MicrobiologyOpen. 2018;e597.  | 1 of 12https://doi.org/10.1002/mbo3.597

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Received:8September2017  |  Revised:2January2018  |  Accepted:9January2018DOI: 10.1002/mbo3.597

O R I G I N A L R E S E A R C H

Responses of fungal community composition to long- term chemical and organic fertilization strategies in Chinese Mollisols

Mingchao Ma1,2  | Xin Jiang1,3 | Qingfeng Wang1 | Marc Ongena2 | Dan Wei4 |  Jianli Ding1,3 | Dawei Guan1,3 | Fengming Cao1,3 | Baisuo Zhao3 | Jun Li1,3

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2018TheAuthors.MicrobiologyOpenpublishedbyJohnWiley&SonsLtd.

1InstituteofAgriculturalResourcesandRegionalPlanning,ChineseAcademyofAgriculturalSciences,Beijing,China2MicrobialProcessesandInteractionsResearchUnit,GemblouxAgro-BioTech,UniversityofLiège,Gembloux,Belgium3LaboratoryofQuality&SafetyRiskAssessmentforMicrobialProducts,MinistryofAgriculture,Beijing,China4TheInstituteofSoilFertilityandEnvironmentalSources,HeilongjiangAcademyofAgriculturalSciences,Harbin,China

CorrespondenceXinJiangandJunLi,InstituteofAgriculturalResourcesandRegionalPlanning,ChineseAcademyofAgriculturalSciences,Beijing,China.Emails:jiangxin@caas.cn;lijun01@caas.cn

Funding informationNationalNaturalScienceFoundationofChina,Grant/AwardNumber:41573066and31200388;NationalKeyBasicResearchProgramofChina,Grant/AwardNumber:2015CB150506;theFoundationforSafetyofAgriculturalProductsbyMinistryofAgriculture,China,Grant/AwardNumber:GJFP201801202;FundamentalResearchFundsforCentralNon-profitScientificInstitution,Grant/AwardNumber:1610132017010

AbstractHowfungirespondtolong-termfertilizationinChineseMollisolsassensitiveindica-torsofsoilfertilityhasreceivedlimitedattention.Tobroadenourknowledge,weusedhigh-throughputpyrosequencingandquantitativePCRtoexploretheresponseofsoilfungalcommunitytolong-termchemicalandorganicfertilizationstrategies.Soilswerecollected ina35-year fieldexperimentwith four treatments:no fertilizer, chemicalphosphorus,andpotassiumfertilizer(PK),chemicalphosphorus,potassium,andnitro-gen fertilizer (NPK), andchemicalphosphorus andpotassium fertilizerplusmanure(MPK).Allfertilizationdifferentlychangedsoilpropertiesandfungalcommunity.TheMPKapplicationbenefitedsoilacidificationalleviationandorganicmatteraccumula-tion,aswellassoybeanyield.Moreover,thecommunityrichnessindices(Chao1andACE)werehigherundertheMPKregimes,indicatingtheresilienceofmicrobialdiver-sity and stability. With regards to fungal community composition, the phylumAscomycotawasdominant inall samples, followedbyZygomycota,Basidiomycota,Chytridiomycota,andGlomeromycota.Ateachtaxonomiclevel,thecommunitycom-positiondramaticallydifferedunderdifferentfertilizationstrategies,leadingtodiffer-entsoilquality.TheNPKapplicationcausedalossofLeotiomycetesbutanincreaseinEurotiomycetes,whichmightreducetheplant–fungalsymbiosesandincreasenitro-genlossesandgreenhousegasemissions.Accordingtothelineardiscriminantanalysis(LDA)coupledwitheffectsize(LDAscore>3.0),theNPKapplicationsignificantlyin-creasedtheabundancesoffungaltaxawithknownpathogenictraits,suchasorderChaetothyriales,familyChaetothyriaceaeandPleosporaceae,andgeneraCorynespora,Bipolaris,andCyphellophora.Incontrast,thesefungiweredetectedatlowlevelsundertheMPKregime.SoilorganicmatterandpHwerethetwomostimportantcontribu-torstofungalcommunitycomposition.

K E Y W O R D S

fungalcommunitycomposition,illuminamiseqsequencing,inorganicfertilizer,manure,soildegradation

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1  | INTRODUCTION

Mollisols(blacksoilregions)arewidelydistributedinnortheastChinaandareconsideredhighlyfertilesoils.Consequently,theseblacksoilregions have become important agricultural areas for grain produc-tionandcultivation(Zhaoetal.,2015).However,severaldecadesofexcessivecultivationandintensivefertilizationhavecausedsubstan-tial loss of soil and soil productivity (Liu etal., 2015; Singh,Verma,Ansari,&Shukla,2014).Inappropriatechemicalfertilizerapplicationscauseserioussoildegradationandenvironmentalpollution(Yinetal.,2015), especially the overuse of nitrogen (N) fertilizers,which havea litanyof consequences, including climate change, greenhouse gasemission,marineandfreshwatereutrophication,soilacidificationandsoilmicrobialdiversity,activity,andbiomassreduction(Edwards,Zak,Kellner,Eisenlord,&Pregitzer,2011;Guoetal.,2010;Ramirez,Craine,&Fierer,2012;Zhouetal., 2015).Furthermore, excessiveuseofNfertilizercanalterthedynamicsofplantpopulations,causechangesinplantspeciescompositions,andincreasetheconcentrationsofel-ements likemanganese, iron,andaluminum,whichharmplantsatahighconcentration(Clarketal.,2007;Johnson,Wolf,&Koch,2003).

TheabovementionedproblemswillbedifficulttosolveaslongasexcessiveNfertilizationinputscontinue,thusreductionsinchemicalfertilizer application have been advocated (Williams, Börjesson, &Hedlund,2013).OrganicamendmentscansupplyNtocropsandarebeneficialforsoilquality,causingresidualNeffectstheyearaftertheirapplication(Schröder,Uenk,&Hilhorst,2007).Manure,asanimport-antsourceoforganicmatter(OM),isaneffectivesubstituteforchem-icalNinputsanditsusecouldsolvetheproblemswithoutdecreasingcropyields(Dingetal.,2017).However,weknowlessabouttheef-fectsofmanureapplicationonsoilmicroorganisms,whicharevaluableindicatorsofsoilqualityandare involved instabilizingsoilstructure(Chuetal.,2007;Romaniuk,Giuffré,Costantini,&Nannipieri,2011).Comparedwithbacteria,soilfungaldiversityismoresensitivetosoilfertility(He,Zheng,Chen,He,&Zhang,2008),duetotheirorganicNandphosphorus(P)acquisitioncapabilities(Behie&Bidochka,2014;Näsholm,Kielland,&Ganeteg,2009)andimportantrolesinnutrientcycling(Cairney,2011;Szuba,2015).Astableandappropriatefungalcommunity composition is alsobeneficial for soil biochemical cycle,and also leads to a healthy and stable surrounding ecosystem forplants (Sun,Liu,Yuan,&Lian,2016).Thus, it isofcrucial interesttoinvestigatesoilfungalcommunities.

In our previous studies, the impacts of long-term fertilizationson bacterial community composition have been examined (Zhouetal.,2015).Inorganicfertilizationledtoasignificantdecreaseinthebiodiversity and abundance of bacteria, and the influence of moreconcentrated fertilizer treatments was greater than that of lowerconcentrations. However, a comprehensive understanding of thefungal responses is still unclear, especiallywhen organicmanure issubstitutedforchemicalNfertilizer.Inthisstudy,soilswerecollectedduringa35-yearfieldexperimentintheChineseMollisols,andhigh-throughputpyrosequencingandquantitativePCR(qPCR)technologywere performed to analyze soil fungal community composition andabundance.Here,wehypothesize that: (1) thedifferences in fungal

communitycompositionandabundancearearesultoflong-termfer-tilizationstrategiesthatinduceschangesinsoilproperties;(2)manurehelpsshiftthesoilfungalcommunitytoagoodstatus,whereaschemi-calfertilizerapplicationsexhibittheoppositepattern;and(3)theshiftsoffungalcommunitymaymainlyresultfromchangesinthesoilpHandOM.Insummary,understandingtheresponsesoffungalcommunitycompositiontodifferentfertilizationstrategiesisnotonlyaneffectivewaytorevealtherelationshipbetweenintensivefertilizationandblacksoil degradation but is alsomeaningful for determining appropriatefertilizationapplicationstoimproveandmaintainsoilfertility.

2  | MATERIALS AND METHODS

2.1 | Field experiments and soil sampling

Thisstudyhasbeenperformedinanexperimentalfieldwithawheat–maize–soybeancroprotationsince1980inHarbinCity,HeilongjiangProvince,China (45°40′N, 126°35′E). The climate for this region ischaracterized as typical temperate monsoon, with an annual meanairtemperatureof3.5°C,evaporationof1,315mmandprecipitationof533mm.Thefieldexperimentwassetupasablockdesignwiththreereplicates,witheachblockcomprisedofadifferenttreatmentrandomized inplotsof9×4m.Chemical fertilizerswereappliedasurea(75kg/hm2),calciumsuperphosphateplusammoniumhydrogenphosphate(150kg/hm2),andpotassiumsulfate(75kg/hm2),respec-tively.Thehorsemanurewasusedatapproximately18,600kg/hm2. Moredetailson theexperimental fieldwere shown inourpreviousstudy(Weietal.,2008).

SoilswerecollectedamongplantrowsafterthesoybeanharvestinSeptember2014.Fourtreatmentswiththreereplicateswerechosen:nofertilizer (CK),chemicalPandpotassium(K)fertilizer (PK),chem-icalN,PandK fertilizer (NPK), andchemicalPandK fertilizerplusmanure (MPK). Foreach replicateplot inevery treatment, six coreswererandomlycollectedintheploghedsoillayer(5–20cm)afterre-movingplantresiduesandgravels.Coreswerecombinedandmixeduniformlytoobtainahomogeneousblendandsubsampledintothreeparts.Onepartwasreservedat−80°C,andtheothertwowereusedastwosubsamples.Atotalof24soilsubsampleswereobtained.Soilchemicalpropertiesandmolecularanalyseswereperformedforeachsubsample.

2.2 | Analyses of soil chemical properties and soybean yield

Soilchemicalproperties,includingsoilpH,OM,TotalN(TN),nitratenitrogen(NO3

−–N),ammoniumnitrogen(NH4+–N),TotalP(TP),avail-

ableP(AP),TotalK(TK),andavailableK(AK)wereanalyzedafterbeingairdriedatroomtemperatureandpassedthrougha2.0-mmsieve.SoilpHwasmeasuredwithapHmeterusinga1:1sample:waterextract.SoilOMwasassayedbyapplying theK2Cr2O7-capacitancemethod(Strickland & Sollins, 1987). TN was measured using the Kjeldahlmethod(Huangetal.,2007).NH4

+–NandNO3−–Nwereextractedby

2mol/LKClsolutionandsubjectedtoflowinjectionanalysisaccording

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toHart, Stark,Davidson, and Firestone (1994).AmodifiedmethodofresinextractionwasusedfortheAPanalysis (Hedley&Stewart,1982),andTPwasdeterminedusingthecolorimetricmethod (Garg& Kaushik, 2005). TK andAKwere analyzed by atomic absorptionspectrometerand flamephotometry, respectively, as recommendedbyHelmkeandSparks (1996)andHabib, Javid,Saleem,Ehsan,andAhmad (2014). Soybean yields under different conditions were re-cordedafterharvest.

2.3 | Total DNA extraction

TotalDNAwas extracted from0.25g soil in each subsampleusingaMOBIOPowerSoilDNA IsolationKit (Carlsbad,CA,USA)accord-ing to themanufacturers’ protocol withmodifications (Fierer etal.,2012). Briefly, six successive replicate extractionswere taken fromeachsubsampleandfixedtogetherasoneDNAtemplatetoprovideenoughtotalDNA(Zhouetal.,2016).DNApurificationfollowed,andthen,DNAconcentrationandquality(A260/A280)oftheextractswereestimatedvisuallyusingaNanoDropND-1000UVevis spectropho-tometer(ThermoScientific,Rockwood,TN,USA).

2.4 | qPCR analysis

ThesoilfungalabundancelevelswerequantifiedusingtheqPCRde-tectionsystem(AppliedBiosystems7500,CA,USA).Theinternaltran-scribedspacer(ITS)primersITS4F(5′-TCCTCCGCTTATTGATATGC-3′)andITS5(5′-GGAAGTAAAAGTCGTAACAAGG-3′)wereusedtoamplifythefungalITSregionofribosomalRNAgeneasrec-ommendedbySchochetal.(2012).Thecomponentsofthereactionmixture (25μl) and the optimized conditions for amplificationwereaspreviouslyreported(Zhouetal.,2016).TheqPCRwascarriedoutwiththreereplicatesforeachsoilsubsample.Thestandardcurvewasgeneratedusing10-fold serial dilutionsof a plasmid containing theITSgeneinsert.Theabundancesofthebacterial16S rRNA gene cop-ieswerequantifiedusingthesamemethodasfortheITSgene,withprimers515Fand806R(Lauber,Ramirez,Aanderud,Lennon,&Fierer,2013),andpresentedinTableS1.Thevalueofthefungi/bacteriaratio(F/Bratio)wascalculatedbydividingtheITSgenecopynumberbythe16S rRNAgenecopynumber(Wurzbacher,Rösel,Rychła,&Grossart,2014).

2.5 | Illumina MiSeq sequencing

ThefungalITS1regionwasamplifiedusingtheprimersITS1F(5′-CTTGGTCATTTAGAGGAAGTAA-3′)andITS2(5′-GCTGCGTTCTTCATCGATGC-3′)aspreviouslydocumented(Bueeetal.,2009;Degnan&Ochman,2012;Dingetal.,2017).TheITS1F/ITS2primersarecon-sideredastheuniversalDNAbarcodemarkersforthemoleculariden-tificationoffungi(Blaalidetal.,2013;Schochetal.,2012).Barcodeswere connectedwithprimers andwereused to separate rawdata,allowingmultiplesamplestobepooledintoonerunofIlluminaMiSeqsequencing.TheconditionsofthePCRreactionwereasfollows:94°Cfor2min;32cyclesof94°Cfor30s,55°Cfor30sand72°Cfor30s;

and72°Cfor5min.PCRproductsweremixed(equimolarratio)afterpurification, creating aDNApool. Sequencing librarieswere gener-ated.Finally,thelibrariesweresequencedonIlluminaMiSeqplatformatPersonalBiotechnologyCo.,Ltd.(Shanghai,China).

2.6 | Data processing and statistical analyses

Barcode sequences were removed according to the methods ofEdgar,Haas,Clemente,Quince,andKnight(2011).TherawsequencereadswereprocessedusingQIIME(version1.7.0,http://qiime.org/)(Caporaso etal., 2010) and referring to the default parameters toobtain valid tags (Bokulich etal., 2013). Singletons, non-bacterialandnon-fungalOTUswere removed, and theOTUabundance lev-elswerenormalizedbasedon the samplewith the leastnumberofsequences.Toperforma faircomparisonbetweensamples,all sub-sequentanalyseswereperformedaccording to thenormalizeddata(Zhouetal.,2016).Then,operationaltaxonomicunitsdefinedbyclus-teringat the97%similarity levelweregeneratedandtaxonomicallyclassified using a BLAST algorithm against theUNITE database re-lease5.0(Koljalgetal.,2014)withaminimal80%confidenceestimate(Bokulich&Mills,2013).TheUNITEandINSDCfungalITSdatabaseswere used as references for classification (Abarenkov etal., 2010).ThesequenceswereuploadedanddepositedintheNationalCenterforBiotechnologyInformation (NCBI)SequenceReadArchive (SRA)undertheaccessionnumberSRP092759.

Thefungalα-diversityindex(includingShannon,Simpson,Chao1,andACE)wasanalyzedusingMothursoftware(version1.31.2,http://www.mothur.org/)(Schochetal.,2012).TheunweightedFastUniFracmetric was calculated to construct distance matrices using QIIME(Caporasoetal.,2010).Aprincipalcoordinateanalysis (PCoA)basedon theunweightedFastUniFracmetricwascarriedout tocomparebetween-samplevariationsinfungalcommunitycomposition(Marsh,OSullivan,Hill,Ross,&Cotter,2013).A lineardiscriminantanalysiscoupledwitheffectsize(LEfSe)wasperformedtodistinguishsignifi-cantly different fungal taxa betweenMPKandNPK regimes to thegenusorhighertaxonomylevel(Segataetal.,2011).ThesoftwareofCANOCO5.0wasusedforribosomaldatabaseproject(RDP)analysiswithaminimal60%threshold toexplorepossible linkagesbetweenfungalcommunityandsoilproperty,followedthemethodofBraakandSmilauer(2012).Ananalysisofvariancewasperformedonallexperi-mentaldatausingSPSS(v.19).Inalltests,ap-value<.05wasconsid-eredstatisticallysignificant.

3  | RESULTS

3.1 | Soil properties and soybean yields under different fertilization regimes

SoilpropertiesunderdifferentfertilizationregimesareshowninTable1.PKandNPKapplicationssignificantlydecreasedsoilpH,whereastheMPKapplicationalleviatedsoilacidification.TheMPKapplicationalsohad an accumulative effect on soilOM.Comparedwith theCK, thethreefertilizationstrategiessignificantly increasedtheconcentrations

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ofAKandTK,aswellasAPandTP.TheNPKapplicationsignificantlyin-creasedtheTNconcentration,whereastheconcentrationsofNO3

−–NandNH4

+–NwerelowerthanundertheMPKregime.Inaddition,soy-beanyieldsweresignificantlyhigherunderthefertilizationregimes,withtheMPKapplicationbeingthemosteffectivestrategy(2702kg·ha−1).

3.2 | Fungal ITS gene copy number under different fertilization regimes

ThevaluesofthefungalITScopynumberrangedfrom1.62×106 to 6.67×106 g−1soilwithsignificantdifferences(Figure1a).Comparedwith theCK,PK,andNPKapplications increasedthe ITS gene cop-ies,resultinginasignificant increaseintheF/Bratio,whereasMPKapplications exhibited the opposite pattern (Figure1b). In addition,therewere significant positive correlations between ITS gene copy numberandTP(r=.613,p<.01)andAP(r=.435,p<.05),referringtoPearson’scorrelations(TableS2).Moreover,theF/Bratioshowedsignificantly negative correlations with soil pH (r=−.912, p<.01)andOM (r=−.572, p<.01), but was positively correlatedwith TN(r=.795,p<.01)andTP(r=.523,p<.01).

3.3 | Fungal diversity analysis under different fertilization regimes

A total of 1,399,128 raw sequence reads were obtained from theIlluminaMiSeqplatformanalysisof24soilsubsamples,and1,179,936effectivesequenceswereproducedafterprocessing.Thehigh-qualitypercentagewasmorethan82%,withameanreadlengthof280bp.MorestatisticaldataofsequencingindifferentsamplesaredetailedinTableS3.Rarefactionanalysis (FigureS1)displayedsimilartrendswherebythegreatestOTUoccurredundertheMPKregime,andthelowest value occurred in theNPK treatment. TheGood’s coveragevalues(0.991–0.992)indicatedthatthereweresufficientreadstoob-tainthefungaldiversity.Withregardstofungalcommunityrichnessindices(Table2),PKandNPKapplicationsreducedChao1andACEin-dices,whereastheMPKapplicationledtothegreatestindices.Inad-dition,Pearson’scorrelations(TableS2)showedthattheChao1indexwassignificantlypositivelycorrelatedwithOM(r=.564,p<.01).

APCoAwasperformedtoanalyzetheimpactsoffertilizationstrat-egiesonfungalcommunitystructure(Figure2).Thetwoaxes,PC1andPC2,explained32.78%and19.84%ofthetotalvariation,respectively.TheNPKplotslocatedinthelowerrightcornerandwerefarfromtheCK;whereasPKandMPKplotswereclusteredtogetherandlocatedinthemiddle.ComparedwiththeCK,long-termfertilizationstrategiesclearlychangedthefungalcommunitycompositionduetotheeffectsofchemicalNinputs.

3.4 | Fungal community compositions and relative abundance under different fertilization regimes

Phyla Ascomycota, representing 70.83%–76.16% of the total se-quences,wasdominant,followedbyZygomycota(15.56%–19.22%),Basidiomycota (6.14%–10.72%), Chytridiomycota (0.94%–3.37%),T

ABLE 1 Soilpropertiesandsoybeanyieldunderdifferentfertilizationregimes

Fert

iliza

tion

regi

mes

pHO

M (g

·kg−1

)A

K (g

·kg−1

)TK

(g·k

g−1)

NH

4+ (mg·

kg−1

)N

O3− (m

g·kg

−1)

TN (g

·kg−1

)A

P (g

·kg−1

)TP

(g·k

g−1)

Soyb

ean

yiel

d (k

g·ha

−1)

CK6.43±0.08c

24.39±0.37a

0.17±0.03a

6.30±0.89a

35.01±1.16a

2.45±0.88a

1.20±0.05a

0.02±0.01a

0.44±0.03a

1812.67±141.99a

PK6.18±0.04b

25.51±0.30c

0.24±0.01b

28.57±2.25b

37.80±2.95a

3.62±0.57b

1.26±0.03a

0.89±0.04c

0.73±0.02c

2377.33±118.85bc

NPK

5.54±0.04a

24.88±0.25b

0.23±0.03b

30.36±1.02b

37.30±6.29a

4.53±0.91bc

1.43±0.08b

0.94±0.06d

0.70±0.02bc

2241.33±186.11b

MPK

6.38±0.05c

27.47±0.41d

0.23±0.03b

28.43±3.93b

39.36±6.95a

5.17±0.67c

1.20±0.03a

0.66±0.01b

0.61±0.05b

2702.67±169.39c

Valuesaremeans±standarddeviations(n=6).Valueswithinthesamecolumnfollowedbydifferentlettersindicatesignificantdifferences(p<.05)accordingtoTukey’smultiplecomparison.

Fertilizationregimes:CK,nofertilizer;PK,chemicalphosphorusandpotassiumfertilizer;NPK,chemicalphosphorus,potassium,andnitrogenfertilizer;MPK,chemicalphosphorusandpotassiumfertilizerplus

manure.

Soilproperties:AP,availablephosphorus;AK,availablepotassium;NH

4+ ,ammoniumnitrogen;NO

3− ,nitratenitrogen;TK,totalpotassium;TP,totalphosphorus;TN,totalnitrogen;OM,organicmatter.

     |  5 of 12MA et Al.

andGlomeromycota(0.28%–0.83%)(Figure3).ComparedwiththeCK,theMPKapplicationsignificantly increasedtherelativeabun-danceof thephylaAscomycota,whichdecreasedunder theNPKregime.Sordariomyceteswasdominantatclass level, followedbyIncertae_sedis_Zygomycota,Leotiomycetes,andDothideomycetes(shown in Figure4, at least one groupwith a relative abundance>0.1%). NPK and MPK applications significantly increased therelative abundance of Sordariomycetes, but decreased those ofLeotiomycetes and Dothideomycetes. The abundances of theclasses Eurotiomycetes and Tremellomycetes were significantlyhigherundertheNPKregimethanundertheothers.All theferti-lization treatments hadpositive effects on thePezizomycetes.Atthegenuslevel(Figure5),allthefertilizationstrategiessignificantlydecreasedtherelativeabundancesofMortierella,Chaetomium,andEpicoccum, butPenicilliumwas increased.Periconia and Ilyonectria were lower under the NPK and MPK regimes. In particular, thechemical N fertilizer significantly increased the abundances ofChaetomidiumandCorynespora.

3.5 | Significantly different fungal taxa occurred under the NPK and MPK regimes

TheLEfSeanalysisdistinguishedthepresenceofsignificantlydifferentfungaltaxaundertheNPKandMPKregimes(averagerelativeabun-dance>0.01; Figure6). The linear discriminant analysis score was

greater than3.0.TheMPK-treated sampleshad significantlyhigherabundanceofthephylumAscomycota,andgeneraMycothermusandPericonia, whereas the phyla Basidiomycota and Chytridiomycota,the order Chaetothyriales,thefamiliesChaetomiaceae,Pleosporaceae, and Chaetothyriaceae, and genera Chaetomidium, Bipolaris, andCyphellophorawereoverrepresentedundertheNPKregime.

3.6 | Correlation between fungal community composition and soil properties

Basedontheredundancyanalysis(Figure7),alltheselectedsoilprop-ertiesaccountedfor56.8%oftheexplanatoryvariablesinthefungalcommunity composition among the samples. The primary contribu-torsinshiftingthefungalcommunityweresoilOM(F=4.5,p =.002)andpH(F=4.1,p=.002),whichindividuallyaccountedfor14.9%and15.7%ofthevariation,respectively.Theothersoilpropertiesaffectedfungalcommunitycompositioninthefollowingorder:AK>AP>TN>TP>NO3

−–N=NH4+–N>TK.Inaddition,theplotsofCK,PK,NPK

andMPKwerewellgroupedandseparatedfromtheNPKplot.Pearson’scorrelations(TableS4)showedthatAscomycotawaspos-

itivelycorrelatedwithOM(r=.709,p<.01)andpH(r=.508,p<.05),whereasZygomycotawasnegativelycorrelatedwithAP,AK,OM,TK,TP,andNO3

−–N(p<.01).SoilpHhadnegativeimpactsonBasidiomycota;however,theeffectofTNwaspositive.BothAPandTPwerepositivelycorrelated(p<.01)withChytridiomycotaandGlomeromycota.

F IGURE  1 ResultsofthequantitativePCR.(a)TheabundanceoffungiasindicatedbythenumberofITScopies;(b)Fungi/Bacteriaratiosunderdifferentfertilizationregimes.Samelettersabovecolumnsindicatenosignificantdifference(p <.05,Tukey’stest)

TABLE  2 Estimatednumbersofobservedoperationaltaxonomicunits(97%similarity)anddiversityofsoilindifferentfertilizationregimes

Fertilization regimes Observed species Chao1 Ace Simpson Shannon Goods coverage

CK 895.83±59.44a 1050.1±46.2a 1084.1±80.8ab 0.979±0.016a 7.22±0.27ab 0.992±0.0015a

PK 877.33±97.73a 1028.6±37.9a 1040.9±57.8a 0.987±0.002a 7.46±0.21b 0.992±0.0019a

NPK 812.00±40.87a 1034.1±54.5a 1049.0±41.9a 0.985±0.003a 7.19±0.14a 0.992±0.0020a

MPK 914.17±167.33a 1140.2±101.7b 1164.8±101.6b 0.986±0.004a 7.40±0.14ab 0.991±0.0038a

Valueswithinthesamecolumnfollowedbydifferentlettersindicatesignificantdifferences(p<.05)accordingtoTukey’smultiplecomparison.

6 of 12  |     MA et Al.

4  | DISCUSSION

4.1 | Improvements in soil acidification, OM accumulation, and soybean yield

Long-termchemicalfertilizerapplications,especiallytheNPKapplica-tion,significantlyincreasedsoilacidification;however,manurecouldeffectively alleviate soil acidification, perhaps due to the bufferingfunctionsoforganicacids,carbonates,andbicarbonates (García-Gil,Ceppi, Velasco, Polo, & Senesi, 2004; Whalen, Chang, Clayton, &Carefoot,2000).Furthermore,manurehadmacronutrientstatus,con-tributingtothesignificantaccumulationofsoilOM(Xieetal.,2014).Inturn,thehighproductivityresultingfromorganicmanureincreasestheamountsofOMinthesoil,intheformofrootexudates,decayingrootsandabovegroundresidues,whicharebeneficialforsoilOMac-cumulation(Geisseler&Scow,2014).Inaddition,soybeanyieldsweresignificantlyhigherunderthefertilizationregimes,withtheMPKap-plication being themost effective fertilization strategy. The resultsagreedwellwithotherfindings(Zhaoetal.,2014).

4.2 | Changes in the ITS gene’s abundance and F/B ratio

ComparedwiththeCK,ITSgenecopieswereincreasedunderbothPKandNPKregimes,whichconfirmedthepositivestimulatoryeffectsofchemicalfertilizeronfungalpopulations(Zhouetal.,2016).Moreover,Pearson’s correlations showedpositive correlationsbetween fungalabundanceandAP(r=.435,p<.05)andTP(r=.613,p<.01),whichwerequitesimilartootherfindings(Kuramaeetal.,2012).

Inaddition, theF/B ratiowasconsideredan indicatorofeco-systemprocesses,asthechangesinratiowerelikelytoberelatedtodecomposition,nutrientcycling,C-sequestrationpotential,andecosystemself-regulation(Strickland&Rousk,2010).Inthisstudy,the F/BratiosunderCKandMPKregimeswerelowerthanthoseofPKandNPK,probablydue to theacidificationof soil inducedby chemical inputs.As documented by Joergensen andWichern(2008) and Rousk, Brookes, and Bååth (2009), fungi have beenfoundtobemoreacidtolerantthanbacterialeadingtoincreasedfungaldominanceinacidicsoils.Moreover,theF/Bratiowashigh-est under thePK regimeprobably due to thebetter adaptabilityoffungalspeciestoNlimitationcomparedwithbacteria(Rousk&Frey,2015).TheF/BratioundertheMPKregimewassignificantlylowerthanothers,indicatingahigherturnoverrateofeasilyavail-able substrates (Rousk, Brookes, & Bååth, 2010) and highly pro-ductivecropsoils(Strickland&Rousk,2010).Additionally,theF/B ratio was significantly positive correlatedwith soil pH (r=.648,p<.01), thismightbedue to thedifferent responsesofbacteriaand fungi to lower pH levels, namely the significant suppressiveeffectofbacteriaandwelltoleranceoffungi(Coyne,1999;Rousk,Bååth,etal.,2010).

4.3 | Effects on fungal α- diversity

Microbialdiversity insoilwasclosely relatedtosoilqualityandthenutrient cycling rate (Nevarezetal.,2009).The richer thebiodiver-sity,themorestablethesoil(Chaer,Fernandes,Myrold,&Bottomley,2009).Thelowerbiodiversityoffungialsocausedunsustainablecropproduction and an unstable ecosystem (Maček etal., 2011). In this

F IGURE  2 PCoAofthepyrosequencingreadsbasedontheunweightedFastUniFracmetric

     |  7 of 12MA et Al.

study,PKandNPKapplicationsreducedthefungalcommunityrich-ness indices (Chao1andACE),whereastheMPKapplicationsignifi-cantlyincreasedthem.Thismightbeexplainedbythecomplexorganiccompounds present inmanure requiring variousmicroorganisms todegrade.Theresultsconfirmedpreviousfindingsthatahighmicrobialdiversitywasalwaysfoundunderorganicamendmentregimesrather

than chemical regimes (Esperschütz, Gattinger, Mäder, Schloter, &Fließbach,2007).Comparedwithsoilnutrients,theChao1indexwaspositivelycorrelatedwithsoilOM(r=.564,p<.01),whichprobablyprovidedmacronutrient for fungi and stimulated themicrobial bio-massanddiversity(Peacocketal.,2001).Inconclusion,thesubstitu-tionofchemicalNfertilizerwithorganicmanure,whichisbeneficial

F IGURE  3 Relativeabundanceofphylogeneticphylaunderdifferentfertilizationregimes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

CK PK MPK NPKre

lativ

e ab

unda

nce

treatments

Others

Glomeromycota

Chytridiomycota

Basidiomycota

Zygomycota

Ascomycota

F IGURE  4 Relativeabundanceofphylogeneticclassesunderdifferentfertilizationregimes.Atleastonegroup’srelativeabundanceismorethan0.1%ofthetotalsequences

0%

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e

Treatments

other

Incertae_sedis_Ascomycota

Chytridiomycetes

Pezizomycetes

Agaricomycetes

Eurotiomycetes

Tremellomycetes

Dothideomycetes

Leotiomycetes

Incertae_sedis_Zygomycota

Sordariomycetes

F IGURE  5 Relativeabundanceofphylogeneticgeneraunderdifferentfertilizationregimes.Atleastonegroup’srelativeabundanceismorethan1%ofthetotalsequences

0%

2%

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6%

8%

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14%

CK PK MPK NPK

rela

tive

abun

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Chaetomium

Periconia

Epicoccum

Ilyonectria

Bipolaris

Chaetomidium

Corynespora

Penicillium

Mor�erella

8 of 12  |     MA et Al.

for the resilienceofmicrobialdiversity (Naeem&Li,1997)andsoilproductivity(Sapp,Harrison,Hany,Charlton,&Thwaites,2015),wasagoodwaytoreduceanthropogenicNinputs.

4.4 | Impact on fungal community composition

The phylum Ascomycota was dominant in all the fertilization re-gimes. Similar results have been observed in other studies (Xiongetal., 2014; Li, Ding, Zhang, & Wang, 2014). The abundance ofAscomycotaundertheNPKregimewaslowerthanunderthePKandMPKregimes,whichcontrastedwithotherfindingsthatAscomycotawas enhanced by relatively high N inputs (Nemergut etal., 2008;Paungfoo-Lonhienneetal.,2015).ThiscouldbeexplainedbythefactthatmembersoftheAscomycotaareadaptedtotheappropriateNcontent(Klaubaufetal.,2010)butwerevulnerabletoexcessNlevels(Wangetal.,2015).

Attheclasslevel,Sordariomyceteswasthemostdominantmem-ber,inlinewithotherfindings(Zhouetal.,2016).ComparedwiththeCKandPKapplication,therelativeabundancesofSordariomycetesweresignificantlyhigherundertheNPKandMPKregimes,proba-blyduetosufficientnutrients insoil (Dingetal.,2017).However,Dothideomycetesshowedtheoppositepattern,namely,theyweresignificantlylowerundertheNPKandMPKregimes,indicatingpos-itiveeffectsonsoilquality,asmanyofthetaxaappearedtobeplantpathogens(Lyons,Newell,Buchan,&Moran,2003).LeotiomycetesdominancewaslowestundertheNPKregime,indicatinganegative

F IGURE  6 HistogramofthelineardiscriminantanalysisscorescomputedforfeaturesdifferentiallyabundantbetweenNPKandMPKsamplesidentifiedbyLEfSe(LDAscore>3)

F IGURE  7 Redundancyanalysisofsoilbacterialcommunitiesandsoilcharacteristicsforindividualsamples.Soilfactorsindicatedinredtextincludeavailablephosphorus(AP),availablepotassium(AK),pH,soilconcentrationofNH4

+(NH4+),soilconcentrationofNO3

− (NO3

−),totalnitrogen(TN),totalpotassium(TK),totalphosphorus(TP),andorganicmatter(OM)

     |  9 of 12MA et Al.

correlation with the chemical N input (Freedman, Romanowicz,Upchurch, & Zak, 2015; Zhou etal., 2016). The decline ofLeotiomycetesunderNPKregimeprobablycausedalossofplant–fungalsymbiosesunderhighNinputconditions(Deanetal.,2014).Additionally,theNPKapplicationproducedahigherabundanceofTremellomycetes,whichprobablybenefitedinorganicmatterdecay(Freedmanetal.,2015).TheabundanceofEurotiomyceteswasalsohigher under theNPK regime, probably causingN loss in the soiland greenhouse gas emissions due to its N2O-producing activity(Jasrotiaetal.,2014;Mothapo,Chen,Cubeta,Grossman,&Fuller,2015). The abundances of Pezizomycetes were significantly highunderall the fertilization regimes,whichmaybe the resultof soilOMaccumulationduetodecayingwood,dung,leaflitter,andtwigs(Stajich,2015).

Athoroughinvestigationatthegenusorhighertaxonomiclevelshowed differences among the treatments. More harmful fun-gal taxa with known pathogenic traits were also overrepresentedunder theNPK regime, suchas theorderChaetothyriales, familiesChaetothyriaceae, Pleosporaceae, and Chaetomiaceae, and generaCorynespora, Bipolaris, and Cyphellophora Chaetomidium. The order Chaetothyriales, familyPleosporaceae and genusChaetomidium arewell-known for their animal and human opportunistic pathogens(Arzanlou, Khodaei, & Saadati Bezdi, 2012; Sajeewa etal., 2015;Winka,Eriksson,&Bång,1998).AndfamilyChaetomiaceaeincludesnumeroussoil-born,saprotrophic,endophytic,andpathogenicfungi(Zámockyetal.,2016),andalso,severalCyphellophoraspecieshavealso been associated with potential pathogens (Decock, Delgado-Rodríguez, Buchet,& Seng, 2003).Moreover, some isolateswithinCorynesporaarepathogenictoawiderangeofhosts(Dixon,Schlub,Pernezny, & Datnoff, 2009) and Bipolaris causes significant yieldlosses as a foliar disease constraint (Road, 2002).Obviously, long-termNPKapplicationsmayinducetheincidenceratesoffungaldis-eases.Incontrast,thesefungiweredetectedatlowlevelsundertheMPK regime.Meanwhile, the genusMycothermuswas also signifi-cantlymoredominantundertheMPKregime,whichbenefitsthede-compositionofcellulosebecauseofitsappreciabletitersofcellulasesandhemicellulases(Basotra,Kaur,DiFalco,Tsang,&Chadha,2016).Thus,manurehelpsshiftthesoilfungalcommunitytoagoodstatus,whereaschemicalfertilizerapplicationsexhibittheoppositepattern.

4.5 | Soil properties effects on fungal community composition

Inlinewithpreviousfindings(Liuetal.,2015;Dingetal.,2017),weconcludedthatsoilOMandpHwerethemostimportantcontributorstothevariation inthefungalcommunitycomposition,basedontheredundancyanalysis.AsdocumentedbyBroeckling,Broz,Bergelson,Manter,andVivanco (2008), themajorityof fungiareheterotrophsanddependonexogenousC forgrowth, thus labileOMhasapro-foundinfluenceontheirabundance.Moreover,soilpHalsoplayedakeyroleinshapingfungalcommunitycomposition(Dingetal.,2017;Kimetal.,2015).ThiscouldbeexplainedbythemoresensitivityoffungitoapHchange(Liuetal.,2015).Additionally,soilpHmayaffect

fungalcommunitycompositionbyrespondingtoothervariablesandmayprovideanintegratedindexofsoilconditions.Hydrogenioncon-centration varies bymanyorders ofmagnitude across various soilsand, as numerous soil properties are related to soil pH, these fac-torsmayhavedriventheobservedshiftsincommunitycomposition(Rousk,Bååth,etal.,2010;Shenetal.,2013;Xiongetal.,2012).Thus,soilmicroorganismscouldrapidlyrespondtothechangesintheenvi-ronmental conditions (Eilers,Debenport,Anderson,&Fierer,2012),suchassoilchemicalorphysicalpropertiesinducedbyfertilization.Inturn,shiftsinmicroorganismcompositioncouldinfluencesoilqualityandplantgrowth.

Inaddition,long-termdifferentfertilizationstrategieshadsignifi-canteffectsonbacterialβ-diversityandshapedvariantmicrobialcom-positionsinthesoil(Zhouetal.,2017).Inthisstudy,aPCoArevealedtherelationshipbetweensoilfertilizationandthefungalcommunity.TheNPKplot,locatedinthelowerrightcorner,wasfarfromtheCKplot,indicatingastrongeffectofchemicalfertilizer;however,thePKandMPKplotswereclusteredtogetherandneartheCKplot,suggest-ing theeffective resilienceoforganicmanureon fungal communitystructure,inlinewithDingetal.(2017).

5  | CONCLUSION

Ourfindingsdeterminedtheresponsesofsoilfungalcommunitycom-positiontolong-termfertilizationstrategiesinblacksoil.SuchshiftsmaymainlybederivedfromchangesinthesoilpHandOM.Comparedwithchemicalfertilization,manureapplicationsalleviatedsoilacidifi-cation,accumulatedsoilOM, increasedsoilnutrients, improvedsoilfungalcommunitycomposition,andrestoredsoilmicrobialalterations,leadingtoimprovementsofsoilqualityandsoybeanyield.Theresultshighlightedthepotentialoforganicmanureasasubstituteforchemi-calNfertilizersinthesustainabledevelopmentofChineseMollisols.

ACKNOWLEDGMENTS

ThisworkwassupportedbytheNationalNaturalScienceFoundationofChina(No.41573066andNo.31200388),theNationalKeyBasicResearch Program of China (973 Program: 2015CB150506), theFoundationforSafetyofAgriculturalProductsbyMinistryofAgriculture,China (GJFP201801202), and the Fundamental Research Funds forCentral Non-profit Scientific Institution (No. 1610132017010). WethanktheUniversityofLiège-GemblouxAgro-BioTechandmorespe-cificallytheresearchplatformAgricultureIsLifeforthefundingofthescientificstayinBelgiumthatmadethispaperpossible.

CONFLICT OF INTEREST

Noconflictofinterestisdeclared.

ORCID

Mingchao Ma http://orcid.org/0000-0003-2609-321X

10 of 12  |     MA et Al.

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How to cite this article:MaM,JiangX,WangQ,etal.Responsesoffungalcommunitycompositiontolong-termchemicalandorganicfertilizationstrategiesinChineseMollisols. MicrobiologyOpen. 2018;e597. https://doi.org/10.1002/mbo3.597