Arbuscular mycorrhizal fungal communities in the rhizosphere of a continuous cropping soybean system at the seedling stage

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Environmental

Microbiology

Arbuscular

mycorrhizal

fungal

communities

in

the

rhizosphere

of

a

continuous

cropping

soybean

system

at

the

seedling

stage

Jiaqi

Cui

a,c

_,

_Li

_Bai

a,c

_,

_Xiaorui

_Liu

a

_,

_Weiguang

_Jie

a

_,

_Baiyan

_Cai

a,b,∗

a_Heilongjiang_University,_College_of_Life_Sciences,_Harbin,_China b_Key_Laboratory_of_{Microbiology,}_Harbin,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received20September2016 Accepted1March2017

Availableonline14November2017 AssociateEditor:JerriZilli.

Keywords: Soybean Continuouscropping Arbuscularmycorrhizae PCR-DGGE Geneticdiversity

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Arbuscularmycorrhizae(AM)fungiplayacrucialroleinthegrowthofsoybean;however, theplantingsystememployedisthoughttohaveaneffectonAMfungalcommunitiesin therhizosphere.Thisstudywasperformedtoexploretheinfluenceofcontinuoussoybean croppingonthediversityofArbuscularmycorrhizal(AM)fungi,andtoidentifythe domi-nantAMfungusduringtheseedlingstage.Threesoybeancultivarswereplantedundertwo andthreeyearscontinuouscropping,respectively.ThediversityofAMfungiinthe rhizo-spheresoilattheseedlingstagewassubsequentlyanalyzedusingpolymerasechainreaction (PCR)-denaturinggradientgelelectrophoresis(DGGE).Theresultsshowedthatanincrease incroppingyearsimprovedthecolonizationrateofAMinallthreesoybeancultivars. More-over,thedominantspecieswerefoundtobeFunneliformismosseaeandGlomusspecies.The resultsofclusteranalysisfurtherconfirmedthatthenumberofyearsofcontinuouscropping significantlyaffectedthecompositionofrhizosphericAMfungalcommunitiesindifferent soybeancultivars.

licenses/by-nc-nd/4.0/).

Introduction

Soybean isthefourth mostimportantgraincultivarinthe world,andanimportantoilcropinChina.Soybeancontains activesubstancesthatarebeneﬁcial tohumanhealthsuch assoyproteins,isoﬂavones,saponinsandoligosaccharides.1

∗ _{Corresponding}_author_at:_College_of_Life_Sciences,_Heilongjiang_University,_Harbin_150080,_China.

E-mail:caibaiyan@126.com(B.Cai).

c _Jiaqi_Cui_and_Li_Bai_contributed_equally_to_this_work_and_share_the_ﬁrst_authorship.

Considerableattentionhasrecentlybeendirectedtowardthe increasingly severe problem of root rot in soybean under intensiﬁed cultivation.Continuouscroppinghasresultedin an increase in diseases and pests, poor soilchemical and physicalproperties,adrasticreductioninyieldand,insome cases,totalcropfailure.2–4_Tackling_the_yield_reductions

result-ing from continuous croppinghas therefore becomea key

https://doi.org/10.1016/j.bjm.2017.03.017

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issue inthe soybean industry.Recent studies suggest that continuouscroppingofsoybeanleadstodrasticchangesin the rhizosphere microﬂora, witha gradual transition from bacterial-dominanthighlyfertilesoiltofungal-dominantlow fertilesoil, and therefore, microbiota dominatedby fungi.5

Moreover,theconcentrationofrootexudatessuchas pheno-licacidsissigniﬁcantlycorrelatedwithfungalquantity,while increasedlevels of soilorganic compounds (sugars, amino acidsand organicacids)asaresultofcontinuouscropping playafacilitativerole inthe growth ofroot rotpathogens. Sincesoilsterilizationtreatments havenoeffecton fungal spores,agradualtransitiontoadominantfungalcommunity issubsequentlyobserved.6

Arbuscularmycorrhizae(AM)fungiarethemostimportant typeofendotrophicmycorrhiza,andareassociatedwiththe majorityofterrestrialplants.AMfungiareagroupof ubiq-uitousmicrobesinnature, and are oneofthemajor types intherhizosphere,7–10_{signiﬁcantly}_improving_the_absorption

andutilization ofnutrientsbyhostplants.AMfungusalso stimulategrowth,increasestressanddiseaseresistance,and promote community succession and ecosystem stability.11

AMfungiare knownforseveraleffectsandcanbeusedas bio-fertilizer.12–16 _Application _of_AM _fungi _is _widely

recog-nized as important formany crops including soybean.17,18

AMfungiappliedtosoybeancanimprovetheabsorptionof nutrients inthe hostplants, improve the nitrogenﬁxation capabilityinRhizobiaandalsothecolonystructureofthe rhi-zosphericeconiche,thusincreasingtheyieldandeconomic beneﬁtsofsoybean.19,20 _The_antagonistic_effects_of_AM

fun-gus on the problems associated withcontinuous cropping havethereforebeenexamined.21,22_However,_the_diversity_of

AMfungiinrhizosphericsoilwithdifferentsoybeancultivars underdifferentcontinuouscroppingregimesremainslargely unknown.

In this study, three typical soybean cultivars (Heinong 37, Heinong 44 and Heinong 48) grown widely in Hei-longjiang province, China, were selected to examine the diversityofAMfungiinthe rhizosphereduringcontinuous cropping.Polymerasechainreaction-denaturinggradientgel electrophoresis(PCR-DGGE) was employedwith the aimof identifyingdominant AMFungicommunities. Theﬁndings provideatheoreticalbasisforfurtherstudiesofthe relation-shipbetweenAMfungiandsoil-bornepathogens.

Materials

and

methods

Experimentalplots

Thisstudy was carried out at the Experimental Station of theResearchInstituteofSugarIndustry,HarbinInstituteof Technology,China(125◦42–130◦10E,44◦04–46◦40N).Soybean cultivarswereplantedinthesameplotandsubjectedtotwo orthreeyearscontinuouscropping,respectively.Management techniquesinthetrialfieldwerethesameasthoseinstandard productionfields.Organicmatter,potassium,phosphorusand nitrogencontentsofthesoilweredeterminedusingthe Igni-tion method, NaOH melt-flame photometry method, alkali fusion-Mo-SbAntispectrophotometricmethodandKjeldahl method,respectively.

Soybeancultivars

ThreetypicalsoybeancultivarsplantedwidelyinHeilongjiang Provincewereused:‘Heinong44’(highoil,36%averageprotein content,23%averagefatcontent,designatedHN44),‘Heinong 37’(intermediatecultivar,40%averageproteincontent,20% averagefatcontent,designatedHN37),and‘Heinong48’(high protein, 45%average proteincontent,19% averagefat con-tent, designatedHN48). Eachcultivarwassubjected totwo andthreeyearscontinuouscropping,respectively,asfollows: 2yearscontinuouscroppingofHN37(designatedC2HN37),2 yearscontinuouscroppingofHN44(C2HN44),2years contin-uouscroppingofHN48(C2HN48),3yearscontinuouscropping of HN37 (C3HN37), 3 years continuous cropping of HN44 (C3HN44)and3yearscontinuouscroppingofHN48(C3HN48). The proteincontent ofthe soybeanseeds wasdetermined using theKjeldahlmethodand thefatcontentdetermined usingtheSoxhletextractionmethod.

Harvestingandprocessingofsamples

Soybean sampleswere collectedafter30 d(atthe seedling stage) along with soil samples. Three soybean plants per cultivarwererandomlyselectedfromeachtreatmentby dig-gingtoadepthof10–20cm. Theplantswereshakengently and any soiladhering tothe rootsurface1–3mm removed usingabrushasrhizospheresoil.Thethreesoilsamplesper treatmentwere subsequentlymixedasacollectivesample. Samples were then air driedthen stored at4◦C untiluse. Therootswerethenwashedforsoilsamplecollection, and threerandomlyselectedsegmentsremovedwithscissorsand used as a collective sample. All treatments were sampled intriplicate.Rootswerethenwashedindistilledwaterand placedinFAAﬁxativeand thelevelofAMfungal coloniza-tion determined microscopically. Roots were stained using alkalihydrolyzableandacidfuchsin,thenstoredat4◦Cuntil use.Rootandsoilsamplesfromeachcroppingsystemwere denotedRL2andRL3,andSL2andSL3torepresent2and3 yearscontinuouscropping,respectively.

DeterminationoftherateofAMfungalcolonization

Fifty ﬁbrous root segments from each root sample were selectedrandomly,stained,preparedonslidesandexamined microscopicallytoobservethelevelofAMcolonization. Col-onizationratesofeachﬁbrous rootsegmentwere assigned valuesof0,10,20,30,40and100%basedonthemicroscopic observationsandthecolonizationfrequency(F)ofeach culti-varundereachcroppingsystemcalculatedasfollows:

F(%)=numberofcolonizedrootsegments/totalnumber ofrootsegments.

AMcolonizationintensity(M)wasthencalculated statis-ticallyforeachﬁbrousrootsegmentasfollows23_:

M(%)=(0%×numberofrootsegments+10%×numberof rootsegments+20%×numberofrootsegments+...100%×

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numberofrootsegments)/totalnumberofrootsegments Mean colonizationrates and standard deviations were calculatedusingExcelsoftwaretogiveavariationruleof col-onizationfrequencyandintensity.

PCR-DGGE

Total DNA in the soybean roots and rhizosphere soil was extracted using the cetyl trimethyl ammonium bromide (CTAB)methodandtheOmegagenomicDNAextractionseries E.Z.N.A.® SoilDNA kit.Thenested-PCRprocedure was per-formedasdescribedbyJie.24

In the first amplification, primers GeoA2 (5-CCAGTAGTCATATGCTTGTCTC-3) and Geo11 (5 -ACCTTGTTACGACTTTTACTTCC-3) were used to amplify thefungal18SrDNAsequenceasfollows:initialdenaturation at94◦C(4min)followedby30cyclesofdenaturationat94◦C (1min), annealing at 54◦C (1min), and extension at 72◦C (2min),endingwithfinalextensionat72◦C(7min).

Productsfrom the first amplification were subsequently diluted 1/100 and used as template for a second ampli-fication using the AM fungal specific primer AM1 (5 -GTTTCCCGTAAGGCGCCGAA-3)combinedwiththeuniversal eukaryoticprimerNS31-GC(5 -TTGGAGGGCAAGTCTGGTGCC-3).Amplificationwasperformedinathermalcyclerasfollows: initialdenaturationat94◦C(2min)followedby30cyclesof denaturation at94◦C (45s), annealing at65◦C (1min) and extensionat72◦C(45s),endingwithfinalextensionat72◦C (7min).AllPCRamplificationreactionswereconductedina 20␮Lvolumecontaining2.0␮L10×PCRbuffer (withMg2+_),

2.0␮L2.5mMdNTPs,0.5␮Lprimer1(10␮M),0.5␮Lprimer2 (10␮M),0.1␮LTaqDNApolymerase,and2␮LDNAtemplate. Ampliﬁcationproductswereseparatedbygelelectrophoresis on1.0%(W/V)agarosegelinTAEbuffer.

Productsfrom the second PCR amplificationwere again diluted 1/100 and used as template for a third reaction usingtheprimersNS31-GC(5 -TTGGAGGGCAAGTCTGGTGCC-3)and Glol (5-GCCTGCTTTAAACACTCTA-3). Amplification wasperformedinathermalcyclerasfollows:initial denatur-ationat94◦C(2min)followedby30cyclesofdenaturationat 94◦C(45s),annealingat55◦C(1min)andextensionat72◦C (45s),endingwithfinalextensionat72◦C(7min). Amplifica-tionproductswereseparatedbygelelectrophoresison1.2% (w/v)agarosegelinTAEbuffer.

A total of 3.0␮L of the third PCR products of root and soilsamplesfromeachsoybeancultivarwasusedforDGGE analysis.DGGE conditionswere optimizedbyrepetitive tri-alson8%polyacrylamidegelcontainingdenaturingagents. DGGEconditionswereasfollows:denaturantrange,40–65%; electrophoresistemperature,60◦C;pre-runningvoltage,50V; electrophoresisvoltage,130V;electrophoresistime,9h;and silverstaining,15min.

Phylogeneticanalysis

The PCR products were cloned and transformed into a vector, and positive clones selected and sequenced. The genetic relationships between the sequences were subse-quentlydetermined,andthesequencescomparedtoknown

sequencesofsimilarstrainsinGenBank.MEGA5.1wasused toconstructaphylogenetictreebasedontheneighbor-joining method. Bootstrap resampling analysis of 1000 replicates was performed to determine the conﬁdence of the tree topologies.

DGGEandclusteranalysis

Diversityisoftenindicatedbysamplerichness,dominance, and thediversityindex.Therichness oftheDGGE patterns wascomparedusingaGel-ProAnalyzer,anddiversity calcu-latedusingtheequationbelow.ClusteranalysisoftheDGGE bandswassubsequentlyperformedusingQuantityOne4.62 software.

Richness:numberofbandsperlaneintheDGGEpatterns. Diversity index (H value): comprehensively reﬂected by species richness and species evenness, indicated by the Shannon-Weinerindex(largeHvaluesindicatinghigher diver-sity): H=− s

i=1 (Pi)(log2Pi)

whereSisthenumberofbandsinthesampleDGGEﬁngerprint patternandPithedominanceoftheithband.25

Statisticalanalysis

Allvaluesareexpressedasthemean±SD.Analysisof vari-ance (ANOVA) was used to determine the signiﬁcance of differencesbetweenAMFcommunitiesofthethreesoybean cultivarsusingSPSS19.0.Valuesofp<0.05wereconsidered statisticallysigniﬁcant.

Results

Physicochemicalparametersoftheanalyzedsoil

Major physical and chemical soil indicators in the plots included: organic matter, 26.13gkg−1; total N, 1.69gkg−1; total K,25.4gkg−1; totalP,5.5gkg−1; alkali-hydrolyzableN, 133.1mgkg−1;rapidlyavailableP,13.14mgkg−1,rapidly avail-ableK,206mgkg−1;andpH,7.0.

EffectofyearsofcontinuouscroppingonAMfungal colonizationofeachsoybeancultivar

AMfungalcolonizationratesofthesoybeanseedlingsineach plotundereachcontinuouscroppingsystemwerecalculated statistically(Table1).Colonizationratesofeachcultivarwere higherunderthreeyearscontinuouscroppingthantwoyears, suggestingthatanincreaseincroppingyearsisbeneﬁcialfor AM fungi colonizationofsoybean roots. This wasperhaps relatedtotransitionofthesoilcommunitystructurefrom bac-terialtofungalduringcontinuouscropping.TheAMfungal populationwillsubsequentlyincreasewithanincreasing pro-portionoffungiintherhizospherecommunity,thusleading toanaturalincreaseinAMfungalcolonizationrates.

Colonization ratesat the seedlingstage differed signiﬁ-cantly between cultivarsunder the same cropping system

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Table1–Arbuscularmycorrhizal(AM)fungal

colonization(%)ofseedlingsofthreesoybeancultivars undercontinuouscropping.

Yearsofcontinuous cropping

HN37 CultivarHN44 HN48

Two 28.9±2.5a 19.9±1.3a 17.8±1.2a Three 39.5±2.8b 33.1±2.2b 31.2±2.5b Note:Lowercaselettersinthesamecolumnindicateasigniﬁcant differenceatp<0.05.

Table2–Varianceanalysisoftheeffectsoftwoand threeyearscontinuouscroppingonAMfungal colonization(%)ofthreesoybeancultivars. Yearsofcontinuous

cropping

HN37 CultivarHN44 HN48

Two 28.9±2.5c 19.9±1.3b 17.8±1.2a Three 39.5±2.8c 33.1±2.2b 31.2±2.5a Note:Lowercaselettersinthesamelineindicateasigniﬁcant dif-ferenceatp<0.05.

(Table 2), with Heilong 37 showing the highest

coloniza-tion rate. Nutrient use differs between soybean cultivars, andcontinuouscroppingampliﬁesthedifferencesin rhizo-spherenutrients used. Rhizosphere microﬂorapopulations thereforealso differwith differentsoybean cultivars, lead-ing to interspecies differences in AM fungal colonization rates.

DGGEanalysis

PCRproductsfromtheNS31/Glolregionofrootandsoil sam-plesofthethreesoybeancultivarswere subjectedtoDGGE analysis.DGGEandsilverstainingrevealeddiversebanding patterns (Fig. 1), whichwere clearlyseparated bydifferent fingerprintpatternsanddifferentdegreesofbrightness. Six-teenbands,M1through M16(Mrepresentsmicrobial),that occurredatahighfrequencyandwereclearlyseparatedwere selectedforfurtherisolation.Therecoveredbandswere re-amplifiedandreanalyzedbyDGGE.AmplifiedPCRproducts were thenligatedtothepGEM-Tvector(Tiangen, GER)and transformed into EscherichiacoliDH 5(AS,CHN) competent cells. Positive cloneswere screened, re-amplified and ana-lyzedoncemoreusingDGGEtoverifythattheDNAsegment insertedintotheplasmidrepresentedsingle-bandDNA. Posi-tiveclonescomplyingwiththeDGGEdetectionrequirements wereselectedforsequencing.

Phylogeneticanalysis

Sequencingrevealedthatthe16DNAfragmentswere234bpin length.Theyweresubsequentlycomparedwithknownstrains in the GenBank sequence database. The results revealed >97% similarity, preliminarilysuggesting the presenceofa dominantstrain.Microﬂorarepresentedbythe16bands all belonged to AM (Fig. 2). M2 and M11 fragments belonged to Funneliformis mosseae with similarities of >98%, while those represented by the remaining 14 bands belongedto speciesinthegenusGlomus.M6andM15belongedtoGlomus viscosumwithsimilarityofupto99%.Bandsrepresentingthese

1 2 3 M3 M8 M10 M15 M16 M13 M14 M11 M12 M6 M7 M5 M1 M4 M2 M9 4 5 6 7 8 9 10 11 12

Fig.1–Denaturinggradientgelelectrophoresis(DGGE)bandingpatternsofrootandsoilsamplesfromthreesoybean cultivarsaftertwoandthreeyearscontinuouscropping.Lanes1–6:rootsamples;lanes7–12:soilsamples.

1:HN37,twoyearscropping;2:HN37,threeyearscropping;3:HN44,twoyearscropping;4:HN44,threeyearscropping;5: HN48,twoyearscropping;6:HN48,threeyearscropping;7:HN37,twoyearscropping;8:HN37,threeyearscropping;9: HN44,twoyearscropping;10:HN44,threeyearscropping;11:HN48,twoyearscropping;12:HN48,threeyearscropping.

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M9(JX203238) Glomus sp. (AM946844) Glomus sp. (EU332711) Glomus sp. (HQ323464) Glomus sp. (AB630081) Glomus sp. (AM946840) Glomus sp. (AM946881) Glomus sp. (FR693424) Glomus viscosum Funneliformis mosseae Funneliformis mosseae Funneliformis sp. (HE775329) 52 52 97 0.05 29 63 32 44 65 35 33 Glomus sp. (GU353594) Glomus sp. (GU353595) Glomus sp. (AJ563896) M7 (JX203236) M4 (JX203233) M3 (JX203232) M1 (JX183559) M13 (JX203242) M14 (JX203243) M12 (JX203241) M10 (JX203239) Group I Group II M5 (JX203234) M8 (JX203237) M6 (JX203235) M15 (JX203244) M2 (JX203231) M11 (JX203240) (AJ505812) (JX026802) (JX026803) M14 (JX203245)

Fig.2–Phylogenetictreebasedonthenucleotidesequencesof16denaturinggradientgelelectrophoresis(DGGE)-derived DNAfragmentsandsimilararbuscularmycorrhizae(AM)-derivedDNAsequencesdepositedinGenBank.

dominantAMfunguswerestatisticallyassignedto16strains. Basedonthesesequencingresults,thedominantAMfungi intherhizospherecommunitiesweredeemedtobeGlomus

andFunneliformismosseae.MEGA5.1wassubsequently used toconstructaphylogenetictree(Fig.2).

Fig. 2 shows that the 16 bands generated by DGGE are mainly representative of two types of microﬂora: Glomus

spp.andFunneliformismosseae.Thetwotypesare dominant AMfungi.TheﬁrstgroupbelongstothegenusGlomus,and includesGlomus viscosum; the second group comprises the speciesFunneliformismosseae.

Effectofcontinuouscroppingonthepopulationstructure ofAMfunguscommunitiesintherhizosphereofthree soybeancultivars

Statisticalanalysisofrichnessdataofboththerootandsoil samplesrevealedthatunderthreeyearcroppingallthree cul-tivarsshowedhigherrichnessofAMfungicomparedtotwo

yearscropping.Theseresultssuggestthatincreasedyearsof continuouscroppingisbeneﬁcialforimprovingtherichnessof AMfungalcommunities.Analysisofvariancefurtherrevealed thattherelativerichnessofAMfungalcommunitiesin soy-beancultivarsattheseedlingstageforagivencroppingyear weresigniﬁcantlydifferent(Table3).

Under threeyearscropping,rootand soilsamplesofall threecultivarshadahigherAMfungaldiversityindex com-pared to two years cropping based on statistical diversity index data. These findings suggest that continuous crop-ping is beneficial for improving the diversity index of AM fungal communities. Analysis of variance further revealed that the diversity indexes of AM fungi of each soybean cultivar for the same cropping years were significantly different(Table4).Forexample,RL3ofHN37didnot signif-icantlydifferfromRL3ofHN48,butbothweresignificantly different from HN44. This finding suggests that the AM fungal communities in HN48 roots had a higher diversity index,andtherefore,amorecomplexAMpopulation struc-ture.

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Table3–Richnessofarbuscularmycorrhizalfungiinthe rhizosphereofthreesoybeancultivarsondifferent croppingyears. Sample HN37 HN44 HN48 RC2 8a 10b 11c RC3 10a 10a 11b SC2 10a 12b 14c SC3 15b 15b 14a

RC2:rootsample,2yearscontinuouscropping;RC3:rootsample, 3yearscontinuouscropping;SC2:soilsample,2yearscontinuous cropping;SC3:soilsample,3yearscontinuouscropping.

HN37:Heinong37;HN44:Heinong44;HN48:Heinong48. Note:Lowercaselettersinthesamerowindicateasigniﬁcant dif-ferenceatp<0.05.

Table4–Analysisofvarianceoftheeffectsoftwoand threeyearscontinuouscroppingonarbuscular mycorrhizae(AM)fungalcommunitydiversityinthree soybeancultivars. Sample HN37 CultivarHN44 HN48 RL2 2.95±0.01a 3.48±0.02bc 3.47±0.02b RL3 3.42±0.02ab 3.63±0.02c 3.39±0.02a SL2 3.53±0.02a 4.20±0.03b 4.35±0.03c SL3 4.12±0.03b 4.03±0.03a 4.19±0.03bc Note:Lowercaselettersinthesamerowindicateasigniﬁcant dif-ferenceatp<0.05.

ClusteranalysisoftheDGGEproﬁlesshowedthattheAM

fungalcommunitieswereclusteredintoeightgroups(Fig.3).

GroupIincludedtherootsamplesofHN37undertwoyears

cropping);GroupIItherootsamplesofHN37underthreeyears croppingandHN44undertwoyearscropping;GroupIIIthesoil samplesofHN44underthreeyearscropping;GroupIVthesoil samplesofHN48undertwoandthreeyearscropping;Group VthesoilsamplesofHN37undertwoyearscropping;Group VIthesoilsamplesofHN37underthreeyearscroppingand HN44undertwoyearscropping;GroupVIItherootsamples ofHN44underthreeyearscropping;andGroupVIIItheroot samplesofHN48under twoandthree yearscropping.The ﬁndingssuggestthatAMfungalcommunitieswereaffected bythesoybeancultivar,croppingdurationandmicro-habitat (soilorroot).Thatis,thesecommunitieswerecultivar-speciﬁc anddependentonthecroppingduration.

Discussion

Inthisstudy,morphologicalanalysisandmoleculargenetics werecombinedtoinvestigatetheeffectofcontinuous crop-pingonAMfungalcolonizationandpopulationstructurein roots and rhizosphere soilof three soybean cultivars. The dominantspecieswerefoundtobeFunneliformismosseaeand

Glomus species. A signiﬁcant effect of years of continuous croppingonAMfungiofthedifferentsoybeancultivarswas alsorevealed,supportingfurtherstudiesoftherelationships betweenAMfungiandsoil-bornediseasepathogensresulting fromcontinuouscropping.21,22

ThenumberofcontinuouscroppingyearsaffectedtheAM fungalpopulationstructureofboththerootsandrhizosphere, whileDGGEprofilesshowedthatthepopulationstructurewas morecomplexunderthreeyearscroppingcomparedtotwo. Thesefindingssuggestthatcontinuouscroppingisbeneficial

0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

HN37 two years of continuous cropping root sample

HN44 three years of continuous cropping root sample

HN44 two years of continuous cropping root sample HN37 three years of continuous cropping root sample

HN48 three years of continuous cropping root sample HN37 three years of continuous cropping root sample

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toAMfungaldiversity.However,DGGEpatternsoffungal rich-nessdifferedatdifferent stagesofsoybean growth.Atthe branchingstage,bands M10,M11,M12,M14 andM15 were observedinsamplesfrombothtwoandthreeyearscropping. Thissuggeststhat themicroﬂorarepresented bythese ﬁve bandswasrelativelystableinthepopulationstructureofthe rhizospheresoil.26_However,_at_the_seedling_stage,_bands_M2,

M5,M7andM11were observed.Itfully illustratedthatthe twostudiescouldbecomplimentarytoeachother.Resultsat thepoddingstage,thethirdmostimportantstageofsoybean growth,nowneedtobeconﬁrmed.

Underdifferentcroppingyears,theAMfungialsoshowed different colonization rates, with rates under three years croppinghigherthanthoseunder twoyears cropping.This findingsuggeststhatcontinuouscroppingisbeneficialtoAM growthand colonization.Anumber ofstudies suggestthat AMfungiformmycorrhizamoreefficientlyinbarrensoil.27–29

Undershort-termcontinuouscropping(twotothreeyears), theamountanddiversityofAMfungiinthesoilincreased, suggestingtheabilitytoeasethedeleteriouseffectsof con-tinuouscroppingonsoybean growth.Populationstructures ofthe AMfungal communitiesin therhizosphere soil sig-nificantlydifferedbetweentwoandthreeyearscontinuous cropping,andthesedifferentAMfungiwerenotcompletely consistent with the proportion ofAM fungal communities in the soil. This suggests that different soybean cultivars have different AM fungi colonization efficiencies. Under-standingthe variationinAMmicroflorainrhizospheresoil withdifferentsoybeancultivarsisthereforeimportantsince thesecommunitiesserve asamicrobialfertilizer, minimiz-ingthenegativeeffectsofcontinuouscroppingandpromoting growth.

ThisstudyrevealedthatthetotalrichnessofAMfungiin rootand soilsamplesofthethreesoybeancultivars(HN37, HN44andHN48)washigherunderthreeyearscroppingthan two.Thissuggeststhatcontinuouscroppingcausesachange insoilnutrients,soilmicroorganismsandsoilenzyme.30–32

For example, continuous cropping may cause a signiﬁcant decreaseinpH,causingachangeinsoilfromneutraltoacidic, therebyfavoringthefungalgrowthandinhibitingthe breed-ingofbacteriaandactinomycete.Overall,thisleadstofungal dominanceinthesoil.

Despite ourﬁndings,the resultsofnested-PCR revealed thatthelengthofthePCRproductsgraduallydecreasedFlaws in the primers used possibly caused the ampliﬁcation of non-AMfungi,eventhoughmanystudiessuggestthe feasi-bilityoftheselectedprimers.30,31_Other_authors_have_reported

thattheAMfungiprimerfailedtoamplifyAMfungi belong-ingtoArchaeosporaceaeand Paraglomaceae.32 _Our_sequencing

resultssuggestthattheAMfungalcommunitieswere dom-inatedbybandsbelongingtoGlomusspecies,withnobands containingspeciesbelongingtotheabovetwofamilies. Glo-mus was therefore considered the dominant genus of AM fungiinthe soybeanrhizosphere, consistentwithprevious studies.33,34

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

ThisworkwassupportedbygrantsfromtheNational Natu-ral ScienceFoundationofChina(No.31570487)andtheKey ProjectofHorizontalSubjectofHeilongjiangEastUniversity (No.HDFHX160103).

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