h ttp : / / w w w . b j m i c r o b i o l . c o m . b r /
Environmental
Microbiology
Rhizobial
characterization
in
revegetated
areas
after
bauxite
mining
Wardsson
Lustrino
Borges
a,∗,
Yves
Prin
b,
Marc
Ducousso
b,
Christine
Le
Roux
b,
Sergio
Miana
de
Faria
caEmbrapaAmapá,RodoviaJuscelinoKubitschek,Macapá,AP,Brazil
bCIRAD,UMR82,LaboratoiredesSymbiosesTropicalesetMéditerranéennes(LSTM),CampusInternationaldeBaillarguet,Montpellier,
France
cEmbrapaAgrobiologia,Seropédica,RJ,Brazil
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r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received26March2014 Accepted23August2015 Availableonline2March2016 AssociateEditor:CynthiaCanêdoda Silva Keywords: Rhizobia Rep-PCR Clusteranalysis Nodules Mining
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b
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t
Littleisknownregardinghowtheincreaseddiversityofnitrogen-fixingbacteriacontributes totheproductivityanddiversityofplantsincomplexcommunities.However,someauthors haveshownthatthepresenceofadiversegroupofnodulatingbacteriaisrequiredfor dif-ferentplantspeciestocoexist.Abetterunderstandingoftheplantsymbioticorganism diversityroleinnaturalecosystemscanbeextremelyusefultodefinerecoverystrategiesof environmentsthatweredegradedbyhumanactivities.ThisstudyusedARDRA,BOX-PCR fingerprintingandsequencingofthe16SrDNAgenetoassessthediversityofrootnodule nitrogen-fixingbacteriainformerbauxiteminingareasthatwerereplantedin1981,1985, 1993,1998,2004and2006andinanativeforest.Amongthe12isolatesforwhichthe16S rDNAgenewaspartiallysequenced,eight,threeandoneisolate(s)presentedsimilaritywith sequencesofthegeneraBradyrhizobium,RhizobiumandMesorhizobium,respectively.The rich-ness,Shannonandevennessindiceswerethehighestintheareathatwasreplantedthe earliest(1981)andthelowestintheareathatwasreplantedmostrecently(2006).
©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction
Nitrogen-fixing bacteria are an extremely importantgroup ofmicroorganismsforvariousecosystemsbecausethey pro-motetheentryofnitrogeninto thesoil.Thecapacitytofix atmosphericnitrogeniswidelydistributedamong microor-ganismswithdifferentlevels ofphylogeneticrelationships,
∗ Correspondingauthor.
E-mail:wardsson.borges@embrapa.br(W.L.Borges).
including representatives ofArchaea and Eubacteria. How-ever, the capacity to fix atmospheric nitrogen and induce noduleformationinleguminousplantsisrestrictedto mem-bers of the proteobacteria phylum.1–4 Legume nodulating
nitrogen-fixingbacteria,whicharecommonlyknownas rhi-zobia, are abundant in the soil of many ecosystems5 and
have a high diversity and variability regarding symbiotic efficiency.6–8
http://dx.doi.org/10.1016/j.bjm.2016.01.009
1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Theimportanceofsymbioticbiological nitrogenfixation (BNF) in agricultural systems is well-documented in plant speciessuchassoybeans,commonbeansandpeanuts.6,9,10
However,theroleofthisgroupofmicroorganismsinnatural ecosystemsispoorlyunderstood.11,12 Little isknownabout
thecontributionoftheincreaseddiversityofnitrogen-fixing bacteriatotheproductivity anddiversityofplantslivingin naturalcommunities.Mellonietal.13reportedthatagreater
diversityofbacteriainthe soilresultsingreater resilience ofthesystem andthat ahigher diversityoflegume nodu-latingbacteriacanfavorsymbiosiswithvariousleguminous plantspeciesandmaximize thebiologicalfixationof nitro-genindegradedareas.PreviousresearchbyvanderHeijden etal.11demonstratedthatsymbioticnitrogen-fixingbacteria
promoteevenness,productivityandnitrogencapturein sys-temsthatarerichinleguminousspecies,whichsuggeststhat thepresenceofnodulatingbacteriaisnecessaryfordifferent speciesofleguminousandnon-leguminousplantstocoexist. Althoughminingactivitiesgenerallyalteraproportionally smallerarea than other humanactivities, suchas farming and planting pastures for livestock, the level of environ-mental degradation is very high because of the intense disturbanceofthesoil.Thismakesitnecessarytotake meas-urestorestorethesedegradedareasattheendofthemining operations. In Brazil,to promote the rapid revegetation of highly degraded mined areas, the planting of leguminous speciesinoculatedwithnitrogen-fixingbacteriaand arbuscu-larmycorrhizalfungihasbeensuccessfullyemployed.14The
plantingofleguminousspecieswithselectedisolatesofthese microorganismsenablestheinitialcolonizationofsubstrates thathavebeensubjectedtohighchemical,physicaland bio-logicaldegradation.15Thecolonizationwithlegumesleadsto
thedepositionoflitterandincreasesthe concentrationsof nutrientsinthesoilsurface,enablingthereplantedsitesto entertheinitialstagesofplantsuccession.15
Withinthis context and inthis study, weassessed the diversityofthesebacteriainareasthatwererevegetatedafter bauxiteminingtobetterunderstandtheir roleindegraded ecosystems underthe recoveryprocess. Theareas studied wererevegetatedbetween1981and2006onsoilconsistingof overburdenortailingsandusedmixesofnativespeciesand inoculatedleguminousspecies.
Materials
and
methods
Studyareaandcollectionprocedure
Thecompany Minerac¸ão Rio do Norte (MRN) operates the Saracá,Almeidas and Avisos mines (all withinthe Saracá-TaqüeraNationalForest,whichislocatedinthemunicipality of Oriximiná, Pará state/Brazil, at 1◦21S – 56◦22W, 180m elevation).16Inthesemines,oreisfoundatanaveragedepthof
8mandiscoveredbydensevegetationandalayercalled over-burden,whichiscomposedoforganicsoil,nodularbauxite and ferruginous laterite. Tomine the reserves, it is neces-saryto remove the overburden to reveal the economically exploitablebauxiteore.Thisoperationisconducted sequen-tiallyinwhichtheoverburdenisdepositedinanadjacentpit thatwaspreviouslymined.Intheseareas,thereplantingis
performed on the overburden. The bauxiteore iscrushed, cyclonedandfiltered.Attheendofthisprocess,27%ofthe solidmassistailings,whicharedepositedinponds.16These
tailingspondareasarethenrevegetatedwhentheybecome full.
Thereplantingoftheoverburdenareasinvestigatedinthis studywasconductedbyacompanyusingavailableseedsof variousspecies(Parkiamultijiga,Parkiapendula,Parkia oppositifo-lia,Ormosiaholerythra,Ormosiaexcelsa,Sclerolobiumpaniculatum andAcosmiumnitens)in1981,1985,1993,1998,2004and2006. The revegetationof the twotailings pondswas conducted usingthespeciesAcaciamangiumin1993.Inpartofthisarea, theplantingwasconductedwithoutrhizobiainoculation (Tail-ingsWaste1),andtheotherpartwiththerhizobiainoculation consistedofamixtureofall oftherecommendedrhizobial strainsfromEmbrapaAgrobiologia(TailingsWaste2).
Ineacharea,twentysimplesamples(0–0.20m)were har-vestedtocomposeacompoundsampleinplotsmeasuring 250m2.Ninecompoundsampleswerecollectedin2007:sixin
plotsthathadbeenrevegetatedonoverburden,twointailings plotsandoneinanativeforestplot(Table1).Thechemical analysisofthesoilsampleswasconductedaccordingtothe ManualonSoilAnalysisMethods.17
Obtainingthenodulesandbacterialisolation
TheexperimentwasconductedusingsterilizedLeonardjars containingsandandvermiculiteataproportionof1:1(v/v)and inarandomizedblockdesignwiththreerepetitions.The treat-mentsconsistedoftheinoculationofasuspensionofsoilfrom eachplotonthehosttrapplantsMacroptiliumatropurpureum (siratro)andMimosaacutistipula.
The seeds were treated with concentrated sulfuric acid (H2SO4)for10mintobreakthedormancyandwith30%
hydro-genperoxide(H2O2)for3minforsurfacedisinfection.They
were thengerminatedinPetridishescontainingmoistened filterpaperandcotton.Threeseedlingsofeachspecieswere transplanted toeachjar, and eachseedlingwas inoculated with1mLofthesoilsuspensioninsalinesolution.The sus-pension was prepared using 10g of soil in 90mL of NaCl solution(0.145M)andkeptunderorbitalagitationfor30min. Theexperimentlasted90 days,duringwhichtheplants receivedwaterandanutrientsolution18intercalatedevery15
days.Toisolatethebacteriapresentinthenodules,theywere washedinethanol(70%,v/v–1min),externallydisinfected with30%hydrogenperoxidefor3min,washedfivetimesin steriledistilledwaterandcrushedinPetridishescontaining YMAmedium.19
Molecularcharacterization
The restriction analysis of the 16S rDNA gene was con-ducted according to Laguerre et al.20 and Teixeira et al.8
using HinfI, MspI and DdeI endonucleases. The DNA was extractedaccordingtoDoyleandDoyle21usingthedetergent
CTAB.The16SrDNAgenewasamplifiedusingtheuniversal primers Y1(5-TGGCTCAGAACGAACGCTGGCGGC-3)and Y3 (5-CTGACCCCACTTCAGCATTGTTCCAT-3).22
Weselected isolates representing12 distinct clusters in the ARDRA dendrogram for partial sequencing of the 16S
Table1–Chemicalcharacteristicsofthesoilsamples. Plot pH Al Ca+Mg Ca Mg P K C O.M. cmolcdm−1 Mg(dm−3) % 1981 5.2 1.3 0.7 0.4 0.3 4.7 60 2.85 4.91 1985 5.1 1 2.3 1.8 0.5 2.8 53 2.76 4.76 1993 4.6 1.7 0.3 0.15 0.15 2.8 39 2.19 3.78 1998 4.5 0.9 0.2 0.1 0.1 3.3 11 0.99 1.71 2004 4.9 0.6 1.8 1.4 0.4 4.2 18 2.1 3.62 2006 4.6 1.2 0.2 0.2 0 8.3 9 1.32 2.28 Waste1 5.2 0.1 0 0 0 3.1 1 0.12 0.21 Waste2 5.7 0 0.3 0.2 0.1 3.9 1 0.09 0.16 Forest 4 1.8 0.4 0.2 0.2 7.1 28 2.22 3.83
rDNAgene.ThePCRproductwaspurifiedusingtheWizard®
SVGelandPCRClean-UpSystemkit(PromegaCorporation, Madison,WI,USA)followingthemanufacturer’s recommen-dations.ThereactionswereconductedwiththeDYEnamicTM
ETDyeTerminatorkit (MegaBACETM)andaMegaBACE1000
automaticsequencer(GEHealthcareLifeSciences).The iden-titiesofthesequences(accessnumbersKT29901–KT429912) wereestimatedwiththedatabaseoftheNationalCenterfor BiotechnologyInformationthroughitsBasicLocalAlignment SearchTool.23
The BOX-PCR reactions were conducted according to Kaschuk et al.24 byemploying 50ng oftemplate DNA and
theBOX-A1Rprimer(5-CTACGGCAAGGCGACGCTGACG-3).25
ThesimilaritydendrogramwasconstructedusingtheJaccard similarityindexandtheUPGMA.Weassignedoperational tax-onomicunits (OTUs)based ondistinct clusters inthetree, whichwethenusedtocalculatetherichness,Shannonand evennessindexvaluesbyemployingthePASTprogramand therarefactionanalysisusingtheEstimatesversion8.0 pro-gram(Colwell,2010)26accordingtoMagurran.27
Results
Isolationofthebacteria
Atotalof139isolateswereobtainedandseparatedbyorigin into24,17,13,11,22,3,8,21and20isolatesobtainedfrom theareasreplantedin1981,1985,1993,1998,2004,and2006, andtheForestArea,TailingsWaste1andTailingsWaste2, respectively.Alloftheseisolateswereobtainedfromsiratro nodules.NonodulationwasobservedinM.acutistipula. ARDRAandsequencing
Ofthe139isolatesobtained,twodidnotgrowintheculture mediumafterthestorageperiodandwereexcludedfromthe molecularanalysis(onefromtheareareplantedin2004and onefromTailingsWaste2),andfortwootherisolates(from TailingsWaste1),noPCRproductwasobtainedfromthe16S rDNAgenetoconducttherestriction.Therefore,theARDRA wasconductedwith135isolates.
Theisolatesweredistributedin25differentclusters (thick-est lines inFig. 1), and the number of isolatesper cluster rangedfrom 1to 17.Fifty-six isolatespositioned in differ-entgeneticclusterswerenotdiscriminatedbecausetheyhad
100%similarity.Amongthe12 isolatesfrom whichthe 16S genewaspartiallysequenced,similaritycouldbedetectedin thesequencesofthreegenera.Ofthese,eightbelongedtothe Bradyrhizobiumgenus,threetoRhizobiumandoneto Mesorhi-zobium.Ofallofthe135isolatesanalyzedfromthe16Sgene restriction,96isolateswereclusteredwiththeBradyrhizobium genus,25withRhizobiumand8withMesorhizobium(Fig.1).
BOX-PCRanddiversityanalysis
TheBOXanalysiswasconductedwith137isolates.These iso-latesweredistributedin40clusters(Fig.2),andthenumberof isolatesperclustervariedbetween1and12.Twelveisolates positionedindifferentgeneticclusterswerenotdiscriminated becausetheyhad100%similarity.Basedonthegenerated den-drogram,wecalculatedthediversityoftheisolatesforeach plot.TherichnessoftheOTUsvariedbetween2forthe for-estareaand17fortheareathatwasrevegetatedin1981and TailingsWaste2;theShannonindexrangedfrom0.37forthe forestareato2.78forTailingsWaste2;andtheevennessindex varied between0.54 forthe forest area to0.98 forTailings Waste2(Figs.3and4).
Discussion
Severalstudieshavedemonstratedthatspeciesbelongingto theMimosagenushaveahighspecificityforrhizobiaisolates belongingtothebetaproteobacteriasubdivision,notably, iso-latesoftheBurkholderiagenus.28,29Inaddition,somespecies
ofMimosoideae mustassociate witharbuscular mycorrhizal fungifornodulationtooccur.30Theabsenceorlowdensityof
beta-rhizobiainthesampledareasandtheabsenceof arbus-cularmycorrhizalfungiintheLeonardjarsusedinthisstudy canbepossibleexplanationsforthelackofnodulationinM. acutistipula. However,the majorityofsiratro plantsshowed nodulation,whichdemonstratesthatthedensityofrhizobia presentintheinoculumwassufficienttoinducenodulation. Thisspecies,becauseofitswidehostrange,hasbeen previ-ouslyusedtoassessthediversityofisolatesfromnodules.31,32
ARDRAwasinitiallyproposedasausefultoolfortherapid identification ofthetaxonomicpositionofrhizobiaisolates becauseitisbasedontherestrictionofagenethatcodesfor ribosomalRNA,whichpermitsseparationatthegenuslevel andatthespecieslevelinsomecases.Itismainlyemployed forthispurposeinsituationswherethe16Sgenecannotbe
20 36 52 68 84 100 1981 Mesorhizobium sp. Forest 1981 1981 1981 1981 1981 1981 1981 1993 1993 1993 1993 1993 1993 1993 1993 1981 1993 1998 Waste 2 Waste 2 Waste 2 Waste 1 Waste 1 Waste 1 Waste 1 1981 Waste 2 1981 1981 Rhizobium sp. 1993 Rhizobium sp. 2004 Rhizobium sp. Forest Bradyrhizobium sp. Waste 2 Waste 2 Waste 1 Waste 1 Waste 1 Forest Forest Forest 2006 1981 1981 1981 1981 1981 1985 1985 1985 1985 1985 1985 1985 1985 1985 1985 1985 1981 1981 1981 1981 1998 1998 1998 1998 1998 1998 1998 1998 1998 Bradyrhizobium sp. Waste 1 Waste 1 Waste 2 Waste 1 Waste 1 Waste 1 Waste 1 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 Waste 2 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 1985 2004 2004 2004 2004 2004 1985 1985 1985 1985 1993 Waste 2 Waste 1 Waste 1 Waste 2 1999 2004 Bradyrhizobium sp. 1981 1993 1981 1993 Bradyrhizobium sp. Waste 2 Bradyrhizobium sp. 1985 Bradyrhizobium sp. 1981 Bradyrhizobium sp. 2006 Bradyrhizobium sp. Waste 1 Waste 1 Waste 1 2006 Forest Forest Forest * * * * * * * * * * * * * * * * * * * * * * *
Fig.1–Thegeneticsimilaritydendrogramfortherhizobiaisolatesanalyzedbytherestrictiongenethatcodesforribosomal RNA16S(ARDRA).
0 20 40 60
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80 100 1981 1981 1985 1985 1985 1985 1985 1985 2006 1981 Mesorhizobium sp. 1985 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 2004 Waste 1 Waste 2 Waste 2 Waste 2 Waste 2 Forest Forest Forest Forest Forest Forest Waste 1 Waste 1 Waste 2 Waste 2 Waste 1 Waste 2 Waste 1 Waste 1 Waste 1 Waste 1 Waste 1 Waste 2 Waste 2 Waste 2 Waste 1 Waste 1 Waste 1 Waste 1 Waste 1 Waste 1 Waste 1 Waste 1 Waste 2 Waste 2 Waste 2 Waste 2 Waste 1 Waste 1 Waste 2 Bradyrhizobium sp. Waste 2 Waste 2 1981 1993 1993 1993 1993 1993 1993 1993 1981 1981 1981 1981 1981 1998 1998 1981 1981 1981 1981 1981 1981 1993 1993 1985 1993 Forest 1981 Rhizobium sp. 1993 Bradyrhizobium sp. Waste 2 Waste 1 1981 1981 1981 2006 Bradyrhizobium sp. Forest Bradyrhizobium sp. 1993 2004 1981 Bradyrhizobium sp. Waste 1 2004 Bradyrhizobium sp. 1981 2004 Waste 2 Waste 1 2006 1981 1985 1981 1981 1998 1998 1998 1985 1981 1981 1998 1985 1985 1985 1985 1985 1985 1998 1985 Bradyrhizobium sp. 1998 Bradyrhizobium sp. 2004 Rhizobium sp. 1993 Rhizobium sp.1981 1985 1993 1998 2004 200 6 Forest Waste 2 Waste 1 0 5 10 15 20 0 1 2 3 Richness Shannon Equitability
Ri c hn es s Sha nn on , Eq u it ab ilit y
Fig.3–Richness,Shannonandevennessfromsiratrorhizobiaisolatesfromplotsthatwererevegetatedonoverburdenin 1981,1985,1993,1998,2004and2006,andinTailingsWaste1,TailingsWaste2in1993andtheForestarea,basedon BOX-PCRoperationaltaxonomicunit.
sequenced.8,20,33,34 Inthepresentstudy,themajorityofthe
isolatespresentedsequencesclosetothoseofthe Bradyrhi-zobiumgenus(groups10–22,Fig.1).Agreaterabundanceof Bradyrhizobiumisolateshavebeenobservedbyotherauthors underavarietyofconditions,5,32,35whichsuggeststhatthere
ishighvariabilitywithinthisgenusintheareasstudied.In additiontotheisolatesproximatelyrelatedtothe Bradyrhi-zobiumgenus,wealsoobservedisolatesproximatelyrelated totheRhizobiumandMesorhizobiumgenera.Previousresults showed the capacity of isolatesbelonging to these genera towithstandadverseconditionsofpH,temperatureandthe presenceoftoxicelements.36–38 Thepresenceofthese
gen-era inthe areas studiedconfirms their capacityto survive andestablishsymbiosisfixing-nitrogenunderadverse envi-ronmentalconditions.
TheBOX-PCRanalysisallowsthesimultaneousevaluation ofdistinctgenomicregionstoidentifyintraspecificvariability. Thisfeaturewasdemonstratedinthisstudybecausethe iso-latesweredistributedinto25clustersbytheARDRAand40 clustersbytheBOX-PCRanalysis.UsingARDRA,evenbased ondatafromthreerestrictionenzymes,56isolatespositioned indifferentgeneticclustersshowed100%similarity,whereas thisnumberwasonly12fortheBOX-PCRtechnique.Thisis becausethe16SrDNAgeneisrelativelysmallandpresents highly conserved regions. Additionally, this gene is poorly discriminated within the genus Bradyrhizobium.39 These
findingsshowthattheBOX-PCRtechniquewasmore discrim-inatingbetweenisolates,demonstratingtheapplicabilityof thistooltoseparateisolatesthataretaxonomicallyproximate
40–43anditsutilityinstudiesaimingtocomparethelevelof
diversitybetweendifferentsites.
Amongtheareasthatwererevegetatedonoverburden,the OTUrichness,Shannonandevennessindiceswerehigherin the area thatwas revegetated the earliest (1981). Thearea revegetated morerecently (2006) showed a low number of isolates, which impaired the diversity estimate because it was not possible to perceive stabilization of the Shannon index(Fig.4)whenpresentedwiththelowestOTUrichness,
0 5 10 15 20 25 0 1 2 3 Number of isolates Sh a n n o n 1981 1985 1993 1998 2004 2006 Forest Waste 1 Waste 2
Fig.4–ThevariationoftheShannondiversityindexvalues asafunctionofthenumberofisolatesfromsiratroand fromplotsthatwererevegetatedonoverburdenin1981, 1985,1993,1998,2004and2006,andinTailingsWaste1, TailingsWaste2in1993andtheForestarea.
Shannon andevenness indicesvalue.Theother areas pre-sented intermediatevalues(Fig.3).Theforestarea showed OTUrichness,Shannonandevennessindiceslowerthanall of the overburden areas, and the two tailings waste areas thatwererevegetatedusingAcaciaspeciespresentedsimilar indicestotheoverburdenarearevegetatedin1981andhigher indicesthanintheoverburdenareathatwasrevegetatedin thesameyear(1993)usingamixtureofplantspecies.
Alowerdiversityofnoduleisolatesinforestareas com-paredcultivatedareasorfallowshasbeenpreviouslyreported inseveralstudies.13,30,32Jesusetal.31evaluatedtheeffectof
thetypeoflanduseonthediversityofnoduleisolates cap-turedbysiratroand observedthat thearea cultivatedwith cassava presented a higher diversitythan the forest areas
and those cultivated with peach palm, which did not dif-ferbetweenoneanother.Limaetal.32 alsoinvestigatedthe
communityofnoduleisolatesfromsiratroinareaswith differ-entusesintheAmazonregionandobservedthattherichness indexofthenoduleisolatesintheprimaryforestareawas 12,whichwasthe lowestvalueamongthe areasanalyzed. Thevaluesintheotherareaswere46forthecultivatedarea, 48inthe agroforestryarea,24forthearea identifiedasold secondaryforest,28 innewsecondary forestand 29inthe pasture.
Theseresultsshowthattherevegetationstrategiesusedin theseareasenabledtheestablishmentofaplantcommunity thatwasabletosustainanincreasingdiversityofrootnodule isolates.AccordingtoMellonietal.,13 studiesofthe
diver-sityofkeygroupsofmicroorganismsinreclaimedareasare importantbecausetheysupply anindicationoftheeffects ofdifferentrehabilitationmethodsonthediversityofthese microorganisms.
The tailings pond areas were replanted with Acacia mangium in 1993. Since then, the changes caused by the coverages withleguminousspecieshaveled tothe deposi-tionofplantmaterialonthesoil,whichprovidedconditions fortheestablishmentofadiverse communityofroot nod-ule isolates. A higher diversity of legume-nodulating and nitrogen-fixingbacteria was also foundin areas that were replantedwiththeleguminousspeciesMimosascabrellaand Cajanuscajanafterminingactivities.13Thecapacityof
legu-minousspeciestocolonizedegradedenvironmentshasbeen reportedby other authors 14,15 and has been linkedtothe
ability of these species form symbiotic associations with nitrogen-fixingbacteriainthesoil,aswellaswith arbuscu-larmycorrhizalfungi.Thisthree-wayinteraction favorsthe nutritionofplantsbyenhancing their uptakeofnutrients. Asaresult oftheincrease ofnitrogeninthe biomassand theabsorptionofimportantnutrients,suchasphosphorous, leguminousplantsfavorplantsuccessionandthediversityof microorganisms.
Conflicts
of
interest
Theauthorsdeclarenoconflictsofinterest.
Acknowledgements
WewouldliketothankMinerac¸ãoRiodoNorteforallowing access to the study area; the National Council for Scien-tific and Technological Development (CNPq) for individual support (Borges, W.L.received a doctoral scholarship, pro-cess no. 142315/2007–9; de Faria, S.M. received a research grant)and projectfunding (no. 492683/2004-2), and IBAMA (029/2007 02001.006557/2005) and the GraduateProgram in Agronomy–SoilScienceofUFRRJandEmbrapaforthe infras-tructurethatwasmadeavailable.Wewouldalsoliketothank TelmoFelixdaSilva,CarlosFernandodaCunha,Cândido Bar-retodeNovaisandAdrianaSantosdoNascimentoforhelp inthegreenhouseexperimentandwiththebacterial isola-tion.
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