h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Polyphasic
characterization
of
bacteria
obtained
from
upland
rice
cultivated
in
Cerrado
soil
夽
Lívia
Fabiana
Braga
a,
Fênix
Araújo
de
Oliveira
a,
Eva
Aparecida
Prado
do
Couto
a,
Karina
Freire
d’Ec¸a
Nogueira
Santos
a,
Enderson
Petrônio
de
Brito
Ferreira
b,∗,
Claudia
Cristina
Garcia
Martin-Didonet
aaUniversidadeEstadualdeGoiás(UEG),Anápolis,GO,Brazil bEmbrapaArrozeFeijão,SantoAntôniodeGoiás,GO,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:Received29November2016 Accepted27April2017 Availableonline5August2017 AssociateEditor:FernandoAndreote
Keywords: Oryzasativa PGPR Associativebacteria Bacterialdiversity
a
b
s
t
r
a
c
t
Thisworkaimedtocharacterize20isolatesobtainedfromuplandriceplants,basedon phe-notypic(morphology,enzymaticactivity,inorganicphosphatesolubilization,carbonsource use,antagonism),genotypicassays(16SrRNAsequencing)andplantgrowthpromotion. Resultsshowedagreatmorphological,metabolicandgeneticvariabilityamongbacterial isolates.Allisolatesshowedpositiveactivityforcatalaseandproteaseenzymesand,90% oftheisolatesshowedpositiveactivityforamylase,catalaseand,nitrogenase.Allisolates wereabletometabolizesucroseandmalicacidincontrastwithmannitol,whichwas metab-olizedonlybyoneisolate.Fortheothercarbonsources,weobservedagreatvariabilityin itsusebytheisolates.MostisolatesshowedantibiosisagainstRhizoctoniasolani(75%)and
Sclerotiniasclerotiorum(55%)and,50%ofthemshowedantibiosisagainstbothpathogens. Sixisolatesshowedsimultaneousabilityofantibiosis,inorganicphosphatesolubilization andproteaseactivity.Basedonphylogeneticanalysisofthe16SrRNAgenealltheisolates belongtoBacillusgenus.Undergreenhouseconditions,twoisolates(S4andS22)improved toabout24%,25%,30%and31%theTotalN,leafarea,shootdryweightandrootdryweight, respectively,ofriceplants,indicatingthattheyshouldbetestedforthisabilityunderfield conditions.
©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/
licenses/by-nc-nd/4.0/).
夽PartoftheMaster’sDissertationofthefirstauthorapprovedbytheMolecularSciencePost-GraduationProgramoftheGoiásState
University.
∗ Correspondingauthor.
E-mail:enderson.ferreira@embrapa.br(E.P.Ferreira).
https://doi.org/10.1016/j.bjm.2017.04.004
1517-8382/©2017SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Beneficialendophyticmicroorganismscanliveinsideplants without causing damage to the host. Among them, many bacteria colonize the intercellular spaces throughout the whole plant, including seeds.1 Intercellular spaces contain
sources of carbohydrates and minerals, such as: nitrogen, phosphorus, calcium, potassium, and chlorine, as well as othermetabolitesasorganicacids,facilitatingbacterial devel-opmentandcolonizing.2
Among several microorganisms colonizing plant tis-sues,plantgrowth-promotingrhizobacteria(PGPR)werealso found.3ThesePGPRsprovidebeneficialeffects,suchas:
min-eralnutritionimprovement,increaseonthetoleranceofbiotic andabioticstresses,rootdevelopmentpromotion,and sup-pressionofsoilbornediseases.2
Also,thesebacteriacanbeinvolvedonnitrogenfixation, inorganicphosphatesolubilization,ironcomplexation, phy-tohormonessynthesisandplantpathogencontrol,involving many enzymes on these processes.2,4 Thus, plants under
influence of PGPR can be greater, stronger, more produc-tiveandhealthier.3,5,6Besides,theassessmentofbiochemical
characteristicsinbacteriaallowsinferencesabouttheir adap-tivecapacitytotheenvironmentandabouttheplant–bacteria interaction,sinceitinvolvesseveralgenesandmechanisms, suchaschemoattractionandbiofilmformation,amongother activities.3
AssociationbetweenPGPRsandrootofriceplantshasbeen studied and many isolates showingeffects on rice growth werefound.5Theapplicationofthistechnologyonricechain
productioncanprovidebenefitsforcropgrowthandreduce productioncosts,aswellashelpingonthereductionof envi-ronmentalrisks.5,7
Furthermore, the identification and characterization of bacteriaasPGPRprovideinsightsforunderstandingthe com-positionofbacterialcommunitiesassociatedwithriceplants grownunderCerradoconditions.However,studieswithfocus ontheisolationandcharacterizationofbacteriafromCerrado soilsabletopromoteplantgrowthofuplandricearestillscarce aswellas,necessaryforthiscrop.Thus,thisstudyaimedto characterizebacterialisolatesobtainedfromricerootsbased onbiochemicalandgeneticcharacteristicsand,todetermine theirabilitytopromoteplantgrowthaimingtheiruseas inoc-ulantforuplandrice.
Material
and
methods
Bacterialisolates
Theisolatesevaluatedinthisstudywereobtainedfromrice roots by Rezende8 and are available at the Collection of
MicroorganismsandMultifunctional FungiofEmbrapaRice andBeans.
MorphologicalcharacterizationandGramcoloringassays
Morphologicalcharacterizationwas performedaccordingto Vermelhoetal.9basedonshape,border,surface,consistence
andelevationofthecolonies.Gramcoloringwasperformed accordingtoLouvetetal.10
Biochemicalassays
All biochemicaltrialswereperformedonacompletely ran-domizeddesignintriplicateunderlaboratoryconditions.For theevaluationofcarbonsourcesuse(maleicacid,malicacid, nicotinic acid,d-arabinose,d-fructose, d-glucose,mannitol, mio-inositol, sucroseand sorbitol),each isolatewas inocu-latedinPetridishescontainingsolidKingBmediumadded witheachdifferentcarbonsourcesandincubated(28◦C;2d). Afterincubation,wecheckedoutthegrowthoftheisolates accordingtoHungriaetal.11
The activity of the enzymes citrate lyase and urease wasmeasuredthroughqualitativeassaysbyinoculatingthe isolates on solid Simmons citrate and urea medium and observing the blue and red colors of the culture medium, respectively.9 Nitrate reductase and nitrogenase enzymes
werequalitativelyassayedinsemi-solidmedium.12Catalase
activitywasassayedaccordingtoVermelhoetal.9 For
amy-laseandproteaseactivities,weobservedtheformationofa translucid haloaroundthe colonies,formedbytheisolates inoculatedonM9solidmediumcontaining1%ofstarchand 2%ofmilkprotein,respectively.13Cellulaseactivitywas
deter-minedaccordingtoCattelanetal.14
Solubilizationofinorganicphosphateassaywasperformed using three replicates. Solubilization abilitywas confirmed based on the observation of a translucid halo around the coloniesformedbytheisolatesinoculated onNBRI-P15 and
Pikovskayamedium.16
Antagonistictest
Theantagonisticabilityoftheisolateswasperformedusing three replicates,underin vitroconditions againstthefungi
Sclerotinia sclerotiorum and Rhizoctonia solanion Petri dishes containingBDAmediumaccordingtoHammamietal.17
Clusteringanalysisoftheisolatesbasedon morpho-physiologicaldata
Morphological, enzymatic activity, carbon source use and antibiosis datawere transformed into abinary matrix and submitted to cluster analysis performed by the software NTSYSpc®23usingUPGMAasgroupingalgorithmandJaccard
assimilaritycoefficient.
16SrRNAgenesequencinganalysis
Based on the clustering analysis of the isolates based on morpho-physiologicaldata11isolates(S2,S4,S6A,S17,S22, S26,S29,S37,S41,S63andS105)wereselectedfor16SrRNA gene sequencing. The DNA of the isolates was obtained accordingtoLaranjoetal.18The16SrRNAgenewas
ampli-fiedbyPCRusingtheprimersY1andY3.18,19Theamplicons
were purifiedandusedonthe sequencingreaction accord-ing toLaranjoet al.18Sequences coding forthe partial16S
rRNAgeneoftheisolatesS2,S4,S6A,S17,S22,S26,S29,S37, S41,S63andS105wasobtainedandwhensubmittedtothe
GenBank database (www.ncbi.nlm.nih.gov) received the accession numbers KX855959.1, KX855958.1, KX197211.1, KX197210.1,KX855960.1,KX197209.1,KX855957.1,KX197208.1, KX197214.1,KX197206.1andKX197207.1,respectively.
Evaluationofplantgrowthpromotion
Based on genotypic characterization, four isolates were selectedtobeevaluatedundergreenhouseconditionsas com-pared to a no-inoculated treatment (NI). Seeds of rice cv. Aimoréwere sownin8Lpotsfilled withsoiland arranged inarandomblockdesignwithfourreplicates.Atsowingtime, seedswereinoculatedwithpeat-basedinoculantcontaining 1×108cellg−1ofeachisolateandreferencestrain.Riceplants
wereharvestedat40daysafteremergence(DAE)todetermine leafarea(LA),rootvolume(RV),rootdryweight(RDW),shoot dryweight(SDW)accordingtoFerreira20,21 and,thetotalN
(N-Total)usingtheKjedahlmethod,asdescribedbySilvaand Queiroz.22
Dataanalysis
For the 16S rRNA-based phylogeny study, the sequences obtained were submitted to NCBI BLAST against a non-redundant nucleotide database for getting homologous sequences.24 Sequences showingdegree of similarity were
aligned using the CLUSTAL W program.25 The
evolution-aryhistory wasinferred byusing themaximum likelihood method,26withtreeconsensusbeinginferredfrom500
repli-cates using bootstrap.27 All positions containing gaps and missing data were eliminated. There were a total of 1312 positionsinthefinaldataset.Evolutionaryanalyseswere con-ductedinMEGA6.28
Dataobtainedfromthegreenhouseexperimentwere sub-jected toanalysis ofvariance.When F was significant,the Scott–Knotttestofmeanswasappliedwitha5%probability usingSISVARstatisticalsoftware.
Results
Analysisbasedonthemorphologicalcharacteristicsofthe iso-latesresulted infour differentgroups(A, B,Cand D). The groupswerecomposedby5,9,2and4isolates,respectively
(Table1).IsolatesofgroupsAandBshowthesame
morpho-logicalcharacteristics,exceptforGramcoloration,bywhich theisolatesofgroupAareGram−,whileisolatesofgroupBare Gram+.Similarly,isolatesofthegroupCareGram−,and iso-latesofgroupDareGram+.Additionally,isolatesofgroupsA andBshowdifferentmorphologicalcharacteristicscompared toisolatesofgroupsCandD(Table1).
The results of carbon source use and enzyme activ-ity showed a great metabolic diversity amongthe isolates
(Table2).
Allthe isolateswereable touse sucroseandmalicacid ascarbonsource,whileonlytheisolateS22wasabletouse mannitol.Also,45%and35%oftheisolateswereabletouse fructoseandmio-inositol,respectively.Besides,theisolateS22 wasabletousenineofthetencarbonsourcesevaluated,while theisolateS97usedjusttwoofthesesources(Table2).
G1 0.40 0.50 0.60 0.70 Jaccard coefficient 0.80 0.90 1.00 S2 S5 S4 S8A S35 S37 S39 S29 S41 S32 S6 S25 S26 S97 S6A S105 S8B S22 S17 S63 G2 G3 G4 G5 G6
Fig.1–Consensusdendrogramobtainedbycombiningthe morphological,enzymaticactivity,carbonsourceuseand antibiosisdataamong20endophyticisolatesobtained fromuplandriceplants.Dendrogramwasgeneratedbythe algorithmUPGMAandsimilaritymatrixobtainedfromthe useofJaccardcoefficient.
Alltheisolatesshowedcatalaseactivity,while90%ofthe isolatesshowedamylase,phosphataseandnitrogenase activ-ity. TheisolatesS8A, S22,S35 and S105showed activityof sevenenzymes,andisolatesS5andS19 showedactivityof
four(Table2).
For the phosphate solubilization assay, only the results obtainedfromthePikovskayamediumareshown,sincewe didnotobservegrowthoftheisolatesintheNBRI-Pmedium. About 75% ofthe isolateswere abletosolubilize inorganic phosphate(Table3).TheseisolatesshowedaSIofabout111% inaverage, withthe greatestvaluesbeen observedfor iso-latesS2(133.86%)andS5(133.6%).IsolatesS8bandS63did notpresentsolubilizationability.
ThegeneralmeanobservedforPEIwasabout1.92.Six iso-lates(S6,S4,S29,S17,S37andS2) showedPEIgreater than 2,whiletheisolateS105showed thesmallestvalueforPEI
(Table3).
OntheantibiosisassaysagainstS.sclerotiorum,11isolates were able toinhibitthe growth ofthe fungi,whileagainst
R.solani14 isolatesinhibitedthefungigrowth.Besides, the isolatesS6A,S25,S26,S29,S32,S35,S37,S41,S97andS105 showedantibiosisagainstbothfungi(Table3).
Thesimilarityanalysisbasedonmorphological character-istics, enzymatic activity,carbon sourceuse and antibiosis assayrevealedsixclustersformationat60%ofsimilarity, indi-catinggreatdiversityamongthe20isolatesregardingthese traits(Fig.1).
TheisolateS63,ongroupG6,showeda40%similarityto the otherisolates,followedbytheisolatesofthe groupG1. Thegreatestsimilaritywasabout90%,observedbetweenthe isolatesS5andS37.Theseisolatesgroupedwithothereight isolatesongroupG2(Fig.1).
The16SrRNAgenesequencinganalysisrevealedthatall theevaluatedisolatesshowedhighsimilaritywiththegenus
Bacillusascomparedwithotherplantgrowth-promoting gen-era(Fig.2).
ThephylogenyanalysisconsideringonlythegenusBacillus
showedthattheisolatesS6A,S17,S26,S37andS22areclosely relatedwiththestrainsEGE-B-Li-6andSt05ofB.subtilisand
Table1–MorphologicalcharacteristicsonKingBmediumofendophyticisolatesobtainedfromriceplants.
Groups Isolate Shape Border Surface Consistence Elevation Gram
A S2 I W R D F − A S4 I W R D F − A S6A I W R D F − A S39 I W R D F − A S97 I W R D F − B S5 I W R D F + B S6 I W R D F + B S8A I W R D F + B S8B I W R D F + B S17 I W R D F + B S32 I W R D F + B S35 I W R D F + B S37 I W R D F + B S105 I W R D F + C S41 C S G A H − C S25 C S G A H − D S22 C S G A H + D S26 C S G A H + D S29 C S G A H + D S63 C S G A H +
I,irregular;C,circular;W,wrinkled;S,smooth;R,rough;G,gentle;D,dry;A,aqueous;F,flat,H,high.
Table2–Metaboliccharacteristicsofendophyticisolatesobtainedfromriceplants.
Evaluations Isolates S2 S4 S5 S6 S6A S8A S8B S17 S22 S25 S26 S29 S32 S35 S37 S39 S41 S63 S97S105 Carbonsources Sucrose + + + + + + + + + + + + + + + + + + + + Glucose + + + + + + + + + + + + + + + + + + − − Fructose − − − − − − + − + + − − − − − − − − − + Mannitol − − − − − − − − + − − − − − − − − − − − Sorbitol − − − + − − + + + − − − − − − − − + − + Mio-inositol + + + − − + + − − − − − − + + + − − − − Arabinose − − − − + − + + + − − − − − − − − + − + Maleicacid + + + + − − − − + − − − − − − − − + − − Nicotinicacid − − + − − − + + + + − − − − − − − − − − Malicacid + + + + + + + + + + + + + + + + + + + + Enzymeactivity Cellulase + − + − + + + − + − − − − − − − − − + + Amylase + + + + + + + + + + − + + + + + + − + + Protease + + + + + + + + + + + + + + + + + + + + Phosphatase + + + + + + − + + + + + + + + + + − + + Urease + − − − − − − − − − − − + − − − + + − − Nitrogenase + + − + + + + + + + + + + + + + + + − + Reductasenitrate − + − + + + + − + + + − + + + − − − + + Citratelyase − + − − − + − + + − + + − + − − − + + − Catalase + + + + + + + + + + + + + + + + + + + +
B.methylotrophicus,respectively,whiletheisolatesS2andS41 areclosely relatedwithdifferentstrainsofB. methylotrophi-cusandB.valezensis.Ontheotherhand,theisolatesS63and S105clusteredwiththestrainFBZ42ofB.valezensis.Also,the isolateS29showedhighsimilaritywithB.vallismortisandB. amyloliquefaciens.Thisanalysisalsorevealedthattheisolate S4isanoutgroup,showinglowsimilaritywiththedifferent strainsofthegenusBacillus.EvenbelongingtoBacillusgenus (Fig.2)andshowing98%ofidentityontheBLASTanalysiswith thestrainSL8ofB.amyloliquefaciens,thespecieofthisisolate isstilluncertainneedingfurtherstudiestoitsdetermination (Fig.3).
Based on the results of the phylogenetic analysis, the isolates S4, S22, S29 and S105 were chosen to be evaluated as plant growth promoters under greenhouse conditions. Results showed a clear effect of the isolates on the root development of upland rice plants cv. Aimor é(Fig.4).
Theplantgrowthpromotionabilityoftheisolateswasalso observedoverrootdryweight(RDW),leafarea(LA),shootdry weight(SDW)andtotalN(N-Total)accumulatedontheshoots ofriceplantsasobservedinTable4.TheisolatesS4andS22 promotedhighervaluesofRDW,LA,SDWandN-Totalas com-paredtoNItreatment(Table4).
Table3–Solubilizationindex(SI)forinorganicphosphateassayedinPikovskayamedium,proteaseenzymaticindex (PEI)andantibiosisassaysagainstSclerotiniasclerotiorum(Ss)andRhizoctoniasolani(Rs)ofendophyticisolatesobtained fromriceplants.Meanvaluesofthreereplicates.
Isolate SI(%) PEI Antibiosis
Ss Rs S2 133.86(±16.9) 2.03(±0.15) − − S4 126.94(±13.7) 2.52(±0.22) − + S5 133.60(±14.5) 1.98(±0.04) − − S6 130.56(±4.8) 3.04(±0.06) − + S6A 116.78(±9.8) 1.88(±0.18) + + S8A 119.63(±2.8) 1.57(±0.10) − − S8B 0 1.88(±0.05) − − S17 117.10(±7) 2.21(±0.22) − + S22 131.02(±7.9) 1.67(±0.01) + − S25 129.17(±19) 1.91(±0.17) + + S26 114.95(±4.3) 1.96(±0.13) + + S29 123.15(±11.2) 2.36(±0.15) + + S32 119.39(±10) 1.96(±0.34) + + S35 120.20(±6.1) 1.61(±0.13) + + S37 117.78(±1.9) 2.19(±0.07) + + S39 116.06(±5.3) 1.96(±0.13) − − S41 128.52(±5.7) 1.89(±0.07) + + S63 0 1.57(±0.10) − + S97 116.74(±8.4) 1.73(±0.07) + + S105 126.52(±9.1) 1.35(±0.08) + +
Table4–Effectofdifferentisolatesontheleafarea(LA),rootdryweight(RDW),shootdryweight(SDW)andtotalN (N-Total)ofuplandricecv.Aimoréascomparedtoano-inoculatedtreatment(NI).Meanvaluesoffourreplicates.
Treatments RDW(gplant−1) LA(cm2plant−1) SDW(gplant−1) N-Total(mgplant−1)
S4 3.57a 678.63a 6.88a 217.48a S22 2.79a 601.01a 5.92a 218.02a S29 2.01b 462.35b 4.46b 161.72b S105 2.05b 561.68a 5.44a 219,06a NI 2.16b 475.15b 4.45b 164.87b CVa(%) 8.95 8.08 7.55 7.90
ValuesfollowedbythesameletterwithineachcolumnarenotsignificantlydifferentbytheSkott–Knotttest(p<0.05).
a CV=coefficientofvariation.
Discussion
The morphological analysis revealed four different mor-phological groups with the prevalence ofisolates showing irregularshape,wrinkledborderandGram+bacteriaaswell
(Table1).ThegenusBacillussp.comprisesGram+bacteriaand
severalspeciesofthisgenusshowcolonymorphologyquite similartothosefoundinthisstudy.29
All isolateswere able touse sucrose and malicacid as acarbonsource(Table2).Theabilitytometabolizesucrose indicatesthat theseisolateshaveenzymeinvertase,which catalysesthehydrolysisofsucrose,allowingthoseisolatesto usethiscompoundasanenergysource.30Malate,theionized
formofmalicacid,isasecondpreferredcarbonsourceforB. subtilis.31
Wealsofoundtheenzymeamylaseinmostoftheisolates
(Table2).Amylasesarewidelydistributedinbacteriaandfungi
andareclassifiedbytheactionofexoenzymes,endoenzymes andenzymedebranching.4
Bacteriautilizecitratethroughtheenzymecitratelyase. Inaddition,those bacteriacanproduceother organicacids
derivatives,whichcanactchelatingaluminumand phospho-rusmolecules.32Inourstudy,amongthepositivebacteriafor
citratelyaseonlyonedidnotshowthecapacitytosolubilize inorganicphosphate,indicatingthatfortheotherbacteriaa synergybetweentheseactivitiesmayoccur.
Amongtheisolates,tenofthemshowedactivityfornitrate reductase and nitrogenase, and three for urease activity
(Table2).Theseenzymes aredeterminingfactorsrelatedto
biological nitrogen fixationand nitrogenmetabolism.2 The
presenceoftheseenzymesindicatesthattheseisolatesshow metabolicpathwaysinvolvedinNassimilationand,thusthey cancontributetoplantgrowth.7
Ontheotherhand,onlytwoisolatesdidnotshowinorganic phosphate solubilizationcapacity(Table3).Besides,among thebacteriastudiedinthisstudy,thehighestinorganic phos-phatesolubilizationindex(SI)wasabout 134%.Severalsoil bacteriacansolubilizeinorganicphosphateandprovidePfor plantgrowth,increasingPutilizationefficiency.33Bacterialike
PseudomonasandAcetobactercanshowSIof260%and483%, respectively.34
On studies with bacteria isolated from Brazilian soils, theaverageSIwas170.5%andthebacteriaLMG1222,which
73 100 100 100 100 100 60 99 0.02 S63(KX197206.1) S2(KX855959.1) S105(KX197207.1) S4(KX855958.1) S6A(KX197211.1) S41(KX197214.1) S17(KX197210.1) S37(KX197208.1) S22(KX855960.1) S26(KX197209.1) S29(KX855957.1) Bacillus valezensis FZB42 (KX898131.1)
Bacillus valezensis GEN11 (KU245537.1) Bacillus methylotrophicus St05 (JN700065.1)
Bacillus methylotrophicus NS 1-29 (HQ831404.1)
Bacillus methylotrophicus IHB B 7249 (KJ767354.1)
Bacillus methylotrophicus SL 8 (KP058543.1)
Rhizobium tropici CIAT899 (NC020059.1)
Bradyrhizobium japonicum GMC438 (AB741460.1) Azospirillum amazonense NBRC 107773 (AB682665.1)
Azospirillum brasilense Gr54 (FR667893.1) Azospirillum lipoferum NBRC 102290 (AB681746.1)
Burkholderia amabifaria L3 (LN889999.1) Pseudomonas fluorescens N07 (FJ972536.1) Pseudomonas putida HR5.1 (FN395007.1) Bacillus vallismortis A7-8 (JF496368.1) Bacillus amyloliquefaciens HR62 (JF700435.1) Bacillus subtilis EGE-B-1i-6 (JF926531.1)
Bacillus subtilis pspsg (KU956005.1) Bacillus valezensis G-5 (KY021155.1)
Fig.2–Maximumlikelihoodphylogenyofthe16SrRNAgeneshowingtherelationshipsamongendophyticisolates obtainedfromriceplants(inbold)withdifferentgeneraofplantgrowth-promotingrhizobacteria.GenBankaccession numbersareshowninparentheses.Bar,20ntsubstitutionsper1000nt.
belongstothegenusBurkholderiawasthemostefficient,with aSIof251%.35ThemeanSIobservedforthetestedisolatesin
thisstudywas122.9%,withthehighestvaluesaround133% forisolatesS2andS5(Table3).
Anotherimportanttoolforplantgrowth-promoting bacte-riaistheir abilityto produceproteases.The productionof proteasebybacteriaisoftenstudiedandhasgreatimportance forindustrial use and on the understanding ofthe activi-tiesoccurringinthesoilenvironment.3AccordingtoOliveira
etal.,13bacteriashowingproteaseenzymaticindex(PEI)≥2
areconsideredgoodproteaseproducers.
Ourresultsrevealed fivegoodproteaseproducers
bacte-ria(Table 3). IsolateS6 showed PEI about 3, which isvery
close to the PEI values showed by Bacillus megaterium and
Corynebacteriumrenale(3.1and4.3,respectively)isolatedfrom
Jacarandadecurrensplants,isolatedfromsoilsofthestateof Goiás,Brazil.36
The enzymatic diversitydisplayed byendophytic bacte-riaisolatedfromuplandricegrowninCerradoindicatesthat it is possibleto developa strategyto benefitthe selection of microorganismsas biofertilizers, improving their poten-tialasplantgrowthpromotersforricecrop.Thus,thegreater theextentofenzymaticpathwayspresentedbythe microor-ganisms,thegreaterwillbethegeneratedbenefit.Also,the successoftheplant–bacteriuminteraction andcompetition withothermicroorganismsfavorstheestablishmentofthose microorganismsinsuchenvironments.3
Theabilityof endophyticbacteria strainstogrow using carbon sourcepresent insoiland plantsassubstrate indi-catestheirabilitytosurviveandcompeteintheenvironment. Organicacidssuchasmaleicandnicotinicacidcanalsobe exudedbytherootsofplants,playinganimportantrolein theselectionoftheassociatedmicrobiota.Nicoticandmaleic acids showed suppressive effect over most of the studied
B. subtilis EGE-B-1i-6 (JF926531.1) 66 63 96 0.002 B. subtilis pspsg (KU956005.1) B. methylotrophicus St05 (JN700065.1) B. methylotrophicus NS 1-29 (HQ831404.1) B. valezensis GEN11 (KU245537.1)
B. valezensis FZB42 (KX898131.1) B. methylotrophicus IHB B 7249 (KJ767354.1) B. valezensis G-5 (KY021155.1) B. vallismortis A7-8 (JF496368.1) Bacillus amyloliquefaciens HR62 (JF700435.1) B. methylotrophicus SL 8 (KP058543.1) S6A(KX197211.1) S17(KX197210.1) S26(KX197209.1) S37(KX197208.1) S2(KX855959.1) S41(KX197214.1) S63(KX197206.1) S105(KX197207.1) S29(KX855957.1) S4 (KX855958.1) S22(KX855960.1)
Fig.3–Maximumlikelihoodphylogenyofthe16SrRNA geneshowingtherelationshipsamongendophyticisolates obtainedfromriceplants(inbold)withBacillussp.
referencestrains.GenBankaccessionnumbersareshown inparentheses.Bar,2ntsubstitutionsper1000nt.
S29 NI S22 S105 S4
Fig.4–Rootdevelopmentofuplandricecv.Aimoréas affectedbyendophyticisolatesobtainedfromriceplants.
isolates(Table2),whichcorroboratesthefindingsofFernandes Junioretal.4
Among the evaluated bacteria, 55% and 70% showed antibiosis against S. sclerotiorum and R. solani, respectively
(Table 3). Antibiosis may be triggered by many processes,
includingcompetitionandproductionofmetabolites.37
Iso-lates S4, S6, S22, S25, S29 and S105 showed antibiosis, solubilizationofinorganicphosphateandproteaseactivities
(Table3).AccordingtoParketal.,38thesolubilizationof
inor-ganicphosphateandproteaseactivitymaybeinvolvedinthe antibiosisactivity ofbacteria againstfungi. These bacteria
showedaveryclosesimilaritybasedontheirmorphological andenzymaticactivity,carbonsourceuseandantibiosisdata. Isolates S4,S6,S25 andS29 sharedabout62% ofsimilarity ongroupG2;isolatesS22andS105about65%ofsimilarityon groupG4.ThesimilaritybetweengroupsG2andG4wasabout 52%(Fig.1).
Mostoftheisolatesevaluatedinthisstudy,especiallyS4 and S22,show differentmechanisms involvedinthe plant growthpromotion,besidesthepresenceofenzymesactivity relatedtoadaptationtodifferentenvironmentalconditions,as thoseofCerrado.Inthisway,theresultsofthisstudyrepresent agreatscientificcontributionsincemanymetabolicpathway andactionmechanismscanbestudiedfromafewnumberof isolates.Besides,thecontributionfromtheagriculturalpoint ofviewishighlypromisingfortheBrazilianCerradobecause therearenonebiologicalproductsregisteredfortheupland ricecrop.
Thephylogeneticanalysisofthe16SrRNAgene sequenc-ing,performedfor11isolatesrevealedthatallofthembelong to the genus Bacillus (Fig. 2). The isolate S4 showed low similarity with the different Bacillus spp., remainingas an undefined specie (Fig. 3). The presenceof the genus Bacil-lus associated to rice plants in Brazil was also reported,39
but never with the presence of the B. amyloliquefaciens
species, as observed inthis study.In a study that investi-gated the associatedmicrobiota withtomato, thepresence of B. amyloliquefaciens was related with antibiosis against pathogens.40However,manyotherfactors,suchassoiltype andplantgenotypemayinfluencethisplant–microorganism association.39,41
In our study, the best results for plant growth promo-tion under greenhouse conditions were observed for the isolates S4 and S22 (Table 4). As reported before, these isolatesshowdifferenttraitsrelatedtoplantgrowth promo-tion,suchasantibiosis,solubilizationofinorganicphosphate and protease activity. The isolate S22 increased root dry weight,leafarea,shootdryweightandN-Totalaccumulated on rice shoots in about 23%, 21%, 25% and 24%, respec-tively,ascomparedtononinoculatedtreatment.Considering thesesameparameters, theisolateS4,anunknownspecie, promoted an increase of about 40%, 30%, 35% and 24%, respectively(Table4).
TheBrazilianCerrado isaverychallengingenvironment for cultivated plants, like rice, due to its soil and climate characteristics.Cerradosoilsaregenerallyacidand poorin nutrients,42whiletherainfallregimeiswelldefined,witha
rainyseasonfromOctobertoAprilandadryseasonfromMay toSeptemberwithanaverageannualrainfallof1460mm.43
Then,theuseofmicroorganismsasinoculantshowingagreat variabilityoftraitsrelatedwiththegrowthpromotionofplants isaveryimportantapproachtoimprovetheestablishmentof ricecropsintheBrazilianCerrado,sinceplantsunder influ-ence ofPGPRcanbegreater,stronger,moreproductiveand healthier.3,5,6
Theseresultspointoutforfurtherstudiesonthespecie identificationoftheisolateS4.Also,theisolatesS4andS22 mustbetestedfortheiragronomicefficiencyunderfield con-ditions,aimingtostatetheirpotentialofuseasinoculantfor rice.
Conclusions
Thisstudyrevealsgreatmorphological,metabolicandgenetic variabilityamongbacterialisolatesobtainedfromuplandrice cultivatedintheCerradoregionofthestateofGoiás.Those bacteriashowmanyenzymesactivityrelatedtoadaptationto differentenvironmentalconditions,asthoseofCerrado.All theisolatesbelongtothegenusBacillusandexhibitatleast onemechanismassociatedtoplantgrowthpromotion.Under greenhouseconditionstheisolatesS4andS22showedhigh plantgrowthpromotion,indicatingthattheyshouldbetested forthisabilityunderfieldconditions.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgments
TheauthorsthankCAPES,EmbrapaRiceandBeansandINCT onBiologicalNitrogenFixationforthefinancialsupport.The Department of Biochemistry and Molecular Biology at the FederalUniversityofParaná andGoiás StateUniversity for researcherexchange(BIP).
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