Characterization of rhizobia isolates obtained from nodules of wild genotypes of common bean

h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /

Environmental

Microbiology

Characterization

of

rhizobia

isolates

obtained

from

nodules

of

wild

genotypes

of

common

bean

Aline

Assis

Cardoso

_,

_Michel

_de

_Paula

_Andraus

_,

_Tereza

_Cristina

_de

_Oliveira

_Borba

_,

Claudia

Cristina

Garcia

Martin-Didonet

_,

_Enderson

_Petrônio

_de

_Brito

_Ferreira

a_{UniversidadFederaldeGoiás(UFG),Goiânia,Goiás,Brazil} b_{EmbrapaArrozeFeijão,SantoAntôniodeGoiás,Goiás,Brazil} c_{UniversidadeEstataldeGoiás,Anápolis,Goiás,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received25June2015 Accepted20May2016

Availableonline4October2016 AssociateEditor:JerriÉdsonZilli

Keywords: Phaseolusvulgaris Rhizobium

Symbioticefficiency Carbonsourceuse Tolerancetosalinityand temperature

a

b

s

t

r

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c

t

Thisstudyaimedtoevaluatethetolerancetosalinityandtemperature,thegeneticdiversity andthesymbioticefficiencyofrhizobiaisolatesobtainedfromwildgenotypesofcommon beancultivatedinsoilsamplesfromtheStatesofGoiás,MinasGeraisandParaná.The iso-latesweresubjectedtodifferentNaClconcentrations(0%,1%,2%,4%and6%)atdifferent temperatures(28◦C,33◦C,38◦C,43◦Cand48◦C).Genotypiccharacterizationwasperformed basedonBOX-PCR,REP-PCRmarkersand16SrRNAsequencing.Anevaluationofsymbiotic efficiencywascarriedoutundergreenhouseconditionsinautoclavedLeonardjars.Among 98isolatesabout45%ofthemandRhizobiumfreireiPRF81showedahightoleranceto tem-perature,while24isolatesandRhizobiumtropiciCIAT899wereabletouseallofthecarbon sourcesstudied.Clusteringanalysisbasedontheabilitytousecarbonsourcesandonthe tolerancetosalinityandtemperaturegrouped49isolates,R.tropiciCIAT899andR.tropici H12withasimilaritylevelof76%.Basedongenotypiccharacterization,65%oftheisolates showedanapproximately66%similaritywithR.tropiciCIAT899andR.tropiciH12.About 20%oftheisolatesshowedsymbioticefficiencysimilartoorbetterthanthebestRhizobium referencestrain(R.tropiciCIAT899).Phylogeneticanalysisofthe16SrRNArevealedthattwo efficientisolates(ALSG5A1andJPrG6A8)belongtothegroupofstrainsusedascommercial inoculantforcommonbeaninBrazilandmustbeassayedinfieldexperiments.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Thecommonbean(PhaseolusvulgarisL.)isaleguminousplant ofworldwidesocialandeconomicimportance,providingmost ofthedailyrequirementsofproteinandcarbohydratesforthe poorestpopulationsofSouthandCentralAmerica,Africaand

∗ _{Correspondingauthor.}

E-mail:enderson.ferreira@embrapa.br(E.P.Ferreira).

India.1_{Withrespecttointernationalagriculture,Brazilisthe}

world’sthirdlargestproducerofcommonbean,accountingfor 12.7%ofworldwideproduction.2InBrazil,thecommonbean iscultivatedonatotalareaof3.1millionhectareswithatotal grainproductionofapproximately2.8milliontons,3_forwhich

highamountsofnitrogen(N)arerequired.

http://dx.doi.org/10.1016/j.bjm.2016.09.002

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Despiteitsabundanceintheatmosphere, Nisscarcein tropicalsoilsduetothefastmineralizationoforganicmatter intropicalconditions.Althoughthedecompositionoforganic matterisanimportantsourceofNforcrops,theadequate supplyofNtocropsdependslargelyontheuseofnitrogen fertilizers.4_{However,biologicalnitrogenfixation(BNF)is}

con-sideredamoresustainableapproachforsupplyingNtothe productionsystem.

BNFisakeyprocessfortheconversionofnitrogengas(N2) intoammonia(NH3)performedbybacteriabelongingtothe groupofrhizobia.ThereductionreactionofN2toNH3is car-riedoutbyN-fixingbacteriaordiazotrophicmicroorganisms containingtheenzymaticcomplexinwhichnitrogenasetakes part.5_{AmongN-fixingbacteriaoftherhizobiagroup,a}

vari-etyofRhizobiumand Ensiferspeciesisable tocolonizeand establishasymbioticpartnershipwithcommonbean.6,7

ToimproveBNFefficiency,moreefficientrhizobiastrains areneeded.Manyisolatingworkshavebeenperformedusing soilfromdifferentsites;however,astrapplantusuallyisused acommercialvarietyofcommonbean.Thestrategyusedin ourworkwastocollectsoilindifferentsitesandtousewild genotypesofcommonbeanastrapplantlookingforabetter explorationoftherhizobialcommunity,sincewildgenotypes showabroadergeneticbase.Thisworkaimedto character-izeanddeterminethesymbioticefficiencyofrhizobiaisolates obtainedfromtherootnodulesofwildgenotypesofcommon bean.

Materials

and

methods

Bacterialstrainsandrhizobiaisolates

TheisolatesevaluatedinthisworkwereobtainedbySampaio FB8_{andareavailableattheCollectionofMicroorganismsand}

MultifunctionalFungiofEmbrapaRiceandBeans.Strainsof Rhizobiumtropici(CIAT899andH12),Rhizobiumfreirei(PRF81) wereusedasreferencestrainsinallanalysesand,Rhizobium etlibv.phaseoli(CFN42)usedintheBOX-andREP-PCRanalyses. Carbonsourceuse(CSU)andtolerancetosalinityand temperature(TST)assays

CSU was assayed for 98 isolates and for the R. tropici ref-erence strains. Bacteriawere kept for growth on modified YMA(Yeast MannitolAgar) culturemedium,without man-nitol,addedwithindividualcarbonsourcessucrose,glucose, malicacid,maleicacid,nicotinicacid,inositol,sorbitol, arabi-nose,fructoseandglycerol.Afterincubationat28◦C,bacterial growthwasverifiedfrom48to96hateach24h.

Thesameisolatesandreferencestrainswereassessedfor TSTonYMAculturemediumonafactorial(5×5)arrangement (concentrationsofNaCl–0%,1%,2%,4%,6%andtemperature –28◦C,33◦C,38◦C,43◦C,48◦C)incubatedforaperiodof48h.

Genotypiccharacterizationbasedonmolecularmarkers Based on CSU and TST, 55 isolates were selected for genotypic characterization. Genomic DNA was extracted accordingtoAusubeletal.9_{DNAquantitywasestimatedby}

spectrophotometry(NanoDrop®_{,Thermoscientific,} Wilming-ton,USA),andDNAconcentrationwasadjustedto50ng␮L−1 for all samples. BOX-PCR was performed using the primer BOX A1R (5-CTACGGCAAGGCGACG-3), while REP-PCR was performedusingtheprimersREP-1(5-IIIICGICGICATCIGGC-3) and REP-2 (5-ICGICTTATCIGGCCTAC-3)according to Versa-lovicetal.10

TheBOX-PCRreactionwasperformedinafinalvolumeof 15␮L,containing2.9␮Lofmilli-Qwater,7.5␮Lof2×QIAGEN MultiplexPCRMasterMix(3mMMg2+_{),1.6␮LofprimerBOX} A1R(50pmol␮L−1_{)and3␮LofDNAtemplate(50ng␮L}−1_).The REP-PCRreactionwasperformedinafinalvolumeof15␮L, containing2.0␮Lofmilli-Qwater,7.5␮Lof2×QIAGEN Multi-plexPCRMasterMix(3mMMg2+_{),1.25␮LofeachprimerREP1} andREP2(10pmol␮L−1)and3␮LofDNAtemplate(10ng␮L−1). The amplification program was designed according to Kaschuketal.11_{PCRamplificationconsistedofaninitial}

dena-turingstep(95◦C;7min);followedby35cyclesofdenaturation (94◦C;1min),annealing(55◦C;1minforBOX-PCRand40◦C; 1minforREP-PCR)andextension(65◦C;8min);followedby afinalextensioncycle(65◦C;15min).ThePCRprogramwas performedinathermocyclerBiocycler®_{(AppliedBiosystems).} PCR products were subjected to electrophoresis on an agarosegel1%(50V;7h)inTAEbuffer0.75×12_using1kbDNA

Ladder®_{(Norgen)asaDNAbandpositionmarker.Theagarose} gelwasstainedwithSYBR®_{green(LifeTechnologies)and} visu-alizedwithaMultiDoc-it®_system.

Symbioticefficiencyundergreenhouseconditions

Basedongenotypiccharacterization,30isolateswereselected toevaluatetheirsymbioticefficiency.Inadditiontothe iso-lates,thetreatmentswerecomposedoftwoR.tropicistrains (CIAT899andH12),oneR.freireistrain(PRF81),twonitrogen fertilizedtreatments(NT1=60andNT2=120kgha−1ofN)and onecontroltreatment(CT–withoutinoculationandwithout N).

Seedsofcommonbeancv.Pérolaweresowninautoclaved Leonardjarsinarandomblockdesignwiththreereplicates. Atfivedaysafteremergence(DAE),plantletswereinoculated withacellsuspensioncontaining1×109_cellmL−1_ofeach iso-late andreference strain. Onceaweek,200mLofnutritive solution withoutNwere added.13 _{Tothenitrogenfertilized}

treatmentsNT1andNT2,1and2mL,respectively,ofasolution containing106.68mgmL−1ofureawereadded.

Plants were harvested at 35 DAE. Roots were carefully washed,driedinapapertowel,andthenodulesweredetached andcountedtodeterminethenumberofnodules(NN).The leavesweredetachedfromshootstodeterminetheleafarea (LA)usingaleafareameterLI-CORmodel3100.Shootsand noduleswere dried(65◦C;72h)todeterminethe shootdry mass (SDM)and nodule dry mass (NDM). Subsequently,to determineSDM,shootofplantsweremilledtodeterminethe totalN(N-Total)usingtheKjedahlmethod,asdescribedby SilvaandQueiroz.14

16SrRNAsequencinganalysis

Based on the symbiotic efficiency five isolates (ALSG5A1, JPrG1A1, JPrG6A8, JPrG8A7 and PCG4A2) were selected for

16SrRNAsequencing.TheDNAoftheisolateswasobtained accordingtoLaranjoetal.15_{The16SrRNAregionwas}

ampli-fiedbyPCRreactionusing theprimersY1 and Y3.15,16 _The

ampliconswerepurifiedandusedonthesequencingreaction accordingtoLaranjoetal.15_{Sequencescodingforthepartial}

16SrRNAgenes ofthe isolatesALSG5A1,JPrG1A1,JPrG6A8, JPrG8A7andPCG4A2wereobtainedandwhensubmittedto theGenBankdatabase (www.ncbi.nlm.nih.gov)receivedthe accessionnumbersKU598665,KU598663,KU598662,KU598664 andKU598661,respectively.

Statisticalanalyses

CSU, TST and genotypic characterizationdata were trans-formedintoabinarymatrix.Thebinarymatriceswereused fortheconstructionofasimilaritymatrixusingtheJaccard coefficient.TheUPGMA(Unweightedpair-groupmethod)was appliedtotransformthe similaritymatrix into asimilarity dendrogramusingNTSYSpc®_software.17

Dataobtainedfromthegreenhouseexperimentwere sub-jected to analysisof variance; when F was significant,the ScottKnotttestofmeanswasappliedwitha5%probability usingSISVARstatisticalsoftware.18_{Pearsoncorrelation}

anal-yseswerealsoperformedforNDMandSDM,LAandN-Total usingRstatisticalsoftware.19

Forthe16SrRNA-basedphylogeny,thesequencesobtained were submitted to NCBI BLAST against a non-redundant nucleotide database for getting homologous sequences.20

Sequencesshowingdegreeofsimilaritywerealignedusingthe CLUSTALWprogram.21_{Theevolutionaryhistorywasinferred}

usingtheMaximum-likelihoodmethod,22_withtree

consen-susbeinginferredfrom500replicatesusingbootstrap.23_The

evolutionarydistanceswerecomputed usingtheMaximum CompositeLikelihood method proposed byTamura et al.24

Allpositionscontaininggapsand missingdatawere elimi-nated.Therewereatotalof989positionsinthefinaldataset. EvolutionaryanalyseswereconductedinMEGA6.25

Results

CSUandTSTclusteringanalysis

CSUevaluationrevealedthedistributionof98isolatesamong 28groups,whileforTSTtheisolatesweredistributedinto27 groups.Amongthereferencestrains,R.tropiciCIAT899grew inall carbon sources, showingthe same reference ofCSU observedtothe24isolates(TableS1).RegardingTST,R.freirei PRF81showedgreatertoleranceamongthereferencestrains; however,18isolateswereabletogrowundermorestringent conditionsascomparedtoR.freireiPRF81(TableS2).

SimilarityanalysisbasedontheCSUandTSTdataresulted insimilaritiesamongisolatesvaryingfrom60%to100%,and fivephysiologicalgroups(PG)wereformedconsideringa sim-ilaritylevelof75%(Fig.1).PG1comprisesthereferencestrain R.freireiPRF81andthreeisolates,representing3%ofthetotal isolates.ThereferencestrainsR.tropiciCIAT899andR.tropici H12clusteredwith49isolatesonPG4,representing48.5%of theevaluatedisolates.Moreover,38,7and1isolatesformed PG2,PG3andPG5clusters,respectively(Fig.1).

60 70 75% 80 90 ALSG10A8 R.freirei PRF81 NVSG7A7 NVSG8A2 ALSG2A10 NVSG4A1 UnPaG3A6 NVSG3A4 ALSG5A4 UnPaG4A6 JPrG10A10 NVSG7A11 UnPaF3A17 UbALG8A9 NVSG11A3 NVSG2A6 NVSG1A1 NVSG3A3 UnPaG3A2 UnPaG7A6 UnPaG11A5 UnPaG6A3 NVSG2A1 JPrG1A9 ALSG2A5 UnPaG8A2 JPrG6A8 PCG5A8 UnPaG7A3 UnPaG3A5 JPrG1A1 JPrG9A9 UbALG3A5 UnPaG8A1 ALSG6A1 JPrG10A6 UbALG3A4 JPrG11A7 UnPaG6A2 JPrG5A7 ALSG5A6 UnPaG2A9 NVSG2A4 UnPaG8A7 NVSG5A3 UnPaG8A8 UbALG7A7 UnPaG8A4 NVSG8A9 ALSG11A2 JPrG4A10 PCG3A3 UnPaG6A5 ALSG5A1 JPrG8A7 PCG7A8 JPrG2A5 JPrG4A4 ALSG7A7 LASG9A8 UnPaG2A5 UnPaG11A9 PCG2A5 PCG10A7 PCG7A1 JPrG3A7 JPrG6A2 JPrG7A2 UbALG6A3 PCG4A2 PCG8A2 UnPaG8A12 UbALG4A6 ALSG2A9 JPrG8A6 UnPaG1A12 UnPaG4A13 PCG1A6 PCG4A6 PCG2A2 JPrG4A9 UnPaG4A9 PCG5A6 ALSG3A5 JPRG9A3 NVSG4A5 NVSG7A9 UbALG8A1 UnPaG6A7 UnPaG1A9 UnPaG3A19 R. tropici H12 UnPaG1A10 NVSG2A2 JPrG7A5 R. tropici CIAT899 UnPaG6A12 NVSG4A6 UnPaG1A2 NVSG6A2 JPrG5A1 100 PG5 PG4 PG3 PG2 PG1

Fig.1–ConsensusdendrogramobtainedbycombiningCSU andTSTdataof98isolatesfromwildgenotypesof

commonbean.Thedendrogramwasgeneratedusingthe UPGMAalgorithm,andthesimilaritymatrixwas determinedusingtheJaccardcoefficient.

GenotypiccharacterizationbasedonBOXandREP-PCR markers

Basedontheresultsofphysiologicalclustering(Fig.1),55 iso-lates were selectedforgenotypiccharacterizationwith the REP-andBOX-PCRmarkers.Usingthisapproach,isolateswere distributed among11 differentgenotype groups(GG)when consideringa65%similarity(Fig.2).

FingerprintinganalysisbasedonBOX-andREP-PCR mark-ers showed that most of the isolates (65.45%) clustered with the R. tropici reference strains R. tropici CIAT899 and R. tropici H12, forming GG1 with an approximately 65% similarity. The isolates JPrG4A9, UbALG7A7 and PCG4A2 showedanapproximately69%similaritywiththereference strainR. freireiPRF81(GG3). Moreover,the isolateALSG9A8 showedanapproximately 72%similarity withthe strainof R. etli bv. phaseoli CFN 42 (GG2). Even with a large frac-tionofisolatesclusteringwithR.tropicireferencestrains,no instancesofidenticalfingerprintingwereobservedamongthe bacteriastudied, indicating highpolymorphism amongthe isolates. ALSG10A8 PCG4A6 ALSG5A4 ALSG7A7 JPrG10A10 UbALG4A6 ALSG2A10 UnPaG2A9 NVSG2A6 JPrG7A2 NVSG11A3 UnPaG7A3 NVSG4A1 JPrG5A7 ALSG2A5 ALSG2A9 NVSG2A2 UnPaG1A9 NVSG6A2 UnPaG1A10 UnPaG3A2 JPrG1A1 UnPaG3A19 ALSG5A1 JPrG2A5 NVSG7A7 JPrG6A8 UbALG3A5 UnPaG8A2 JPrG8A6 PCG2A2 R. tropici H12 R. tropici CIAT899 JPrG3A3 UnPaG8A12 UnPaG2A5 PCG10A7 UnPaG4A9 ALSG9A8 CFN42 JPRG4A9 R.freirei PRF81 UbALG7A7 PCG4A2 JPrG1A9 JPrG4A10 UnPaG1A2 PCG1A6 PCG2A5 PCG5A6 JPrG6A2 UnPaG4A6 JPrG3A7 UnPaG8A1 JPrG9A3 UbALG8A9 UnPaG6A2 JPrG10A6 UnPaG11A9 GG1 GG2 GG3 GG4 GG5 GG6 GG7 GG8 GG9 GG10 GG11 100 85 70 55 65%

Fig.2–Consensusdendrogramobtainedbycombiningthe REPandBOX-PCRdataof55isolatesfromwildgenotypesof commonbean.Thedendrogramwasgeneratedusingthe UPGMAalgorithm,andthesimilaritymatrixwas determinedusingtheJaccardcoefficient.

Table1–Nodulenumber(NN–n◦plant−1),noduledry mass(NDM–mgplant−1),shootdrymass(SDM– gplant−1),leafarea(LA–cm2_plant−1_{)andtotalnitrogen}

(N-Total–gkgplant−1)ofcommonbeaninoculatedwith differentrhizobiaisolates.

Treatments NN NDM SDM LA N-Total R.tropiciCIAT899 110b 260a 1.5b 261b 21.6b R.freireiPRF81 52c 95b 0.5c 86c 12.3d R.tropiciH12 96b 150a 1.2b 184b 13.0d ALSG5A1 85b 131b 0.5c 100c 11.4d ALSG5A4 133a 279a 0.9c 150c 12.9d ALSG7A7 88b 150a 0.3c 61c 16.0c JPrG1A1 39c 23c 0.4c 72c 15.7c JPrG2A5 63b 243a 0.9c 135c 16.7c JPrG3A3 55b 73b 0.7c 123c 11.0d JPrG5A7 42c 101b 0.5c 83c 8.6d JPrG6A8 174a 372a 1.0b 154c 14.6c JPrG8A7 60b 331a 0.9c 154c 9.2d JPrG9A3 11d 22c 0.5c 83c 10.8d NVSG11A3 170a 341a 1.3b 220b 14.6c NVSG2A2 61b 303a 1.3b 247b 22.0b NVSG2A6 75b 196a 0.6c 220b 16.0c NVSG7A7 81b 271a 1.4b 206b 11.0d PCG10A7 30c 38c 0.4c 51c 10.2d PCG1A6 75b 53c 0.5c 77c 12.0d PCG2A5 95b 336a 1.5b 230b 13.2d PCG4A2 96b 292a 0.7c 126c 18.1c PCG4A6 43c 85b 0.5c 109c 17.0c UbALG3A5 37c 100b 1.2b 150c 13.1d UbALG4A6 89b 197a 1.1b 155c 14.0c UnPaG11A9 154a 334a 0.8c 197b 12.7d UnPaG1A10 52b 132b 0.8c 131c 9.0d UnPaG2A5 13d 21c 0.4c 54c 9.8d UnPaG2A9 28c 32c 0.6c 77c 9.9d UnPaG3A19 32c 101b 0.8c 108c 10.1d UnPaG3A2 74b 138a 0.6c 89c 11.1d UnPaG4A9 59b 174a 0.9c 138c 14.9c UnPaG6A2 98b 279a 0.8c 146c 18.3c UnPaG8A12 169a 384a 1.8b 285b 19.9b NT1 0d 0c 1.6b 242b 37.5a NT2 0d 0c 4.6a 486a 32.9a CT 0d 0c 0.3c 63c 14.8c Valuesfollowedbythesameletterinthesamecolumnwerenot sig-nificantlydifferent,asdeterminedbytheSkott–Knotttest(p<0.05).

Symbioticefficiencyundergreenhouseconditions

Toevaluatesymbioticefficiencyingreenhouseconditions,30 isolateswereusedbasedongenotypeclustering(Fig.2).The inoculationsignificantlyaffectedalloftheevaluated

parame-ters(Table1).

The NN showed significant difference among the iso-lates. The Significant greater values varied from 133 to 174nodulesplant−1 and, it were found for the isolates ALSG5A4,JPrG6A8,NVSG11A3,UnPaG11A9and UnPaG8A12. Amongthereferencestrains,thebestresultswereobserved for R. tropici CIAT899 and R. tropici H12, with 110 and 96nodulesplant−1, respectively. Those same isolates also showedhighNDMvalues;however,therewasnosignificant differencebetweenthereferencestrainsR.tropiciCIAT899and R.tropiciH12.Moreover,17isolatesshowedgreaterNDMthan theR.freireiPRF81(Table1).

RegardingSDM,greatervalueswereobservedforthe treat-mentcorrespondingto120kgha−1ofN(NT2).However,the isolates JPrG6A8,NVSG11A3, NVSG2A2, NVSG7A7, PCG2A5, UbALG3A5,UbALG4A6andUnPaG8A12showedvaluesofSDM similarto those obtainedbythe referencestrains R.tropici CIAT899andR.tropiciH12andbythetreatmentcorresponding to60kgha−1ofN(NT1)(Table1).

TheeffectofthetreatmentsonLAwassimilartothose on SDM, except for the isolates JPrG6A8, UbALG3A5 and UbALG4A6,whichhadlowervaluesofLAcomparedtothe ref-erencestrainsR.tropiciCIAT899andR.tropiciH12.Additionally, theisolatesNVSG2A6andUnPaG11A9showedvaluesequalto thoseobservedforthereferencestrainsR.tropiciCIAT899and R.tropiciH12(Table1).

Similar to SDM and LA, higher N-Total values were observed for NT1 and NT2 and between inoculated treat-ments. Among the inoculated treatments, the isolates NVSG2A2andUnPaG8A12hadN-Totalvaluesthatwere sta-tisticallysimilartothatobservedforthereferencestrainR. tropiciCIAT899(Table1).

Correlation analysis of NDM with parameters of shoot growth (SDM, N-Total and LA) allowed for the identifica-tion of different classes of symbiotic efficiencies, such as fewnodulation/fewgrowth,fewnodulation/highgrowth,high nodulation/fewgrowthandhighnodulation/highgrowth.The distributionoftheisolatesoccurredindifferentquartersof thegraphicdefinedinfunctionofthegeneralmeanofthe treatments(Fig.3).

CorrelationanalysesbetweenNDM×SDM,NDM×N-Total andNDM×LAshowedhighlysignificantresults,with corre-lationcoefficients(r)of0.7,0.48and0.78,respectively.This analysisshowed that forthe threecorrelations performed, theisolatesUnPaG8A12, NVSG11A3,NVSG2A2, JPrG6A8and UbALG4A6 and, the reference strain R. tropici CIAT899, were above the generalmean (Fig. 3).Remarkably the iso-latesUbALG3A5 and UnPaG4A9 (Fig. 3A), JPrG1A1,PCG4A6, ALSG7A7andUnPaG4A9(Fig.3B)andUbALG3A5(Fig.3C)also showedhighvaluesofSDW,N-TotalandLA,respectively,even withlowvaluesNDM.

RegardingthecorrelationbetweenNDMandSDM,10 iso-lates and the reference strain R. tropici CIAT899 appeared on the upper right quarter, indicating that these bacteria showahighcapacityforbothnodulationandaccumulation ofSDM.Incontrast,theisolatesUbALG3A5and UnPaG4A9, and thereference strainR.tropici H12,accumulated ahigh SDM,evenwithfewnodulations (Fig.3A).Theisolatesand the reference strainR. freireiPRF81 appearingin the lower leftquarterhadasymbioticperformancebelowthegeneral mean, and for those in the lower right quarter, the vege-tal growth was weak, even with high nodulation. For the NDM×N-Total correlation, nine isolates and the reference strainR.tropiciCIAT899showedahighsymbioticefficiency, promotinghighvaluesforbothNDMandN-Total.Inaddition, theisolatesJPrG1A1,PCG4A6,ALSG7A7andUnPaG4A9were ableto promotehigh N-Total accumulation withlow NDM (Fig.3B).

Regarding the NDM and LA correlation, twelve isolates andthe referencestrain R.tropici CIAT899appearedonthe upperrightquarter,indicatingthatthesebacteriashowhigh capacityforbothnodulationandLAgrowth,andtheisolate

UbALG3A5 andthe referencestrainR.tropici H12promoted highLAwithlowNDM(Fig.3C).

Sequencing analysis revealed the grouping ofthree iso-lates(ALSG5A1,JPRG1A1andJPrG6A8)intotheR.tropiciand R.freireicluster,whiletheothertwoisolatesgroupedwith Rhi-zobiumleguminosarumbv.viciae,R.leguminosarumbv.phaseoli, Rhizobiumfabae,RhizobiumpisiandRhizobiumphaseolistrains (Fig.4).

Discussion

TheclusteringanalysisbasedonCSUandTSTshowedthatthe Rhizobiumreferencestrainsclusteredwith52isolates,forming

1.9 1.5 R. freirei PRF81 R. freirei PRF81 R. tropici H12 R. tropici H12 R. tropici CIAT899 R. tropici CIAT899 UbALG3A5 UnPaG4A9 UnPaG8A12 UnPaG6A2 UnPaG4A9 UnPaG8A12 UbALG4A6 UbALG4A6 JPrG2A5ALSG5A4 y=0.0023x+0.4154 r=0.70∗∗ y=0.015x+10 912 r=0.48∗∗ y=0.4297x+62 655 r=0.78∗∗ ALSG7A7 JPrG8A7 JPrG2A5 JPrG1A1 JPrG6A8 JPrG6A8 NVSG2A2 NVSG7A7 NVSG2A2 NVSG11A3 NVSG2A6 PCG2A5 PCG4A2 PCG4A6 R. freirei PRF81 R. tropici H12 R. tropici CIAT899 UnPaG11A9 UnPaG6A2 UnPaG8A12 UbALG3A5 UbALG4A6

ALSG5A4 JPrG8A7JPrG6A8

NVSG2A2 NVSG7A7 NVSG11A3 NVSG2A6 PCG2A5 NVSG11A3 1.1 0.7 0.3 25 20 15 10 5 300 200 100 0 0 100 200 NDM (mg plant–1₎ LA (cm 2 plant –1 ) N-total (g kg plant –1 ) SDM (g plant –1) 300 400

A

B

C

Fig.3–Pearsoncorrelationanalysisfor(NDM)noduledry massand(SDM)shootdrymass.(A)NDMand(N-Total) totalnitrogen,(B)NDMand(LA)leafarea,and(C)common beaninoculatedwithdifferentrhizobiaisolates.Dashed linesrepresentthecorrelationtendency,andredlines representthegeneralmeanforeachparameter.**p<0.01.

R.freirei PRF81 (EU488742) R.trocipi H2 (EU488740) R.trocipi H12 (EU488743)

R.trocipi CIAT899 (U89832) R. leucaenae CFN299 (X67234) R. hainanense 166 (U71078) R. calliandrae CCGE542T (JX855162) R. multihospitium CCBAU83401 (EF035074) R. rhizogenes ATCC11325 (AY945955)

R. jaguaris CCGE525 (JX855169) R. vallis CCBAU65647T (FJ839677) R. lusitanum P-17 (AY738130)

R. mayense CCGE526T (JX855172) R. miluonense CCBAU41251 (EF061096)

R. mesoamericanum CCGE501 (JF424606) R. endophyticum CCGE2052 (EU867317) R. tibeticum CCBAU85039 (EU256404)

R. grahamii CCGE502 (JF424608) R. tubonense CCBAU85046 (EU256434)

R. etli CFN42 (U28916)

R. fabae CCBAU33202 (DQ835306) R. pisi DSM30132 (AY509899) R. phaseoli ATCC14482 (EF141340)

0.002

R. leguminosarum bv. viciae (U229386)

R. leguminosarum bv. phaseoli SED23-2 (KJ634555) PCG4A2 (KU598661)

JPrG8A7 (KU598664)

ALSG5A1 (KU598665) JPrG1A1 (KU598663) JPrG6A8 (KU598662)

Fig.4–Maximum-likelihoodphylogenyofthe16SrRNAgeneshowingtherelationshipsbetweenrhizobiaisolatesobtained fromwildgenotypesofcommonbean(inbold)andRhizobiumsp.referencestrains.GenBankaccessionnumbersareshown inparentheses.Bar,2ntsubstitutionsper1000nt.

thegroupsPG1andPG4. IsolatesgroupedinthesetwoPGs accountedformorethan50%oftheisolatesevaluated(Fig.1). The reference strain R. freirei PRF81 and the isolates NVSG7A7,ALSG10A8andNVSG8A2weregroupedinthePG1 cluster and showed a high TST. However, these bacteria showed weakCSU (Table S2).High TSTmay be associated withaspecificgeneticcontrol,conferringsuchabilitytothese bacteria.Gomesetal.26_{showedthatthePRF81(SEMIA4080)}

strainiscapableofdifferentiallyexpressingsomeconserved heat-responsiveproteins,suchasDnaKandGroEL.Theyalso reportedtheup-regulationofproteinsinvolvedinavarietyof metabolicpathways,includingtranslationfactorsand oxida-tivestress-responsiveproteins,indicatingthattheresponses ofR.freireistrainPRF81toheatstressgobeyondtheinduction ofheatshockproteins.

ReferencestrainsR.tropiciCIAT899andR.tropiciH12formed PG4togetherwith48.5%oftheevaluatedisolates.These bacte-ria were not able to grow under temperatures of 43 and 48◦C;however,theygrewuntil2%NaClcontentontheother testedtemperatures.Moreover,thesebacteriashowedgreater CSU than the bacteria ofthe PG1 (TableS1). These results

corroboratethoseofDall’Agnoletal.,6_{whoreportedagreater}

capacityofR.tropiciCIAT899touseCsourceascomparedto R.freireiPRF81.

In our work, R. tropici CIAT899 and R. tropici H12 were abletogrowonculturemediumcontainingglucoseand glyc-erol.According toCastellane and Lemos,27 _{theseCsources}

favorgrowthandexopolysaccharide(EPS)productioninthese strains. EPSproductionisrelatedtotheprocess ofrhizobia adaptationtolimitingenvironmentalconditions,28_allowing

thesebacteriatogrowunderconditionsofhightemperature and soil salinity.Thus, the evaluation oftemperature and salinitytoleranceisakeystepintheselectionprocessof rhi-zobiabecausesuchfactorscaninhibittheirgrowth.6,29,30

OnthefingerprintinganalysisbasedonBOX-andREP-PCR markersthehighestsimilarity(95.5%)wasidentifiedbetween theisolatesNVSG11A3andUnPaG7A3(GG1).Theseresults cor-roborate thework ofGrange,31 who reportedhighdiversity amongrhizobiaisolatesobtainedfromcommonbeannodules cultivatedinCerradosoils.

These markers are used to characterize and determine genotypicdifferencesamongbacterialstrainstodescribenew

species32–34_{andidentifyingtherhizobiastrainsusedto}

com-posetheBraziliancommercialinoculantsforcommonbean.35

Among the 30 isolates evaluated under greenhouse conditions, the isolates JPrG6A8, NVSG11A3, UnPaG8A12, UnPaG11A9andALSG5A4producedhighervaluesofNNthan the reference strains and,higher NDMthan the reference strainR.freireiPRF81.AlthoughNNisnotadetermining fac-torfortheefficiencyofBNF,36_{itmaybeindicativeofthehigh}

nodulationefficiencyofbacteria.Moreover,NNandNDMare measurementsfrequentlyusedasindicatorsofnodulation.37

WhenNNandNDMwereevaluatedsimultaneously, ver-ified that 80% of the isolates had better or equal results compared to those of the R. tropici and R. freirei reference strains.BecauseNDMshowsabettercorrelationwith symbi-oticperformance,36_{ourresultsdemonstratedthat17isolates}

producedsimilaramountsofNDMcomparedtothereference strainsR.tropiciCIAT899andR.tropiciH12,indicatingthehigh symbioticefficiencyoftheseisolates.

Resultsofnodulationhadadirecteffectonthe accumula-tionofdrymatterontheshootsoftheplants.Thetreatments withhigherNNandNDMalsoshowedhigh valuesofSDM andLA.Amongtheinoculatedtreatments,greatervaluesof SDM andLAwere found forthe referencestrains R.tropici CIAT899and R.tropici H12 and for the isolatesNVSG11A3, NVSG2A2,NVSG7A7,PCG2A5andUnPaG8A12.Measurements ofSDMand LAprovide importantinformationabout plant growth38 _{because these parameters are good indicators of}

plantnutritionalstatus,whichhasadirectinfluenceoncrop production.39,40 _{Moreover, this approachhasbeen used for}

strainselectiontocomposebacterialinoculants.40,41

Thenitrogencontentofplants(N-Total)wasalsoaffected by inoculation; however, only two isolates, NVSG2A2 and UnPaG8A12,showedN-Totalcontentsimilartothatofthe ref-erencestrainR.tropiciCIAT899.GreatervaluesofN-Totalwere observedtothenitrogentreatments.Similarresultsreported byGonzálesetal.41_{inafieldexperimentshowedthatnitrogen}

treatmentresultedingreaterN-Totalcomparedtoinoculation. TheuseofcorrelationofNDMwithparametersofshoot growth(SDM,N-TotalandLA)allowstoidentifymoreefficient isolates,whicharelocatedattheupperleftandupperright quarters(Fig.3).Interestingly,ourresultsrevealedahigh sym-bioticefficiencyoftheisolatesJPrG6A8,NVSG11A3,NVSG2A2, UbALG4A6andUnPaG8A12,showingNDM,SDM,N-Totaland LAvaluessimilartothebestreferencestrain,R.tropiciCIAT899. Moreover,theisolateUnPaG4A9alsohadpromisingresults, withhighvaluesofSDMandN-TotalevenunderlowNDM. Theseareveryinterestingfeaturesforrhizobialisolates,since itcan allowtoprovidebetterproducing resultsunderfield conditions.26,31,34,39

Sequencinganalysisofthe16SrRNAgenerevealedthatthe isolatesALSG5A1,JPrG1A1andJPrG6A8areverycloselyrelated withthereferencestrainsofR.tropiciCIAT899,R.tropiciH12 andR.freireiPRF81,whichareusedinBrazilascommercial inoculantforcommonbean42_{;however,theisolateJPrG1A1}

showedlowefficiencyinthegreenhouseexperiment(Table1). Consideringthatmanystudieshavediscussedthe impor-tance of investigating the efficiency of biological nitrogen fixationinthe selectionofisolates forthe developmentof inoculants,43,44 _{the isolatesALSG5A1 and JPrG6A8must be}

testedfortheiragronomicefficiencyunderfieldconditions,

aimingtostatetheirpotentialuseasinoculantforcommon bean.

Conclusions

Forty-fivepercentoftheisolatesevaluatedandthereference strain R. freirei PRF81 show high tolerance to temperature, while24% ofthe isolatesand thereferencestrain R.tropici CIAT899areabletouseallofthecarbonsourcesstudied. Clus-teringanalysisbasedonphysiologicalparametersgroup50% oftheisolates,R.tropiciCIAT899andR.tropiciH12witha simi-laritylevelof76%.REP-andBOX-PCRmarkersgroupabout65% oftheisolates,R.tropiciCIAT899andR.tropiciH12witha sim-ilaritylevelof66%.About20%oftheisolatesshowsymbiotic efficiencysimilartoorbetterthanthebestRhizobium refer-encestrain(CIAT899).Bythephylogeneticanalysisofthe16S rRNAtheisolatesALSG5A1andJPrG6A8belongtothegroup ofstrainsusedascommercialinoculantforcommonbeanin Brazil.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

The authors thank the National Council for Scientific and TechnologicalDevelopment(CNPq)andtheBrazilian Agricul-tural ResearchCorporation (Embrapa)bythe technical and financialsupport.

Appendix

A.

Supplementary

data

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,atdoi:10.1016/j.bjm.2016.09.002.

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