The Effect of Root Exudate 7, 40-Dihydroxyflavone and Naringenin on Soil Bacterial Community ...

16 Pages·2015·0.49 MB·English

RESEARCHARTICLE
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The Effect of Root Exudate 7,4 -
Dihydroxyflavone and Naringenin on Soil
Bacterial Community Structure
MártonSzoboszlay1,AlisonWhite-Monsant2,LukeA.Moe1*
1 DepartmentofPlantandSoilSciences,UniversityofKentucky,Lexington,Kentucky,UnitedStatesof
America,2 DepartmentofAnimal,PlantandSoilScience,CentreforAgriBioscience,LaTrobeUniversity,
Melbourne,Australia
*[email protected]
a11111
Abstract
Ourgoalwastoinvestigatehowrootexudateflavonoidsinfluencethesoilbacterialcommu-
nitystructureandtoidentifymembersofthecommunitythatchangetheirrelativeabun-
danceinresponsetoflavonoidexudation.Usingamodelsystemthatapproximates
OPENACCESS flavonoidexudationofMedicagosativaroots,wetreatedasoilwith7,40-dihydroxyflavone
Citation:SzoboszlayM,White-MonsantA,MoeLA andnaringeninintwoseparateexperimentsusingthreedifferentrates:medium(equivalent
(2016)TheEffectofRootExudate7,40- totheexudationrateof7,40-dihydroxyflavonefromM.sativaseedlings),high(10×the
DihydroxyflavoneandNaringeninonSoilBacterial
mediumrate),andlow(0.1×themediumrate).Controlsreceivednoflavonoid.Soilsamples
CommunityStructure.PLoSONE11(1):e0146555.
weresubjectedtoATPassaysand16SrRNAgeneampliconsequencing.Theflavonoid
doi:10.1371/journal.pone.0146555
treatmentscausednosignificantchangeinthesoilATPcontent.Withthehigh7,40-dihy-
Editor:GabrieleBerg,GrazUniversityofTechnology
droxyflavonetreatmentrate,operationaltaxonomicunits(OTUs)classifiedasAcidobac-
(TUGraz),AUSTRIA
teriasubdivision4increasedinrelativeabundancecomparedwiththecontrolsamples,
Received:September26,2015
whereasOTUsclassifiedasGaiellales,Nocardioidaceae,andThermomonosporaceae
Accepted:December19,2015
weremoreprevalentinthecontrol.Thenaringenintreatmentsdidnotcausesignificant
Published:January11,2016 changesinthesoilbacterialcommunitystructure.Ourresultssuggestthattherootexudate
Copyright:©2016Szoboszlayetal.Thisisanopen flavonoid7,40-dihydroxyflavonecaninteractwithadiverserangeofsoilbacteriaandmay
accessarticledistributedunderthetermsofthe haveotherfunctionsintherhizosphereinadditiontonodgeneinductioninlegume—rhizo-
CreativeCommonsAttributionLicense,whichpermits
biasymbiosis.
unrestricteduse,distribution,andreproductioninany
medium,providedtheoriginalauthorandsourceare
credited.
DataAvailabilityStatement:16SrRNAgene
ampliconsequencingdataareavailableatthe
Introduction
NationalCenterforBiotechnologyInformation
SequenceReadArchive(www.ncbi.nlm.nih.gov/sra)
Flavonoidsareplantsecondarymetabolitessynthesizedviathephenylpropanoidpathway.
undertheaccessionnumberPRJNA295777.
Theyarepresentinalltissuesofhigherplantsandsomeareexudedfromtherootsintotherhi-
Funding:Thisworkwassupportedbygrant2011- zosphere[1].Theirmoststudiedfunctionsinthesoilarethoseassociatedwithlegume—rhizo-
67020-30195fromtheNationalInstituteofFoodand
biasymbiosis,wherebytheyactivateorrepressbacterialnodgeneexpression[2]andtrigger
AgricultureoftheUnitedStatesDepartmentof
chemotaxisinnitrogen-fixingrhizobia[3].Asidefromtheirassociationwithrhizobia,flavo-
AgriculturetoLAM.Thefundershadnoroleinstudy
noidsarepresentinrootexudatesofnon-legumeplants[1],andagrowingbodyofdatasug-
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript. geststhattheyinfluencethegrowthandactivityofvarioussoilbacteria.Forexample,some
PLOSONE|DOI:10.1371/journal.pone.0146555 January11,2016 1/16
FlavonoidsandSoilBacterialCommunityStructure
CompetingInterests:Theauthorshavedeclared flavonoids,particularlyisoflavonoids,areconsideredphytoalexinsorphytoanticipinsdueto
thatnocompetinginterestsexist. theirantimicrobialeffect[4].Furthermore,theexperimentsofHartwigetal.[5]implythat
rootexudateflavonoidsmaycontroltheproliferationofsomerhizospherebacteria.They
foundthatthedoublingtimeofEnsifermelilotiandPseudomonasputidadecreasedwhen
exposedtoluteolinorquercetininmicromolarconcentrationsinlaboratorycultures.Flavo-
noidscanalsobeutilizedasasourceofcarbonandhaveotherdirectandindirecteffectson
soilnutrientcycles[6].RootextractswithahighflavonoidcontentfromLupinusalbushave
beenshowntodecreasesoilrespirationwithoutasignificantchangeinmicrobialbiomass
basedonsoilATPcontent,andtodecreasephosphataseandincreaseureaseactivity[7].Flavo-
noidsmayalsoaffectbacterialactivityintherhizospherebyinfluencingquorumsensing.Van-
deputteetal.[8,9]foundthatcatechin,apigenin,eridictyol,kaempferol,luteolin,myricetin,
naringenin,naringin,quercetin,taxifolin,andchalconehadsomeeffectontheproductionof
quorum-sensing-dependentfactorsinPseudomonasaeruginosa.Further,Pérez-Montañoetal.
[10]showedthatacyl-homoserine-lactoneproductionbyEnsiferfredii,Rhizobiumetli,andR.
sullaestrainswasenhancedinthepresenceofflavonoidsthatareknowntoinduceexpression
oftheircognatenodgenes.
Thesefindingsindicatethatrootexudateflavonoidsinfluenceadiverserangeofsoilbacte-
ria.Toimproveourunderstandingofhowrootexudateflavonoidsinfluencethesoilmicrobial
communitystructure,wedesignedamodelsystemthatapproximatesflavonoidexudationof
Medicagosativaroots.Ourgoalwastoidentifymembersofthesoilbacterialcommunitythat
changetheirrelativeabundanceinresponsetoflavonoidexudation.Toachievethis,we
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exploredtheimpactofsimulatedexudationof7,4-dihydroxyflavone,themostabundantnod
geneinducingflavonoidamongtherootexudatesofM.sativaseedlings[11],andnaringenin,
whichisalsoanodgeneinducingflavonoidpresentintherootexudatesofvariouslegumes
[1].
MaterialsandMethods
Soilproperties
ThesoilchosenfortherhizospheremodelsystemstudieswasfromapastureattheUniversity
ofKentuckySpindletopFarm(38°06'51.7"N,84°29'41.7"W)thathadnotbeenfertilizedor
plantedforthelast5years;butpriortothis,M.sativahadbeengrownforthestudybyProbst
andSmith[12].TheMaurysiltloam(fine,mixed,semiactive,mesicTypicPaleudalf)wascol-
lectedfromthesurface10–15cm,sieved(4mm),driedatroomtemperaturewithregularmix-
ing,andthenstoredatroomtemperatureinaclosedplasticcontainerforover3months.This
storageperiodwastodecreasetheeffectofrootexudateflavonoidsinthesoilfromthevegeta-
tionatthecollectionsite.SamplesweresenttotheUniversityofKentuckyRegulatoryServices
SoilTestingLaboratorytodeterminebasicsoilproperties(http://soils.rs.uky.edu/tests/
methods.php).
Thesoiltexturewas18.5%sand,64.1%silt,and17.4%clay.ThepHwas5.95(1:1solution:
soilusing1MKCl)andthebufferedpHwas7.03(withSikorabuffer[13]).Thesoilcontained
4.49%organicmatterand0.25%totalnitrogen.Thecationexchangecapacitywas25.95meq/
100g,thebasesaturation87.77%andtheexchangeableK,Ca,Mg,andNawere0.36,17.85,
4.55,and0.02meq/100g.MehlichIIIextractableP,K,Ca,Mg,andZnwere243.5,110.0,
2925.5,486.5,and1.2mg/kg.
Beforeusingthesoilitwasmixedwith30%m/msand(previouslybleached,washed,and
ovendried)tofacilitatedrainage.
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FlavonoidsandSoilBacterialCommunityStructure
Therhizospheremodelsystem
Thesoil—sandmixturewasmoistenedwithdistilledwater(60.0gwaterto400.0gsoil—sand
mixture)andthoroughlymixedinplasticbagsbeforefilling60.0ginto50-mlpolypropylene
conicalcentrifugetubes(FisherScientific,USA).Theconicalbottomportionsofthetubeswere
previouslycutoffandreplacedwithplasticmeshtoallowdrainage.Eachtubewaswrappedin
aluminumfoiltoprotectthesoilfromlight.Weusedrhizonsoilmoisturesamplers(Rhizon
MOM,RhizosphereResearchProducts,Netherlands)tomodeltherootexudationprocess.The
rhizonshad5-cmlongporousparts,2.5mmdiameter,poresize0.12–0.18μm,aglassfiber
strengthener,andpolyethylene/polyvinylchloridetubing.Tothesoilineachtube,6.00mldis-
tilledwaterwasaddedbeforeinsertingthreerhizonsverticallyandequidistanttoeachother
andthewallofthetube.Therhizonswerepreviouslybleachedandthenwashedinsteriledis-
tilledwater.
Initialexperimentsstudyingtheeffectofrootexudatesonthesoilmicrobialcommunity
deliveredexudatestothesoilviaartificialrootssuchasmembranefilters[14,15]orcylindrical
tubesorwickspreparedfromsuchfilters[16,17].Inlaterexperiments,rhizonsoilmoisture
samplerswereemployedasrootexudationmodels[18–20]byusingtheminthereversedirec-
tion:topumpasolutionintothesoilinsteadofsamplingthesoilsolution.Analternative
approachdevelopedbyZiegleretal.[21]usedglassslidescoatedwithagarosecontainingthe
exudatecompoundsthatwereinsertedintothesoil.Ourgoalwastostudythemicrobialcom-
munityinavolumeofsoilhomogenouslyexposedtotheexudates;therefore,weadoptedthe
useofrhizonsoilmoisturesamplersinourrhizospheremodelsystems.
Treatmentsolutionsandapplications
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Therhizospheremodelsystemsreceived7,4-dihydroxyflavoneornaringeninathigh(24.00
nmol/day),medium(2.40nmol/day),low(0.24nmol/day),ornoflavonoid(control)rates.The
mediumtreatmentratewasdeterminedfromthecalculationsofCescoetal.[1]whichwere
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basedontheresultsofMaxwellandPhillips[11]tomatchtheexudationrateof7,4-dihydrox-
yflavonefromM.sativaseedlingsassumingthat0.4g(freshweight)rootbiomassoccupiesthe
soilinarhizospheremodelsystem.Wedeterminedtherootbiomassvalueexperimentallyby
growingM.truncatulausingthesamesoilandtubesetupasthepresentexperiment(unpub-
lished).Thetreatmentsolutionswereappliedin1.2mlaqueousvolumeslowlypumped
throughtherhizons(400μlperrhizon)intothesoilusing1-mlsyringes,onceevery24hours.
Aftereachapplication,approximately0.2mlofairwasinjectedintotherhizontoensurethat
theentirevolumeofthetreatmentsolutionhadreachedthesoilandthatnoliquidremainedin
thetubing.
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Stocksolutionsof7,4-dihydroxyflavone(Indofine,USA)andnaringenin(MPBiomedicals,
USA)werepreparedindimethylsulfoxideandstoredat−20°Cuntilthestartofthetreatments.
Thefinaltreatmentsolutionseachcontained0.0625%v/vdimethylsulfoxide.Thetreatment
solutionsforeachflavonoidtreatmentrateandthecontrolweresupplementedwiththemajor
carbohydrates,aminoacids,andotherorganicacidspresentintherootexudatesofM.sativa
[22–24]assuming0.4g(freshweight)rootbiomassintherhizospheremodelsystem:1396μM
glucose,223μMarabinose,115μMmaltose,108μMmannose,690μMserine,250μMglycine,
26μMmalate,24μMcitrate,and4μMsuccinate.Stocksolutionswerepreparedinwater,filter
sterilized(0.22μm),andstoredat4°C.Thecontroltreatmentsreceivedthecarbohydrates,
aminoacids,andotherorganicacids,anddimethylsulfoxide,butnoflavonoid.Thetreatment
solutionswereadjustedtopH7withKOH.
Sixrhizospheremodelsystemsweresetupforeachofthehigh,medium,low,andcontrol
treatmentrates,andthenplacedonplastictraysandincubatedinadarkcabinetatroom
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FlavonoidsandSoilBacterialCommunityStructure
temperature(21–23°C).A400-μlaliquotofdistilledwaterwaspumpedthrougheachrhizon
onceadayfor5daysbeforethetreatmentsstarted.Inthefirstdaysofincubationafewseed-
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lingsgerminatedinthesoil.Thisaffected3rhizospheremodelsystemsreceivingthehigh7,4-
dihydroxyflavonerate,1receivingthemediumrate,3receivingthelowrateand2receivingthe
controltreatment.Fromthenaringenintreatments2rhizospheremodelsystemsreceivingthe
highrate,1receivingthemediumrate,1receivingthelowrate,and2receivingthecontrol
treatmentwereaffected.Seedlingswereremovedassoonastheyemergedfromthesoil.The
lastseedlingappeared6daysbeforesampling.Thetreatmentswereappliedfor10days.Every
2daystherhizospheremodelsystemswereweighedbeforeadministeringthetreatmentsand
thensufficientdistilledwaterwasappliedtothesoiltomaintainaconstantwatercontent,and
theywererandomlyrearrangedonthetrays.
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Theexperimentwasfirstconductedwith7,4-dihydroxyflavoneandthenrepeatedwithnar-
ingeninusingthesameprocedures.
Sampling
Soilsampleswereharvestedapproximately3hoursafterthelasttreatmentapplication.The
rhizonswereremovedandthesoilcolumnwaspushedoutofthetube.Thetop1cmofsoil
wasdiscardedandthenext4cmwascollectedinasterileplasticbagusingasterilespatula,
mixed,andthenfrozeninliquidnitrogen.Sampleswerestoredat−80°Cpriortoanalysingall
samplesformicrobialbiomassandmicrobialcommunitystructure.
Somerhizospheremodelsystemscontainedgerminatingseedsthatdidnotreachthesur-
face,andthuswereonlydetectedduringsampling.Thesesystemswereremovedfromthe
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studyleavingthenumberofreplicatesto5high,4medium,5low,and4controlinthe7,4-
dihydroxyflavoneexperiment,and5high,5medium,4low,and5controlinthenaringenin
experiment.
ATPassays
ToassesstotalmicrobialbiomassATPwasextractedfrom1.0gofthesoilsampleswith
dimethylsulfoxideandtrisodiumphosphateasdescribedbyBaietal.[25],butwith1:10
insteadof1:100dilutioninglycine-EDTAbuffer.Dilutedextractsweretreatedwithbenzalko-
niumchloridefollowingtheprotocolofMartens[26]bymixinga100-μlaliquotwith100μl
0.05%m/mbenzalkoniumchlorideinTris-Mg2+buffer(50mMTris,10mMMgSO ,pH7.8)
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ina12mm×50mmautoclavedglasstube.Thetubewassonicatedfor5sand190μlTris-
Mg2+bufferwasadded,followedby10μlStayBriteHighlyStableLuciferase/LuciferinReagent
(BioVision,USA).Thesamplewasquicklymixedwithapipetandtheluminescencewasmea-
suredfiveconsecutivetimesusingaTurnerDesign20/20luminometerwith10-sintegration
periodsand54.8%sensitivity.Outofthefivemeasurements,generallythefirsttwowerehigher
thanthelastthree,whichshowedlessvariation.Therefore,theaverageofthethird,fourth,and
fifthreadingswasusedinthecalculation.Blankswereusedtomeasurethebackgroundlumi-
nescenceusingglycine-EDTAbufferinsteadofasoilextract.Astandardcurveusinglog
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(ATPconcentrationinnM)vs.log (luminescence)wasobtainedbymeasuringATPstan-
10
dardscomprisingblanksamplessupplementedwith0.1,0.5,1,5,and10nMATP.TheATP
stocksolutionwaspreparedfromATP-Na salt(SERVAElectrophoresisGmbH,Germany)in
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Tris-Mg2+bufferandthenfiltersterilized(0.22μm)andstoredat−80°C.Toestimatethe
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extractionefficiency,soilsamplesfromthe7,4-dihydroxyflavone(n=3)andthenaringenin
(n=3)experimentswererandomlychosen,andATPextractswerepreparedfromthemby
adding20μl0.1mMATPsolutionwhenaddingthedimethylsulfoxide.
PLOSONE|DOI:10.1371/journal.pone.0146555 January11,2016 4/16
FlavonoidsandSoilBacterialCommunityStructure
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Samplesfromthe7,4-dihydroxyflavoneandnaringeninexperimentswereprocessedsepa-
rately.ThreeblanksandasetofATPstandardsweremeasuredforbothexperiments.Theaver-
ageoftheblankswassubtractedfromthereadingsandtheATPcontentpergramofsoil(dry
weight)wascalculatedaccordingtothestandardcurvesinMicrosoftExcel.Theresultswere
analyzedinJMP10.0.0(SASInstitute,USA)usingANOVAandTukey’sHSD.
16SrRNAgeneampliconsequencinganddataprocessing
DNAwasextractedfrom250mgofsoilfromeachsamplewithPowerSoilDNAIsolationKit
(MOBIOLaboratories,USA).TheV4regionofthe16SgenewasamplifiedwithPCRusing
theprimersofKozichetal.[27].Thereactionscontained22.5μlAccuPrimePfxSuperMix
(Invitrogen,USA),7.5ngDNA,and7.5pmolforwardandreverseprimers(IntegratedDNA
Technologies,USA)in25μlfinalvolume.AmplificationwascarriedoutinaBio-RadMy
Cyclerversion1.065thermocycler(Bio-Rad,USA)with4mininitialdenaturationat95°Cfol-
lowedby30cyclesof20sat95°C,15sat55°C,and2minat68°C,andafinalextensionfor10
minat68°C.ThesizeandqualityofthePCRproductswerecheckedusingagarosegelelectro-
phoresis.ThePCRproductswerecleaned,quantified,pooled,andsequencedonanIllumina
MiSeqinstrumentwitha500cyclev2kit(Illumina,USA)attheUniversityofKentucky
AdvancedGeneticsTechnologiesCenteraccordingtotheprotocolofKozichetal.[27].Sam-
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plesfromthe7,4-dihydroxyflavoneandthenaringeninexperimentsweresequencedsepa-
rately.ThesequencedataisaccessibleattheNationalCenterforBiotechnologyInformation
SequenceReadArchive(www.ncbi.nlm.nih.gov/sra)undertheaccessionnumber
PRJNA295777.
Forwardandreversesequencereadswerejoined,andthensequencesthatwerelowquality,
chimeric,mitochondrial,chloroplast,archaeal,eukaryotic,andunclassifiablewereremovedin
mothurv1.34[28]asdescribedintheMiSeqSOP(http://www.mothur.org/wiki/MiSeq_SOP,
accessedDecember2014)usingtheSILVAalignment[29]release119,andtheRibosomal
DatabaseProject[30]release10.Sequenceswerebinnedtooperationaltaxonomicunits
(OTUs)usingminimumentropydecomposition(MED)[31]withthefollowingsettings:
m=0.0965,c=4,M=(numberofsequencesinthedataset/10000),andV=3.Arepresenta-
tivesequencefromeachOTUwasclassifiedaccordingtotheRibosomalDatabaseProject
release10usingmothurwith70%bootstrapcutoff.
Insteadofrarefying[32],centeredlog-ratio(CLR)transformation[33,34]wasappliedto
thedatamatricesusingthecompositionspackage[35]inRversion3.2.1(www.R-project.org).
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Thedatamatricesfromthe7,4-dihydroxyflavoneandnaringeninexperimentscontained13
and43zeroes,respectively.Thesewerereplacedwithonestoallowthistransformation.Ordi-
nationplotsweremadewithnon-metricmultidimensionalscaling(NMS)inPC-ORD6.0
(MJMSoftwareDesign,USA)withEuclideandistances:250runswereperformedwithrandom
startingconfigurationsinonetosixdimensionswitha10−7instabilitycriterionand500maxi-
mumiterationswith0.2initialsteplengthtofindthebeststartingconfigurationsineach
dimensionality.Statisticsforthefinalstressforeachdimensionalitywereobtainedfrom250
runswithrandomizeddata.Dimensionswereonlyacceptediftheydecreasedthestresstoa
lowervaluethanthatfrom95%oftherandomizedruns.Basedontheresults,two-dimensional
solutionswereselected.Thefinalrunwasconductedusingthedeterminedbeststartingconfig-
uration.Multi-responsepermutationprocedure(MRPP)[36]wasusedontheCLRtrans-
formeddatasetswithEuclideandistancestotestthesignificanceofthedifferencesbetweenthe
high,medium,low,andcontroltreatments.TofinddifferentiallyabundantOTUsinthetreat-
ments,weusedDESeq2(withoutCLRtransformation)[37]asrecommendedbyMcMurdie
etal.[32]withtheDESeq2packageversion1.8.1[37]inR.Toaccountforthehighnumberof
PLOSONE|DOI:10.1371/journal.pone.0146555 January11,2016 5/16
FlavonoidsandSoilBacterialCommunityStructure
Table1. SoilATPconcentrations,andestimatedATPextractionefficiency.
7,40-dihydroxyflavone(μg/gsoildryweight) Naringenin(μg/gsoildryweight)
High 1.557±0.116 1.212±0.195
Medium 1.698±0.306 1.211±0.164
Low 1.780±0.101 0.983±0.062
Control 1.621±0.187 1.121±0.164
Extractionefficiency(%) 86.3±1.47 87.3±6.85
Numbersrepresentaverage±SD.ATPconcentrationsarenotcorrectedforextractionefficiency.
doi:10.1371/journal.pone.0146555.t001
simultaneoustests,q-valueswerecalculatedaccordingtothepositivefalsediscoveryrate
method[38]withthesmootheroptioninQVALUE(http://genomine.org/qvalue).Theq-value
ofatestestimatestheproportionoffalsepositivefindingsinthedatasetiftestswithequalor
lowerp-valuesareacceptedassignificant.
Results
ATPassays
TherewasnosignificantdifferenceinthesoilATPcontentbetweenthetreatmentsineither
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the7,4-dihydroxyflavoneorthenaringeninexperiment(Table1).Thestandardcurvesare
showninS1Fig.
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16Ssequencingresultsfromthe7,4-dihydroxyflavoneexperiment
Thedatasetcontained117,276–359,427sequencespersamplewith253bpaveragereadlength,
whichwerebinnedto1580OTUs.OntheNMSplot(Fig1A)thehigh,medium,andlowtreat-
mentsamplesoverlappedeachother,butwereseparatefromthecontrolsamples,indicatinga
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differenceinbacterialcommunitystructurebetweenthecontroland7,4-dihydroxyflavone
treatments.Thisdifferencehowever,wasnotsignificantintheMRPPaftercorrectingformul-
tiplecomparisons(Table2).
WeusedDESeq2tofinddifferentiallyabundantOTUsinthecontrolcomparedwiththe
mediumandhightreatments.ThesecomparisonsgavethehighestA-valuesintheMRPP
(Table2).TheDESeq2resultsincludingthenormalizedmeanabundance,abundancefold
change,p-andq-values,andthetaxonomicclassificationforallOTUsarelistedinS1Table
(comparingthecontrolandhightreatments)andS2Table(comparingthecontroland
mediumtreatments).Therewere37differentiallyabundantOTUsbetweenthecontroland
hightreatmentswithq-valuesbelow0.1.Thisq-valueindicatestheproportionoffalsepositives
iftheabundancesofalltheseOTUsareacceptedassignificantlydifferent.Thenormalized
meanabundance,abundancefoldchange,p-values,andthetaxonomicclassificationofthese
37OTUsareshowninTable3.
Fromthe37OTUs,14hadahigherrelativeabundanceinthehightreatmentthaninthe
control.HalfoftheseOTUswereclassifiedasAcidobacteriasubdivision4(Table3).These
sevenOTUscover18.9–22.8%oftheAcidobacteriasubdivision4sequencesinthehightreat-
mentand14.1–17.2%inthecontrols.MostoftheOTUsinTable3thathadalowerrelative
abundanceinthehightreatmentcomparedwiththecontrolwereclassifiedasActinobacteria.
SixoftheseActinobacteriaOTUsbelongtogenusGaiella,togethercovering13.0–14.5%and
15.0–18.7%ofthesequencesfromthisgenusinthehighandcontroltreatments,respectively.
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FlavonoidsandSoilBacterialCommunityStructure
Fig1.Non-metricmultidimensionalscaling(NMS)ordinationplots.(A)7,40-Dihydroxyflavoneexperiment,(B)naringeninexperiment.Pointsrepresent
samples,crossesaregroupcentroids.Samplesofthesametreatmentareenclosedinconvexhulls.Stressiscalculatedonascaleof0to100.(Treatments:
●high,♦medium,&low,and▲control).
doi:10.1371/journal.pone.0146555.g001
ThreeOTUswereclassifiedasNocardioidaceae,encompassing37.8–47.9%and46.3–59.5%of
thesequencesfromOTUsclassifiedintothisfamilyinthehighandcontroltreatments,respec-
tively.ThermomonosporaceaewasalsorepresentedbythreeOTUs,whichcontain69.3–77.6%
and72.4–75.9%ofthesequencesfromthisfamilyinthehighandcontroltreatments,
respectively.
Inacomparisonofthecontrolwiththemediumtreatment,onlysixdifferentiallyabundant
OTUshadq-valuesbelow0.1(S2Table).ThreeoftheseOTUs(OTU#886,10591,and12806)
werealsodifferentiallyabundantbetweenthecontrolandthehightreatment(Table3).Twoof
theotherthreeOTUswereclassifiedasMicromonosporaceaeandAcidobacteriasubdivision
16,andonecouldnotbeclassifiedatphylumlevelwithhigherthan70%bootstrapsupport.
SeveralOTUslistedinTable3thathadsignificantlydifferentabundancesbetweenthecontrol
andhightreatmentshowedasimilarresponseinthemediumtreatmentcomparedwiththe
control.FromtheAcidobacteriasubdivision4OTUslistedinTable3thatweresignificantly
moreabundantinthehightreatmentthaninthecontrol,OTU#10591,13198,and11314were
alsomoreabundantinthemediumtreatmentcomparedwiththecontrolandhadlowq-values
(0.049,0.115,and0.115,respectively)(S2Table).OTU#11842and14169wereclassifiedas
Gaiella,OTU#4243and9952wereclassifiedasNocardioidaceae,andOTUs11413and9555
wereclassifiedasThermomonosporaceae(Table3).Similartothecontrolversushightreatment
comparison,theseOTUshadhigherrelativeabundanceinthecontrolthaninthemedium
treatmentandhadq-valuesof0.176,0.209,0.115,0.163,0.171,and0.202,respectively(S2
Table).
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Table2. Multi-responsepermutationprocedure(MRPP)results.
Comparison A p Correctedp
7,40-dihydroxyflavone control—low -0.00039 0.4435 2.6608
control—medium 0.02922 0.0446 0.2674
control—high 0.02306 0.0333 0.1997
low—medium 0.00497 0.3274 1.9642
low—high -0.00054 0.4464 2.6781
medium—high -0.01322 0.9229 5.5374
Naringenin control—low 0.00323 0.3482 2.0890
control—medium -0.01677 0.7711 4.6265
control—high -0.02264 0.9584 5.7502
low—medium 0.00028 0.4185 2.5113
low—high 0.00403 0.3345 2.0068
medium—high -0.01294 0.7590 4.5542
TheA-valuesarethechance-correctedwithin-groupagreementsanddescribetheeffectsize.Correctedp-valueswerecalculatedusingtheBonferroni
method.
doi:10.1371/journal.pone.0146555.t002
16Ssequencingresultsfromthenaringeninexperiment
Thedatasetcontained149,471–339,727sequencespersamplewith253bpaveragereadlength.
TheMEDresultedin1589OTUs.Interestingly,thecontrolsamplesshowednoseparation
fromthehighormediumtreatmentsintheNMSplot,butthelowtreatmentappearedtosepa-
rate(Fig1B).Thisdifferencehowever,wasnotsignificantintheMRPP(Table2).Thecompar-
isonsbetweenthelowandhightreatmentsandbetweenthecontrolandlowtreatments
resultedinthelargesteffectsizesintheMRPP(Table2);thustheywereinvestigatedwith
DESeq2tofinddifferentiallyabundantOTUs.Thenormalizedmeanabundance,abundance
foldchange,p-andq-values,andthetaxonomicclassificationforallOTUsarelistedinS3
Table(comparingthecontrolandlowtreatments)andS4Table(comparingthelowandhigh
treatments).OnlythreeOTUshadsignificantlydifferentabundancesbetweenthecontroland
thelowtreatmentandhadq-valuesbelow0.1(S3Table).OneOTUwasclassifiedasBacillus,
oneasGaiella,andonewasnotpossibletoclassifyatthephylumlevelwithhigherthan70%
bootstrapsupport.ComparisonofthelowandthehightreatmentsresultedinfourOTUswith
significantlydifferentabundancesandwithq-valuesbelow0.1(S4Table).Thesewereclassified
intoOxalobacteraceae,Intrasporangiaceae,Acidobacteriasubdivision6,andChitinophagaceae.
Discussion
SoilATPcontentisameasureofthetotallivingmicrobialbiomassincludingactiveanddor-
0
mantorganisms[39].The7,4-dihydroxyflavoneandnaringenintreatmentshadnosignificant
effectontheATPcontentofthesoil.Thissuggeststhatthechangeinrelativeabundanceof
OTUsfoundinDESeq2isduetoagrowthresponseofthoseparticularbacterialgroupsandis
notduetoageneralantimicrobialorgrowthpromotingeffectontheothermembersofthe
bacterialcommunity.HoweverwenotethatsoilATPcontentreflectsthebiomassofallsoil
organisms,includingfungi,othereukaryoticmicrobes,andArchaea,whicharenotrepresented
inour16Ssequencingresults.
NaringeninhasbeenshowntoinhibitthegrowthofavarietyofGrampositiveandnegative
bacteriaaswellasSaccharomycescerevisiaewithminimuminhibitoryconcentrationsranging
PLOSONE|DOI:10.1371/journal.pone.0146555 January11,2016 8/16
FlavonoidsandSoilBacterialCommunityStructure
a s
Genus Nocardioides Pseudonocardi Janibacter Actinoallomuru Gaiella Gaiella Conexibacter Gaiella Gaiella Kribbella Gaiella Gaiella Adhaeribacter Flavisolibacter Onlytaxonomic econtrol.
h
0<qentsforthe7,4-dihydroxyflavoneexperiment(-values0.1). ClassisOrdoFamilia ActinobacteriaActinomycetalesNocardioidaceae ActinobacteriaActinomycetalesThermomonosporaceae ActinobacteriaActinomycetalesPseudonocardiaceae ActinobacteriaActinomycetalesIntrasporangiaceae ActinobacteriaActinomycetalesThermomonosporaceae ActinobacteriaGaiellalesGaiellaceae ActinobacteriaGaiellalesGaiellaceae ActinobacteriaSolirubrobacteralesConexibacteraceae BacilliBacillales ActinobacteriaActinomycetalesNocardioidaceae ActinobacteriaGaiellalesGaiellaceae ActinobacteriaSolirubrobacterales ActinobacteriaGaiellalesGaiellaceae ActinobacteriaActinomycetalesNocardioidaceae ActinobacteriaActinomycetalesThermomonosporaceae AlphaproteobacteriaRhizobiales ActinobacteriaGaiellalesGaiellaceae Subdivision17 AlphaproteobacteriaRhodospirillales ActinobacteriaGaiellalesGaiellaceae Subdivision4 Subdivision4 Subdivision4 CytophagiaCytophagalesCytophagaceae Subdivision4 Subdivision6 Subdivision4 Subdivision4 SphingobacteriiaSphingobacterialesChitinophagaceae SphingobacteriiaSphingobacterialesChitinophagaceae Subdivision4 fiTUclassiedaccordingtotheRibosomalDatabaseProjectrelease10. geofthenormalizedabundanceinthehightreatmentcomparedwitht
m O an
ntrolandhightreat Phylum Actinobacteria0 Actinobacteria5 Actinobacteria2 Actinobacteria4 Actinobacteria1 Actinobacteria1 Actinobacteria5 Actinobacteria8 Actinobacteria8 Actinobacteria3 Firmicutes2 Actinobacteria1 Actinobacteria4 Actinobacteria1 Actinobacteria6 Actinobacteria5 Actinobacteria3 Proteobacteria5 Actinobacteria5 Acidobacteria3 Proteobacteria1 Actinobacteria0 Acidobacteria6fi1unclassied Acidobacteria7 Acidobacteria9 Bacteroidetes7 Bacteroidetes5 Acidobacteria4 Acidobacteria2 Acidobacteria7fi4unclassied Acidobacteria2 Bacteroidetes5 Bacteroidetes3 Acidobacteria1 uencesfromeach angeisthefoldch
o 2 1 2 1 8 0 2 2 4 3 8 1 1 5 4 5 7 2 4 6 2 1 9 2 3 8 3 5 8 2 1 2 6 5 9 3 q h
c 0 1 1 3 3 5 5 5 7 2 2 4 4 6 9 0 0 5 6 6 7 8 0 2 6 2 3 5 5 6 7 8 0 4 6 0 e c
weenthe p 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.002 0.002 0.002 0.002 0.000 0.000 0.000 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.002 0.002 0.002 0.003 ntatives ed.Fold
bet ge 7 1 1 2 5 2 4 6 2 5 0 6 5 3 4 6 8 1 3 9 9 3 1 5 2 5 1 6 8 1 3 9 4 7 5 7 ese list
ntOTUs FoldChan 0.5 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.6 0.6 0.6 0.5 0.5 0.6 0.6 0.6 0.5 0.6 0.6 0.6 0.5 0.6 1.9 2.0 1.6 1.4 1.8 1.8 1.4 1.7 1.5 1.7 1.4 1.6 1.4 1.4 donrepr pportare
a e u
d s s
abun edce 2.3 7.7 8.4 9.0 7.8 6.3 9.8 1.8 1.7 1.0 9.9 1.2 0.5 3.2 3.5 4.3 9.6 3.7 7.4 4.3 6.2 6.2 7.6 9.0 2.8 7.0 8.5 4.7 2.3 3.4 2.7 0.5 0.7 7.4 1.3 3.9 asba strap
Table3.DESeq2results.Differentially OTU#MeanNormalizAbundan Moreabundantin424314thecontrol1280619treatment1141333 999338 85354 316015 955517 1184269 1365711 903015 412131 99524 57953 327316 598515 1011214 95574 74515 141699 547739 38196 141707 Moreabundantin131985thehightreatment29451 1131415 1354722 8862 123405 1059168 133585 1136414 503 13562127 111095 1232317 564920 ficationoftheOTUswTaxonomicclassi assignmentswithhigherthan70%boot doi:10.1371/journal.pone.0146555.t003
PLOSONE|DOI:10.1371/journal.pone.0146555 January11,2016 9/16
FlavonoidsandSoilBacterialCommunityStructure
from250to1000μg/ml[40–42].Abroad-spectruminhibitionofgrowthwouldlikelycausea
decreaseinsoilATPcontent,butthisdidnotoccur.Thehighflavonoidtreatmentsolutionin
ourexperimenthadaconcentrationof5.45μg/ml,whichisabouttwomagnitudeslowerthan
thereportedminimuminhibitoryconcentrationsfornaringenin.Itseemslikelythatsomeroot
exudateflavonoidsthathavebeenconsideredantimicrobialbasedonlaboratoryculturestudies
donotfunctionasinhibitorsofbacterialgrowthinthesoil.Thisissimplybecausethedeter-
minedminimuminhibitoryconcentrationsarehigherthanthoseexpectedintherhizosphere,
ortheyonlyhaveanantimicrobialeffectadjacenttothesiteofexudation,suchasintherhizo-
planewheretheirlocalconcentrationsmaybehigh.
ForsoilswithasimilartextureandpHtotheMaurysiltloamusedinthepresentstudy,Bai
etal.[25]reportedasoilATPcontentof0.76–2.20μg/g.Ourresultsusingthesameextraction
methodalsooccurredwithinthisrange(Table1).However,Baietal.didnotincubatetheir
soilswithcarbonsourcesbeforeextraction,whereasoursamplesweretreateddailyfor10days
withamixtureofcarbohydratesandorganicacids,whichshouldpromotethegrowthofhet-
erotrophicmicroorganisms,andthusincreasethemicrobialbiomassandATPcontent.In
theirreview,BlagodatskayaandKuzyakov[39]concludedthatanATPcontentbelow1–2μg/g
soiliscommonwhenthesoilmicrobialcommunityisnotactivated,andthattheadditionof
readilyavailablesubstrates,suchasglucose,causesseveral-foldincreases,whichimpliesthat
ourresultsarelowerthanexpected.AlikelyexplanationfortherelativelylowATPcontentof
oursamplesisthatthetreatmentsolutionaddedonly0.0040mgcarbonpergsoildailyasglu-
coseandothersubstrates,whichisaboutthreemagnitudeslessthantheamountusedinother
studiestoactivatethesoil[43,44].TheconcentrationsofcarbonsourcesandtheC:Nratiosin
ourtreatmentsolutionswereclosertothoseusedbyDrakeetal.[18]intheirC+Ntreatment
(carbonat500mg/lversus172.3mg/linthepresentstudy,C:Nof10versus13.1inthepresent
study),whichdidincreasesoilmicrobialbiomasssignificantly,butnotseveral-foldasreported
byBlagodatskayaandKuzyakov[39].
0
7,4-Dihydroxyflavoneisknownforitsabilitytoinducetheexpressionofnodgenesinspe-
cieslikeRhizobiumleguminosarumbv.trifolii[45],Ensifermeliloti[46],andBradyrhizobium
japonicum[47].TherearenumerousOTUsinourdatasetclassifiedasRhizobiumandBradyr-
hizobium(S1Table),andthesoiloriginatedfromasitewhereM.sativahadbeengrownfor
0
years.Therefore,itislikelythatrhizobialspeciesabletoreactto7,4-dihydroxyflavoneasanod
geneinducerwerepresentinthesoilmicrobialcommunity.However,weonlyfoundasingle
OTUclassifiedasRhizobialesthatsignificantlychanged(q-value<0.1)itsrelativeabundance
0
inthehigh7,4-dihydroxyflavonetreatmentcomparedwiththecontrol(Table3),andthe
abundanceofthisOTUdecreasedintheflavonoidtreatment.Thisimpliesthatnodgene
0
inductioninbacteriaby7,4-dihydroxyflavoneisnotnecessarilycoupledwithsignificant
growthpromotion.OurresultsconcurwithHartwigetal.[5],whofoundthat10μM7,40-dihy-
droxyflavonedidnotaffectthedoublingtimeintheculturesofR.leguminosarumbv.trifolii
andE.meliloti.
OurresultsshowthatalargeportionoftheAcidobacteriasubdivision4communityinthe
0
soilincreaseditsrelativeabundanceinthehigh7,4-dihydroxyflavoneflavonoidtreatment.
Basedonculture-independentstudies,Acidobacteriaisoneofthemostabundantbacterial
phylaintherhizosphere[48],andsubdivision4isamongthemostdominantofthe26subdivi-
sionsinawidediversityofsoils,especiallyinsoilswithlowacidity[49].However,thereare
onlythreevalidlydescribedspeciesinthistaxon[50–52],whichimpedesourunderstandingof
theirmetabolismandactivity.InclonelibrariesconstructedfromtherhizosphereofNoccaea
caerulescensandfromunplantedbulksoilthatwascontaminatedwithheavymetals,fewer
clonesfromsubdivision4werefoundintherhizospherecomparedwiththebulksoil[53].The
abundanceofvariousacidobacterialsubdivisionsinunplantedsoilandintherhizospheresof
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