Rhizobacterial characterization for quality control of eucalyptus biogrowth promoter products

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brazilian journal of microbiology47(2016)973–979

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

Genetics

and

Molecular

Microbiology

Rhizobacterial

characterization

for

quality

control

of

eucalyptus

biogrowth

promoter

products

Talyta

Galafassi

Zarpelon

a

_,

_Lúcio

_Mauro

_da

_Silva

_Guimarães

a

_,

_Poliane

_{Alfenas-Zerbini}

b

_,

Eli

Sidney

Lopes

c

_,

_Reginaldo

_Gonc¸alves

_Maﬁa

d

_,

_Acelino

_Couto

_Alfenas

a,∗

a_Federal_University_of_Vic¸osa,_Department_of_{Phytopathology,}_Vic¸osa,_MG,_Brazil

b_Federal_University_of_Vic¸osa,_Department_of_{Microbiology,}_Vic¸osa,_Minas_Gerais_State,_Brazil c_Bio_Soja_Indústrias_Químicas_e_Biológicas,_São_Joaquim_da_Barra,_SP,_Brazil

d_Fibria_Centro_de_Tecnologia,_Aracruz,_ES,_Brazil

a

r

t

i

c

l

e

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o

Articlehistory:

Received15June2015 Accepted14March2016 Availableonline26July2016 AssociateEditor:JerriÉdsonZilli

Keywords: Formulation PGPR Eucalyptus Biologicalproduct

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b

s

t

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t

Plantgrowth-promotingrhizobacteriastrainsfromspecialformulations havebeenused to optimizeeucalyptuscuttingproduction.Toundertake qualitycontrol forthe formu-latedproducts,therhizobacterialstrainsshouldbecharacterizedtoassesstheir purity andauthentication.Inthepresentstudy,wecharacterizedninestrainsofrhizobacteria, includingthreeBacillussubtilis(S1,S2and3918),twoPseudomonassp.(MF4andFL2),P.putida

(MF2),P.fulva(Ca),Frateuriaaurantia(R1),andStenotrophomonasmaltophilia(CIIb).Thestrains weredifferentiatedbycolonymorphologyafter24hofincubationinthreedifferentsolid stateculturemedia(glucose-nutritiveagar,523mediumandyeastextract-mannitolagar), sensitivitytoapanelof28antibiotics(expressedaccordingtotheformationofinhibition halosofbacterialgrowthinthepresenceofantibiotics),andPCR-RFLPproﬁlesofthe16S rDNAgeneproducedusingninerestrictionenzymes.Itwaspossibletodifferentiateallnine strainsofrhizobacteriausingtheirmorphologicalcharacteristicsandsensitivityto antibi-otics.ThemolecularanalysisallowedustoseparatethestrainsCIIb,FL2andR1fromthe strainsbelongingtothegeneraBacillusandPseudomonas.Byusingthesethreemethods concomitantly,wewereabletodeterminestrainpurityandperformtheauthentication.

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

Introduction

Free-livingbacteria orbacteria associatedwith roottissues prevail in the plant rhizosphere.1 _Plant _{growth-promoting} rhizobacteria(PGPR),the beneﬁcgroupofthese microorgan-isms,areaclassofnon-pathogenicsoilmicroorganisms.2–4

∗ _{Corresponding}_author.

E-mail:aalfenas@ufv.br(A.C.Alfenas).

Rhizobacteria are natural inhabitants ofsoil that are able tocolonizetherootsystemsofplants,therebycontributing several important characteristics. For example, enhanced growthcanoccurdirectlythroughtheproductionofgrowth promoters,oritcanbeinhibitedbytheactionofpathogenic microorganisms.5

http://dx.doi.org/10.1016/j.bjm.2016.07.013

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PGPRbacteriamaydirectlyinﬂuenceplantgrowthbyeither synthesizing plant hormones, such as indol-3-acetic acid (IAA),6,7 _or _favoring _the _uptake _of _nutrients _from _the _soil

through different mechanisms, such as nitrogen ﬁxation,8

phosphorusandpotassiumsolubilization9_and_the_synthesis

ofsiderophoresforironsequestration.10_PGPR_can_also

indi-rectlyaffectplantsthroughantagonismbetweenthebacteria andsoil-bornepathogens11 _and_by_inducing_systemic

resis-tanceinplantsagainstbothrootandfoliarpathogens. Many studies have explored the biocontrol capacity of theseorganisms.Additionally,theirabilitytoproduce antibi-oticsmakesthematargetforthebiologicalcontrolofplant diseases.StrainsofrhizobacteriaisolatedfromEucalyptusspp. have been shown to promote rooting through anincrease inrootbiomassandgrowth ofeucalyptuscuttings12,13 _and

the reduction of Cylindrocladium cutting rot, rust infection (PucciniapsidiiWinter)inthenursery,14,15 _and_bacterial_wilt

(Ralstoniasolanacearum).16_Based_on_the_results,_a_bioproduct

namedRizolyptus®17_formulated_with_selected_{rhizobacterial}

strainshas beenused ineucalyptuscuttingnurseries. The Rizolyptus® _is_an _inoculant _based_on _only_one

rhizobacte-riastraininaliquidformulation.However,itisessentialto know the intrinsiccharacteristics ofeach selected growth-promotingrhizobacteriastrainpriortomasspropagationto ensuretheproduct’squality.

Bacterialcharacterizationiscurrentlybasedon biochemi-caltests,antibioticsensitivity,microscopicobservationsand molecular analysis. Restriction fragment length polymor-phism (RFLP) analysisofthe ribosomalDNA region (rDNA) associatedwithpolymerasechainreaction(PCR)isan appro-priateandinexpensivemolecularmethod.1,2,18–20_Thus,_in_the

present work,wecharacterizedtheRizolyptus® _production

fromninerhizobacterialstrainsbasedontheirmorphology, antibioticsensitivity,andPCR-RFLPprofiles.Thefindingsand methodspresentedinthisstudyrepresentimportanttoolsto ensurethepurity,qualityandauthenticationofstrainsinthe finalproductRizolyptus®_for_{commercialization.}

Material

and

methods

Rhizobacteriumstrains

Ninestrains ofrhizobacteriaisolated from eucalyptusthat werepreviouslyselectedfortheircapacitytopromoterooting

andthegrowthofeucalyptuscuttings14_were_{characterized.}

Thestrainswereidentiﬁedandcodedasfollows:S1,S2and 3918(Bacillus subtilis Cobn); Ca(Pseudomonasfulva Lizuga & Komagata);CIIb(StenotrophomonasmaltophiliaHugh,Palleroni &Bradbury);R1(FrateuriaaurantiaSwingsetal.);MF2(P.putida

Migula);andFL2andMF4(Pseudomonassp.Migula).The molec-ular identiﬁcation, based on homology (>98%) of the 16S rDNAwasperformedaspreviouslydescribed.21_The_cultures

of rhizobacteriaare stored inthe Forest Pathology Labora-tory/Bioagro ofthe Universidade Federal de Vic¸osa, Minas Gerais,Brazil.

Morphologicalcharacterization

The strains were grown on 523 medium,22 _yeast

extract-mannitolagar(YMA)23_and_{glucose-nutritive}_agar_(ANG)24_for

24hat28◦Candwerecharacterizedaccordingtotheircolony shape,elevation,edgetype(Fig.1),consistency(i.e.,mucosus, ﬂuidormycelial),aspectofthecolonysurface(i.e.,smoothor rough),brightness(i.e.,bright,translucentoropaque),color, size(i.e.,<1mm,1–2mm,2–3mmor>3mm),andgrowthspeed (i.e., veryfast: visibletothe nakedeyeafterless than 24h ofincubation;fast:visiblewithin24–48h;intermediate: vis-iblewithin24–48h;slow:visiblewithin36–96h;orveryslow: visibleonlyafter96h).

Antibioticsensitivity

Strain sensitivity to 28 antibiotics was assessedusing the standard antibiogram method.25 _An _inoculum _sample _of

0.1mLwasevenlyspreadinaPetridish(9cmdiameter) con-taining523 medium,and fourWhatman® _No.1_ﬁlter_paper

disks(Ø=0.7cm)thatwerepreviouslysoakedintheantibiotics tobetestedweredistributedoverthemedium.Acompletely randomdesigncontainingthreereplicatesperantibioticwas used.Aftera48-hincubation,thepresenceorabsenceofan inhibitionhalowasobserved.

Molecularcharacterization

GenomicDNAfromtherhizobacterialstrains26_was_ampliﬁed

byaPCRreactionconsistingof10–20ngDNA,2mMMgCl2,

50mMKCl,10mMTris–HCl(pH8.3),0.1mMofeach deoxynu-cleotide(dATP,dTTP,dCTPanddGTP)(Invitrogen),0.1␮Mof

Tala Rhizoid Irregular Convex-high Colonies Shape Elevation Edges

Central protusion Umbilical

Dented Filamentous Curly Flat Spreaded Convex

Filamentous Circular

Pointed

Waved

Complete Lobed

Fig.1–Patternsusedforthemorphologicalcharacterizationofrhizobacterialstrainsbasedontheircolonyshape,elevation andedgetype.

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eacholigonucleotide, 1unitofTaqpolymerase(Phoneutria) enzymeandsterilewater(MilliQ)toreachthefinalvolumeof 50␮L.ThespecificoligonucleotidesP1(5-AGAGTTTGATCC TGGCTCAG-3)andP2(5-AAGGAGGTGATCCAGCCG CA-3)27wereusedtoamplifyafragmentofapproximately1.6kb fromthebacterial16SrDNA16Sregion.Theamplificationwas performedinaMastercycler®_thermocycler_(Eppendorf)_under

thefollowingconditions:94◦Cfor3min,then35cycles com-prising1minat94◦C,30sat60◦Cand1minand30sat72◦C, andafinalextensionat72◦Cfor7min.Subsequently,15␮Lof theamplifiedproductwascleavedseparatelywiththeEcoRI, RsaI,Sau3AI,SphI,MspI,BamHI,DdeI,TaqIandHinfIrestriction enzymes.Avolumeof1␮Lofrestrictionenzymein3␮Lof 10×bufferwasaddedtotheamplifiedproductandincubated underthespecifictimeand temperatureconditions recom-mendedforeachenzymebythemanufacturer(Promega).The cleavedfragmentswereseparatedinanagarosegel(1.2%)and photodocumented.

Results

Morphologicalcharacterization

Themorphologicalcharacterizationvariedaccordingtothe culturemedium(523,YMA,or ANG)and therhizobacterial strainassessed.Thecolonycolorin523mediumallowedfor thedifferentiationofeightoftheninestrainstested,whereas thecolonyelevationinANGmediumallowedforthe differ-entiationofsixstrains (Table1).Allofthestrainscould be differentiatedon 523mediumwhenthesetwo characteris-ticswerecombined.Consistency,surface,andgrowthspeed provided minor contributions to the differentiation of the assessedrhizobacterialstrains(Table1).

TheR1strainwasdifferentiatedfromtheothersbyitsfast growth(within24h)followingincubationinallthreegrowth media(Table1).ColoniesoftheCIIbisolatedifferedfromthe othersbasedontheirpointedshapeandcolonysizeofless than1mminthethreemediatested.AlthoughtheS1,S2and 3918strainsbelongtoB.subtilis,theywereindividually differ-entiatedbytheircolorin523mediumandbytheiredgeshape inANGandYMAmedia(Table1).Thesestrainswereseparated fromtheothersbytheirbrightnessinANGandYMAmedia andbytheirsurfacecharacteristicsinYMAmedium.TheCa, FL2,MF2andMF4strainsweredifferentiated fromthe oth-ersbycolonycolor.Additionally,FL2wastheonlystrainthat showedatranslucentbrightnessinthethreeculturemedia evaluated(Table1).

Antibioticsensitivity

Pefloxacinwastheonlyantibiotictestedthatinhibitedallof therhizobacterialstrains(Table2).Alloftheremaining antibi-oticsinhibitedatleastonestrain.TheS2andR1strainswere themostsensitivetotheantibioticstestedandwereresistant toonlytwo(penicillinandcephalexin)andthree(oxacillin, rifampicin,andaztreonam)antibiotics,respectively.In con-trast,FL2 wasthe leastsensitive,withonlyfive antibiotics (pefloxacin,pipemidic acid,streptomycin, tetracycline, and sulfonamide)inhibitingitsgrowth.TheMF2andFL2strains

wereeasilyseparatedfromeachotherandtheotherstrains using a single antibiotic (e.g., sulfonamide and amikacin, respectively). A minimumof twoantibiotics were required to distinguish between the S1, CIIb, and MF4 strains (e.g., for the1st strain: ceftazidime and tetracycline; 2nd strain: streptomycinandneomycin;and3rdstrain:amoxycillinand rifampicin). Theother strains couldbedistinguished using atleastthreeantibioticssequentiallyintheculturemedium (e.g.,forS2:cephalexin,sulphazotrim,andstreptomycin;3918: ampicillin,sulfonamide,andneomycin;andCa:clindamycin, sulfonamide,andamikacin).

Molecularcharacterization

Theampliﬁcationof16SrDNAgeneratedfragments approx-imately 1.6kb insizeforall strains.Thecleavageforthese fragmentswithrestrictionenzymesallowedusdifferentiate two groups ofstrains (Bacillus (3918,S1, and S2) and Pseu-domonas (MF2, MF4, and Ca)), from the remaining strains (Fig.2).However,itwasnotpossibletodistinguishstrainsfrom thesamegenus.Alloftheotherstrainscouldbe differenti-atedfromeachotherusingatleastonerestrictionenzyme. CIIbwasthemostuniqueamongthetestedstrains,withsix oftheninetestedenzymesresultinginrestrictionproﬁlesthat allowedforitsseparationfromtheotherstrains.AlthoughCa andFL2belongtothesamegenus,theywereeasily differenti-atedbycleavagewiththeSau3AI,MspI,DdeI,BamHI,andTaqI

enzymes.Theserhizobacteriabelongtodifferentspeciesof

Pseudomonas,whichmakestheiridentiﬁcationeasier;similar resultswere observedforStenotrophomonasmaltophilia(CIIb) and Frateuria aurantia(R1). TheMspIenzymeallowedusto discern morestrains, separatingBacillus (3918, S1,and S2),

Pseudomonas(MF2,MF4,andCa),FL2,R1,andCIIb.Incontrast, theuseofBamHIonlydifferentiatedFL2fromtheotherstrains. Theremainingenzymesallowedustoseparatethestrainsinto 3–5groups(Fig.2).

Discussion

The use of pre-selectedbacterial strains to induce rooting and growth of eucalyptus cutting requires authentication of the rhizobacteria employed in the formulated product (Rhizolyptus®₎ _without_mixture _between_the _strains_of

rhi-zobacteriaorcontamination.Inthisstudy,wecharacterized nine strains of rhizobacteria based on their morphology, antibioticsensitivity,andPCR-RFLPproﬁles.

Allstrainsweredistinguishedbasedontheir morphologi-calcharacteristicsincultureandsensitivityto28antibiotics. Their morphologicalcharacteristics variedaccording tothe culturemediumandstrainused.Colorandcolonyelevation in523mediumwerethemostefficientmorphologicalfeatures for strain identification, allowing the differentiation of all rhizobacterialstrainsstudied.However,theuseofthese char-acteristicsdemandedexpertiseandskilltodistinguishsubtle differencesamongthestraincolonies.Specialcarewasalso requiredtorecognizeintermediateshapesthatmayhamper the accuracyoftheidentification.Therefore, adding antibi-oticstotheculturemediumcouldaidintheidentificationof bacterialstrains.Forexample,theantibioticsensitivityprofile

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b r a z i l i a n j o u r n a l o f m i c r o b i o l o g y 4 7 (2 0 1 6) 973–979

Table1–Morphologicalcharacterizationofrhizobacterialstrainsassessedafter24hofincubationinsolidstateglucose-nutritiveagar(ANG),523mediumandyeast

extract-mannitolagar(YMA).

Medium Strain Morphologicalcharacteristics

Elevation Shape Edges Color Brightness Growth Size Consistency Surface

ANG S1 Spread Circular Complete LightBeige Opaque Veryfast 1–2mm Mucosus Smooth

S2 Spread Irregular Dented Beige Opaque Veryfast 1–2mm Mucosus Rough

3918 Flat Irregular Waved LightBeige Opaque Veryfast 2–3mm Dried Rough

Ca Convex–low Irregular Complete LightBeige Bright Veryfast 1–2mm Mucosus Smooth

CIIb Convex–high Pointed Complete LightBeige Bright Veryfast <1mm Mucosus Smooth

R1 Flat Pointed Complete LightBeige Bright Fast 1–2mm Mucosus Smooth

FL2 Flat Circular Complete Yellow-orange Translucent Veryfast >3mm Mucosus Smooth

MF2 Umbilical Circular Complete DarkBeige Bright Veryfast 1–2mm Mucosus Smooth

MF4 Centralprotrusion Circular Complete Beige-yellow Bright Veryfast 1–2mm Mucosus Smooth

523 S1 Convex–low Circular Complete Beige Bright Veryfast >3mm Mucosus Rough

S2 Convex–low Circular Complete LightBeige Bright Veryfast >3mm Fluid Smooth

3918 Umbilical Circular Complete White-ice Bright Veryfast >3mm Fluid Rough

Ca Umbilical Circular Waved Beige-green Bright Veryfast >3mm Mucosus Smooth

CIIb Convex–medium Pointed Complete White Bright Veryfast <1mm Mucosus Smooth

R1 Flat Circular Dented Beige Bright Fast 1–2mm Mucosus Smooth

FL2 Flat Circular Complete Green-ﬂuorescent Translucent Veryfast >3mm Mucosus Smooth

MF2 Convex–low Circular Complete Yellow-green Bright Veryfast 1–2mm Mucosus Smooth

MF4 Umbilical Circular Complete Beige-yellow Bright Veryfast 2–3mm Mucosus Smooth

YMA S1 Flat Circular Lobed White Opaque Veryfast >3mm Dried Rough

S2 Flat Irregular Dented Beige Opaque Veryfast >3mm Dried Rough

3918 Flat Irregular Waved White Opaque Veryfast >3mm Dried Rough

Ca Flat Irregular Complete Beige-yellow Bright Veryfast >3mm Mucosus Smooth

CIIb Convex–high Pointed Complete White Bright Veryfast <1mm Mucosus Smooth

R1 Flat Pointed Complete LightBeige Bright Fast 1–2mm Mucosus Smooth

FL2 Flat Circular Complete Yellow-orange Translucent Veryfast 2–3mm Mucosus Smooth

MF2 Centralprotrusion Circular Complete Beige Bright Veryfast 1–2mm Mucosus Smooth

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Table2–Rhizobacterialstrainsensitivityexpressedaccordingtotheformationofinhibitionhalosofbacterialgrowthin thepresenceofantibiotics.

Antibiotic Rhizobacterialstrains

Commercialname Concentration S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4

Peﬂoxacin 5mcg + + + + + + + + +

PenicillinG 10U − − − − − + − − −

Amikacin 30␮g + + + + + + − + +

Amoxycillin+clavulanicacid 30mcg + + + + + + − + +

Cefepime 30␮g + + + + + + − + + Cefotaxime 30mcg + + + + + + − + + Norﬂoxacin 10␮g + + + + + + − + + Sulphazotrim 25␮g + + + − + + − − − Clindamycin 2␮g + + + − + + − − − Vancomycin 30mcg + + + − + + − − − Oxacillin 1␮g + + + − − − − − − Rifampicin 5␮g − + − − + − − + + Tobramycin 10␮g − + − + − + − + + Aztreonam 30␮g − + + − − − − + + Cefalotin 30␮g − + + − − + − − − Ceftazidime 30␮g − + + + + + − + + Cephalexin 30mcg + − + + − + − − − Ampicillin 10␮g + + − + + + − − − Chloramphenicol 30␮g + + − + + + − − − Erythromycin 15␮g + + − − − + − − − Nitrofurantoin 300␮g + + − − − + − + − Amoxycillin 10␮g + + − + + + − + − Pipemidicacid 20mcg + + − + + + + − + Streptomycin 10mcg + + + + − + + − + Neomycin 30mcg + + + + − + − + − Tetracycline 30␮g − + − + + + + − +

Ticarcillin+clavulanicacid 85mcg + + + + + + − − +

Sulfonamide 300␮g + + + + + + + − +

Absenceofinhibitionhalo(−). Presenceofinhibitionhalo(+).

andtriagebyPCRofthetoxinshowedthatBacilluspumiluswas thepredominantspeciesintheseashoreareaaroundCochin, India.28 _In_addition_to_help_the_{rhizobacterial}_{identiﬁcation,}

the supplementation of the culture medium used to pro-ducetheinoculantswithantibioticsavoidedcontamination orstrainmisusebecauseitallowedonlyantibiotic-resistant straingrowth.Forexample,theP.putida(MF2)straincouldbe differentiatedbytheuseofsulfonamidetoensurestrainpurity becausethisantibioticinhibitedalloftheotherrhizobacterial strainstested.Similarly,amikacin,amoxycillinandclavulanic acid,cefepime,cefotaxime,andnorﬂoxacindidnotaffectthe growthoftheFL2isolate.

Recently, molecular techniques have been broadly employed in taxonomy and biodiversity studies of bacteria.1,19,20,29–32 _The _PCR-RFLP _analysis _results _may

vary among genera and species along with the 16S rRNA and 23S rRNA sequences.1,32 _We_used _the _16S _gene _in_the

PCR-RFLP technique for the molecular characterization of rhizobacterialstrainsbecauseityieldsconsistentresultsand isaneasytouse,reliable,andsensitivemethod.Theresults from the molecular analysis in this study were similar to theﬁndingsofotherauthors.19,31–34 _The_cleavage_of_partial

sequencesofthe16S regionoftheribosomalDNA allowed fordifferentiationofthegenusBacillus(3918,S1,andS2)and

Pseudomonas(MF2,MF4,andCa)fromotherstrains(CIIb,R1,

and FL2).However,theobservedrestrictionproﬁlesdidnot differamongstrainsS1,S2,and3918(thespeciesofB.subtilis)

or amongMF2, MF4, and Ca (the species of Pseudomonas),

most likely because the 16S region of the ribosomal DNA ofthese specieswashighly conserved.Tassaand Duarte20

were also not successful in separating Pectobacterium caro-tovorumsubsp.brasiliensis,P.carotovorumsubsp.carotovorum,

and the other pectobacteria available in GenBank due to the lowspeciﬁcitypresentedbythe enzymaticdigestionof the ampliﬁed recA gene. However, cleavage with the HhaI

and TasI enzymes allowed for the separation of 13 differ-ent groups and the discrimination ofP.carotovorum subsp.

brasiliensis.

ThesamemolecularmethodcouldbeusedtoseparateB. subtilisfrom thePseudomonasstrains, butit requires ampli-ﬁcationoftheITSrDNAregions.Becausemutationsinthis regionaremorefrequentthanintheribosomalgenes,which areveryusefulforintraspeciﬁcseparation.29,35_The_intergenic

space(ITS)regionhasbeenusedtodifferentiateindividuals, includinggeneticallyrelatedspecies.29,30,35_For_example, Xan-thomonasaxonopodispv.citriTypeAcanbedifferentiatedfrom

X.axonopodispv.aurantifoliiTypesBandCandX.axonopodispv.

citrumelaTypeEbyamplifyingtheITSregionbetweenthe16S and23Sregions,followedbyposteriorcleavagewiththeDdeI, AluI,andSau3AIrestrictionenzymes.30

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M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 CaCIIb R1 FL2 MF2 MF4

M’ S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 Ca CIIbR1 FL2 MF2 MF4

M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 M S1 S2 3918 Ca CIIb R1 FL2 MF2 MF4 12 000 5000 2000 1650 1000 850 650 500 400 300 200 100 12 000 5000 2000 1650 1000 850 650 500 400 300 200 12 216 3054 2036 1636 1018 506 396 bp

G

H

A

B

C

D

E

F

I

Fig.2–Restrictionproﬁleofampliﬁedfragmentsofthe16SregionfromtherDNAofninerhizobacterialstrains.The fragmentswerecleavedwiththefollowingrestrictionenzymes:(A)EcoRI,(B)RsaI,(C)Sau3AI,(D)SphI,(E)MspI,(F)BamHI,(G)

DdeI,(H)HinfI,(I)TaqI,and(M)restrictionenzyme–1kbPlusDNALadder.

Conclusions

Morphologicalcharacterizationofthecolonies,assessmentof antibioticsensitivity,and PCR-RFLPanalysiscanbeusedto separatetheninestrainsofrhizobacteriatested.

Theconcomitant useofthe threemethodspresentedin thisworkreducesthelikelihoodofacontaminantbeing mul-tipliedwiththeselectedrhizobacteriastrain.

Conﬂicts

of

interest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgments

ThisresearchwassupportedbyCNPq,FAPEMIGandCAPES.

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