠŠ Š 0.55 Š Niastella Bacteroidetes Š Š 0.70 ŠŠ Š 0.63 ŠŠ Š 0.53 Š Burkholderia Proteobacteria Š 0.55 Š 0.67 Š Š 0.50 Š 0.64 Š Š 0.48 Š 0.52 Š Pedomicrobium Proteobacteria Š Š 0.63 ŠŠ Š 0.70 ŠŠ Š 0.49 Š Spartobacteria Verrucomicrobia Š Š 0.62 ŠŠ Š 0.63 ŠŠ Š 0.50 Š Actinoplanes Actinobacteria Š 0.56 ŠŠ Š 0.31 ŠŠŠ 0.17 ŠŠ Kribbella Actinobacteria Š 0.58 ŠŠ Š 0.71ŠŠ Š 0.51 ŠŠ Phenylobacterium Proteobacteria Š 0.53 ŠŠ Š 0.64 ŠŠ Š 0.63 ŠŠ Solirubrobacter Actinobacteria Š 0.53 ŠŠ Š 0.39 ŠŠŠ 0.20 ŠŠ FrontiersinMicrobiology |TerrestrialMicrobiologyMay2013|Volume4|Article104|6

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Zhalninaetal. Keysignaturesforthelanduse nitrogeninagriculturalplots.NH3contentwassignicantly higherinagriculturalplotsforBRRandEAA,butatKBSthe levelofNH3didnotchangewithagriculturalmanagementdue tolowpH.CORRELATIONSBETWEENSOILPARAMETERS, Ca. NITROSOSPHAERA AND BradyrhizobiumTherelationshipsbetweeneachmeasuredsoilparameterand therelativeabundanceof Ca. Nitrososphaeraatthethree sitesweredeterminedbyusingSpearmancorrelation( Table5 ). NH+ 4,SOM,totalN,andmoistureweresignicantlynegatively FIGURE2|Proportionof16SrRNAgenereadsof Ca. Nitrososphaera and Bradyrhizobium atRothamstedResearch(BRR),theEverglades AgriculturalArea(EAA)andtheKelloggBiologicalStation(KBS). Proportionof16SrRNAreadswasnormalizedbyarcsinesquareroot. correlatedwiththeabundanceof Ca. Nitrososphaera.However, NH3andpHhadthemostsignicantandhighestpositivecorrelationwiththerelativeabundanceofthisgenus( Figures6A and 7A ).Moreover,atallsites,therelationshipsbetweenthe Ca. NitrososphaeraandeitherpHorNH3,werestrongerin agriculturalplots( Figures6A and 7A ). Bradyrhizobium wasstronglynegativelycorrelatedwithpH andthelevelofNH3insoil( Figures6B and 7B ).Moisture andNOŠ 3alsoweresignicantlynegativelycorrelatedwith Bradyrhizobium, buttheireffectwaslessnegativethantheeffect ofNH3andpHlevel( Table5 ). Nocorrelationwasobservedbetweentherelativeabundance of Ca .Nitrososphaeraand Bradyrhizobium overthecourse of13monthsofmonthlymeasurementsattheEverglades AgriculturalArea.Similarly,thedifferencesinthesetwogenerainagriculturalandnon-agriculturalareasremainedthe sameoverthese13months.AtBroadbalk,theproportionof Ca .Nitrososphaerato Bradyrhizobium didnotchangeduring 5monthsofmonthlymeasurementsintheagriculturalplots. However,inthenon-agriculturalplotsatBroadbalk,therewas astatisticallysignicantpositivecorrelationacrosstimeinthe proportionof Ca .Nitrososphaerato Bradyrhizobium ( r2= 0 252, p = 0 004).DISCUSSIONNopreviousworkshowsthedramaticeffectsoflandusemanagementonsoilmicrobialdiversityonadjacentplotsusing long-termeldexperiments.Inaddition,theobservationthat theseeffectsarereversible,asseenthroughthestudyofthe Kelloggsuccessionalplotsisalsonovel.Inaddition,nopreviousworkidentiesspecicmarkersoflandusechangeashas beendoneherewiththediscoveryof Ca .Nitrososphaeraasan abundantorganisminagriculturalsoilsand Bradyrhizobium as anabundantorganisminnon-agriculturalsoilsandthatthese FIGURE3|Barchartrepresentingtheproportionof16SrRNAgene readsfor Ca. Nitrososphaera(A)and Bradyrhizobium (B)atthe KelloggBiologicalStation(KBS)siteatagriculturalplots,andplots withoutagriculturalmanagement. Errorbarsindicatestandarderror. Differentletterdesignationsindicatestatisticallydifferentproportionsof microorganism. www.frontiersin.orgMay2013|Volume4|Article104|7

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Zhalninaetal. Keysignaturesforthelanduse FIGURE4|Representationoftotal Archaea domainfoundinagriculturalplots(A)andinnon-agriculturalplots(B)attheBroadbalkExperimentat RothamstedResearch(BRR),theEvergladesAgriculturalArea(EAA)andtheKelloggBiologicalStation(KBS). landusemarkersarereproducibleacrossasuiteofdiversesites andhighlystatisticallysignicant.Andnopreviousworkhas shownthatammoniaisthedriverofarchaealrelativeabundance insoils.Thesemajorndingsarediscussedbelowinthecontext ofpreviouswork. Thisworkwasinspiredbytheresultsof Roeschetal.(2007) whofoundahighrelativeabundanceofarchaeainthreeagriculturalsoils(412%)andavanishinglylownumberinaboreal forestsoil(0.01%).Thisresultledtoseveralquestions.Whywere thearchaeasolowintheborealcomparedtotheotherssites? WasitthecolderclimateintheborealforestsitefromnorthernOntario,Canadacomparedtotheothersites?Wasitthe likelylowernutrientstatusoftheforestsitecomparedtothe agriculturalsites?Justpriortothepublicationof Roeschetal. (2007) ,thediscoveryofammonia-oxidizingarchaeainsoilwas made( Leiningeretal.,2006 ).Itwasreasonabletoassumethat nitrogenfertilizationofagriculturalsitesmightcontributetothe higherrelativeabundanceofarchaeainagriculturalsoils.Totest thisnotion,threelong-termexperimentalsiteswerechosenthat metthefollowingthreeimportantcriteria.First,thesiteshad tobelong-termsiteswiththeavailabilityofnutrientandother environmentaldata.Second,thesiteshadtohaveadjacentplots thatwerecultivatedanduncultivated.Third,collectivelythesites hadtodiffersignicantlyinmeanannualtemperaturetobe abletotestaclimateeffectonsoilarchaea.Usingthesecriteria, threesiteswerechosen:theBroadbalkexperimentatRothamsted ResearchintheUK,theKelloggBiologicalStationinMichigan USA,andtheEvergladesAgriculturalAreainSouthFlorida,USA. TheBroadbalkandKelloggsiteshadtheaddedadvantageof havingexperimentalplotswithv aryingamountsofNfertilizer appliedannually. Inadditiontothearchaea,bacterialtaxawerealsoexaminedfortheirchangeswithlanduse.Onlyabout24%of allreadscouldbeclassiedtoknowngenera.Ofthemore than700knowngenerafoundateachsite,anaverageof20 generaateachsitewereofreasonablyhighabundancethat alsodifferedinrelativeabundancebetweenagriculturaland non-agriculturalplots.Ofthose,onlytwodifferedinrelativeabundancebylanduseatallthreesites.Oneofthese, Bradyrhizobium ,isinvolvedinammoniaproductionandisbest knownforitsroleasanitrogen-xingsymbiontonlegume roots.Theother, Ca .Nitrososphaera,isbestknownforitsrole FrontiersinMicrobiology |TerrestrialMicrobiologyMay2013|Volume4|Article104|8

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Zhalninaetal. Keysignaturesforthelanduse FIGURE5|Relativeabundanceof Ca .Nitrososphaera(A)and Bradyrhizobium (B)insoilsattheBroadbalkExperimentat RothamstedResearch(BRR),theEvergladesAgriculturalArea(EAA) andtheKelloggBiologicalStation(KBS),separatedintoagricultural (Ag)andnon-agricultural(Non-ag)plots. Therectangularboxrepresents the2575thpercentiles,thewhiskersrepresentthe1090thpercentiles, thelineinsidetheboxrepresentsthemedian,andtheopencircles representtheoutliers.Thesameletterabovetheboxindicatesno signicantdifferencebetweenmeansbyDuncan'stestatthe95% condenceinterval. Table4|Soilpropertiesofagriculturalandnon-agriculturalsoilsat BroadbalkExperimentatRothamstedResearch(BRR),theEverglades AgriculturalArea(EAA)andtheKelloggBiologicalStation(KBS). PlotpHMoistureOrganicmatterTotalNNOŠ 3-NNH+ 4-Nlog[NH3] %%%mgkgŠ 1mgkgŠ 1 BRR Agricultural**7.3716.322.620.159.161.86**Š 2.21 Non-agricultural6.24*24.08**8.28**0.416.141.29 Š 2.99 EAA Agricultural**7.98*122.6471.10ND53.648.84**Š 0.44 Non-agricultural5.63102.07*83.31ND88.819.63 Š 2.85 KBS Agricultural**5.81ND1.49*0.13**9.571.3 Š 3.32 Non-agricultural5.2ND**2.430.091.38**6.18 Š 3.24 *Signicant,p-value 0.05 **Signicant,p-value 0.001 ND-notdetermined.inammoniaoxidation.Thetwogenera, Bradyrhizobium and Ca .Nitrososphaera,arenegativelyandpositivelycorrelatedwith ammonialevels,respectively.Thisalsomakessensebiochemically.AdditionofxedNinhibitsnitrogenaseandnitrogen xationinthelabandeld( Sekhonetal.,1987;Peoplesetal., 1995;HalbleibandLudden,2000 ).Inaddition,astheproduct ofnitrogenase,ammoniaisaproductinhibitorofnitrogenase andalsoblockstranscriptionofthe nif regulon( Halbleiband Ludden,2000 ).Asaresult,higherlevelsofNwouldreducethe rolefornitrogen-xingorganismsinnon-legumeagricultureat eachofthethreesitesstudiedhere.Theobservationthatthe proportionof Ca .Nitrososphaerato Bradyrhizobium increases slightly,butsignicantlystatistically,overtimeatBroadbalk supportsthenotionthat Ca .Nitrososphaera'srelativenumbers comparedto Bradyrhizobium increasewithincreasingammonia concentrationsinsoil.Thismayoccurbecause Bradyrhizobium isprovidingmoreammoniatosoil,whichencouragesapopulationincreaseamongtheammoniaoxidizingarchaeaina non-agriculturalsoil.InanagriculturalsoilwhereNfertilizeris applied,thenumberoffree-livingbradyrhiozbiadeclinessince www.frontiersin.orgMay2013|Volume4|Article104|9

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Zhalninaetal. Keysignaturesforthelanduse xedNinhibitsN2xation.Sothenumbersofammoniaoxidizingarchaeaincreaseinanagriculturalsoilasaresultofadded NandahighersoilpH.Bothfactorsresultintheincreased availabilityofammonia. Theabovehypothesisdependsonnitrogenxationbyfreeliving Bradyrhizobium insoilsincethesamplescollectedinthis workwerefromsoilanddidnotcontainroots.Thereareseveralexamplesoffree-livingnitrogenxationby Bradyrhizobium (formerlyreferredtoasslow-growing Rhizobium ).Thiswasrst shownby Paganetal.(1975) whodemonstratedfree-livingnitrogenxationbystrain32H1aswellasotherstrains.Thisworkwas followedshortlybyexperimentsthatoptimizedtheconditions fornitrogenaseactivitybyfree-livingbradyrhizobiaincluding O2requirementsaswellasthecellmorphologychangesthat occurundernitrogenxationconditions( Gibsonetal.,1976; Table5|Spearmancorrelation(rho)forBroadbalkexperimentat Rothamstedresearch(BRR),theEvergladesAgriculturalArea(EAA) andtheKelloggBiologicalStation(KBS)betweenrelativeabundance of Ca. Nitrososphaera, Bradyrhizobium andsoilfeatures(TN,total nitrogen;NH+ 4,ammonium;NH3,ammonia;NOŠ 3,nitrate;Moisture; SOM,organicmatterandpH). Variables Ca .Nitrososphaera Bradyrhizobium pH*0.63*Š 0.53 NH+ 4*Š 0.27 Š 0.03 NH3*0.53*Š 0.51 SOM*Š 0.24 Š 0.09 TN*Š 0.37*0.42 NOŠ 3Š 0.08*Š 0.21 Moisture*Š 0.41*Š 0.24 Agriculturalandnon-agriculturalplotswereanalyzedtogether(n = 158). *Signicant,p-value 0.001.KeisterandEvans,1976;vanBrusseletal.,1979 ).Theseobservationsweresoonexpandedtostillmorestrainsof Bradyrhizobium ( Subba-Rao,1977;Skotnickietal.,1979 ).Nevertheless,attempts toobtainfree-livingnitrogenxationinmanybradyrhizobiahave failed( Paganetal.,1975;Skotnickietal.,1979 ).However,this maybecausedbythelackofaspecicnutrientinmedium. Forexample,itwasrecentlyshownthatsymbioticrhizobialack nifV ,ageneessentialfortheprodu ctionofhomocitrate,anecessarycomponentoftheFeMocofactorpresentindinitrogenase andthatthehostplantprovideshomocitratetothenodule bacteriatocompensateforthelackof nifV ( Hakoyamaetal., 2009 ). Nevertheless,free-livingbradyrhizobiahavebeenshownto x15N2insoilusingstableisotopeprobing.15Nlabelwas foundinbradyrhizobial16SrRNAsequencesafterfeeding15N2tosoilmesocosms( Buckleyetal.,2007 ).Inaddition thegenomeofafree-living Bradyrhizobium strainsisolated fromaricepaddywasrecentlysequencedandfoundtocontainthesamecomplementofnitrogenxationgenesfoundin thegenomeofanitrogen-xingsymbiontof Bradyrhizobium However,thispaddysoil Bradyrhizobium straindidnotnodulateanylegumetested,lackedasymbiosisislandofgenesoften foundinN2-xinglegumesymbionts,anddidnotpossessany ofthenodulationgenes.Alloftheseresultstakentogether withtheresultspresentedhereareexpectedtoencouragean examinationoffree-livingbradyrhizobiainuncultivatedsoils todeterminetheirabilitytoprovidexedNtounmanaged ecosystems. Thereisalsoasoundbiologicalbasisfor Ca .Nitrososphaera toberelativelymoreabundantinagriculturalsoilsthannonagriculturalsoils.Asammonia,notammonium,isthesubstrate forammoniamonooxygenase( Suzukietal.,1974;Arpetal., 2002 ),itisnotsurprisingthatammonialevels,notammonia plusamoniumlevels,correlatewellwiththerelativeabundance of Ca .Nitrososphaera,particularlyathigherpHlevels.However, FIGURE6|Relationshipbetweentheproportionof Ca. Nitrososphaera16SrRNAgenereads(%)andNH3concentration(A); Bradyrhizobium and NH3concentration(B)atthreeexperimentalsites. Proportionof16SrRNAreadswasnormalizedbyarcsinesquareroot. FrontiersinMicrobiology |TerrestrialMicrobiologyMay2013|Volume4|Article104|10

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Zhalninaetal. Keysignaturesforthelanduse FIGURE7|Relationshipbetweentheproportionof Ca. Nitrososphaera16SrRNAgenereads(%)andpH(A); Bradyrhizobium andpH(B)atthree experimentalsites. Proportionof16SrRNAreadswasnormalizedbyarcsinesquareroot. discoveringthisrequiredthatammonialevelsbecalculatedfrom thetotalammoniaplusammoniumlevelsandsoilpHascurrentmethodsofmeasuringammonia/ammoniumlevelsinsoil donotdistinguishbetweenionizedammoniumandnon-ionized ammonia.CONCLUSIONTheresultshereshowthatagriculturalmanagementcausessignicantchangesinsoil,whichleadstoanincreaseinAOA abundance. Ca .Nitrososphaera,themostabundantsoilAOA,was presentinagreaterabundanceatallthreesitesinresponsetoagriculture.Ofallfactorsexamined,pHmediatedNH3accumulation wastheprimarydriveroftheAOAcommunityinsoil. Inaddition,thisworkshowstheeffectofagricultureonthe relativeabundanceofotherorganismsinvolvedinthenitrogen cycle.Ateachsite,therelativeabundanceof Bradyrhizobium anitrogen-xingsymbiont,wasstronglynegativelycorrelated withagriculturallanduse,pH,andNH3levels.Thereciprocal responsesof Bradyrhizobium and Ca .Nitrososphaeraappearto beexcellentbiologicalmarkersforlanduse.Forfurthervalidationofthesemicroorganismsasbiologicalmarkers,theseresults shouldencouragethetestingofthesegeneraasmarkersforland useatothersites.ACCESSIONNUMBERSAllsequenceshavebeendepositedintheGenBankdatabasewith AccessionNo.PRJNA191521,RJNA191098,andPRJNA191523.ACKNOWLEDGMENTSThisworkwassupportedbytheNationalScienceFoundation (grantnumberMCB-0454030);andtheUnitedStates DepartmentofAgriculture(grantnumbers2005-35319-16300, 00067345).RothamstedResearchreceivesstrategicfundingfrom theUKBiotechnologyandBiologicalSciencesResearchCouncil.REFERENCESArp,D.J.,Sayavedra-Soto,L.A., andHommes,N.G.(2002). Molecularbiologyandbiochemistryofammoniaoxidationby Nitrosomonaseuropaea.Arch. Microbiol. 178,250255. Bates,S.T.,Berg-Lyons,D.,Caporaso, J.G.,Walters,W.A.,Knight,R., andFierer,N.(2011).Examining theglobaldistributionofdominant archaealpopulationsinsoil. ISMEJ. 5,908917. Black,C.A.(1965). Methodsof SoilAnalysis:PartIPhysicaland MineralogicalProperties. Madison, WI:AmericanSocietyofAgronomy. Bottcher,A.B.,andIzuno,F.T.(ed.). (1994). EvergladesAgricultural Area(EAA)—Water,Soil,Crop, andEnvironmentalManagement Gainesville,FL:UniversityPressof Florida. Bru,D.,Ramette,A.,Saby,N.P.A., Dequiedt,S.,Ranjard,L.,Jolivet, C.,etal.(2011).Determinantsof thedistributionofnitrogen-cycling microbialcommunitiesatthelandscapescale. ISMEJ .5,532542. Buckley,D.H.,andSchmidt,T.M. (2001).Thestructureofmicrobial communitiesinsoilandthelasting impactofcultivation. Microb.Ecol. 42,1121. Buckley,D.H.,Huangyutitham, V.,Hsu,S.-F.,Tyrrell,A.,and Nelson,T.A.(2007).Stableisotopeprobingwith15N2reveals novelnoncultivateddiazotrophsin soil. Appl.Environ.Microbiol. 73, 31963204. Caporaso,G.J.,Kuczynski,J., Stombaugh,J.,Bittinger,K., Bushman,F.D.,Costello,E.K., etal.(2010).QIIMEallowsanalysis ofhigh-throughputcommunity sequencingdata. Nat.Methods 7, 335336. Castro,H.,Newman,S.,Reddy,K.R., andOgram,A.(2005).Distribution andstabilityofsulfate-reducing prokaryoticandhydrogenotrophic methanogenicassemblagesin nutrient-impactedregionsofthe FloridaEverglades. Appl.Environ. Microbiol. 75,26952704. Chu,H.,Fujii,T.,Morimoto,S.,Lin,X., andYagi,K.(2008).Populationsize andspecicnitricationpotential ofsoilammonia-oxidizingbacteriaunderlong-termfertilizermanagement. SoilBiolol.Biochem. 40, 19601963. Clark,I.M.,Buchkina,N.,Jhurreea, D.,Goulding,K.W.T.,andHirsch, P.R.(2012).Impactsofnitrogen applicationratesontheactivityand diversityofdenitrifyingbacteriain www.frontiersin.orgMay2013|Volume4|Article104|11

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