Determination of Factors Associated with Natural Soil Suppressivity to Potato Common Scab

13 Pages·2015·0.23 MB·English

RESEARCHARTICLE
Determination of Factors Associated with
Natural Soil Suppressivity to Potato Common
Scab
MarketaSagova-Mareckova1*,OndrejDaniel1,MarekOmelka2,VaclavKristufek3,
JiriDivis4,JanKopecky1
1CropResearchInstitute,Dept.EpidemiologyandEcologyofMicroorganisms,Prague,CzechRepublic
2FacultyofMathematicsandPhysics,Dept.ProbabilityandMathematicalStatistics,CharlesUniversity,
Prague,CzechRepublic,3BiologyCentreoftheAcademyofSciencesoftheCzechRepublic,v.v.i.,
InstituteofSoilBiology,CeskeBudejovice,CzechRepublic,4FacultyofAgriculture,Dept.PlantProduction,
UniversityofSouthBohemia,CeskeBudejovice,CzechRepublic
* [email protected]
Abstract
OPENACCESS
Commonscabofpotatoesisadisease,whichisdifficulttomanageduetocomplexinterac-
Citation:Sagova-MareckovaM,DanielO,OmelkaM,
KristufekV,DivisJ,KopeckyJ(2015)Determination tionsofthepathogenicbacteria(Streptomycesspp.)withsoil,microbialcommunityandpo-
ofFactorsAssociatedwithNaturalSoilSuppressivity tatoplants.InBohemian-MoravianHighlandsintheCzechRepublictwosites(Vyklantice
toPotatoCommonScab.PLoSONE10(1):
andZdirec)wereselectedforastudyofcommonscabdiseasesuppressivity.Atbothsites,
e0116291.doi:10.1371/journal.pone.0116291
afieldwithlowdiseaseseverityoccursnexttoonewithhighseverityandthesituationwas
AcademicEditor:GabrieleBerg,GrazUniversityof
regularlyobservedoverfourdecadesalthoughallfourfieldsundergoacroprotation.Inthe
Technology(TUGraz),AUSTRIA
fourfields,quantitiesofbacteria,actinobacteriaandthegenetxtBfromthebiosynthetic
Received:August13,2014
geneclusterofthaxtomin,themainpathogenicityfactorofcommonscab,wereanalyzedby
Accepted:December6,2014 real-timePCR.Microbialcommunitystructurewascomparedbyterminalfragmentlength
Published:January22,2015 polymorphismanalysis.Soilandpotatoperidermwerecharacterizedbycontentsofcarbon,
nitrogen,phosporus,sulphur,calcium,magnesium,andiron.Qualityoforganicmatterwas
Copyright:©2015Sagova-Mareckovaetal.Thisis
anopenaccessarticledistributedunderthetermsof assessedbyhighperformanceliquidchromatographyofsoilextracts.Thestudydemon-
theCreativeCommonsAttributionLicense,whichper- stratedthatthesuppressivecharacterofthefieldsislocallyspecific.AtZdirec,thesuppres-
mitsunrestricteduse,distribution,andreproductionin sivitywasassociatedwithlowtxtBgenecopiesinbulksoil,whileatVyklanticesiteitwas
anymedium,providedtheoriginalauthorandsource
associatedwithlowtxtBgenecopiesinthetuberosphere.Thedifferenceswerediscussed
arecredited.
withrespecttotheeffectofabioticconditionsatZdirecandinteractionbetweenpotatoplant
DataAvailabilityStatement:Theauthorsconfirm
andsoilmicrobialcommunityatVyklantice.SoilpH,Casoilcontentorcationconcentra-
thatalldataunderlyingthefindingsarefullyavailable
withoutrestriction.Thesupportinginformationfilesin- tions,althoughdifferentwerenotintherangetopredictthediseaseseverity.Lowseverity
cludingresultsofallanalysesinreplicates,T-RFLP ofcommonscabwasassociatedwithlowcontentofsoilC,N,C/N,CaandFesuggesting
andHPLCprofilesareavailableunderDryadDigital
thatoligotrophicconditionsmaybefavorabletocommonscabsuppression.
Repositoryentrydoi:10.5061/dryad.332dj.
Funding:ThestudywassupportedbytheMinistryof
AgricultureoftheCzechRepublic,grantNo.
QJ1210359andprojectMZeRO0414(http://www.
mze.cz),andCzechScienceFoundation,grantNo.
P201/11/P290(http://www.gacr.cz).Thefundershad
noroleinstudydesign,datacollectionandanalysis,
decisiontopublish,orpreparationofthemanuscript.
PLOSONE|DOI:10.1371/journal.pone.0116291 January22,2015 1/13
NaturalSoilSuppressivitytoPotatoCommonScab
CompetingInterests:Theauthorshavedeclared Introduction
thatnocompetinginterestsexist.
Thecommonscabofpotatoesisanimportantsoil-borndiseasewithworldwideoccurrence.It
hasbeenratedamongthetopfivediseasesofpotatoesbyseedproducersintheUSA[1].The
diseaseaffectstuberqualityduetosuperficialandpittedlesionsthatformaroundthesiteofin-
fection[2].TheinfectioniscausedbyactinobacteriafromthegenusStreptomycesthatpossess
alarge(325–660kb)pathogenicityisland(PAI)intheirgenomes.Themostimportantpatho-
genicitydeterminantisaphytotoxinthaxtominsynthetizedfromtwoamino-acidprecursors
viaacondensationstepcatalyzedbynonribosomalpeptidesynthetasecodedbytxtABgenes.
Thesegenesareusedfordeterminationandquantificationofpathogensresponsibleforthedis-
ease[3,4,5].
Thedistribution,severityandincidenceofcommonscabhavebeenwidelystudiedinrela-
tionshiptophysico-chemicalandmicrobialsoilcharacteristicsbutthediseaseisstilldifficultto
control.TraditionalcontrolstrategiesofcommonscabsuchasirrigationandreducedsoilpH
arenotsufficientandoftenfailbecausetheinvolvedrelationshipsareverycomplex.Several
studiesindicatedthatcontrolmeasuresmaybesoilspecific[5],thoughitisunclearwhether
thisisduetophysicalsoilproperties,orsoilmicrobialflora[1].
Naturallyoccurringdisease-suppressivesoilsprovideavaluableresourceforimplementa-
tionofasustainablealternativetocurrentcontrolstrategies[6].Yetmoreinformationabout
thosesoilsfunctioningneedstobecollectedbecausedifferent,oftenlocallyspecific,mecha-
nismsleadtosoilsuppressiveness.Thoseincludesituations:(i)thepathogendoesnotestablish
orpersistatthesite,(ii)thepathogenestablishesbutcauseslittleornodamage,or(iii)the
pathogenestablishesandcausesdiseaseforawhilebutthereafterthediseaseceases,although
thepathogenmaypersistinthesoil[7].
Fieldssuppressivetocommonpotatoscabwereidentifiedatvariouslocations,forexample
incentralWashington,GrandRapidsandBecker,MN,EastLansing,MI[8].Inallofthose
sitesthesuppressivitywasattributedtobiologicalinteractionsbetweenantagonistic
microfloraandpathogensmediatedviaantibioticproductionorenzymaticactivity[9].That
typeofsuppressivitycanestablishaftermanyyearsofpotato(orothercrop)monoculturebe-
causenewprotectivebioticinteractionsarestimulatedovertimebycontinuousco-occurrence
ofpathogensandcompetingmicroorganisms.However,themechanismsofsuppressionvary
andparticularlyinsoilsundergoingregularcroprotationthesuppressivecharactermaybe
relatedtogeologicaloriginandsoilphysico-chemicalcharacteristics[9].
TheBohemian-MoravianHighlandsrepresentanimportantpotatogrowingareaincentral
Europe,whereparticularlyseedpotatoes,representingproductionofabout70thousandof
metrictonsareproducedanddistributedinEuropeandnearEast.Thecertificationforseed
potatoesrequireslessthan5%ofsurfaceirregularities.Soilconditionsintheareadonotbelong
tothosefavorabletocommonscabbecausetheyareusuallycharacterizedbypHbetween5–6
andrelativelyhighspringandsummerprecipitationwithannualaverageabout800mm.How-
ever,commonpotatoscabiswidelyspreadproducingsignificantdamagetopotatoesandcon-
sequentlycausingeconomiclosses.
Longhistoryofpotatogrowingintheareaenabledtoidentifyfieldswithlowincidenceof
commonpotatoscab.Inthisstudy,twositesVyklanticeandZdirecwhereafieldwithlowpo-
tatoscabseverityliesnexttoafieldwithhighdiseaseseveritywereselected.Thosetwositesun-
dergoaregularfourcroprotation,whilethedifferencesinseverityofthediseasebetweenthe
respectivefieldshavebeenconstantoverfourdecades.
Thegoalofthestudywastodetermineandprovethesuppressiveorconducivecharacterof
thetwonearbyfieldsateachsite.Theapproachwasbasedonafieldexperiment,inwhich
threepotatocultivarssusceptibletocommonscabweregrownatthetwosites(fourfields)ina
PLOSONE|DOI:10.1371/journal.pone.0116291 January22,2015 2/13
NaturalSoilSuppressivitytoPotatoCommonScab
Latinsquaredesignwithfourreplicates.Thethreecultivarswereselectedtoaccountforthe
variabilityofrelationshipsbetweenpotatoesandpathogens.Thesoilsuppressivityorcondu-
civityofthestudiedfieldsweredeterminedbyrelationshipsbetweencommonscabseverity,
quantityofthaxtominebiosyntheticgenetxtB,soilandperidermchemicalcharacteristics,
qualityoforganicmatterandmicrobialcommunity.Differencesbetweenthoserelationships
determinedinfieldswithlowandhighseverityofcommonscabwereusedtopredictmecha-
nismsinvolvedindiseasesuppressivityandtoidentifysoildescriptors,eitherabioticorbiotic,
associatedwithsoilhealthandsuitabilityforpotatogrowing.
Methods
EthicsStatement
Thefieldstudydidnotinvolvevertebrates,endangeredorprotectedspecies;itwasnotcarried
outinaprotectedarea.Theexperimentswerecarriedoutonprivatearablelandwiththeper-
missionoftheowners,thecompaniesVysocinaVyklanticea.s.(fieldsV ,V )andVesaCeska
L H
Belaa.s.(fieldsZ ,Z ).Nootherpermissionswererequired.
L H
Sites
VyklanticeandZdirecaresiteswheretwofields(V ,V ,andZ ,Z ,resp.)about100mdistant
L H L H
differincommonscabseverity(markedbyLandHforlowandhighscabseverityattwosites
Vyklantice,Zdirec(V,Z)byobservationover30years.Thetwositesdifferinenvironmental
factors,whilethetwofieldswithineachsitearesimilaringeologicalcontext,soiltype,climate
andmanagement(Table1).Allstudiedfieldswereregularlyplantedunderfour-fieldcroprota-
tionsystemincludingrape,clover,potatoes,andgrains(wheat,oats)inthepasttwodecades.
Fieldexperiment
Thefieldexperimentwasconductedin2010.PotatoeswereplantedonMay27andthe
samplesofbulksoil,tuberospheresoilandpotatoeswerecollectedafter80days.Threecultivars
susceptibletocommonscabAgria,DavidandValfiwereused.Potatoeswereallcertified
seedtubers(commonscabbelow5%surface).Fourreplicatesofeachcultivarateachfield
wereusedandarrangedinaLatinsquaredesign(3cultivars×4plotsplus4bulksoilsat
eachfieldandsite,16samplesperfield×4fields).Eachplotwasplantedwith3rowsof12
potatoplants(36plants)separatedby50cmofbaresoil.Theeffectofthesiteandfield
(selectivelyalsotheeffectofacultivar)oncommonscabseveritywasassessed.Atallfields,
soilchemicalcharacteristicsweredeterminedbytotalsoilC,N,S,P,Ca,Mg,Fecontent,potato
chemicalcharacteristicsweredeterminedbyN,P,Ca,Mg,Feperidermcontent.Microbial
Table1.Sitecharacteristics.
Vyklantice Zdirec
V V Z Z
L H L H
coordinates 49°33'48"N,15°03'31"E 49°33'43"N,15°03'18"E 49°37'37"N,15°37'56"E 49°37'48"N,15°37'58"E
altitude[MAMSL] 600 590 510 510
clay[%] 3 0 12 6
silt[%] 16 30 56 56
sand[%] 72 62 29 35
gravel[%] 9 8 3 3
CEC[cmol/kg] 13.3 16 15.4 20.1
doi:10.1371/journal.pone.0116291.t001
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NaturalSoilSuppressivitytoPotatoCommonScab
characteristicsweredescribedbyactinobacterialdiversity,quantityofbacteria,actinobacteria
andthaxtominbiosyntheticgenetxtBinbothsoilandpotatoperiderm.
Sampling
Onepotatoplantgrowinginthecenterofeachplotwassampled.Tuberospheresoilsamples
werecollectednofurtherthan3mmfromapotatotuber.Atuberwaslocatedbycarefuluncov-
eringthetopsoilsurroundingtheplant,slightlypressedtotheremainingsoilandtakenout.
Thesocketremaininginsoilafterpotatoextractionwascarefullyscratchedbyasharpspoonto
collectathinlayerofsoil.Soilwasalsocollectedfromthetuberitselfifanysoilremainedat-
tachedonthetuberinathinlayer.Bulksoilwascollectedatadistanceofcca30cmfromthe
closestplantwithineachplotusingasmallsterilespade.Todemonstratetheeffectofpotato
plantsonmicrobialcommunity,resultswerecalculatedeitherforallsoilsamples(identified
“soil”)orseparatelyforbulkortuberospheresoil.Potatoesfromthecentralplantwerecollect-
edandwashedindistilledwater.Allpotatoeswerecarefullypealedbyapotatopeelertaking
approximately1mmthickperidermsamples,thepeelswerehomogenizedandmixedandsub-
samplesweretakenforfurtheranalyses.Commonscabseveritywasevaluatedon20potatoes
perplotusinga9degreescale[10].Potatoesusedforevaluationwerethoseofthecollected
plantandseveralmoreplantsfromeachplottoachieveatleast20measurementsperplot.
Soilandpotatoperidermanalyses
TodeterminetotalsoilC,N,andScontent2-gramaliquotsofhomogenizedsoilsamplesfrom
bothbulkandtuberosphereweredried,milled,andanalyzedusingVarioMAXCNSanalyzer
(ElementarAnalysensysteme,Hanau,Germany).Todetermineallothersoilelementssoilsub-
sampleswereleachedwithboilingnitro-hydrochloricacid(aquaregia)andassessedbyoptical
emissionspectroscopywithinductivelycoupledplasma(ICP-OES)inAquatestcompany
(Prague,CzechRepublic).Theanalysesofpotatoperidermwereperformedbytheservicelabo-
ratoryoftheInstituteofBotany(Trebon,CzechRepublic).Fortotalnitrogenanalysis,1–3mg
driedperidermwasmineralizedbymodifiedKjeldahlmethodinH SO withcatalyzerat360°
2 4
C.Fortotalphosphorusanalysis,20mgdriedperidermwassequentiallydecomposedby
HNO andHClO [11].Inthemineralizedsamples,bothNandPweredeterminedbyflowin-
3 4
jectionanalysiswithspectrophotometricdetectionusingFIALachatQC8500analyzer(Lachat
Instruments,HachCompany,Loveland,CO).Cationcontentsinperidermweredetermined
byatomicabsorptionspectrometryusingAASspectrometerContrAA700(AnalytikJena,
Jena,Germany)aftermineralizationwithnitro-hydrochloricacid.
DNAextraction
Soilsamplesfromtuberosphereandbulksoilwerehomogenizedandsubsamplesof0.5gwere
usedforDNAextractionbymethodSKdescribedbySagova-Mareckovaetal.[12].Briefly,the
methodisbasedonbead-beatingandphenol/chloroformextractionfollowedbypurification
withCaCl andaGeneCleanTurbokit(MPBiomedicals,SantaAna,CA).ForDNAextraction
2
frompotatoperiderm,3gofperidermsampleswerefinecutinsterilePetridish,homogenized,
and0.3gsubsamplewasprocessedinthesamewaytoobtaintotalperidermDNA.
HPLCanalysisofextractablelow-molecular-weightsoilmetabolites
SoilsamplesforHPLCanalyseswerecollectedfromthetuberosphereofAgriacultivarand
bulksoilfromthesameplots(8samplesperfield,32intotal).Approximately50mlofsoil
wereextractedwithapproximately50mlofmethanol-water-aceticacid(80:19:1,vol/vol/vol)
PLOSONE|DOI:10.1371/journal.pone.0116291 January22,2015 4/13
NaturalSoilSuppressivitytoPotatoCommonScab
atroomtemperaturefor1to2days.Theextractswerefilteredandvacuumconcentratedtore-
moveorganicsolvent.TheresultingaqueoussuspensionwaschromatographedoverAmberlite
XAD1180(5gofaqueoussuspension;diameterofglasscolumn,2cm).Fiftymlofwater
(MilliQquality)wasusedaseffluentforthefirst(hydrophilic)fraction;50mlofabsolute
ethanolwasusedforthesecond(lipophilic)fraction.Theethanolicfractionwasevaporated
anddilutedinmethanolataconcentrationof5mgml-1.Theanalysiswasperformedusing
AcquityUPLCsystemwiththe2996PDAdetector(Waters,Milford,MA).Thecolumnwasa
PhenomenexLuna5mmC18(2)100Å,100×4.6mm,equippedwithSecurityGuardcartridge
C18,4×3.0mmID(Phenomenex,Torrance,CA).Theseparationwasruninair-conditioned
roomat20°C,andtheflowratewas1.0mlmin-1.SolventAwasultrapurewater;solventBwas
acetonitrile.Thegradientstartedwith10%ofsolventBfor1minandthenincreasedlinearlyto
100%ofBwithin8min.Thefinalconcentrationwasheldforafurther3.5min.Twentymlof
eachsamplewereinjected.Thespectrawererecordedfrom194to800nmin1nmintervals
anddatapointsrecordedoncepersecondwereexportedfromMassLynxsoftware(Waters)in
textformatforfurtherprocessing.
Terminalrestrictionfragmentlengthpolymorphismanalysis(T-RFLP)
SoilandperidermDNAsampleswereanalyzedbyamplificationofgenesfor16SrRNAusing
universalforwardprimer16Seu27f(5´-AGAGTTTGATCMTGGCKCAG-3´)[13]5-endla-
belledwith6-FAM(6-carboxyfluorescein)andActinobacteria-specificreverseprimer
16Sact1114r(5´-GAGTTGACCCCGGCRGT-3´)[14].Reactionmixturecontainedinatotal
volumeof50ml:1×polymerasebuffer,1.5mMMgCl ,400nMofeachprimer,0.2mMofeach
2
dNTP,0.6mg/mlBSA,1UTaqpolymerase(TopBio,Prague,CzechRepublic).PCRamplifica-
tionwasperformedinC1000ThermalCycler(Bio-Rad,Hercules,CA,USA)andthePCRpro-
gramconsistedofaninitialdenaturationat94°Cfor2min,followedby35cyclesof94ºCfor
45s,annealingat57ºCfor45s,extensionat72ºCfor1min30s,andafinalextensionstepat
72ºCfor5min.LabelledPCRproductswerepurifiedusingQIAquickPCRpurificationkit
(Qiagen,Hilden,Germany).LabelledPCRampliconsofthe16SrRNAgeneregionwere
cleavedbyAluIrestrictionendonucleasefor4hat37ºCwithsubsequentadditionsoftheen-
zymeintwoequalaliquotsatthebeginningandinthemiddle.Afterinactivationoftherestric-
tionenzymesandpurificationwithSigmaSpinPost-ReactionClean-UpColumns(Sigma-
Aldrich,St.Louis,MO),thesamplesweresubjectedtofragmentanalysisona96-capillaryse-
quencer(AppliedBiosystems,FosterCity,CA)inGenomaccompany(Prague,CzechRepub-
lic).T-RFLPanalyseswereperformedinonerunforallsamples.Crudeprofileswerefilteredto
thelargestT-RFpeaksrepresenting95%ofthetotalofpeakheights(areas)toeliminatethe
noiseintermittentlyexceedingthecut-offlimit.
QuantitativePCR
Quantificationswereperformedwithprimerseub338f(5’-ACTCCTACGGGAGGCAGCAG-3)
[15]andeub518r(5’-ATTACCGCGGCTGCTGG-3’)[16]amplifyinga197bpfragmentofthe
16SrRNAgenefromBacteria,act235f(5’-CGCGGCCTATCAGCTTGTTG-3’)[17]andeu518r
yielding280bpproductforActinobacteria,andStrepF(5’-GCAGGACGCTCACCAGGTAGT-
3’)andStrepR(5’-ACTTCGACACCGTTGTCCTCAA-3’)yielding72bpampliconofthethax-
tominbiosyntheticgenetxtB[18],respectively.TheanalysesweredoneonaStepOnePlusReal-
TimePCRSystem(AppliedBiosystems,FosterCity,CA)using96-wellplateswithGoTaqqPCR
MasterMix(Promega,Madison,WI)containingSYBRGreenasadouble-strandedDNAbind-
ingdye.Thereactionmixturecontainedinatotalvolumeof15ml:1×GoTaqqPCRMasterMix,
0.2mMprimers,and0.2–2ngdilutedDNAsample.ForallofthementionedtargetsthePCR
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NaturalSoilSuppressivitytoPotatoCommonScab
cyclingprotocolconsistedofinitialdenaturationat95°Cfor10min,followedby45cyclesof
95°Cfor15s,60°Cfor30sand72°Cfor30s.MeltingcurveswererecordedtoensureqPCRspeci-
ficity.BaselineandthresholdcalculationswereperformedwiththeStepOnev.2.2.2software.
TheinhibitionwastestedbyserialDNAdilutionfromeachsite.AllqPCRmeasurementswere
doneinduplicate.
TheqPCRstandardswerepreparedbycloningthefragmentsofthetargetgenesin
pGEM-TEasyvectorsystem(Promega).AfterPCRverificationandisolationofthecloned
constructsbyPureYieldPlasmidMiniprepSystem(Promega),alinearstandardwasprepared
bycleavingwithSalIenzyme(NewEnglandBiolabs,UK)ina200mlreactionmixturecontain-
ing1×reactionbuffer,2mgcircularplasmid,and20Urestrictionendonucleasefor2hin37°C.
ThelinearizedplasmidDNAwaspurifiedbyphenol-chloroformextraction.Thealiquotsof
linearizedandpurifiedstandarddilutedto20ng/mlwerestoredin-70°C.
Dataanalysis
ThedifferencesbetweensuppressiveandconducivefieldsweretestedbyANOVAandWelch’s
twosamplet-test(allowingdifferencesbetweenvariabilityofvariables),whichaimsatdetec-
tionofdifferencesinmeanvalues.Allvariableswerelog-transformedtomaketheirdistribu-
tionmoresimilartoanormaldistribution.TheHPLCabsorptionsignalsrecordedatthe
wavelength210nmwerebaselinecorrectedandnormalizedbydivisionthroughthemedianof
theabsorptionvalues.FortheT-RFLPandHPLCprofiles,theManhattanmetric(sumofabso-
lutedifferences)wasusedtocalculatethedistancematrices.Thedistancematriceswereplotted
bySammon’sMultidimensionalScaling[19].Twotestsbasedondistancematriceswasused.
WhilePERMANOVA[20]teststhatthewithingroupdistancesareonaverageshorterthan
thebetweengroupdistances(aimingatdetectionofdifferentmeanprofiles),dispersiontest
(denotedlateras‘disp’)introducedinGijbelsandOmelka[21]teststhattheaveragewithin
groupdistancesdifferamongthegroups(aimingatdetectingofdifferentdispersionsofthe
samples).Thecorrelationcoefficientsatdifferentfieldswerecomparedthroughthepermuta-
tiontestintroducedinOmelkaandPauly[22].AllstatisticalcalculationsweredoneintheR-
computingenvironment[23].
Results
Commonscab
Atbothsites,Vyklantice(V)andZdirec(Z),thetwofields(V ,V ,andZ ,Z )differedsignif-
L H L H
icantlyincommonscabseverity(V xV p<0.001,Z xZ p<0.001)(Table2).Thefields
L H L H
V andZ hadhighercommonscabseveritythantherespectivefieldsV andZ atthesame
H H L L
site(Table2).
Soilandperidermbiologicalcharacteristics
Inbulksoil,thefieldV hadsignificantlyhighertxtBgenecopiesthanV (p=0.002),whileZ
L H L
hadsignificantlylowertxtBgenecopiesthanZ (p=0.020)(Table2).Intuberospheresoil,the
H
fieldV hadsignificantlylowertxtBgenecopiesthanthefieldV (p=0.003),whileZ hadsig-
L H L
nificantlyhighertxtBgenecopiesthanZ (p=0.015).Inperiderm,thefieldV hadsignifi-
H L
cantlylowertxtBgenecopiesthanthefieldV (p<0.001),whileZ didnotdifferfromZ .
H L H
Also,thefieldV hadsignificantlyhigheractinobacteriainsoilthanthefieldV ,whilethe
H L
fieldZ hadsignificantlyhigheractinobacteriainperidermthanthefieldZ (Table2).
H L
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NaturalSoilSuppressivitytoPotatoCommonScab
Table2.Biologicalcharacteristics.
VL VH ZL ZH p-values
N mean S.E. mean S.E. mean S.E. mean S.E. overallANOVA VLvs.VH ZL.vs.ZH
Soil
bacteria 16 10.30 0.06 10.10 0.04 10.10 0.06 10.20 0.04 0.073 0.059 0.058
actinobacteria 16 9.57 0.05 9.53 0.03 9.57 0.03 9.75 0.04 0.001 0.534 0.001
txtB.total(bulkandtuberosphere) 16 4.38 0.07 4.49 0.12 4.93 0.19 5.57 0.14 <0.001 0.436 0.013
txtB.bulk 4 4.53 0.04 3.97 0.07 4.00 0.22 4.93 0.06 <0.001 0.002 0.02
txtB.tuberosphere 12 4.33 0.09 4.67 0.13 5.80 0.14 5.27 0.15 <0.001 0.044 0.015
Periderm
actinobacteria 12 7.82 0.12 8.74 0.17 9.40 0.13 9.27 0.11 <0.001 <0.001 0.45
txtB.periderm 12 6.10 0.05 7.18 0.16 7.31 0.15 7.51 0.12 <0.001 <0.001 0.312
Commonscabseverity 12 2.34 0.14 4.06 0.14 4.23 0.15 6.65 0.15 <0.001 <0.001 <0.001
Quantityofbacteria,actinobacteria(16SrRNAgene)andtxtBgeneinsoilandperiderm.Meansandrespectivestandarderrors(genecopiespergram
soil,log10transformed).Significantlyhighervaluesareinbold.
doi:10.1371/journal.pone.0116291.t002
Soilandperidermchemicalcharacteristics
ThefieldV hadsignificantlyhigherpHandcontentsofsoil(bulkandtuberosphere)C,N,P,
H
Ca,FeandperidermP,Ca,Fe,whileithadsignificantlylowercontentofsoilMgthanthe
fieldV .ThefieldZ hadsignificantlyhigherpHandcontentsofsoil(bulkandtuberosphere)
L H
C,N,S,Mg,Ca,FeandperidermCa,whileithadsignificantlylowersoilPthanthefieldZ
L
(Table3).
Actinobacterialcommunities
T-RFLPprofilesofactinobacteriainsoilsamplesdifferedsignificantlybetweensitesVyklantice
andZdirec(p-permanova<0.001,p-disp=0.003,notpresentedinafigure)andbetweenthe
Table3.Chemicalcharacteristicsinsoilandperiderm.
VL VH ZL ZH p-values
N mean S.E. mean S.E. mean S.E. mean S.E. overallANOVA VLvs.VH ZL.vs.ZH
Soil
pH 16 5.61 0.03 6.43 0.03 6.87 0.03 7.01 0.03 <0.001 <0.001 <0.001
C(%) 16 1.28 0.03 2.04 0.03 1.67 0.04 2.33 0.05 <0.001 <0.001 <0.001
N(%) 16 0.146 0.003 0.214 0.003 0.178 0.003 0.221 0.003 <0.001 <0.001 <0.001
S(mg.kg-1) 16 744.0 23.1 702.0 16.8 244.0 6.5 305.0 3.3 <0.001 0.151 <0.001
P(mg.kg-1) 16 921.0 13.8 1180.0 12.8 858.0 20.3 722.0 7.4 <0.001 <0.001 <0.001
Mg(g.kg-1) 16 12.40 0.22 11.80 0.18 3.78 0.18 4.56 0.06 <0.001 0.043 0.001
Ca(g.kg-1) 16 1.89 0.10 3.20 0.13 2.34 0.11 4.15 0.10 <0.001 <0.001 <0.001
Fe(g.kg-1) 16 43.00 0.63 46.80 0.87 22.50 0.89 27.10 0.38 <0.001 0.001 <0.001
Periderm
N(g.kg-1) 12 23.40 1.01 25.80 0.73 23.30 0.83 23.50 0.80 0.105 0.068 0.835
P(g.kg-1) 12 1.95 0.10 2.19 0.12 2.71 0.10 3.18 0.20 <0.001 0.136 0.054
Ca(mg.kg-1) 12 0.57 0.06 1.04 0.10 0.68 0.07 1.00 0.09 <0.001 0.001 0.008
Mg(mg.kg-1) 12 1.09 0.03 1.04 0.02 1.12 0.03 1.07 0.04 0.264 0.146 0.433
Fe(mg.kg-1) 12 0.37 0.03 0.51 0.05 0.70 0.09 0.71 0.15 0.007 0.035 0.966
Meansandrespectivestandarderrors.Significantlyhighervaluesareinbold.
doi:10.1371/journal.pone.0116291.t003
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NaturalSoilSuppressivitytoPotatoCommonScab
fieldswithineachsite(Fig.1;p-permanova<0.001bothsites,p-disp=0.042Zdirec).T-RFLP
profilesofactinobacteriainperidermsamplesdidnotdifferbetweensitesVyklanticeand
Zdirecbutdifferedsignificantlybetweenfields(Vyklantice:p-permanova=0.009,Zdirec:
p-permanova=0.019andp-disp=0.017).T-RFLPprofilesofperidermsampleswere
significantlydifferentbetweencultivarsatbothsites(Vyklantice:p-disp=0.005;Zdirec:
p-permanova=0.022;notpresentedinafigure).
Figure1.T-RFLPprofilesofthecommunitiesofactinobacteriasampledatVyklanticeandZdirecexperimentalsitesfromtuberospheresoil(A,B)
andpotatoperiderm(C,D).Sammon’smultidimensionalscalingwasbasedonManhattandistancematricescalculatedforthebaselinefilteredand
normalizedT-RFLPprofiles.Thesymbolsrepresenttheprofilesofactinobacteriaatthefieldswithlow(opensymbols)andhigh(closedsymbols)common
scabseverity.
doi:10.1371/journal.pone.0116291.g001
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NaturalSoilSuppressivitytoPotatoCommonScab
Low-molecular-weightcompounds
TheHPLCprofilesofextractablelow-molecular-weightcompoundsofsoilsampleswere
significantlydifferentbetweensites(p-permanova=0.009)andfieldsinVyklantice
(p-permanova=0.033)andZdirec(p-disp=0.049)(Fig.2).
Discussion
Commonscabseveritydifferedbetweenthetwofieldsateachsite,whilethepathogenidenti-
fiedbythethaxtominbiosyntheticgenetxtBwasalwayspresent.Therefore,weconsideredthe
fieldswithlowcommonscabseveritysuppressivetothedisease.Bothproposedsuppressive
fieldsrepresentthelong-term/naturaltypeofdiseasesuppressivityfunctioningoverlongtime
inspiteofregularcroprotation[24].
Thistypeofsuppressionislikelyafunctionofthebroadphysicalandchemicalcharacteris-
ticsofthesoilnotonlyofsoilmicrobialcommunities[25].Inourstudy,thefieldswithhigher
severityofcommonscabhadincommonsignificantlyhighercontentofsoilC,N,C/N,Ca,Fe
andhigherpH.HighsoilpHandCawereassociatedwithhighdiseaseseverityinotherstudies
[5,26,27]butitwasdemonstratedthateventhisrelationshipwassitespecific.Theobserved
thresholdofpHforlowdiseasesoilswasestablishedbyLaceyetal.[28]asalimittocommon
scabseveritybelow5.0–5.2butthesoilpHofallstudiedfieldswasabovethisvalue.Also,com-
monscabwasnotobservedonpotatoesgrowninsoilwithcombinedexchangeableCa,Mgand
Kat12cmol/kgorless[28]butinourfieldsonlytheconducivesoiloftheZ fieldhadcation
H
concentrationabovethisvalue.So,thelowersoilpHorcationconcentrationwerenotlikely
thecauseofsuppressivityinourfieldswithlowdiseaseseverity.
ThetwosuppressivefieldsatbothsiteshadlowerCainperiderm,thefieldinVyklantice
alsoperidermFe.ForaccumulationofCainplantcellsinducedbythaxtominitwasestablished
thatincreaseininfectedcellsisratheraneffectthanacauseofinfectionbecausethistoxin
Figure2.HPLC-UVprofilesofextractablelow-molecular-weightcompoundsatVyklantice(A)andZdirec(B)sites.Sammon’smultidimensional
scalingwasbasedonManhattandistancematricescalculatedfromnormalizedsignalat210nm(fordetails,seeMaterialsandMethods).Thesymbols
representtheprofilesofcompoundsextractedfromthefieldswithlow(opensymbols)andhigh(closedsymbols)commonscabseverity.
doi:10.1371/journal.pone.0116291.g002
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NaturalSoilSuppressivitytoPotatoCommonScab
inducedarapidCa2+influxandcelldeathinArabidopsisthalianacellsuspensions[29].Fe
ionsrepresentoneofredoxtransformationsbrokers[30].Increasedironmaybearesultof
crosstalkbetweenironandmanganese,ironandzinc,manganeseandphosphate,andcopper
andzinc,amongthemanydocumentedinteractions,butmaybealsorelatedtoreactiveoxygen
species(forironandmanganese)asaresultofpathogenstress[30].SinceCaiscloselyrelated
tosoilpHandFemayberatheraresultofinfection,justlowsoilC,N,andC/Nremainpoten-
tiallyassociatedwithsuppressivitytocommonscabatoursites.Thatresultwascomplemented
byspecificallyhighsoilMgandPcontentsinthesuppressivefieldsatVyklanticeandZdirec,
respectively.
Soilorganicmatter(SOM)canenhancedisease-suppressiveactivitiesofsoilmicrobialcom-
munities[31–33].Inourstudy,soilorganicmattercontentwashighinconducivesoilsbutthe
HPLCprofilesoflowmolecularweightcompoundsdifferedbetweenthefieldswithproposed
suppressivity.ThereforeitshowedthatqualityratherthanquantityofSOMwasrelatedtosup-
pressivityinourfields.
Soilactinobacterialcommunitiesweresignificantlydifferentbetweenthetwofieldswith
lowandhighcommonscabseverityatbothsites.Thatisconsistentwithpreviousfindingsat
similardiseasesuppressivesites,wheredifferencesbetweenmicrobialcommunitystructures
wereassignedtheprimarycauseofdiseasesuppressivity[6,8].TheT-RFLPmethodwhichwas
usedherecandemonstrategeneraldifferencesbetweenactinobacterialcommunitiessowepro-
posethatthedetermineddifferencesmustbeduetomanytaxaatvarioustaxonomiclevels.
Thesuppressivecharacterofsoilswasoftenassociatedwithmultiplespecies[6,8,34].Yet,
sincesoilmicrobialcommunitiesaresitespecific[35,36]itmaybedifficulttoshowthetaxa
particularlyresponsibleforsoilsuppressivity[8,34].
Peridermactinobacterialcommunitiesdifferedsignificantlybetweenthefieldsandpotato
cultivarsatbothsitesbutnotbetweenthetwosites.Thatshowedtheimportanceoftheeffect
ofcultivarsinspiteofthedifferencesinthesoilmicrobialcommunities,whichwasfoundpre-
viously[37].Itwasalsoproposedthatthecultivardistinctivemicrobialcommunitymaybere-
latedtocultivars’specifictraitsincludingresistancetopathogens[38,39].
Inourstudyitseemedthatthecharacterofsuppressivitydifferedbetweenthetwosites.
Firstly,thebulksoilatthesuppressivefieldinZdirec(Z )hadlowernumberoftxtBgenecop-
L
iesthantheconducivefield(Z ),whileinVyklanticethesuppressivefield(V )hadhigher
H L
numberoftxtBgenecopiesthantheconducivefield(V ).Secondly,thetuberospherewasthe
H
oppositebecausethesuppressivefieldinZdirec(Z )hadsignificantlyhighertxtBgenecopies
L
thantheconducivefield(Z ),whilethesuppressivefieldinVyklanticehadsignificantlylower
H
txtBgenecopiesthantheconducivefield(V ).ThereforeitseemedthatatZdirecthesoilcon-
H
ditionsdirectlysuppressedthepathogendevelopmentbecauseinthesuppressivefielditwas
lowinbulksoilbutincreasedinthetuberosphere.Thismaybepossiblyrelatedtoincreased
phosphorusconcentrationinthesuppressivesoiltherebecausePcontentwaspreviouslyoften
associatedwithlowdiseaseseverity[40,41].InthecontraryatVyklanticetheinteractionbe-
tweenthepotatoplantandmicrobialcommunitymayberesponsibleforthesuppressionbe-
causethepathogenpopulationsdecreaseinthetuberosphere.Differentmicrobialtaxawere
foundtoparticipateinsuppressionofdiseaseseitherdirectlyorindirectlybyenhancingplant
nutritionbyacquiringlimitingnutrientsorproducingbeneficialenzymes(e.g.[24,42])and
consequentlysupportingtheplantself-protectioncapabilities.Alsoactinobacteria,namely
nonpathogenicstreptomycetes,weresuggestedtoparticipateincommonscabsuppression
[43].
Inconclusion,wedemonstratedthatindeedthesuppressivecharacteroffieldsislocallyspe-
cific.InourstudyitseemedthatsoilpH,calciumcontentorcationconcentrationswerenot
predictingthediseaseseverity.Microbialcommunitieshadasitespecificcharacter
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