Phenotypic, genetic and symbiotic characterization of Erythrina velutina rhizobia from Caatinga dry forest

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

Environmental

Microbiology

Phenotypic,

genetic

and

symbiotic

characterization

of

Erythrina

velutina

rhizobia

from

Caatinga

dry

forest

Dalila

Ribeiro

Rodrigues

_,

_Aleksandro

_Ferreira

_da

_Silva

_,

Maria

Idaline

Pessoa

Cavalcanti

_,

_Indra

_Elena

_Costa

_Escobar

_,

Ana

Carla

Resende

Fraiz

_,

_Paula

_Rose

_de

_Almeida

_Ribeiro

_,

_Reginaldo

_Alves

_Ferreira

Neto

_,

_Ana

_Dolores

_Santiago

_de

_Freitas

_,

_Paulo

_Ivan

_{Fernandes-Júnior}

a_{UniversidadeEstadualdaParaíba,CampinaGrande,PB,Brazil} b_{UniversidadeFederalRuraldePernambuco,Recife,PE,Brazil} c_{UniversidadeFederaldaParaíba,Areia,PB,Brazil}

d_{UniversidadeFederaldoValedoSãoFrancisco,Petrolina,PE,Brazil} e_{UniversidadeFederaldePernambuco,Recife,PE,Brazil}

f_{UniversidadedoEstadodaBahia,Juazeiro,BA,Brazil} g_{EmbrapaSemiárido,Petrolina,PE,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Received22June2017 Accepted5September2017 Availableonline2February2018 AssociateEditor:IedaMendes

Keywords:

Biologicalnitrogenfixation “Mulungu”

Diversity Inoculant 16SrRNA

a

b

s

t

r

a

c

t

Erythrinavelutina(“mulungu”)isalegumetreefromCaatingathatassociateswith rhizo-biabutthediversityandsymbioticabilityof“mulungu”rhizobiaarepoorlyunderstood. Theaimofthisstudywastocharacterize“mulungu”rhizobiafromCaatinga.Bacteriawere obteinedfromSerraTalhadaandCaruaruinCaatingaundernaturalregeneration.The bacte-riawereevaluatedtotheamplificationofnifHandnodCandtometaboliccharacteristics. Tenselectedbacteriaidentifiedby16SrRNAsequences.TheyweretestedinvitrotoNaCland temperaturetolerance,auxinproductionandcalciumphosphatesolubilization.The symbi-oticabilitywereassessedinangreenhouseexperiment.Atotalof32bacteriawereobtained and17amplifiedbothsymbioticgenes.Thebacteriashowedahighvariablemetabolic pro-file.Bradyrhizobium(6),Rhizobium(3)andParaburkholderia(1)wereidentified,differingfrom theirgeographicorigin.Theisolatesgrewupto45◦Cto0.51molL−1ofNaCl.Bacteriawhich producedmoreauxininthemediumwithl-tryptophanandtwoRhizobiumandone Bradyrhi-zobiumwerephosphatesolubilizers.AllbacterianodulatedandESA90(Rhizobiumsp.)plus ESA96(Paraburkholderiasp.)weremoreefficientsymbiotically.Diverseandefficientrhizobia inhabitthesoilsofCaatingadryforests,withthebacterialdifferentiationbythesampling sites.

©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

∗ _{Correspondingauthor.}

E-mail:paulo.ivan@embrapa.br(P.I.Fernandes-Júnior).

https://doi.org/10.1016/j.bjm.2017.09.007

1517-8382/©2018SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

Caatinga(etymology:whiteforest)isthemain phytophysiog-nomyoccurringinBrazilianSemi-Aridregion,occupyingmore than70%ofBrazilianNortheastern.Theselandsencompasses severaldry foreststhatshows, asthe maincharacteristics, low rainfall (below 800mmyr−1) concentrated in the firsts 3–4monthsoftheyearandhightemperatureaverages.1_The

plantbiodiversityinCaatingaisveryhighwiththe predomi-nanceofFabaceae(Leguminosae)family,with82generaand 617alreadycatalogedspecies.2_{Thehistoricaluseoflandsin}

Caatingaforshiftingagriculturepracticesleadedtoseveral landsinfallow,wherethenaturalregenerationoccur. Nowa-daysCaatingadryforestsareagreatmosaicoffallowlands withdifferentstagesofnaturalregeneration.3,4 _Inthis

pro-cess,plantsbelongingtoFabaceaearethepioneerscolonizers andcanpresentanimportantecologicalrole.3–6

SeveralleguminouspioneerscolonizingtheCaatinga nat-uralregenerationlandsareabletoassociatewithindigenous rhizobia from the soils of the region. Rhizobia are root and/orstem nodulating,nitrogen-fixing, classifiedwithin␣ and␤-proteobacteriasubclasses.Theyholdthenitrogenase enzymatic complex, able to reducethe atmosphericN2 to

ammonium.Thebiologicalnitrogenfixation(BNF)isthemain sourceof nitrogenincorporation in the food webs, mainly bythe association between legumes and rhizobia.7 _{BNF is}

exploitedinagricultureand forestrybytheproductionand application of rhizobial inoculants that reduces the costs and environmental impacts of plant production, in addi-tion to yield increase.8 _{The continuous selection of new}

and more efficientrhizobial strains is importantto obtain bacteriawithhigheragronomicperformanceandagricultural applications.8,9_{Innaturalregenerationlands,theselectionof}

nativeandadaptedrhizobiacanhelptoimprovethe regenera-tionprocessduetotheiruseintheproductionandapplication ofmoreadaptedandhealthyseedlings.

Besidesthenitrogenfixation,rhizobiaareabletodirectly inducetheplantgrowth byother mechanisms,suchasthe phytohormoneproduction(auxin,forexample)and release ofsoilinsolublenutrients(e.g.phosphorusandiron)among severalothers.10,11 _{Thesemechanismscaneasilybein vitro}

evaluatedbysimpleandlowcoststechniques,showing cor-relationwiththebacterialperformanceinplantinoculation experiments.12,13 _{Atthesame way,theevaluationof}

rhizo-bialtolerancetoenvironmentalstresses,suchasthesalinity and temperature tolerance, also can be easily performed

invitro14_{andcanbecorrelatedtotheresultsofplant}

inocula-tionexperiments.13_{Theevaluationofinvitroenvironmental}

stressesare importanttothe selectionofbacteriathat are (likely)moretolerantandalsotobetterunderstandthe bacte-riabiology.15_{Thesefeaturesareparticularlyinterestingtobe}

evaluatedinstudiesofrhizobiafromtropicaldrylandswith lowrainfallandhightemperatureaverages.

Recent studies showed that Erythrina velutina Willd., commonly known as “mulungu”, is able toestablish sym-bioticassociation with rhizobia in soilsfrom the Brazilian Semi-Arid.16,17_{E.velutina,classifiedwithinthePapilionoidae}

clade and Phaseoleae tribe, is a naturally inhabiting tree from the Northeast region of Brazil. This species is used

as wood, timber, ornamental applications and sources of bioactivecompoundswithpharmacologicalapplications.18,19

Inaddition,duetothefastgrowthandadaptivecharacteristic totheSemi-Aridenvironment,“mulungu”seedlingsshould beappliedinlandregenerationprojects.20

Despite the potentialuse of thisnative speciesfor sev-eral applications, there are not official recommendations of rhizobial strains for E. velutina in Brazil. The selection ofnativenitrogen-fixingisolatescanhelptotheinclusionof the“mulungu”bacteriainthelistofofficiallyrecommended strains toinoculant productionbytheBrazilianMinistryof Agriculture,LivestockandFoodSupply(MAPA).The applica-tionofselectedefficientrhizobiaisusefultothedevelopment of improved “mulungu” seedlings, more healthy and with betternutrition,increasingtheprobabilityofthesuccessful establishmentinthefields,especiallyinnon-favorable envi-ronments,suchastheCaatingadegradedlands.21 _Thus,we

hypothesizedthattheCaatingasoilsundernatural regener-ation inthe Semi-Arid region ofPernambuco State harbor a diversityofefficient “mulungu”rhizobia. Theaim ofthe present study was to characterize the “mulungu” rhizobia fromthesemi-aridregionofPernambucoStateregardingtheir phenotypical,molecularandsymbioticcharacteristics.

Materials

and

methods

Originofthebacterialisolates

The bacteria were isolated from soils collected from the 0 to 20cm superficial layer in areas of Caatinga vegetation undernaturalregenerationinCaruaru,intheAgreste zone ofPernambucostate,andSerraTalhada,intheSertãozoneof thesamestate.Theplantcoveranbothzonesaredifferentas alreadypointedbySilvaandRodal.22_{Thelocationand}

edapho-climaticconditionsofbothareasaredescribedinTable1. For bacterial isolation, a trap-host pot experiment was implemented using “mulungu” (E. velutina) as trap plant. Polystyrene pots (500mL) were filled withthe soilsamples andfourseedsweresowedperpot.Tendaysafterthe emer-gence(DAE)asingleplantwasleftperpot.Thepotsweredaily irrigatedwith100mLofdistilledwaterandtheexperiment was harvestedat85DAE. Therootsand shootswere sepa-ratedandthenodulesdetached.Forisolation,noduleswere superficiallydisinfectedwith96◦GLethanolfor30s;sodium hypochlorite(2.5%,v/v)forfiveminutesfollowedby10washes insteriledistilledwater.Thenoduleswerecrushedinyeast extract-mannitol-agar(YMA)mediumwithcongored23 _and

incubated inagrowth chamberat28◦C.Therise oftypical rhizobialcoloniesweredailyevaluatedduringtendays.The colonieswere purified inYMAmedium withbromothymol blueandstoredinYMmedium+glycerol(2.5%v/v)at−80◦_C

anddepositedintheCultureCollectionofAgricultural Inter-estsMicro-organismsatEmbrapaSemiárido(CMISA).

Amplificationofsymbioticgenes

The bacterial isolates were grown in liquid YM medium for three days for the fast-growing isolates and six days fortheslow-growing.Analiquot of1mLofeachbrothwas used for the DNA extraction using the Wizard® Genomic DNA Purification System (Promega, USA) according to the

Table1–CharacteristicsofCaatingaundernatural regenerationandtheirsoilsinCaruaruandSerra Talhada(PernambucoState,Brazil).

Characteristics Caruaru SerraTalhada

Mesoregion Agreste Sertão

Regenerationtime (underfallow) 21years 24years Mostabundant legumespecies Bauhiniacheilantha Sennacf.spectabilis Poincianella pyramidalis Anadenanthera colubrina Mimosaarenosa Piptadeniastipulacea Poincianella pyramidalis Anadenanthera colubrina Mimosa ophtalmocentra Mimosatenuiflora Amburanacearenses Coordinates 08◦1354Sand 35◦5513W 7◦5729Sand 38◦2337W Altitude(masl) 561 443 Averageannual rainfall(mm) 764 686 Averageannual temperature(◦C) 21.7 26.0

Soiltype Ultisol Luvisol

pH(H2O) 5.96 6.22 P(mgdm−3) 8.78 10.9 Ca2+_(cmol cdm−3) 2.78 3.64 Mg2+_(cmol cdm−3) 1.42 2.85 Na+_(cmol cdm−3) 0.04 0.05 K+_(cmol cdm−3) 0.50 0.81 Al3+_(cmol cdm−3) 0.00 0.00 Sand(gkg−1_{) 684 599} Silt(gkg−1) 229 232 Clay(gkg−1) 87 169

manufacturer’s instructions. nifH and nodCgenes were co-amplifiedinaduplex-PCRreactionasdescribedbyFernandes Júnior et al.24 _{For the nifH, the primers PolF}

(TGCGAYCC-SAARGCBGACTC) and PolR (ATSGCCATCATYTCRCCGGA)25

were used. For nodC, the primers NodCF (AYGTHGTYGAY-GACGGTTC) and NodCR(I) (CGYGACAGCCANTCKCTATTG)26

were applied. For a single isolate that showed positive amplification of the nifH amplicon, a complemen-tary uniplex-PCR was performed with the primers nodCForB (CTCAATGTACACARNGCRTA) and nodCRevB (GAYATGGARTAYTGGYT)57_{targetingthenodCamplificationof}

␤-rhizobia.

ThereactionswereperformedinaVeriti96-well thermo-cycler(AppliedBiosystems,USA)andthePCRproductswere submittedtohorizontalelectrophoresisinagarosegel(0.8%, w/v).ThegelwasstainedwithGelRed(Biotium)and visual-izedinaUVchamber.Thebacterialisolatespositiveforboth ampliconswereselectedforthenextsteps.

MetaboliccharacterizationusingtheAPI20NE® kit

Enzymaticactivitiesandthecarbonsourcesutilization pro-fileswereperformedusingtheAPI20NE®strips(BioMérieux, France) according to the manufacturer’s instructions. The incubationtimewasthreedaysforthefast-growingisolates, andsixdaysfortheslow-growing,at28◦C.

The results were transformed in a binary matrix and the bacteria clustered according to the enzymatic activity

characteristicsandutilizationofdifferentcarbonsourcesin asimilaritydendrogramobtainedapplyingtheJaccard sim-ilaritycoefficientandtheUPGMAclustering algorithmwith theaidoftheBioNumericsv.7.5softwarepackage(Applied Maths,Belgium).Theclusteringanalysiswasusedtoselect thebacterialisolatesforfurtherevaluations.

16SrRNAgenesequenceanalyses

The 16S rRNA gene was amplified using the univer-sal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACTT).27_{Toevaluatethesuccessof}

PCR, the productswere submittedto a gel electrophoresis as described above. The PCR products were purified with the Wizard® SV Gel and PCR Clean-Up System (Promega, USA)commercialkit,followingmanufacturer’sinstructions. Theproductsweresequencedwithbothforwardandreverse primers ina 3730xl geneticanalyzer (Applied Biosystems, USA)atMacrogen(Seoul,SouthKorea).

The quality of the sequences was verified using the SequenceScannerSoftwarev.2.0(AppliedBiosystems,USA). Good quality sequences were used to construct the con-tigs for the bacterial identification through comparison with the sequences available at the EzBioCloud database

(http://www.ezbiocloud.net/).28_{Sequences fromclosesttype}

strains were downloaded and used to perform the phylo-genetic tree. The alignment of the sequences was carried out by MUSCLE and the Neighbor-Joining tree made with the Jakes-Cantormodel usingthe bootstrapphylogenytest with 1000replications. Thesequence alignments and phy-logenetic tree construction were carried out in MEGA 6.0 software.29 _{Thesequences were depositedinthe GenBank,}

databaseoftheNationalCenterforBiotechnologyInformation

(www.ncbi.nlm.nih.gov/Genbank),undertheaccession

num-bersMF288754toMF288763.

Saltandtemperaturetolerance

Strainswereanalyzedforsaltandhightemperaturetolerance, following the methodology described by Fernandes Júnior etal.14_{withsomemodifications,brieflydescribedbelow.To}

theevaluationofsalttolerance,thestrainswereinoculatedin PetridisheswithYMAmediumsupplementedwithzero (con-trol),0.085,0.17,0.34and0.51molL−1ofNaClandincubated inagrowthchamberat28◦C.Toevaluatethegrowthability ofthestrainssubmittedtodifferentincubationtemperatures theisolateswereinoculatedinPetridishescontainingoriginal YMAmediumandincubatedat28(control),35,40and45◦C, indifferentgrowthchambers.Inbothexperiments,conducted withinacompletelyrandomizeddesignwiththree replica-tions,thestrainswere incubatedforthreeand sixdaysfor thefast-growingandslow-growing,respectively.

Auxinproductionandsolubilizationofcalciumphosphate

Theinvitroproductionofauxinwasevaluatedaccordingtothe colorimetricmethoddescribedbySarwarandKremer30_with

modifications,brieflydescribedbelow.Thestrainsweregrown inYMAmediumandwerecheckedforpurity.Purecolonies wereinoculatedinYMmedium(3mL),andafterthreedays

forthefast-growingisolatesandsixdaysfortheslowgrowing, thebacterialbrothwereadjustedtoOD540=0.5withdistilled

autoclavedwater(DAW).Analiquotof1mLofeachadjusted brothwere centrifuged(6000×gfor5min),thesupernatant discardedandpelletre-suspendedwith1mLofdistilled auto-clavedwater.

Toevaluatethemetabolicpathwaysenrolledintheinvitro

auxin production, aliquots of 150␮L of the re-suspended bacteria were inoculated in standard liquid YM culture medium,without bromothymolblue, supplemented or not with168mgL−1ofl-tryptophan(l-try).Themediawere incu-batedat28◦C(±1◦C)inanorbitalshakerwithconstantstirring at120rpm, for four and sevendays to the fast and slow-growing bacteria, respectively. Afterthe incubation period, the cultures were adjusted to an OD540=0.5 as described

above. Aliquots of standardized bacterial solutions (1mL) were centrifuged at 6.000×g for 5min. Aliquots of 200␮L of supernatant were placed in 96-well ELISA microplates andmixedwith100␮LofSalkowskisolution(1mLofFeCl3

0.5molL−1+49mLofHClO46molL−1).TheELISAplateswere

incubatedinthedarkfor30min.Theintensityofredcolorwas determinedinaMultiSkanGOspectrophotometer (Thermo Scientific,Germany) at530nm. Theconcentrationofauxin was estimatedusing astandard curve previouslyprepared witharangeof0to500␮gL−1ofsyntheticindole-3-aceticacid (Sigma–Aldrich,USA).

Theisolateswereevaluatedfortheircapacitytoinvitro sol-ubilizecalciumphosphateinthesolidGLmediumsupplied with insoluble CaHPO4.31 The bacteria were grown in

liq-uidYMmedium,ODadjusted,centrifuged,andre-suspended asdescribedabove.Threealiquotsof10␮Lweredroppedin equidistantpointsinthecenterofthePetridishes.The bacte-riawereincubatedat28◦Cforsevendaysforthefast-growing andfifteendaysfortheslow growing.Aftertheincubation period,thediameterofthecoloniesandthetranslucentzone surroundingthecoloniesweremeasuredinmillimeters(mm) witharuler.ThisdatawereusedtocalculatetheSolubilization Index(SI)=diameteroftranslucentzone/diameterofcolony.32

Theexperimentsofinvitroauxinproductionandcalcium phosphatesolubilizationwereperformedinacompletely ran-domizeddesignwiththreereplications.

Symbioticefficiencyin“mulungu”plants

Thesymbioticefficiencyoftheisolateswereevaluatedunder gnotobioticconditionsinagreenhouse.Theexperimentwas carriedoutattheEmbrapaSemiáridofacilitiesinPetrolina, Pernambucostate.Forthisassay,thedormancyof“mulungu” seedswasbrokenbymechanicalscarificationofthetegument. Theseedsweresurfacedisinfectedwithethanol96◦ GLfor 30s,sodiumhypochlorite2.5%(v/v)forfiveminutes,followed byeightwasheswithDAW.23_{Thesubstratewaswashedand}

sterilized sand (autoclaved twice, at120◦C and 1.5atm for 1h,with72hbetweenthesterilizations).Theexperimentwas setupinpolystyrenepots(500mL),whichweredisinfected bywashingwithsodiumhypochlorite2.5%(v/v),followedby threewasheswithDAW.Pots werecarefully filledwiththe sterilesandandfourseedsperpotweresownsoonafterfilling. For inoculation, bacteria were grown in YM medium up to the end of the exponential growth phase

(around 109_cellsmL−1_{), forthree days forthe fast-growing}

strainsandsixdaysfortheslow-growing,inanorbitalshaker at28◦C,asdescribedabove.Rightaftersowing,the inocula-tionwasperformedbythedropof2mLofthebacterialbroth oneachseed.AttwentyDAEathinningwasperformedand asingleplantwasleftperpot.Thepotsweredailysupplied with 100mLofDAW, and afterthecotyledon drop(around 25–28DAE),50mLofnitrogenfreenutrientsolution,described byNorrisandT’Mannetje,33_{wasappliedonceaweek.}16

Theexperimentaltreatmentsconsistedoftheinoculation oftenbacteria(singleinoculationofeachisolate),apositive controlinoculatedwithBradyrhizobiumelkaniiBR5609(SEMIA 6100),strainofficiallyrecommendedbyMAPAinBrazilforuse asinoculantforErytrinavernaandFalcatariamollucanna,and twouninoculatedcontrols:onesuppliedwithNH4NO3(70mg

Nplant−1week−1)appliedafter35DAE;andonewithout nitro-gensupplementation.

Theplantswere harvestedat92DAEforthe determina-tion ofthenodulenumber (NN),noduledrymatter (NDM), shootdrymatter(SDM),rootdrymatter(RDM)andnitrogen accumulationintheshoot(totalN).For theNNevaluation, thenodulesweredetachedfromtherootsandcounted.For theNDM,SDMandRDM,respectively,thenodules,shootsand rootswereseparatelyinpaperbagsanddriedinaforced-air ovenat65◦Cforsevendaysandweighted.Theshootswere groundedfordeterminationofshootnitrogenconcentration bythedrycombustionmethodinaTruSpecCNelemental ana-lyzer(Leco,USA).Thesevalueswereusedforcalculationof totalaccumulationofnitrogenintheshoot(TotalN)through themultiplicationofthenitrogenconcentrationbytheSDM.

Statisticalanalyses

For the solubilization of calcium phosphate, IAA produc-tion, and symbiotic efficiency in greenhouse experiments, thenormaldistributionoftheerrorswereevaluatedbythe Shapiro–Wilk test,thus the dataforthegreenhouse exper-iment were transformed by (x+1)0.5 _{to reach the normal}

distribution.

Theexperimentaldataweresubmittedtovarianceanalysis (ANOVA)andtheaverageswerecomparedbytheScott–Knott’s meanrangetest(p<0.05).Thedatawereanalyzedusingthe statisticalpackageSisvarv5.0.34

Results

Isolationandsymbioticgeneamplification

Theisolationprocessretrieved32bacteria,17fromCaruaru soilsand15fromSerraTalhada.Theduplex-PCRreactionto

nifHandnodCresultedinthepositivereactionforbothgenes toseven(47%)bacterialisolatesfrom SerraTalhadaand 10 (59%)bacteriafromCaruaru,includingESA96,asingle fast-growingisolatethatdidnotamplifythenodCatthe duplex-PCRreactionbut amplifiedthenodCwhenaprimerpairto ␤-rhizobiawasapplied.

Amongthesebacteria,tenwereslow(sevenfromCaruaru andthreefromSerraTalhada) andsevenwerefast-growing (fourfromCaruaruandthreefromSerraTalhada),respectively.

Key 50 60 70 80 90 92.3 2 3 4 5 6 7 8 9 1 92.9 89.4 71.1 66.6 62.6 88.2 85.7 80.4

100 D-glucose D-mannose D-maltose Potassium gluconate Capr

ic acid

Adipic acid Malic acid Trisodium citr

ate

Phen

ylacetic acid

Nitr

ate reductase

Indole GluF ArgDH

er

m

Urease EscHid Gelatinase β-Galactosidase D-mannitol N-acetyl-glucosamine L-ar abinose Origin (location) ESA 93 ESA 92 ESA 105 ESA 90 ESA 97 ESA 102 ESA 104 ESA 101 ESA 99 ESA 89 ESA 96 ESA 100 Serra talhada Serra talhada Serra talhada Serra talhada Serra talhada Serra talhada Serra talhada Caruaru Caruaru Caruaru Caruaru Caruaru Caruaru Caruaru Caruaru Caruaru Caruaru ESA 94 ESA 95 ESA 103 ESA 91 ESA 98

Fig.1–Clusteranalysisofthemetabolicprofileof17bacterialisolatesofErythrinavelutinabasedontheresultsof12carbon sourcesassimilationand8enzymesactivitiesintheAPI20NEstrips.First12columns:carbonsourcesmetabolism.Last8 columns:Enzymaticactivity.Indole,indoleformation;GluFerm,glucosefermentation;ArgDH,argininedehydrogenase.Red squares:positiveactivity;Greensquares:negativeactivity.Numbersinthenodesarethecopheneticcorrelation.Key, Bacterialisolate.

All15isolatesthatdidnotamplifythesymbioticgeneswere fast-growingbacteria.

Metabolicprofileofthebacterialisolates

Regardingmetabolismofthe12carbonsourcesavailableatthe API20NEstrips,alargevariabilityatthemetabolicprofilewere observedinthe17 bacteriaevaluated. Allbacterial isolates grewind-glucose,l-arabinose,d-mannoseand d-mannitol, assolecarbonsources.Noneofthebacteriagrewusingcapric acid.Around83%grewwithN-acetyl-glucosamine,d-maltose andpotassiumgluconate.Around 76and70% used respec-tively,trisodiumcitrateandmalicacidassolecarbonsources. ThebacteriaESA95,ESA103andESA99showedthebest abil-itytometabolizethe carbonsourcessincetheygrewinall sources,exceptingthecapricacid.Thesebacteriawerealso theonlyisolatesthatgrewusingadipicacidandphenylacetic acid(Fig.1).

Forthe enzymaticactivity,theisolatesshoweddifferent profiles. All bacterial isolatesdid not presentedactivity of glucosefermentation.ExceptingtheisolateESA89,all bac-terialisolateswerepositivetoureaseand,exceptingtheESA 99andESA103,allisolateswerepositivetoesculin hydroly-sis.Anamountof65%ofthebacterialisolateswaspositive to␤-Galactosidase.Variableprofileswereachievedfromthe evaluations of nitrate reductase, indol formation, arginine DiHydrolase,andgelatinase.

Allmetabolicresultsweretabulatedinabinarymatrixand usedtotheclusteranalysisofallbacteria.Thesimilarity den-drogram(Fig.1)presentstheformationofnineclustersatthe thresholdof90% (Jaccard coefficient).Basedon the cluster analysis,tenbacterialisolateswereselectedforfurthersteps.

16SrRNAgenesequenceanalyses

Thesequencesof16SrRNAgenewerecomparedwiththose availableatthe EzBioClouddatabase.Thecomparison indi-catedthatamongthefivebacterialisolatesfromCaruaru,four ofthemwereclassifiedas␣-rhizobiaandclusteredwithinthe

Bradyrhizobium inthe B. elkanii cladeII. The isolateESA 96 wasclassifiedasa␤-rhizobiarelatedtoParaburkholderia dia-zotrophica.ThefivebacteriafromSerraTalhadawereclassified as ␣-rhizobia.TheisolatesESA92 and ESA93 were classi-fiedwithinRhizobumintheR.etliclade.ThebacteriaESA90 wasclassified inthe samegenusbut intheR.tropiciclade. The isolatesESA89 and ESA 91 were classified withinthe

Bradyrhizobiumjaponicumclade,closelyrelatedto Bradyrhizo-biumsubterraneum(Fig.2).

Invitrotemperatureandsalttolerance

Two isolates(ESA89and ESA92) grownunderthe incuba-tiontemperatureof40◦C.ThebacterialisolatesESA98and ESA99grewwhenincubatedat45◦C.Theotherbacterial iso-lates grew onlyunder the incubation temperature of35◦C

Bradyrhizobium elkanii USDA 76T (KB900701) 100 100 100 100 100 100 74 74 99 0.02 80 97 85 86 69 92

Bradyrhizobium japonicum USDA 6T (AP012206) Bradyrhizobium kavangense 14-3T (KP899562) Bradyrhizobium subterraneum 58 2-1T (KP308152) Bradyrhizobium pachyrhizi PAC48T (AY624135) ESA 97 caruaru

ESA 100 caruaru ESA 99 caruaru ESA 98 caruaru

ESA 89 serra talhada ESA 91 serra talhada Rhizobium binae BLR195T (JN648932)

Rhizobium paranaense PRFT35T (EU488753) Rhizobium milounense HAMBI 2971T (EF061096) Rhizobium hainanense CCBAU 57015T (U71078)

Rhizobium tropici CIAT 899T (U89832)

Rhizobium multihospitium HAMBI 2875T (jgi-1052913) Paraburkholderia sprentiae WSM5005T (HF549035) Paraburkholderia tuberum STM678T (AJ302311)

Paraburkholderia piptadeniae STM7183T (LN875219) Paraburkholderia diazotrophica JPY461T (HM366717) Rhizobium etli CNF 42T (CP000133)

ESA 92 serra talhada

ESA 90 serra talhada

ESA 96 caruaru ESA 93 serra talhada

Fig.2–Neighbor-joiningphylogenetictreeusingaJukes-Cantormodelbasedonthepartial16SrRNAgenesequence(1165 nt)oftenrhizobialisolatesfromrootnodulesofErythrinavelutinaand16typestrains.Numbersinthenodesofbranches correspondtothebootstrapvaluefrom1000replications.Bootstrapvalueslowerthan60%arenotshown.

Table2–InvitrotemperatureandNaCltolerance,calciumphosphatesolubilizationandauxinproductionoftenrhizobial isolatesfromErythrinavelutinarootnodules,obtainedfrom“Caatinga”dryforestsoils,inPernambucostate,Brazil.

Bacteria Temperatureb _NaClc _{Solubilizationindex}d _Auxine

(◦_{C) molL}−1 _{(SI) −Try +Try}

ESA89 40 0.51 – 10.08b 16.9d ESA96 35 0.085 – – – ESA90 35 0.34 0.15bf _{– –} ESA91 35 0.34 – – 19.11d ESA92 40 0.085 – – – ESA93 35 0.085 0.35a 20.16a 114.0b ESA97 35 0.51 0.07b 23.63a – ESA98 45 0.34 – 11.10b 150.1a ESA99 45 0.51 – – 44.0c ESA100 35 0.085 – 12.00b 163.6a BR3299a _{nd nd 0.62a 14.16b 55.8c}

a _{Referencestrain:MicrovirgavignaeBR3299}T_.

b _{Maximumincubationtemperaturewithpositivegrowth.}

c _{MaximumNaClconcentrationinculturemediumwithpositivebacterialgrowth.}

d_{S.I.,translucentzonediameter(mm)/colonydiameter(mm).}

e _{Auxinproduction(+l-try,addof168mgofl-tryptophanin1Lofculturemedium;−l-try,noaddofl-tryptophan);–,notdetected;nd,not}

determined.

f _{AveragesfollowedbythesameletterwithineachcolumnarenotsignificantlydifferentaccordingtotheScott–Knott’stest(p<0.05).}

(Table2).ForthetolerancetodifferentNaClconcentrations,

the bacteriagrew inthe YMAmediumsupplementedwith 0.085–0.51molL−1ofNaCl.TheisolatesESA90,ESA91andESA 98grewpositively inthemediumsupplementedwithNaCl 0.34molL−1whilethebacteriaESA89,ESA97andESA99,grew inthemediumwithNaCl0.51molL−1.TheisolatesESA100, ESA93,ESA92andESA96werethelesstoleranttosalinityand grewonlywiththelowerNaClconcentrationinthemedium (0.085molL−1).

Solubilizationofcalciumphosphateandproductionof auxininvitro

The isolates ESA 90, ESA 93 and ESA 97, were able solu-bilize calcium phosphate, highlighting the isolate ESA 93,

statisticallysuperior(p<0.05)thantheothertwobacteria.Six andfiveisolatesproducedauxininthepresenceandabsence ofl-trysupplementation,respectively.Inthemediumwith l-try,ESA100andESA98wereinthehighestclusterinthemean rangetestcomparison.Thisgroupwasfollowedbytheisolate ESA93thatwashigherthanothergroupwiththeisolateESA 99andthereferencestrainBR3299T_{.Consideringtheauxin}

productioninYMmediumwithoutl-try,theisolatesESA93 andESA97showedthebestperformance,comparingtothe otherisolatesandthereferencestrain.

Symbioticefficiencyin“mulungu”plants

Atthesymbioticefficiencyexperimentingnotobiotic condi-tions,allreplicationsofthenon-inoculatedcontrols(boththe

Table3–Averagesforthenodulenumber(NN),noduledrymatter(NDM),shootdrymatter(SDM),rootdrymatter(RDM), andaccumulationofnitrogenintheshoot(totalN)ofErythrinavelutinainoculatedwithnewrhizobialisolatesina gnotobioticconditionsexperiment.

Treatments NN NDM SDM RDM TotalN

nodulesplant−1 mgplant−1 gplant−1 mgNplant−1

ESA89 9ca _{12.50d 1.40 1.82 27.12b} ESA96 5d 35.70c 1.96 1.34 72.32a ESA90 10c 44.65c 1.87 1.89 61.53a ESA91 6d 37.90c 1.05 1.29 18.12b ESA92 4d 21.85d 0.84 1.18 18.42b ESA93 4d 28.40c 1.58 1.29 25.46b ESA97 19b 76.70b 1.61 1.61 25.66b ESA98 20b 67.15b 1.52 1.12 37.37b ESA99 14c 48.43c 0.90 1.33 24.55b ESA100 12c 50.47c 1.07 1.16 31.09b BR5609 31a 105.75a 2.22 1.3 48.26a Ncontrol 0e 0.00d 0.98 1.82 49.32a AbsControl 0e 0.00d 1.03 1.6 22.68b CV(%) 19.9 42.9 38.6 47.8 21.8

a _{AveragesfollowedbythesameletterwithineachcolumnarenotsignificantlydifferentaccordingtotheScott–Knott’stest(p<0.05).}

absoluteandthenitrogensuppliedcontrols)didnotnodulate, beinginferredthatcontaminationsdidnotoccurinthe experi-ment.Thepositivecontrolinoculatedwiththereferencestrain ofB.elkanii,andtheother10treatmentsinoculatedwiththe newbacterialisolatesinducednoduleformationin“mulungu” roots(Table3).

NodifferenceswerefoundforthevariablesSDMandRDM, inalltreatments.Forthenodulationvariables,NNandNDM indicatedthatthereferencestrainBR5609washigherthanthe othertreatments.Consideringthenewbacteria,theisolates ESA97andESA98stoodout,showinghighervalues compar-ingtotheothereightisolates(p<0.05).Forthetotalnitrogen, plants inoculated with ESA 90, ESA 96 and BR 5609 were notstatisticallydifferentthanplantssuppliedwithNH4NO3

(p>0.05).

Discussion

Amongthe typicalrhizobialcoloniesobtained,around53% werepositivefortheamplificationofbothsymbioticgenes. ThebacteriathatdidnotshowedpositiveresultsfornifHand

nodC amplification are probably non-rhizobial endophytes, alreadyobserved incowpea nodulesatthe Brazilian semi-arid region.35 _{Thesimultaneous amplification ofsymbiotic}

genesisafeasibletooltoselectthebacterialisolateswhichare probablyrhizobia,indicatingthatthistechniquecanbe suc-cessfullyappliedforpreliminaryrhizobialselection.24_Among

theselectedbacteria,slowandfastgrowthcharacteristicwere observed,whichiscommoninsoilsfromdrylandsthatharbor alargediversityofrhizobia,nodulatingbothcropsandnative species.36,37_{ParticularlyinBrazil,alargeculturaldiversityof}

rootnodulebacteriawere obtainedfrom soilsatthe semi-aridregionsofthePernambuco,38,39_Paraíba36_andBahia17,38,39

states.

The17bacterialisolatesevaluatedbytheAPI20NEstrips metabolized several carbon sources and presented a vari-ableenzymaticactivityprofile.Someisolatesused11ofthe

12carbonsourcesavailableattheAPI20NEstrips,whileother bacteria showedpositive resultsonlyforfour sources. Fur-thermore,somebacterialisolatesshowedpositiveenzymatic activitiesforsixoftheeightavailablesubstrateswhilstother isolates were positiveonlyfor twoenzymes.These results showedalargemetabolicdiversityofmulungurhizobia,and possiblytheiradaptationindifferentenvironments.37

Theclusteringofbacterialisolatesthroughtheanalysisof theirmetaboliccharacteristicswasalsorelatedtotheorigin (location). Thecluster1encompassed four isolates,among whichthreewere isolatedfromSerraTalhadaandonlyone from Caruaru. Thethreebacteria from SerraTalhadawere classifiedwithintheRhizobiumgenus.Evaluatingthecluster 2,allfiveclusteredbacteriawereobtainedfromCaruaruand twoisolateswereclassifiedwithintheBradyrhizobiumgenus andrelatedtoB.elkaniiclade.Thecluster4alsogroupedtwo isolatesfromCaruaru(oneBradyrhizobium).Theotherclusters presentedonlyoneisolateeach.

Themetaboliccharacteristics werecloselyrelatedtothe differentiation of taxonomic clusters, but did not show phylogenetic relationship. Thischaracteristic was probably observedduetothefunctional redundancyofthebacterial isolates,wherebacteriabelongingtodifferenttaxashow sim-ilarmetabolicprofiles,indicatingtheabilityofbacteriafrom different taxonomic cluster to occupy the same ecological niche.40,41 _{This resultsindicate the importancetoevaluate}

the diversityofrhizobia metaboliccharacteristics from the Brazilian Semi-Arid, to better understand their functional diversity,complementarytothemoleculartaxonomic assess-ment. Regarding the technique, the metabolic profiling of rhizobiausingtheAPIstripsiswidelyappliedinstudiesof newspeciesdescription.42–44_{Thisisafastandeasytestthat}

revealsseveralmetaboliccharacteristicsofplant-associated bacteria,45 _{beenappliedtothe characterizationofrhizobial}

collections.

Martins at al.46 _{did notobserved aclear distributionof}

Paraburkholderia isolates from Mimosa caesalpinifolia in dif-ferent regions of the Brazilian semi-arid. The geographic

differentiation of Bradyrhizobium isolates from nodules of fieldgrownChamaecristaspp.intheSemi-Aridregionofthe BahiaState(Northeastern,Brazil)wasalsonotclear.47_Onthe

otherhand,thedifferentiationofBradyrhizobiumisolateswas observedinseveralcropspeciesalong theOkavangoValley, fromBotswanatoAngola.48

For“mulungu”rhizobiaintheSemi-AridregionofBrazil, therearelocaledaphoclimaticpatternsthatdrivesthe occur-rence of “mulungu” rhizobia. Menezes et al.16 _evaluating

“mulungu”rhizobiafromsoilsofJuazeiromunicipality(Bahia, State) from the Brazilian Semi-Arid region, observed the presenceofBradyrhizobiumfromthecladeB.japonicumand Rhi-zobiumwithintheR.tropiciclade,inadditiontoa␤-rhizobia, pointing to the occurrence of certain “mulungu” rhizobia groups accordingto the sampling location at the Brazilian Semi-Arid.TherhizobiafoundinthesoilsofJuazeirobythose authorsarecloselyrelatedtothoseobtainedfromSerra Tal-hada in the present study. These cities are located about 300kmaway. Despitethedistance,theclimatic characteris-ticsofthecitiesare verysimilarregardingthetemperature averages,rainfallandtheplantcoverage.22_{Caruaruislocated}

at260kmfromSerraTalhada,butshowslowertemperatures averagesandhigherrainfall(Table1),changingtheplant com-munities in the Caatingasites,22 _{resulting in different soil}

rhizobialcommunities.Thedataobtainedinthisstudy rein-force the hypothesis that edapho-climaticconditions drive the“mulungu” rhizobialcommunity inBrazilianSemi-Arid soils.

Thebacterialisolatesshowedvariablebehaviorinfaceof thetemperatureandsalinity.TheisolatesESA98andESA99, bothB.elkaniilikeisolates,grewupto45◦Cofincubation tem-perature.TheisolatesESA89(closetoB.subterraneum),ESA97 andESA99(bothclosetoB.elkanii)highlightedduetotheir pos-itivegrowthwheninoculatedinthemediumsupplemented withNaCl0.51molL−1.Surprisingly,thefastgrowingrhizobia wasnotthemosttolerantbacteriatotemperatureand salin-ity. Dataavailableinthe literaturerefersthe bacteriafrom genusRhizobiumandotherfastgrowersasthemosttolerant isolates,13,14_{probablyduetotheircapacitytoproducemucus,}

whichprovidecellularprotection.49

Thetype strains ofB. subterraneum50 _{and B. tropiciagri}43

did nottolerate high incubation temperature grownwhen incubated at37◦C. Regarding theNaCl tolerance, this type of strains tolerated lower than 0.17molL−1. Generally, the bacterialisolatesfromsemi-arid region, includingthe slow growingrhizobia,showhighertolerancetoinvitro environ-mental stresses, as already observed for cowpea rhizobia regarding their tolerance to salt and temperature39 _and

antibiotics.51 Nevertheless, the data obtainedin this study indicates that our Bradyrhizobium were more tolerant to temperatureandsalinitythan theBradyrhizobiumevaluated by Menezes et al.,17 _{strengthening the hypothesis of}

dif-ferences among the “mulungu” rhizobia in the Brazilian Semi-Arid. Temperatures above 40◦C are common in the Brazilian tropical soils, and the evaluation of the in vitro

bacterialtolerancecapacitytothisconditions areuseful to selectbacterialisolateswithhigherresistancetoharshfield conditions.

Severalisolatesdidnotsolubilizethecalciumphosphate

in vitro. Three bacteria were positive tothis characteristic,

pointing out the isolate ESA 93, a Rhizobium sp. from SerraTalhada.Thedataobtainedinthisstudycorroborated with previousresults, sincelowcalcium phosphate solubi-lizationarefoundinrhizobialcollectionsevaluatedinsolid medium.52

The higher rates ofauxin production were achieved in themediumwithl-tryptophan.ThebacteriaESA98andESA 100,bothBradyrhizobiumfromCaruaru,producedsignificantly moreauxininthemediumwithl-trythantheother bacte-ria.Differingfromourstudy,thebacterialisolatesevaluated by Menezes et al.17 _{showed that Burkholderia and}

Bradyrhi-zobium were the best auxin producers. l-try amino-acid is the main precursor of auxin and the supplementation of culturemediumgenerallyinducesthe auxin productionby the bacterialisolates.53,54 _{In themediumwithoutl-try, the}

bacterial isolates can produce auxin if they present other metabolicpathwayforauxinproduction.Thecalcium phos-phatesolubilizersESA93(Rhizobiumsp.fromSerraTalhada) andESA97(Bradyrhizobiumsp.fromCaruaru)producedmore auxinthantheotherbacteriainmediumwithoutl-try, show-ingthatthisisolatespresentsdifferentpathwaystoproduce auxin,adesirablecharacteristicregardingthe plantgrowth promotion.12

Bradyrhizobium,RhizobiumandParaburkholderiageneraare well knownfortheirabilitytoefficientlynodulate legumes in several ecological regions, including the Brazilian Semi-Arid,16,46,47,55,56 _{asobservedinthepresent study.Theplant}

inoculationexperimentshowedthatthereferencestrainBR 5609ofB. elkanii,and the isolatesESA96 and ESA97,two

B.elkaniilikefromCaruaru,pointedoutthenodulation param-eters. Other bacteria, such as the Paraburkholderia sp. ESA 96 and the Rhizobium sp. ESA 90,as well as the reference strainBR 5609,induced thesamenitrogenaccumulationin the shootsthanthe plantssupplied withmineralnitrogen, indicatinghighsymbioticefficiency.Thedatareinforcesthe efficiencyofthestrainBR5609toE.velutinaasalreadyshown forthisspecieandtoErythrinafalcataandE.verna.Thedata obtainedinthepresent study confirmthepresenceof effi-cient“mulungu”rhizobiainthesoilsfromsemi-aridregion of Brazil.16 _{Thedata alsosupport the selectionofthe}

iso-lates ESA 90, ESA 96 and ESA 97, along with BR 5609, for furtherevaluationsaimingthe officialrecommendationfor

E.velutina.

Fewstudieshadbeencarriedoutevaluatingatthesame time thephenotypic diversity,taxonomicclassificationand thesymbioticefficiencyoftreespeciesinBrazilianSemi-Arid. Toourknowledge,thedataobtainedinthepresentstudyare thefirstreportoftheconcomitantlycharacterizationof bacte-riafrom“mulungu”atphenotypic,molecularandsymbiotic levels.Inthiscontext,thedataconfirmourhypothesisthatthe soilsofdifferentdrylandsregionsofPernambucoState,under naturalCaatingaregenerationharboradiverseandefficient nitrogen-fixingbacterialcommunity.

Furtherassaysareneededtogetabetterunderstandofthe taxonomyandthepresentbacteriaandtheirpotentialto pro-motethe“mulungu”growthundernon-sterileconditions.In thesamecontext,moreextensiveisolationexperimentsare neededtounderstandthebio-geographicalpatternsof rhizo-bial distributioninthe semi-aridregionofthePernambuco State.

Conclusions

Both␣and␤-rhizobiaareabletooccupytheE.velutinaroot nodulesinsoilsoftheCaatingadryforestsatthePernambuco state (Northeastern, Brazil). The bacteria obtained showed metabolicversatility,tolerancetoabioticstressesandinvitro

plantgrowthpromotionmechanismsandtheabilitytoinduce highnitrogenaccumulationintheshoots.Thisdatasetpoints tothepotentialofthesebacteriatoactasplantgrowth pro-motersbydifferentways.Furthermore,somebacteriacanbe selectedforfurtherstudiesaimingtheofficial recommenda-tionofthebacterialisolatesforE.velutina,mainlytheisolates ESA96andESA90,duetotheirperformanceinthe invivo

experiment.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

TotheBrazilianCouncilforScientificandTechnological Devel-opment(CNPq–process406327/2013-0and472997/2012-2)and the Brazilian Agricultural Research Corporation (Embrapa) forproviding the financialsupport. Tothe Coordination of ImprovementofHigherEducationPersonnel(CAPES)forthe firsttosixthauthorsscholarships.TotheSciencefoundation ofthePernambucoState(FACEPE),forthescholarshiptothe seventhauthor.

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