Combined Effects of Soil Biotic and Abiotic Factors, Influenced by Sewage Sludge Incorporation

17 Pages·2016·1.75 MB·English

RESEARCHARTICLE
Combined Effects of Soil Biotic and Abiotic
Factors, Influenced by Sewage Sludge
Incorporation, on the Incidence of Corn Stalk
Rot
RaquelGhini1*,NaraLúciaPerondiFortes2,JuanANavas-Cortés3,CarlosAlbertoSilva4,
WagnerBettiol1
1 EmbrapaEnvironment,Jaguariúna,SP,Brazil,2 TaubatéUniversity,Taubaté,SP,Brazil,3 Institutode
a11111 AgriculturaSostenible,ConsejoSuperiordeInvestigacionesCientíficas,Córdoba,Spain,4 Departmentof
SoilScience,FederalUniversityofLavras,Lavras,MG,Brazil
*[email protected](RG)
Abstract
OPENACCESS
Theobjectivesofthisstudyweretoevaluatethecombinedeffectsofsoilbioticandabiotic
Citation:GhiniR,FortesNLP,Navas-CortésJA, factorsontheincidenceofFusariumcornstalkrot,duringfourannualincorporationsoftwo
SilvaCA,BettiolW(2016)CombinedEffectsofSoil
typesofsewagesludgeintosoilina5-yearsfieldassayundertropicalconditionsandtopre-
BioticandAbioticFactors,InfluencedbySewage
SludgeIncorporation,ontheIncidenceofCornStalk dicttheeffectsofthesevariablesonthedisease.Foreachtypeofsewagesludge,thefol-
Rot.PLoSONE11(5):e0155536.doi:10.1371/ lowingtreatmentswereincluded:controlwithmineralfertilizationrecommendedforcorn;
journal.pone.0155536
controlwithoutfertilization;sewagesludgebasedonthenitrogenconcentrationthatpro-
Editor:VijaiGupta,NationalUniversityofIreland— videdthesameamountofnitrogenasinthemineralfertilizertreatment;andsewagesludge
Galway,IRELAND
thatprovidedtwo,fourandeighttimesthenitrogenconcentrationrecommendedforcorn.
Received:January30,2016 Increasingdosagesofbothtypesofsewagesludgeincorporatedintosoilresultedin
Accepted:April29,2016 increasedcornstalkrotincidence,beingnegativelycorrelatedwithcornyield.Aglobalanal-
ysishighlightedtheeffectoftheyearoftheexperiment,followedbythesewagesludgedos-
Published:May13,2016
ages.Thetypeofsewagesludgedidnotaffectthediseaseincidence.Amultiplelogistic
Copyright:©2016Ghinietal.Thisisanopen
modelusingastepwiseprocedurewasfittedbasedontheselectionofamodelthat
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits includedthethreeexplanatoryparametersfordiseaseincidence:electricalconductivity,
unrestricteduse,distribution,andreproductioninany magnesiumandFusariumpopulation.Intheselectedmodel,theprobabilityofhigherdis-
medium,providedtheoriginalauthorandsourceare
easeincidenceincreasedwithanincreaseofthesethreeexplanatoryparameters.When
credited.
theexplanatoryparameterswerecompared,electricalconductivitypresentedadominant
DataAvailabilityStatement:Allrelevantdataare
effectandwasthemainvariabletopredicttheprobabilitydistributioncurvesofFusarium
withinthepaperanditsSupportingInformationfiles.
cornstalkrot,aftersewagesludgeapplicationintothesoil.
Funding:Financialsupportforthisresearchwas
providedbyProject061102237-04“Impacto
ambientaldousoagrícoladolododeesgoto”from
EmpresaBrasileiradePesquisaAgropecuária
(Embrapa).R.Ghini,C.A.SilvaandW.Bettiolare
recipientsofresearchfellows304189/2013-8, Introduction
303899/2015-8and305818/2015-5,respectively,
from“ConselhoNacionaldeDesenvolvimento Organicamendmentshavebeenusedasastrategyforthemanagementofsoilbornepathogens
CientíficoeTecnológico”(CNPq). basedontheircapacitytoinducesuppressiveness[1,2].Suppressivesoilsaredefinedasthose
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SewageSludgeandIncidenceofCornStalkRot
CompetingInterests:Theauthorshavedeclared inwhichdiseasedevelopmentissuppressedbybioticand/orabioticfactorseventhoughaviru-
thatnocompetinginterestsexist. lentpathogenandsusceptiblehostarepresent[3].However,amendmentapplicationcanalso
increasediseaseincidence,inducingthesoiltobecomeconducivetodiseasedevelopment.
Bonanomietal.[4]foundthatorganicmatteramendmentsweresuppressivein45%ofthe
studiedcases,nosignificantchangesoccurredin35%,anddiseaseincidenceincreasedin20%
ofthecases.
Sewagesludgeisawidelyavailableorganicmatteramendmentthatisrichinmacro-and
micronutrients.Theagriculturalandforestrydisposalofsewagesludgecanmodifysoilbiotic
andabioticcharacteristics,forexample,thedynamicsofmicrobialactivity,organicmatter
decomposition,nutrientcyclingandpestanddiseaseseverity[5–8].However,studiesonsew-
agesludgearemainlyconductedtoassessthenutritionaleffectsandissuesrelatedtoheavy
metalaccumulationinsoils.Thelackofknowledgeonthepossibleeffectsofsewagesludgeon
plantpathogensisoneoftheproblemsrelatedtoitsuseinagriculture.Soilincorporationof
sewagesludgereducedtheincidenceorseverityofSclerotinialettucedropcausedbySclerotinia
minor[9];rotandbeanseedlingdamping-offcausedbySclerotiumrolfsii[5,7];rootroton
sugarcanerootscausedbyPythiumarrhenomanes[10];rootrotonpepperscausedbyPhy-
tophthoracapsici[11];citrusdamping-offcausedbyPhytophthoranicotianae[12]anddamp-
ing-offofpeasandcottoncausedbyRhizoctoniasolaniandPythiumultimum[13].An
inducedsuppressivenesswasalsoobservedforFusariumwiltoncucumber,causedbyFusar-
iumoxysporumf.sp.cucumerinum[14];onchrysanthemum,causedbyFusariumoxysporum
f.sp.chrysanthemi[15]andontomato,causedbyFusariumoxysporumf.sp.lycopersici[16].
However,Chellemietal.[17]andGhinietal.[7]observednoeffectofsewagesludgeonsoil
suppressivenesstobacterialwiltandFusariumoxysporumf.sp.lycopersiciontomatoplants,
respectively.Therearereportsofincreaseddiseasesbecauseofsewagesludgeincorporation,
forexample,Gibberellarotoncorn[18],androotrotofbeans,peasandcottoncausedby
PythiumultimumandThielaviopsisbasicola[19].Theorganicamendmentsincorporatedto
thesoilcanbesuppressiveorconducivetoplantpathogensbyaffectingsoilbiological,chemi-
calandphysicalcharacteristics.Themechanismsaredirectlyduetothepresenceoftoxiccom-
pounds(volatilefattyacid,nitrousacid,andammonia),andindirectlybythestimulationof
soilmicroorganisms[10,20–23].Thefactorsthatdeterminetheeffectsofsoilincorporationof
sewagesludgeonsoilsuppressivenesshavetobeidentifiedtoallowthesafeuseofthiswastein
agriculture.
Ingeneral,thesestudiesmentionedabovewereconductedundertemperateclimatecondi-
tionsandperformedovershort-termperiods.However,aninterdisciplinaryandlong-term
fieldstudywasconductedundertropicalconditionstoevaluatetheenvironmentalimpactsof
twotypesandfivedosagesofsewagesludge[24].Thesoilchemical,physicalandbiological
properties,andcorngrowth,pests,diseasesandyieldwereevaluated.Duringthesecondand
thirdyearofthisresearch,Bettiol[25]observedthattheincidenceofcornstalkrotcausedby
Fusariumspp.waspositivelycorrelatedwiththedosageofsewagesludgeincorporatedintothe
soil.
CornstalkrotiscausedbyFusariumspp.,mainlyF.verticillioides,F.proliferatum,F.subglu-
tinans,F.graminearumandF.temperatum[26–28],whichaffectroots,theplantbase,inter-
nodes,kernelsandears.Thediseasecausesreducedplantgrowthandrottenstalksandleaf
sheaths.Inmatureplants,itcausesawhitish-pinktosalmondiscolorationoftheinternalstalk
pithtissues.Thepathogensurvivesincropresiduesonthesoilandcanbetransmittedbyseeds
[29,30].Highsoilmoistureandtemperaturesofapproximately28–30°Carefavorablecondi-
tionsforthedisease[28].
Theobjectivesofthisstudyweretoevaluatethecombinedeffectsofsoilbiotic(fungal,bac-
terialandFusariumspp.populations)andabiotic[pH,organicmatter(OM),P,K,Ca,Mg,H
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+Al,sumofbases(SB),cationexchangecapacity(CEC),basesaturation(V),electricalconduc-
tivity(EC),N-NH +andN-NO -]factorsontheincidenceofcornstalkrotduringfourannual
4 3
incorporationsofsewagesludgeintosoilina5-yearsfieldassayandtopredicttheeffectsof
thesevariablesoncornstalkrot.
MaterialsandMethods
Fieldexperiment
A5-yearsfieldexperimentwascarriedoutatEmbrapaEnvironment,locatedinJaguariúna,
SãoPauloState,Brazil(latitude22°41’S,longitude47°W,altitudeof570ma.s.l.).Theclimate
ishumidsubtropical(CfaaccordingtotheKöppenclassification)withhotrainysummers
(meanairtemperature=22.6°C,meanprecipitation=663mm)andcolddrywinters(mean
airtemperature=16.9°C,meanprecipitation=98mm)[31].Thesoilattheexperimentalarea
wasadarkreddistroferriclatosol(clayeytexture)whichphysicalandchemicalcharacteristics
inthe0–20cmlayer,beforetheonsetofthestudyfollows:pHinwater=5.8;OM=25.5g/kg;
P=3.5mg/cm3;K=1.51,Ca=27.5,Mg=8.5,Al=1,H=35,andCEC=73.5mmol /dm3;
c
SB=50.8%,andclay=450g/kg[24].
SewagesludgeswereobtainedfromwastewatertreatmentplantslocatedinBarueri,São
PauloState,whichtreatsdomesticandindustrialsewage,andinFranca,SãoPauloState,which
treatsonlydomesticsewage.Bothplantsusetheactivatedsludgeprocess.Onaverage,thechar-
acteristicsofBaruerisewagesludgewereC=340.1,N=48.6,P=1.3,Ca=30.1,Mg=3.7,
S=13.5g/kg;Cu=921.5,Zn=3037.2,Fe=39165.8,Mn=341.6,B=21mg/kg;pH=7.1;
humidity=75.2%andvolatilesolids=55.5%;andFrancasewagesludgewereC=381.8,
N=59.9,P=1.1,Ca=19.1,Mg=2.9,S=12.9g/kg;Cu=244.6,Zn=1136.3,Fe=38892.8,
Mn=400.3,B=18.3mg/kg;pH=7.2;humidity=80.2%andvolatilesolids=64.7%.Bettiol
andGhini[24]describedthecharacteristicsofthesetwotypesofsewagesludgeandthe
amountsofsludgeandfertilizersappliedannuallyineachtreatment.
Foreachsewagesludge,Franca(F)andBarueri(B),thefollowingtreatmentswereused:
controlwithmineralfertilization(NPK)recommendedforcorn(N=90kg/ha,P O =90kg/
2 5
haandK O=70kg/ha);controlwithoutfertilization(0N);applieddosageofsewagesludge
2
basedontheNconcentrationthatprovidesthesameamountofNasthemineralfertilizer
treatment(1N);andtwo(2N),four(4N)andeight(8N)timestherecommendedNdosagefor
thecorncrop.ThecalculationofthesewagesludgerateswasperformedasafunctionoftheN
availableforplantsconsideringtheNmineralizationratewas30%.Baruerisludgeamounts
appliedtoobtain1Ntreatmentwas8095,3995,5315,5295and3200kg/hadrymatter,and
Francasludge3014,3504,3766,4432and4300kg/hadrymatter,forthefirst,second,third,
fourthandfifthyearsofthesewagesludgeapplication,respectively.SupplementaryKwas
appliedinthesewagesludgetreatmentswhennecessary[24].Thesewagesludgewasincorpo-
ratedannuallyoverfiveconsecutiveyears.Itwastossdistributedoverthetotalareaoftheplots
andincorporatedtoadepthof20cmusingarotaryharrow,3or4daysbeforesowing.
Afterthesewagesludgeapplication,thecornvarietyCATIAL30(minicrop)wascultivated
inthefirstyear;hybridAG1043inthesecondyear;andinthethird,fourthandfifthyears
hybridSavanna133Swassown(6–7seedspermeterwithinarow).Inthethirdcultivation,the
pHofeachplotwascorrectedtopH5.7,withincorporationofdolomiticlime,onemonth
beforethesludgeapplication,basedonasoil-neutralizingcurve.
Theagriculturalpracticesadoptedwerethosetraditionallyusedlocallyforthecropwithout
irrigation.Thestubblewasremovedfromtheplotsbeforesludgeapplication.
Theexperimentwassetupasarepeatedmeasuresdesignwithyearofapplicationas
betweensubjectfactor,sewagesludgeanddoseofapplicationaswithin-subjectfactorsand
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threereplicationsassubjectfactor.Eachplotmeasured10x20m,with12rowsperplot.
Hedgerowsseparatedplotswithatleast5moneachside.Inthisstudy,assessmentswereper-
formedinthesecond,third,fourthandfifthyearsofthesewagesludgeapplicationandare
referredasyears1,2,3and4,respectively.
Soilchemicalanalyses
Soilsamples(0to20cm)werecollected90daysafterthesewagesludgeapplication.Atleast
fivecompositedsoilsampleswerecollectedperplotforchemicalandbiologicalanalyses.The
soilsampleswereanalyzedtodeterminepHinwater,OM,P,K,Ca,Mg,H+Al,SB,CEC,V
andEC[32,33].Inyears3and4,N-NH +andN-NO -werealsoanalyzed[34].
4 3
Soilmicrobialpopulations
Thecultivablemicrobialpopulationsinthesoilweredeterminedusingtheserial-dilution
methodfollowedbyplatingonaselectiveculturemedium.Thornton[35]andNashand
Snyder[36]mediawereusedtoestimatethebacterialandFusariumspp.populations,
respectively.Inyears3and4,Martinmedia[37]wasusedtoestimatefungalpopulation.
Aliquots(0.1mL)ofthedilutionsofeachsoilsampleweretransferredtoeachculture
mediuminPetridishes;threereplicateswereused.Theplateswereincubatedat28°Cinthe
darkfor3to6days.Assessmentswereperformedbycountingthenumberofcoloniesper
Petridish,expressedascolony-formingunits/gofdrysoil(CFU/gdrysoil).Microscopic
examinationwasusedtoidentifyFusariumspp.basedonthemorphologyoftheirspore
structures.
Diseaseincidence
Theincidenceofdiseasedplantswasevaluatedapproximately90daysaftersowing.Specifi-
cally,thenumberofplantsshowingsymptomsofcornstalkrotcausedbyFusariumspp.inthe
centralrowoftheplotswerecounted.
Inyears3and4,inadditiontotheevaluationofthesymptoms,pathogenisolationwas
performedtoestimatetheincidenceofFusariumcolonization.Segmentsofrootsandstalks
(3to5mm;25segmentsperplant;6plantsperplot)wererinsedwithtapwaterfor30
min.,immersedinsodiumhypochlorite1%(v/v)for1min.,andthenrinsedinsteriledis-
tilledwater.ThedisinfestedplantsegmentsweretransferredtoPDAsupplementedwith
oxytetracyclineandincubatedat25±2°Cundera12-hphotoperiod.Thepercentageof
Fusarium-colonizedsegmentswasassessedusingmicroscopicinspection.Inthesame
plants,thepercentageofearswithFusariumrotsymptomswasalsoevaluated.Isolations
fromsymptomaticrootsandstalksconsistentlyyieldedFusariumsp.identifiedbymorpho-
logicalcharacteristics.Aselectionofeightisolatesbasedonsimilarityoftheisolatesincol-
onymorphologyandmorphometricfungalstructureswereidentifiedbymolecular
methods.Thetranslationelongationfactor-1α(TEF-1α)genewasamplifiedusingprimers
EF1andEF2[38]andthefragmentsobtainedweresubsequentlysequencedinbothdirec-
tionsfollowingproceduresdescribedinJiménez-Fernándezetal.[39].Sequenceswere
depositedintheNCBISequenceDatabase(AccessionNo.KU372137toKU372144).A
BLASTsearchwasdoneagainstsequencesintheFUSARIUM-IDv.1.0database(http://
fusarium.cbio.psu.edu)[40]andtheGenBankdatabasethatrevealed100%sequenceiden-
titytoF.verticillioides.
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Yield
Cornyieldwasevaluatedbyharvestingtheearsfromthesixcentralrowsofeachplot.Thehar-
vestedearsweredriedunderambientconditions,thegrainswereweighed,andyieldwas
expressedaskg/ha.
Dataanalyses
Firstly,theoveralleffectsofexperimentaltreatmentcombinationsontheincidenceofcorn
stalkdiseaseandcornyieldaswellasonsoilbiotic(bacterialandFusariumspp.populations)
andabiotic[pH,OM,P,K,Ca,Mg,H+Al,SB,CEC,VandEC]parameterswasexplored
usinganalysisofvariance(ANOVA)todeterminetheextentofexplainedvariabilitydueto
mainfactorsinthestudy(i.e.,sewagesludgeoriginanddose)andhowtheseeffectsarecon-
sistentthoroughthe5-yearsofthestudyusingtheGenerallinealmodelprocedureofSAS
software(version9.3;SASInstitute,Cary,NC,USA).Lateronclusteranalyseswereusedto
establishfunctionalgroupsofcorrelatedexperimentaltreatments.Forthat,theagglomera-
tiveclusteringbasedontheSpearmancorrelationmatrixwasperformedusingtheWard
clusteringmethod[41].Aheatmapwasdevelopedtovisualizethevaluesofthedifferent
treatmentsandparametersusedintheanalysis.Allclusteranalysiscalculationswereper-
formedusingtheRsoftware,version3.0.2(RFoundationforStatisticalComputing,http://
www.R-project.org/)withthecluster[42],gplots[43]andvegan[44]packages.
Therelationshipbetweentheclassesofcornstalkdiseaseseverityandthestress-related
parameterswasestimatedusinglogisticregressionanalysis.Logisticmodelsaredirectprobabil-
itymodelsthatarestatedintermsoftheprobabilityoftheoccurrenceofanevent(i.e.,disease
incidenceclass)underagivensetofconditions(i.e.,bioticorabioticparameters)[45].Inthis
study,amultinomiallogisticregressionmodelwasfittedtoeachbioticandabioticparameter
asexplanatoryvariables,anddiseaseincidenceclasswasusedastheresponsevariable,using
thelowestdiseaseincidenceclassasthereferencecategory[46].Threediseaseincidence
homogenousgroupswithlow,moderateandhighdiseaseincidenceweredeterminedusingthe
FASTCLUSprocedureofSASusingthenearestcentroidsortingalgorithminwhichobserva-
tionswereclassifiedaccordingtotheirdiseaseincidencevalues.Toassessthestatisticalsignifi-
canceofeachexplanatoryvariable,eachmodelwascomparedwiththenullmodelusinga
likelihoodratiotest.Theproportionofthevarianceexplainedbyeachmodelwasevaluated
usingthemaximumrescaledR2coefficientofdeterminationandtheclassificationaccuracy.
ThiswasperformedusingtheLOGISTICprocedureofSASsoftware.Toassessthecombined
effectsofallbioticandabioticfactors,amultiplelogisticregressionmodelwasfittedusingthe
stepwiseprocedurethatallowedselectingonlythebioticorabioticfactorswithasignificant
contributiontothemodel.
Results
Overall,bioticparametersweresignificantlyinfluenced(P<0.05)byyearoftheexperiment,
sourceanddosageofsewagesludge,andtheirinteractions(S1Table,Fig1).Stalkrotincidence
tendedtoincreaseovertime,toreachamaximum31.42%intheyear3,beingnegativelycorre-
latedwithcornyieldthatshowedmaximumvaluesof5943.7kg/hainyear2(Fig1A–1D),as
wellaswithFusariumandbacterialpopulationsthatshowedtheirmaximumvaluesof118.67x
104and96.43x104CFU/g,respectively,inyear2(Fig1G–1J).Typeofsewagesludgedidnot
influencedsignificantly(P(cid:1)0.05)bioticparametersexceptforcornyieldandbacterialpopulation
thatshowedhighervalues(P<0.05)inFrancaandBarueri,respectively(S1Table,Fig1).Increas-
ingthedosageofsewagesludgethatwereincorporatedintothesoilresultedinanincreasein
cornstalkrot,andinFusariumandbacterialpopulationsthatweresignificantlyhigher(P<0.05)
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SewageSludgeandIncidenceofCornStalkRot
Fig1.IncidenceofFusariumcornstalkrot,yield,organicmattercontent,Fusariumandbacterial
populationsinsoilamendedwithtwotypesofsewagesludge.(A,B)Cornstalkrotincidence,(C,D)corn
yield,(E,F)organicmattercontent(OM),(G,H)Fusariumand(I,J)bacterialpopulationsinsoiltreatedwith
sewagesludgefromFranca(A,C,E,G,I)andBarueri(B,D,F,H,J)atdifferentdosages[0N,1N,2N,4Nand
8N,basedontheNconcentrationthatprovidedthesameamountofNasthemineralfertilizer(NPK)
recommendedforcorn]forfouryears(1,2,3and4).ErrorbarsrepresenttheSDofthreereplicates.
doi:10.1371/journal.pone.0155536.g001
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SewageSludgeandIncidenceofCornStalkRot
intreatedplotscomparedtothateithernontreatedorthosewithmineralfertilization(S1Table,
Fig1).Thiseffectwastrueforbothsewagesludgetypesexceptforcornstalkrotincidencethat
showedhighervalues(P<0.05)forBarueriatthelowerdosagesof1Nand2N,butsignificantly
higher(P<0.05)forFrancawiththehighestdosage8N(S1Table,Fig1).Inanycase,highsludge
dosagesdidnotresultinincreasedcornyield,mainlybecauseofincreaseddiseaseincidenceand
nutritionalimbalanceresultingfromcumulativeapplications(Figs1–3).
Abioticparametersshowedclearlydistinctpatternsovertime(Figs1E,1F,2and3).A
decreaseduringthefouryearsofexperimentswasobservedforOM,KandH+Al,butthe
oppositeoccurredforP.Theremainingparametersreachedtheirmaximumvaluesonyear3
todecreasetominimumvaluesonyear1(Ca,Mg,SBandV),year2(EC)oryear4(pHand
CEC)(S2andS3Tables,Figs2and3).Allabioticparametersinthestudyweresignificantly
influenced(P<0.05)bysewagesludgesourceanddosage,exceptforOM,ECandMgthat
showednosignificantdifferences(P(cid:1)0.05)duetoeithersewagesludgetypesordosage(Mg).
Moreoverasignificantinteraction(P<0.05)betweenbothexperimentalfactorswasdetected
forEC,H+Al,PandCa(S2andS3Tables,Figs1–3).Sewagesludgeorigindidnotdetermine
significantdifferences(P(cid:1)0.05)forOM,MgandEC,butsignificantlyhigher(P<0.05)values
forP,Ca,CEC,pH,SB,andVwerereachedwhenBaruerisewagesludgewasappliedcompared
toFranca,andtheoppositeoccurredforKandH+Al(S2Table,Figs1–3).Dosageofthesew-
agesludgeamendmentalsohadaneffectontheestimatedabioticparameters.Thus,forboth
typesofsewagesludge,OM,CECandSB,increasedwiththeincreaseindosageoftheamend-
ment,butK,pHandVshowedanegativetrendwiththeincreaseinthedosage(Figs1–3).Due
tothesignificantsewagesludgexdosageinteraction(P<0.05),P,Ca,ECandH+Alshowed
distinctdosagepatternsforeachsewagesludgetype(S2andS3Tables,Figs1–3).
TobetterdecipheringthecomplexinteractionsbetweenFusariumstalkrotandcornyield
withthe13soilparameters[11abiotic(pH,OM,P,K,Ca,Mg,H+Al,SB,CEC,VandEC)and
2bioticparameters(bacterialandFusariumpopulations)],amultivariatehierarchicalcluster
analysiswasperformedandthevariableswereorderedbydiseaseincidence(Fig4).Thisglobal
analysisconfirmedresultsofANOVAanalyseshighlightingacleareffectoftheyearofthe
experiment,followedbythesewagesludgetreatments,i.e.,sludgedosages(Fig4).Overall,high
valuesofdiseaseincidencewererelatedtoEC,V,CEC,SB,Mg,CaandtheFusariumpopula-
tion.LowdiseaseincidencewasrelatedtohighvaluesofthebacterialpopulationandH+Al
(Fig4).Separateanalysesoftheresultsforeachyearoftheexperimentindicatedthatsludge
dosagesof8Nand4Nforbothtypesofsludgeresultedinahigherincidenceofdiseasedplants
(S1Fig).
Inyears3and4,inadditiontotheincreaseddiseaseincidence,highersludgedosages
resultedinahigherfrequencyofpathogenisolationfromrottenstalksandsymptomaticears.
ThefungalpopulationandtheN-NH +andN-NO -contentswerealsorelatedtohighsewage
4 3
sludgedosagesanddiseaseincidence.Thecumulativeeffectofthesewagesludgeapplications
wasparticularlyevidentinyears3and4whenalmostallanalyzedvariableshadthelowestval-
uesinresponsetothe0NdosageofeithertheBarueriortheFrancasludge(S1Fig).
Threedistinctgroupsofdiseaseincidencewereobserved,rangingfromlowdiseaseinci-
dence(0to9%,class1)tomoderate(10to27%,class2)andhighincidence(28to50%,class
3).Adirectrelationshipwasobservedbetweenthediseaseincidencegroupandthesoilparam-
etersP,Ca,Mg,SB,CEC,V,EC,N-NH +,N-NO -,aswellaswithFusariumstalkrotand
4 3
symptomaticears(S2Fig).
Todeterminetherelationshipbetweenthediseaseincidencegroupsofthefouryearsof
evaluationandsoilabioticandbioticparameters,amultinomiallogisticregressionanalysiswas
performed.Logisticregressionmodelsfittedforeachvariableindividuallyexhibitedsignificant
differencesbetweenthediseaseincidenceclassesinallabioticandbioticparameters(pH,P,
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Fig2.Chemicalattributes(P,K,Ca,MgandCEC)ofsoilamendedwithtwotypesofsewagesludge.
(A,B)Phosphorus,(C,D)potassium,(E,F)calcium,(G,H)magnesiumcontentsand(I,J)cationexchange
capacity(CEC)ofsoiltreatedwithsewagesludgefromFranca(A,C,E,G,I)andBarueri(B,D,F,H,J)at
differentdosages[0N,1N,2N,4Nand8N,basedontheNconcentrationthatprovidedthesameamountofN
asthemineralfertilizer(NPK)recommendedforcorn]forfouryears(1,2,3and4).Errorbarsrepresentthe
SDofthreereplicates.
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SewageSludgeandIncidenceofCornStalkRot
Fig3.Chemicalattributes(pH,EC,H+Al,SB,V)ofsoilamendedwithtwotypesofsewagesludge.(A,
B)pH,(C,D)electricalconductivity(EC),(E,F)hydrogenandaluminumcontent(H+Al),(G,H)sumofbases
(SB)and(I,J)basesaturation(V)ofsoiltreatedwithsewagesludgefromFranca(A,C,E,G,I)andBarueri
(B,D,F,H,J)atdifferentdosages[0N,1N,2N,4Nand8N,basedontheNconcentrationthatprovidedthe
sameamountofNasthemineralfertilizer(NPK)recommendedforcorn]forfouryears(1,2,3and4).Error
barsrepresenttheSDofthreereplicates.
doi:10.1371/journal.pone.0155536.g003
PLOSONE|DOI:10.1371/journal.pone.0155536 May13,2016 9/17
SewageSludgeandIncidenceofCornStalkRot
Fig4.Dendrogramshowingresultsofclusteranalysesandheatmaprepresentationofdisease,yield,
andsoilabioticandbioticparametersamendedwithtwotypesofsewagesludge.Dendrogram
showingtheresultsoftheclusteranalyses(redclustersrepresenthighdiseaseincidence,orangeclusters
representintermediateincidenceandgreenclustersrepresentlowdiseaseincidence)andaheatmap
representationofcornstalkrotincidenceandyieldandtheirrelationshipwithsoilabiotic(pH,OM,P,K,Ca,
Mg,H+Al,SB,CEC,V,EC)andbioticparameters(bacterialandFusariumpopulations)ofsoiltreatedwith
sewagesludgefromFranca(F)andBarueri(B)atdifferentdosages[0N,1N,2N,4Nand8N,basedontheN
concentrationthatprovidedthesameamountofNasthemineralfertilizer(NPK)recommendedforcorn]for
years1(purple),2(orange),3(brown)and4(black).Theblueintensityrepresentsthemeanvalueofthe
variable(highervaluesarerepresentedbydarkerblueshades).
doi:10.1371/journal.pone.0155536.g004
Ca,Mg,H+Al,SB,CEC,V,EC,bacteriaandFusariumpopulations),exceptOMandK.EC,
MgandtheFusariumpopulationthatwerethebestexplanatoryparametersandhadthehigh-
estcorrectclassificationrates,i.e.,81.94,83.33and66.67%ofthecases,respectively,correctly
classified(Table1andFig5A–5C).
PLOSONE|DOI:10.1371/journal.pone.0155536 May13,2016 10/17