h tt p : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Genomic
identification
and
characterization
of
the
elite
strains
Bradyrhizobium
yuanmingense
BR
3267
and
Bradyrhizobium
pachyrhizi
BR
3262
recommended
for
cowpea
inoculation
in
Brazil
Jakson
Leite
a,
Samuel
Ribeiro
Passos
b,
Jean
Luiz
Simões-Araújo
c,
Norma
Gouvêa
Rumjanek
d,
Gustavo
Ribeiro
Xavier
d,
Jerri
Édson
Zilli
d,∗aDepartamentodeSolos,UniversidadeFederalRuraldoRiodeJaneiro,23851-970Seropédica,RJ,Brazil
bDepartamentodeCiênciasAmbientais,UniversidadeFederalRuraldoRiodeJaneiro,23851-970Seropédica,RJ,Brazil
cLaboratóriodeGenéticaeBioquímica,EmbrapaAgrobiologia,23851-970Seropédica,RJ,Brazil
dLaboratóriodeEcologiaMicrobiana,EmbrapaAgrobiologia,23851-970Seropédica,RJ,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received3October2016 Accepted12January2017 Availableonline31March2017 AssociateEditor:FernandoAndreote
Keywords: Vignaunguiculata Rhizobia MLSA DDH ANI
a
b
s
t
r
a
c
t
Theleguminousinoculationwithnodule-inducingbacteriathatperformbiologicalnitrogen fixationisagoodexampleofan“eco-friendlyagriculturalpractice”.Bradyrhizobiumstrains BR3267andBR3262arerecommendedforcowpea(Vignaunguiculata)inoculationinBrazil andshowedremarkableresponses;neverthelessneitherstrainwascharacterizedatspecies level,whichisourgoalinthepresentworkusingapolyphasicapproach.Thestrains pre-sentedthetypicalphenotypeofBradyrhizobiumwithaslowgrowthandawhitecolonyon yeastextract-mannitolmedium.StrainBR3267wasmoreversatileinitsuseofcarbon sourcescomparedtoBR3262.ThefattyacidcompositionofBR3267wassimilartothetype strainofBradyrhizobiumyuanmingense;whileBR3262wassimilartoBradyrhizobiumelkaniiand
Bradyrhizobiumpachyrhizi.Phylogeneticanalysesbasedon16SrRNAandthree
housekeep-inggenesplacedbothstrainswithinthegenusBradyrhizobium:strainBR3267wasclosest
toB.yuanmingenseandBR3262toB.pachyrhizi.Genomeaveragenucleotideidentityand
DNA–DNAreassociationconfirmedthegenomicidentificationofB.yuanmingenseBR3267
andB.pachyrhiziBR3262.ThenodCandnifHgeneanalysesshowedthatstrainsBR3267and
BR3262holddivergentsymbioticgenes.Insummary,theresultsindicatethatcowpeacan establisheffectivesymbiosiswithdivergentbradyrhizobiaisolatedfromBraziliansoils.
PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia. ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.
org/licenses/by-nc-nd/4.0/).
∗ Correspondingauthor.
E-mail:jerri.zilli@embrapa.br(J.É.Zilli).
https://doi.org/10.1016/j.bjm.2017.01.007
1517-8382/PublishedbyElsevierEditoraLtda.onbehalfofSociedadeBrasileiradeMicrobiologia.Thisisanopenaccessarticleunderthe CCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Bacteriacollectively known as rhizobia forman important partofthe soilmicrobiota and performbiological nitrogen fixation (BNF) through nitrogenase activity when in sym-biosiswithleguminousplants.Thisecologicalphenomenon hasgreatbiotechnologicalimpactonbiomassandgrain pro-duction. For example, in the soybean crop production in Brazil it isestimated to save over US$ 10 billion annually
bytheuseofBradyrhizobiuminoculationinsteadofchemical
fertilization.1
TheBrazilianMinistryofAgriculturehasalistofrhizobial strains recommended formore than 50 leguminous grain-producingcrops,forageandgreenmanure.2 Inrecentyears
effortshavebeen madetobettercharacterizethesestrains recommendedforinoculationinBrazilandatleastfivenew specieshavebeendescribed.3–6
Brazilistheworld’sthirdleadingcowpeaproducer, with anestimatedproductionof500,000tonsperyear.7Thecrop
yieldvariesfrom400to2000kgha−1,dependingonthesystem andtheregionofcultivation.7Thisyieldhasbeenrisingin
recentyearswithimprovementsincultivationmanagement, suchastheinoculationofseedswithnitrogen-fixingbacteria, apracticeappliedtoapproximately100,000hectares.
ThestrainsBR3267andBR3262areconsideredas“elite” forinoculationofcowpeaplantsinBrazil2andseveral
stud-iesundercontrolledandfieldconditionshaveshownthatboth strainsmakesignificantcontributionstocropyields,including morethan50%NaccumulationviaBNF.8BR3267strainwas
isolatedfromthesemiaridnortheasternregionofthecountry, usingcowpeaastrapplants,whileBR3262strainwasisolated fromanAtlanticForestareainsoutheasternBrazilusingthe samestrategy.1,9 Althoughthesestrainswereisolatedmore
than a decade ago, they were only partially characterized throughassessmentofthegrowth rate,colonymorphology and16SrRNAphylogeny,11 butnotclassifiedatthespecies
level.
Zilli and colleagues11 have characterized the 16S rRNA
genes of the BR 3267 and BR 3262 strains and concluded that both are members of the genus Bradyrhizobium. This genuswascreated inthe early1980stoaccommodate root nodule-inducing bacteria with slowgrowth on media con-taining mannitol and yeast extract.12 Since the turn of
the century, two major subgroup divisions (I and II) have been recognized within this genus based on DNA–DNA hybridization.12–14TheBradyrhizobiumjaponicumand
Bradyrhi-zobium elkaniiwere the firstspeciesassigned tosubgroupI
andII,respectively.15,16AccordingtoZilliandcolleagues,10BR
3267clusteredwithinthesubgroupB.japonicumandBR3262 withinthe subgroupB.elkanii.However,inthe lightof cur-rentknowledge,thoseresultscanbeconsideredinconclusive becausenewBradyrhizobiumspecieswasrecentlydescribed. Thus, furtherinvestigation employing the latestmolecular techniques is needed for the correct positioning of these strains.
In the past five years, the use of housekeeping genes as powerful phylogenetic markers for bacteria has led to the description of over 15 new species within the genus
Bradyrhizobium17 and enabled the separation of genetically
close strains into different species. Examples are the def-inition ofthe species Bradyrhizobiumdiazoefficiens based on
B.japonicumandBradyrhizobiumpachyrhizifromB.elkanii.3,18
Furthermore,newmethodsforgenome-togenome compari-son,likeAverageNucleotideIdentity(ANI)andGenomeBlast DistancePhylogeny(GBDP)havebeenintroducedandare con-tributingtoimprovebacterialtaxonomy.19,20
Therefore,thegoal ofourstudywastocharacterizeata finer thetaxonomiclevelbothBR3267andBR3262strains usingapolyphasicapproach.
Materials
and
methods
Strainsused
The cowpea strains BR 3267 and BR 3262, and the type strain B. elkaniiUSDA 76T were obtainedfrom the Johanna
DöbereinerBiologicalResourceCenter(CRB-JD,Embrapa Agro-biologia, Seropédica-Riode Janeiro,Brazil).Thetypestrains
B.pachyrhiziPAC48T(=LMG24246T)andBradyrhizobium
yuan-mingenseCCBAU10071T(=LMG21827)wereobtainedfromthe
LMGCultureCollection(Belgium).Thestrainsweregrownon yeastextract-mannitolagar medium(YMA)and were incu-batedat28◦C21forsevendaysuntiltheyreachedsufficient colonygrowthlevelstoobservemorphologicalfeaturesand purity.
Phenotypicandphysiologiccharacterizations
Inoculum preparationforbothBR 3267andBR3262strains wascarriedoutusingYMAmediumat28◦C.Carbonsource utilizationwasassessedwithBiologGN2microplates(Biolog Inc., Hayward, CA) following the manufacturer’s instruc-tions, except that cell concentration was adjusted to5 on the McFarland scale. Plates were incubated in the darkat 28◦Cforten days.Thebiochemical featureswereassessed using API 20 NE strips (bioMérieux, Marcy-L’Etoile, France) following a standard protocol that uses a saline solution (0.85%NaCl)forbacterialsuspension.Additionally,the toler-ancetoabioticstress,suchastemperature,pHandsalinity (NaCl), was determined by examining the growth in YMA medium. The temperature tolerance was evaluated at 15, 20, 25, 28, 30, 32 and 37◦C, and the pH tolerance was tested in arange from 4 to10. Thesalinitytolerance was examined at 28◦C in YMA medium supplemented with 0.1, 0.3, 0.5, 1.0, 1.5, 2.0 or 2.5% (w/v) of NaCl. The resis-tance to antibiotics was determined using YMA medium and the disk diffusionmethodforampicillin (25g), chlor-amphenicol(50g),erythromycin(30g),gentamicin(10g), kanamycin(30g),neomycin(10g),penicillin(10g), strep-tomycin(10g)andtetracycline(30g).Alltestswererunin triplicate.
Fattyacidcomposition
The BR 3267and BR 3262and type strains B.elkanii USDA
76T,B.pachyrhiziPAC48TandB.yuanmingenseCCBAU10071T
type strains were characterized based on whole-cell fatty acids,derivedusing themethyl esters’method(FAME) and
analyzedwith aHewlett Packardgaschromatograph fitted with a fused silica capillary column (25m×0.2mm inter-nal diameter). The type strains B. elkanii USDA 76T, B.
pachyrhiziPAC48T and B.yuanmingense CCBAU10071T were
chosenbecausetheywere the closestrelated strainstoBR 3262 and BR 3267 respectively, based on EzTaxon identi-ficationsearch(http://www.ezbiocloud.net/eztaxon/identify). ChemStation A.09.01 software [1206] and a MIDI Micro-bial Identification System 4.0(Sherlock TSBA Library, MIDI ID, Inc., Newark, DE, USA) were used for the fatty acid identification.
DNAextraction,PCRamplificationandsequencingofPCR fragments
Pure cultures ofBradyrhizobiumstrains were grown on YM mediumfor4daysat28◦Cunderstirringof120rpm.Then, 2mL of cell suspension were centrifuged (12,000×g), and the total genomic DNA was extracted using a Wizard® GenomicDNAPurificationKit(Promega,USA)followingthe manufacturer’s instructions. The recA gene was amplified by PCR using the primers TSrecAf and TSrecAr, and the
glnII gene was amplified with the primers TSglnIIf and
TSglnIIr.22 Theprimers NodCfor540and NodCrev1160 were
used to amplify the symbiotic nodC gene, as described by Sarita et al.23 For the nifH, the primers NifHF/NifHI were
used followingthe methods described byLaguerre et al.24
DNA sequences of the PCR products were obtained for bothstrands with the same primers used for gene ampli-fications. The 16S rRNA sequences of the BR 3267 (Gene BankA.no. AY649439)andBR3262(A.no.AY649430)strains obtainedinapreviousstudy10wereretrievedfromGenBank
(http://www.ncbi.nlm.nih.gov/genbank/). For the gyrB gene,
thesequenceswereretrievedfromdraftgenomesequences ofthe BR 3267and BR 3262 strains(Ac. no. KT005410 and KT005409,respectively).
Phylogeneticanalysis
Forward and reverse readings of the 16S rRNA, recA, glnII, nodC and nifH gene fragments were edited and assem-bled using DNABASER (http://www.dnabaser.com). Multiple sequencealignmentswereperformedusingClustalWthrough MEGA6.0.25 Maximumlikelihood(ML)phylogenetic
recons-tructions were completed using MEGA 6 for single gene sequences(16SrRNA,recA,glnII,gyrB,nodCandnifH),anda multilocussequence analysis (MLSA) wasperformed using theconcatenatedsequencesofthehousekeepinggenesrecA,
glnIIand gyrB.Thedistancematriceswerecalculatedusing
the Kimura two-parameter substitution model,26 and the
robustness of the tree nodes was evaluated with a boot-strap analysis27 using 500 pseudoreplicates. The software
default parameters were considered in all of the analy-ses.Sequencesofrhizobialtypestrains usedforalignment and phylogenetic analysis were retrieved from GenBank
(http://www.ncbi.nlm.nih.gov/genbank/),andaMicrovirga
vig-nae BR 3299 strain28 was used as the outgroup in the ML
analysisofthe16SrRNAgene.
DNA–DNArelatednessandaveragenucleotideidentity (ANI)
TheDNA–DNA hybridization(DDH)amongbacterialstrains wasdeterminedbasedonthethermaldenaturation temper-aturesofhybridandhomologousgenomicDNA,asdescribed byGonzalezandSaiz-Jimenez,29,30exceptthattheDNA
con-centrationwasadjustedto2gperreaction.Theexperiments were performed in 96-well optical plates with appropriate optical adhesives in three replicates and including wells without DNA asnegativecontrol.For DNA hybridization,a Bio-RadMyCiclerthermocyclerwasused,andfor fluorimet-ricmeasurementsduringthedenaturationstageanApplied Biosystems7500Real-Timesystemwasused.Thermal con-ditionsforhybridizationconsistedonadenaturationstepof 99◦Cfor10min,followedbyanannealingperiodof8hat79◦ C(optimumtemperatureforrenaturation–TOR).29,31 Itwas
followedbyprogressive10-minsteps,eachat1.8◦Cbelowthe previousone,until25◦Cwhenitwasholdfor30minbefore refrigerationto4◦C.Thefluorescencewasmeasuredusinga denaturationrampsettledatthestepandholdmode.Heating rate was 0.2◦Cs−1 with fluorescence decreasing measure-mentateach0.2◦Cstep,duringa12shold,andbetween25 and99.9◦C.29TheDDHexperimentswereperformedbetween
strainsBR3267andBR3262andtheirphylogeneticallyrelated typestrainsbasedonthe16SrRNAgenesimilarities.BR3267 wascomparedagainstB.yuanmingenseCCBAU10071T,andBR
3262wascomparedagainstB.pachyrhiziPAC48TandB.elkanii
USDA76T.
TheANIestimationwasperformedwithaJSpecies plat-form version 1.2.1,32 and MUMmer was used for genome
alignment.33Allsystemrequirements(BLASTandMUMmer)
weredownloadedandinstalledlocallyinasystemLinux. Bac-terial genome were retrieved from NCBI database as fasta fileandusedtoANIcalculation,consideringJSpeciesdefault parameters.ThesequencedgenomeofstrainBR3262(Ac.no. LJYE00000000.1)34 was comparedtogenomesofthe strains
USDA 76T (B.elkanii – GenBankA.no. ARAG00000000.1)and
PAC48T (B. pachyrhizi–GenBankA.no. LFIQ00000000.1).The
genome ofthe strainBR 3267(A.no. LJYF00000000.1)35 was
comparedtothatofthestrainCCBAU35157(B.yuanmingense
–A.no.AJQL00000000.1).
Results
Phenotypicandphysiologicfeatures
BothBR3267andBR3262strainscangrowinapHrangeof 4–10, tolerate upto 0.5%NaCl inthe mediumand present normal growth in a temperature range from 15 to 32◦C, andBR3267couldalsogrowupto37◦C(Table1).Likewise, bothstrainsshowedpositivereactionstoenzymeureaseand nitratereductase.Theirsusceptibilitiestodifferentantibiotics werevariable(Table1).BR3267wassensitivetostreptomycin andtetracycline,whileBR3262wastolerant.Withrespectto thecarbonsourcesevaluatedwiththeBiologkit,BR3262strain wasabletouse33ofthe95sourcestested,whileBR3267strain wasabletousemorethan50.
Table1–PhenotypicfeaturesofthecowpeaBradyrhizobiumstrainsBR3267andBR3262ascharacterizedbyAPI20NE andBiologGN2microplates.Dataobtainedfrom5daysduplicatereadmeanvalues(+=positive;−=negative,w=weak).
Phenotypicfeature BR3267 BR3262 Phenotypicfeature BR3267 BR3262 Phenotypicfeature BR3267 BR3262
Enzymaticreaction pH4 + + Ester
Catalase + − pH10 + + Methylpyruvate + −
Hydrolysisofgelatine + − 1%NaCl − − Mono-methyl-succinnate + −
Hydrolysisofesculin + + Carbohydrates Aminoacids
ˇ-galactosidase w − d-Arabitol + − d-Alanine − +
Resistanceto(g) ␣-d-Glucose + − l-Alanine − +
Ampicillin(25) − + d-Mannose + − Glycyl-l-asparticacid + −
Chloramphenicol(50) − + d-Psicose + − l-Leucine + −
Kanamycin(30) + − d-Sorbitol + − l-Phenylalanine + −
Neomycin(10) + − Carboxylicacids l-Proline + −
Penicillin(10) − + d-Galacturonicacid + − d-Serine + −
Streptomycin(10) − + d-Glucosaminicacid + − l-Threonine + −
Tetracycline(30) − + ␣-Ketovalericacid + − ␥-Aminobutyricacid + −
Erythromycin(30) + + d,l-Lacticacid + − Polymer
Gentamicin(10) + + Malonicacid + − Dextrin − +
Growthat Quinicacid + − Amide
15◦C + + d-Saccharicacid + − Glucuronamide + −
32◦C + + Sebacicacid + − Amine
37◦C + − Succinicacid + − Phenylethylamine + −
BothstrainsBR3267andBR3262werepositiveto:Oxidase,Urease,Nitratereduction,Hydrolysisofesculin,l-Arabinose,l-Fucose,d-Galactose,
d-Mannitol,l-Rhamnose,Tween40,Tween80,Aceticacid,Citricacid,Formicacid,d-Galactonicacidlactone,d-Gluconicacid,␣-Hydroxybutyric
acid,-Hydroxybutyricacid,␥-Hydroxybutyricacid,p-Hydroxyphenylaceticacid,␣-Ketobutyricacid,␣-Ketoglutaricacid,Propionicacid,
Glyc-erol,l-Alanyl-glycine,l-Asparagine,l-Asparticacid,l-Glutamicacid,Glycyl-l-glutamicacid,l-Pyroglutamicacid,l-Serine,Succinamicacid,
l-AlaninamideandBromosuccinicacid; andnegative to:Tryptophandeaminase,Glucosefermentation,Argininedihydrolase,
N-Acetyl-d-galactosamine,N-Acetyl-d-glucosamine,Adonitol, d-Cellobiose,i-Erythritol,d-Fructose, Gentiobiose,m-Inositol, ␣-d-Lactose,Lactulose,
Maltose,d-Melibiose,-Methyl-d-Glucoside,d-Raffinose,Sucrose,d-Trehalose,Turanose,Xylitol,␣-Cyclodextrin,Glycogen,cis-Aconiticacid,
d-Glucuronicacid,Itaconicacid,2,3-Butanediol,d,l-␣-Glycerolphosphate,Glucose-1-phosphate,Glucose-6-phosphate,l-Histidine,
Hydroxy-l-proline,l-Ornithine,Urocanicacid,Inosine,Uridine,Thymidine,Putrescineand2-Aminoethanol.
Fattyacidcomposition
The fatty acid composition was analyzed for the BR 3262 andBR3267strainsandforB.elkaniiUSDA76T,B.pachyrhizi
PAC48TandB.yuanmingenseCCBAU10071T,theclosesttype
strainsbasedonEzTaxonIdentifysearchofthe16SrRNAgene
(http://www.ezbiocloud.net/eztaxon/identify).Thefatty acid
compositionsofBR3267andBR3262weresimilartothoseof otherstrainswithinBradyhizobiumgenus,especiallybecause ofthepresenceof16:00inapproximately15%andasummed feature8(18:1w7c)betweenapproximately70and80%.40BR
3262hasacompositionclosetothoseofB.elkaniiUSDA76T
andB.pachyrhiziPAC48T,withthepresenceof19:0cyclow8cof
15.98%andasummedfeature8(18:1w7c)of69.32.Instead,BR
3267andB.yuanmingenseCCBAU10071Tdidnotpresent19:0
cyclow8candhadasummedfeature8of86.42and84.63%, respectively.
Phylogeneticanalyses
The Maximum Likelihood (ML) phylogenetic analysis per-formedon the16S rRNA (1227bp) geneplacedthe cowpea strainsBR3267andBR3262intosubgroupsIandII, respec-tively, withinthe genus Bradyrhizobium(Fig.1). Theclosest straintoBR3267wasB.yuanmingenseCCBAU10071T(99.5%
16SrRNAsimilarity),with67%MLbootstrapsupport,followed
byBradyrhizobiumsubterraneum582-1T(99.6%16SrRNA
simi-larity)inaseparatedbranchwith62%bootstrap.Incontrast, BR3262wasplacedwithinsubgroupIIofBradyrhizobiumina
phylogeneticlinagetogetherwithB.pachyrhiziPAC48T(100%
16SrRNAsimilarity),B.elkaniiUSDA76T(99.9%)and
Bradyrhizo-biumtropiciagriCNPSo1112T(99.2%),butwithnodeslowerthan
<60%ofbootstrap.TheBradyrhizobiumferriligniCCBAU51502T
wastheclosesttaxontothecladeofB.tropiciagri–B.elkanii–B.
pachyrhizi–BR3262,asshownbythehighMLbootstrap
sup-port(91%,Fig.1);itshares97.8%of16SrRNAsimilaritywith BR3262.
We usedaligned sequences from the recA (359bp),glnII
(505bp)andgyrB(552bp)housekeepinggenes,whichresulted inaconcatenatedsequenceof1416bp,tobetterassess the phylogeneticaffiliationoftheBR3267andBR3262strains.ML phylogenetic treesofsingleandconcatenatedgenes(Fig.2) weregeneratedandshowedhighcongruence;therefore,we presentedonlytheconcatenatedone.Theconcatenated phy-logeneticanalysisrevealedaclearaffiliationbetweenBR3267
and B. yuanmingense CCBAU 10071T, with high confidence
bootstrapvaluesofthe100%,inalineage thatwasdistinct from the closestsoybean isolatesBradyrhizobiumdaqingense
CCBAU15774T48andBradyrhizobiumhuanghuaihaienseCCBAU
23303T.49 Ontheother hand,BR3262wasphylogenetically
close toB. pachyrhiziPAC48T, whichformed alineage with
high confidence values (bootstrap ML of 98%) and sepa-ratedfromtheclosedtaxaB.ferriligniCCBAU51502T andB.
elkanii USDA76T. Additionally, pairwise comparisonsof the
concatenated sequences revealed that BR 3267shared 99% sequence identity withB.yuanmingense CCBAU 10071T,
fol-lowed byBradyrhizobiumliaoningense USDA 3622T (95%) and
65 72 60 95 60 59 7365 93 90 81 85 50 Bradyrhizobium manausense BR 3351T (HQ641226) Bradyrhizobium guangdongense CCBAU 51649T (KC508867) Bradyrhizobium ganzhouense RITF806T (JQ796661)
Bradyrhizobium cytisi CTAW11T (EU561065) Bradyrhizobium rifense CTAW71T (EU561074)
Bradyrhizobium vignae 7-2T (KP899563)
Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231463) Bradyrhizobium arachidis CCBAU 051107T (HM107167)
Bradyrhizobium kavangense 14-3T (KP899562) Bradyrhizobium iriomotense EK05T (AB300992) Bradyrhizobium ingae BR 10250T (KF927043) Bradyrhizobium guangxiense CCBAU 53363T (KC508877) Bradyrhizobium diazoefficiens USDA 110T (NC_004463) Bradyrhizobium betae LMG 21987T (AY372184)
BR 3267 (AY649439)
BR 3262 (AY649430)
Bradyrhizobium yuanmingense CCBAU 10071T (AF193818)
Bradyrhizobium subterraneum 58 2-1T (KP308152) Bradyrhizobium liaoningense USDA 3622T (AF208513)
Bradyrhizobium daqingense CCBAU 15774T (HQ231274) Bradyrhizobium japonicum USDA 6T (U69638)
Bradyrhizobium canariense BTA-1T (AJ558025) Bradyrhizobium ottawaense OO99T (JN186270)
Bradyrhizobium denitrificans LMG 8443T (X66025) Bradyrhizobium oligotrophicum LMG 10732T (JQ619230) Bradyrhizobium retamae Ro 19T (KC247085)
Bradyrhizobium valentinum LmjM3T (JX514883)
Bradyrhizobium neotropicale BR 10247T (KF927051) Bradyrhizobium embrapense CNPSo 2833T (AY904773) Bradyrhizobium erythrophlei CCBAU 53325T (KF114645) Bradyrhizobium viridifuturi SEMIA 690T (FJ025107)
Bradyrhizobium lablabi CCBAU 23086T (GU433448) Bradyrhizobium icense LMTR 13T (KF896156) Bradyrhizobium paxllaeri LMTR 21T (AY923031)
Bradyrhizobium jicamae PAC68T (AY624134)
Bradyrhizobium pachyrhizi PAC48T (AY624135) Bradyrhizobium elkanii USDA 76T (U35000)
Bradyrhizobium tropiciagri CNPSo 1112T (AY904753) Bradyrhizobium ferriligni CCBAU 51502T (KJ818096) Microvirga vignae BR 3299T (JX504804)
Bradyrhizobium yuanmingense CCBAU 35157 (AJQL01000001.1)
0.02
Fig.1–MaximumLikelihoodphylogenetictreebasedon16SrRNAgenesequencesshowingrelationshipsbetweenthe strainsBR3262andBR3267andthetypespecies(T)ofthegenusBradyrhizobium.Bootstrapvalueswereinferredfrom500 replicatesandareindicatedatthetreenodeswhen≥50%.GenBankaccessionnumbersareprovidedinparenthesis.
MicrovirgavignaeBR3299Twasincludedastheoutgroup.Thebarrepresentstwoestimatedsubstitutionsper100nucleotide
positions.
similar toB. pachyrhizi PAC48T (98.6%), followed by B.
elka-niiUSDA76T (96.9%)andB. ferriligniCCBAU51502T (95.5%).
In an independent analysis of the recA, glnII and a con-catenated recA-glnII sequences (data not shown), BR 3267 and BR 3262 were far from the newest proposal species
Bradyrhizobium kavangense 14-3T,50 Bradyrhizobium vignae
7-2T51 and B. subterraneum 58-2-1T52 isolated from African
soils.
DNA–DNArelatednessandaveragenucleotideidentity
Afterobtainingthephylogeneticresults,weestablishedthe contrastsfortheanalysisoftheDNA–DNAhomology follow-ingtheprotocoldescribedbyGonzalesandSaiz-Jimenez.29BR
3262wascomparedwithB.elkaniiUSDA76TandB.pachyrhizi
PAC48T(thecloseststrainsaccordingtoMLSA),andBR3267
57 80 67 54 85 98 99 100 64 100 95 99 99 99 95 98 99 98 0.02
Bradyrhizobium cytisi CTAW 11T (GU001575, GU001594, JN186292)
Bradyrhizobium ganzhouense RITF806T (JX277144, JX277110, KP420022)
Bradyrhizobium rifense CTAW71T (GU001585, GU001604, KC569466)
Bradyrhizobium canariense BTA-1T (AY591553, AY386765, AB353736)
Bradyrhizobium betae LMG 21987T (FM253174, AB353733, AB353735)
Bradyrhizobium diazoefficiens USDA 110T (NC_004463, NC_004463, NC_004463)
Bradyrhizobium japonicum USDA 6T (AM168341, AF169582, AM418801)
Bradyrhizobium ottawaense OO99T (HQ587287, HQ587750, HQ873179)
Bradyrhizobium liaoningense USDA 3622T (AY494833, AY494803, FM253223)
Bradyrhizobium arachidis CCBAU 051107T (HM107233, HM107251, JX437675)
Bradyrhizobium neotropicale BR 10247T (KJ661714, KJ661700, KJ661707)
Bradyrhizobium iriomotense EK05T (AB300996, AB300995, HQ873308)
Bradyrhizobium ingae BR 10250T (KF927061, KF927067, KF927079)
Bradyrhizobium huanghuaihaiense CCBAU 23303T (HQ231595, HQ231639, JX437672)
Bradyrhizobium daqingense CCBAU 15774T (HQ231270, HQ231301, JX437669)
Bradyrhizobium yuanmingense CCBAU 10071T (AY591566, AY386780, FM253226)
Bradyrhizobium manausense BR 3351T (KF785992, KF785986, KF786000)
Bradyrhizobium guangdongense CCBAU 51649T (KC509269, KC509023, KC509072)
Bradyrhizobium guangxiense CCBAU 53363T (KC509279, KC509033, KC509082)
Bradyrhizobium denitrificans LMG 8443T (FM253196, HM047121, FM253239)
Bradyrhizobium oligotrophicum LMG 10732T (JQ619231, JQ619233,KC569467)
Bradyrhizobium jicamae PAC68T (HM590776, FJ428204, AM418801)
Bradyrhizobium paxllaeri LMTR 21T (JX943617, KF896169, KF896195)
Bradyrhizobium lablabi CCBAU 23086T (GU433522, GU433498, JX437670)
Bradyrhizobium retamae Ro19T (KC247094, KC247108, KF962698)
Bradyrhizobium icense LMTR13T (JX943615, KF896175, KF896201)
Bradyrhizobium valentinum LmjM3T (JX518589, JX518575,NZ_LLXX01000044.1)
Bradyrhizobium erythrophlei CCBAU 53325T (KF114669, KF114693, KF114717)
Bradyrhizobium tropiciagri CNPSo 1112T (FJ391168, FJ391048, HQ634890)
Bradyrhizobium embrapense CNPSo 2833T (HQ634899, GQ160500, HQ634891)
Bradyrhizobium viridifuturi SEMIA 690T (KR149140, KR149131, KR149134)
Bradyrhizobium elkanii USDA 76T (AY591568, AY599117, AB070584)
Bradyrhizobium ferriligni CCBAU 51502T (KJ818112, KJ818099, KJ818102)
Bradyrhizobium pachyrhizi PAC48T (HM590777, FJ428201, HQ873310)
BR 3267 (KF828822, KF828821, KT005410)
BR 3262 (KF828817, KF828816, KT005409)
Fig.2–Unrootedmaximumlikelihoodphylogenetictreebasedonthreeconcatenatedsequences(recA,glnII,gyrB)showing relationshipsbetweenthestrainsBR3262andBR3267andtypespecies(T)ofthegenusBradyrhizobium.Thephylogenetic treewasbuiltusingtheKimuratwo-parametermethod.Bootstrapvalueswereinferredfrom500replicatesandare indicatedatthetreenodeswhen≥50%.GenBankaccessionnumbersareprovidedinparenthesis.Thebarrepresentstwo estimatedsubstitutionsper100nucleotidepositions.
closeststrainintheMLSA).ThehybridizationofBR3262×B.
elkaniiUSDA76TandB.elkaniiUSDA76T×B.pachyrhiziPAC48T
presentedTmvaluesequalto10.1◦Cand3.9◦C,respectively,
and the hybridization of BR 3267×B. yuanmingense CCBAU 10071Tpresentedavalueof3.9◦C(Fig.3).
FortheANIcalculation,weusedtheavailablegenomesof
B.elkaniiUSDA76TandB.pachyrhiziPAC48Tforcomparison
withBR3262and,B.yuanmingenseCCBAU3515754for
compar-isonwithBR3267becausenogenomewasavailableforthe typestrainofB.yuanmingenseCCBAU10071T.TheANIbetween
strainBR3262andB.elkaniiUSDA76TandbetweenstrainBR
3262andB.pachyrhiziPAC48Twas94.6and95.3,respectively.
OnanotherhandtheANIinthecomparisonofBR3267andB.
yuanmingenseCCBAU35157was96.5.
PhylogenybasedonthenodCandnifHsymbioticgenes
Maximum Likelihood phylogenetic analyses of the symbi-otic genes nodC and nifH separated the BR 3267 and BR 3262strainsintotwodivergentphylogeneticlineages(Fig.4A
fornodC, Fig. 4BfornifH).For bothnodC andnifH, BR3267
100 50 BR 3267 BR 3262 PAC 48 USDA 76 BR 3262 CCBAU 10071 Hybrid: BR 3267/CCBAU 10071 Hybrid: BR 3262/PAC 48 Hybrid: BR 3262/USDA 76 ΔTm= 3.9 ΔTm = 3.9 ΔTm = 10.1 0 100 50 0 100 50 0 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100
Temperature (ºC)
Fluorescence (%)
Fig.3–MeltingcurvesgeneratedwithAppliedBiosystems7500Real-TimeSystemforTmdeterminationbetween homologousandhybridDNA.(a)ComparisonbetweenstrainBR3267andB.yuanmingenseCCBAU1007T;(b)betweenBR
3262andB.pachyrhiziPAC48T;(C)betweenBR3262andB.elkaniiUSDA76T.
B.yuanmingenseCCBAU10071Tandthesoybeantypestrains
that represent the symbiovar glycinearum.In contrast, BR 3262was groupedin anearly branchlinkedtothe related strainsB.pachyrhiziPAC48T,B.elkaniiUSDA76TandB.ferriligni
CCBAU51502Tforbothsymbioticgenes(Fig.4AandB).
Discussion
Theanalysesintheculturemediumshowedthatbothstrains, BR3267andBR3262,havesimilarbehaviorsincomparison toothermembersoftheBradyrhizobiumgenusregarding tol-erance to temperature, pH and NaCl concentration in the culturemedium.Thesetraitsindicatetheyaretolerantto dif-ferentedapho-climaticfactorsandmakethemwelladapted totheconditionsofthetropicalsoilsfromwhichtheywere isolated.Thisadaptabilityiscorroboratedbytheirgood per-formanceincowpeainoculations.8,36,37 TheBR3267andBR
3262strains were positive to ureaseand nitrate reductase
enzymesreactions,whichareapparentlycommon character-isticsamongstrainsofthegenusBradyrhizobium.38,39Itseems
tobeacommoncharacteristicamongBradyrhizobiumspecies ofsubgroupI(closetoB.japonicum)tobesensitiveto strepto-mycinandtetracycline,asshowedbyBR3267.Incontrast,BR 3262wastoleranttotheseantibiotics,acommon
characteris-ticofBradyrhizobiumspeciesbelongingtosubgroupII(closeto
B.elkanii).40The50carbonsourcesusedbyBR3267pointsto
greateraccesstodifferentsoilcarboncompounds.Theresults ofthe fattyacid composition analysiscorroborateprevious findingsthatindicatethatthemaindifferencesbetween sub-groupsIandIIofBradyrhizobiumarethe19:0cyclow8cand thesummedfeature8(18:1w7c),whichindicatethatBR3267 belongstothe subgroupofB.japonicumandBR3262tothe subgroupofB.elkanii.40,41
Whenthe16SrRNAgenesofstrainsBR3267andBR3262 were firststudied,11 it wasobservedthatalthoughBR3267
could begrouped withB. japonicum,there were differences withrespecttotheB.japonicumtypestrainpattern.Atthetime,
B. japonicum USDA 6T (AB354632) B. ottawaense OO99T (HQ587980)
B. huanghuaihaiense CCBAU 23303T (KF962706) B. diazoefficiens USDA 110TT(NC_004463) B. daqingense CCBAU 15774TT (HQ231326)
B. yuanmingense CCBAU 10071T (AB354633)
BR 3267 (KF828824)
B. arachidis CCBAU 051107T (KF962705) B. vignae 7-2T (KT362339)
B. kavangense 14-3T (KT033402)
B. pachyrhizi PAC48T (HQ588110)
B. elkanii USDA 76T (AB354631) B. ferriligni CCBAU 51502T (KJ818109)
B. tropiciagri CNPSo 1112T (KP234520) B. embrapense CNPSo 2833T (KP234521) B. canariense BTA-1T (AJ560653)
B. cytisi CTAW11T (EU597844) B. rifense CTAW71T (EU597853) B. erythrophlei CCBAU 53325T (KF114576)
B. jicamae PAC68T (AB573869) B. lablabi CCBAU 23086T (GU433565)
B. paxllaeri LMTR 21T (KF896160) B. retamae Ro19T (KC247112) B. icense LMTR 13T (JX514897) B. valentinum LmjM3T (JX514897) B. neotropicale BR 10247T (KJ661727) B. ingae BR 10250T (KF927054)
B. iriomotense EK05T (AB301000) B. manausense BR 3351T (KF786002)
B. ganzhouense CCBAU 51649TT (JX292035)
Bradyrhizobium sp. ORS285 (AF284858) Bradyrhizobium. sp. VUPME10 (HG940520) BR 3262 (KF828819) 100 99 92 53 69 84 99 9498 67 99 100 100 100 100 100 100 100 100 55 84 97 59 99 71 99 89 95 99 97 93 82 66 95 0.02 89 96 99 0.05 B. huanghuaihaiense CCBAU 23303T (HQ231551) B. liaoningense USDA 3622T (EU818925) B. japonicum USDA 6T (HM047126) B. ottawaense OO99T (JN186287) B. diazoefficiens USDA 110T (NC_004463) B. daqingense CCBAU 15774T (HQ231323)
B. yuanmingense CCBAU 10071T (EU818927) B. arachidis CCBAU 051107T (HM107283)
B. pachyrhizi PAC48T (HM047124) B. elkanii USDA 76T (AB094963) B. ferriligni CCBAU 51502T (KJ818108)
B. tropiciagri CNPSo 1112T (HQ259540) B. embrapense CNPSo 2833T (KP234518) B. viridifuturi SEMIA 690T (KR149137) B. erythrophlei CCBAU 53325T (KF114598) B. lablabi CCBAU 23086T (GU433546)
B. paxllaeri LMTR 21T (DQ085619) B. icense LMTR 13T (KF89616) B. retamae Ro19T (KF670138) B. valentinum LmjM3T (KF806461) B. jicamae PAC68T (HM047127) B. guangdongense CCBAU 51649T (KC509130) B. guangxiense CCBAU 53363T (KC509140) B. denitrificans LMG 8443T (HM047125) B. ganzhouense RITF806T (JX292065)
B. canariense BTA-1T (EU818926) B. cytisi CTAW11T (GU001618)
B. rifense CTAW71T (GU001627)
BR 3267 (KF828825)
BR 3262 (KF828820)
A
B
Fig.4–UnrootedmaximumlikelihoodphylogenetictreebasedonnodC(A)andnifH(B)genesshowingrelationships betweenthestrainsBR3262andBR3267andtypespecies(T)andreferencestrainsofthegenusBradyrhizobium.The phylogenetictreewasbuiltusingtheKimuratwo-parametermethod.Bootstrapvalueswereinferredfrom500replicates andareindicatedatthetreenodeswhen≥50%.GenBankaccessionnumbersareprovidedintheparenthesis.Thebar representsfiveortwoestimatedsubstitutionsper100nucleotidepositions.
however,thespeciesB.yuanmingense,whichalsobelongsto subgroupIofBradyrhizobium,hadnotyetbeendescribed;thus, therewasnospeciesindication.Itwasobservedontheother handthatBR3262hadastrongresemblancetoB.elkaniiby the16SrRNAgene,whichsupportedthatthestrainshouldbe assignedtothatspecies.Atthattime,thespeciesB.pachyrhizi,
whichintermsofbasecompositionofthe16SrRNAgene dif-fersfromstrainsB.elkaniiUSDA76Tonlyontheorderof0.1%,
hadnotyetbeendescribed.16SrRNAsequenceanalysisisnot alwaysfineenoughforspeciesassignment42,43;inthegenus
Bradyrhizobiumsomespeciessharealmostidentical16SrRNA
genesequences.Examplesarethetypestrainsofthespecies
B.japonicumandB.liaoningense,3alongwithB.elkaniiandB.
pachyrhizi.18 Due tothe high conservation ofthe 16SrRNA
geneamongmembersoftheBradyrhizobiumgenus,14,15several
authorshaverecommendedanMLSAofprotein-encoding(i.e., housekeeping) genes.6,22,44–47 In ourcase,the MLSAresults
werecongruenttothe16SrRNAanalysisandclearlyindicate thatBR3267andBR3262asmembersofthespeciesB.
yuan-mingenseandB.pachyrhizi,respectively(Fig.2).TheDNA–DNA
homology resultsconfirmtheresultsobtainedin the anal-ysisofhousekeeping genes, i.e., BR 3262belongs tothe B.
pachyrhizispecies,andBR3267isamember oftheB.
yuan-mingensespecies.Aspreviouslyshown,aTmvalueequalto
5◦Ccanbeusedasthethresholdtodelineateaspecies,where lower valuesindicate equalspeciesandhighervalues indi-catedifferentspecies.29AT
mvalueof5◦Ccorrespondstoa
homologyvalueofapproximately70%obtainedbytraditional DNA–DNAhybridizationtechniques.29,30Moreover,we
calcu-latedtheANI,arobustmethodthatreplacesthetraditional DNA–DNAhybridizationtechniques.19Thismethodinvolves
comparing the sequences oftwo bacterialgenomes, and a valueof95%isconsideredasthethreshold.19,53Inthiscase,
whenthehomologyisgreaterthan95%,thetwostrainsbelong tothesamespecies;ifthehomologyislower,theybelongto differentspecies.Thus,theresultsofthecomparisonsagain confirmedthatBR3262belongstoB.pachyrhizi(ANIgreater
than95%)andthesameBR3267toB.yuanmingense(ANIgreater
than95%).
Symbiotic genes related to nodulation (nod) and nitro-gen fixation (nif)processes hold notaxonomicinformation forspeciesdefinition.17 However,inourstudy,theyshowed
goodphylogeneticcorrespondencetothecoregenomeones. Interestingly,nodCandnifHgenephylogeneticanalyseshave
beenusedtopredictthehostrangenodulationcapacityand for the delineation of symbiovars, i.e., groups of rhizobial strains withsimilar symbiotic behaviors concerning nodu-lation and nitrogen fixation capacity with legumes.55 For
the nodC gene, for example, a BLAST search in the NCBI database (http://blast.ncbi.nlm.nih.gov/)showed the Arachis
hypogaestrainsCCBAU53380(Ac.no.KC509232)andCCBAU
51663(Ac.no.KC509217),isolatedinChina,asthe Bradyrhizo-biumstrainsthathadtheclosestsimilarity(98%)withBR3267, indicatingthatpeanutmightbeapossiblehostforthislatter strain.Instead,inthenodCgene,BR3262had95%similarity withthecowpeastrainBradyrhizobiumsp.VUMPE10(Fig.4A), isolatedfromanEuropeansoilandproposedasanew symbio-var(sv.vignae)withintheBradyrhizobiumgenus.56Thesedata
indicatethatcowpeanodulateswithveryeffective Bradyrhizo-biumstrainsthatareholdingdivergentsymbioticgeneswhich mightrepresentdistinctsymbiovars.
Cowpeacannodulatewithawiderangeofrhizobia geno-types,Bradyrhizobiumbeingthemajorsymbiontgroup.Thies and colleagues57 proposed that cowpea’s promiscuity for nodulation withthe bradyrhizobialpopulation may have a limit.However,aglobalBradyrhizobiumcollectionforcowpea nodulationhasnotyetbeenwellanalyzedtoidentifytrue cow-peasymbiontsinlightofthesymbiovarconcept.Itisnotwell understoodwhetheranyBradyrhizobiumgroupisspecialized toproducenodulesandtofixnitrogenspecificallyincowpea.
Conclusion
TheresultsoftheidentificationsofB.yuanmingenseBR3267
andB.pachyrhiziBR3262canbeconsideredreliable.
Further-more,analysisofthenodCandnifHgenesindicatesthatthese two strains are similar to strains isolated on other conti-nents.StrainBR3262possiblybelongstothevignaesymbiovar, whichwasrecentlyassignedforstrainsisolatedfromcowpea inEurope.Bothstrainsholdsimilarsymbioticgenomeswith
BradyrhizobiumstrainsoutsideofSouthAmerica.
Apparently,cowpeaisabletoestablisheffectivesymbiosis withdivergent bradyrhizobiastrainsisolatedfrom Brazilian soils,confirmingitspromiscuouscapacityfornodulation.
Conflicts
of
interest
Theauthorsdeclaretheyhavenoconflictsofinterest.
Acknowledgements
ThisstudywassupportedbytheConselhoNacionalde Desen-volvimentoCientífico eTecnológico (CNPq). Wewould like tothanktheCoordenac¸ãodeAperfeic¸oamentodePessoalde NívelSuperior(Capes)forthescholarships,likewisetoCNPq forthefellowshipofresearchproductivity(PQ).
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