Cultivated bacterial diversity associated with the carnivorous plant Utricularia breviscapa (Lentibulariaceae) from floodplains in Brazil

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

Environmental

Microbiology

Cultivated

bacterial

diversity

associated

with

the

carnivorous

plant

Utricularia

breviscapa

(Lentibulariaceae)

from

floodplains

in

Brazil

Felipe

Rezende

Lima

_,

_Almir

_José

_Ferreira

_,

_Cristine

_Gobbo

_Menezes

_,

Vitor

Fernandes

Oliveira

Miranda

_,

_Manuella

_Nóbrega

_Dourado

_,

Welington

Luiz

Araújo

a_{DepartmentodeMicrobiologia,InstitutodeCienciasBiomedicas,UniversidadedeSãoPaulo,Av.Prof.LineuPrestes,1374-Ed.}

BiomédicasII,CidadeUniversitária,05508-900SãoPaulo,SP,Brazil

b_{NúcleoIntegradodeBiotecnologia,NIB,UniversidadedeMogidasCruzes,Av.Dr.CândidoXavierdeAlmeidaSouza,200,Centrocívico,}

08780-911MogidasCruzes,SP,Brazil

c_{FaculdadedeCiênciasAgráriaseVeterinárias,UniversidadeEstadualPaulistaUNESP,ViadeacessoProf.PauloDonatoCastellanes/n,}

Centro,14884-900Jaboticabal,SP,Brazil

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received18September2017 Accepted24December2017 Availableonline31March2018 AssociateEditor:FernandoAndreote

Keywords: Utriculariabreviscapa Microbialecology Aquaticmicrobiota Microbialcommunities

a

b

s

t

r

a

c

t

Carnivorousplantspecies,suchasUtriculariaspp.,captureanddigestprey.Thisdigestion canoccurthroughthesecretionofplantdigestiveenzymesand/orbybacterialdigestive enzymes.Tocomprehendthephysiologicalmechanismsofcarnivorousplants,itisessential tounderstandthemicrobialdiversityrelatedtotheseplants.Therefore,inthepresentstudy, weisolatedandclassifiedbacteriafromdifferentorgansofUtriculariabreviscapa(stolonsand utricles)andfromdifferentgeographiclocations(SãoPauloandMatoGrosso).Wewereable tobuildthefirstbacteriumcollectionforU.breviscapaandstudythediversityofcultivable bacteria.TheresultsshowthatU.breviscapabacterialdiversityvariedaccordingtothe geo-graphicisolationsite(SãoPauloandMatoGrosso)butnottheanalyzedorgans(utricleand stolon).Wereportedthatsixgenerawerecommontobothsamplesites(SãoPauloandMato Grosso).Thesegenerahavepreviouslybeenreportedtobebeneficialtoplants,aswellas relatedtothebioremediationprocess,showingthattheseisolatespresentgreat biotechno-logicalandagriculturalpotential.ThisisthefirstreportofanAcidobacteriaisolatedfromU. breviscapa.Theroleofthesebacteriainsidetheplantmustbefurtherinvestigatedinorder tounderstandtheirpopulationdynamicswithinthehost.

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

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

∗ _{Correspondingauthorsat:DepartmentofMicrobiology,InstituteofBiomedicalSciences,UniversityofSãoPaulo,Av.Prof.LineuPrestes,} 1374-Ed.BiomédicasII,CidadeUniversitária,05508-900SãoPaulo,SP,Brazil.

E-mails:mndourado@gmail.com(M.N.Dourado),wlaraujo@usp.br(W.L.Araújo).

https://doi.org/10.1016/j.bjm.2017.12.013

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

Introduction

There are over 700 reported species of carnivorous plants distributed in 5 orders (Caryophyllales, Ericales, Lamiales, OxalidalesandPoales)and10familiesthatoccurindifferent habitatsallovertheworld.1_{Thiscarnivoroussyndromehas}

arisenindependentlyatleastsixtimesintheevolutionary his-toryofangiospermsoveranextensiveevolutionarytimespan andinvolvingdifferentmorphologicaladaptationstocapture anddigesttheprey.2,3

Theplantmustpresentcertainfeaturestobeclassifiedas carnivorous,suchasactivelyattracting,capturingand digest-ing prey, absorbing nutrients from it and obtaining some advantagesingrowthorreproduction.4 _{Thepreycanbean}

additionalsourceofN,P,S,KandMgandcomplementthese photoautotrophicplants.5,6

The genus Utricularia (Lentibulariaceae), with over 220 species,isthemostwidespreadofthecarnivorousplants.One speciesofthisgenus isUtriculariabreviscapa,whichcan be naturallyfoundintheAntilles andSouthAmerica.Itisan aquaticplant,anditssuspendedstructurescontainair-filled parenchymathatallowsittofloat.Itsstolonsarethreadlike, andbearsegmentedleavesand utricules(thetraps).7 Utric-ulariaspeciespresentfoliarstructurescalledutricles,which arehighlyspecializedtrapsthatarecapableofcapturingprey bysuctionthroughtheactionofnegativeinternalhydrostatic pressure.8,9

In many carnivorous plant species, digestion does not necessarily occur through the secretion of plant digestive enzymesbutisperformedbybacterialand/orfungal diges-tiveenzymes.2,10_{Forexample,insomespeciesofcarnivorous}

plants,suchasByblis(Byblidaceae),Brocchinia(Bromeliaceae),

Darlingtonia,HeliamphoraandsomespeciesofSarracenia (Sar-raceniaceae),bacterialenzymesareessentialintheabsence ofplantenzymesecretion,andamongtheplantsthathave suchglands,thebacterialcommunityalsoplaysanimportant roleintheoptimizationprocessofpreydegradation.4,11

Theseendophyteslivinginsidetheplanthaveinnumerous advantages,sincetheinternaltissuesprovideastable envi-ronmentwithoutultravioletrays,temperaturefluctuationsor nutrientcompetitionwithothermicroorganismsaswellasan increasedavailabilityofnutrients.12–14 _This

microorganism-plantinteractionisimportantforthesurvivaloftheplant, sincethesemicroorganismsrepresentseveralbenefitsforthe hostplant,providingprotectionagainstpathogens, promot-inggrowth,increasingtheabilitytocapturetraceelements andseveralotherbenefitsthatareextremelyimportantfor themaintenanceofplantbalance.15,16

Therefore, it is important to understand the microbial diversityrelatedtocarnivorousplants,particularlybacterial

Table1–Samplinglocationsandcharacteristics.

Generallocation City Coordinates River/lake Characteristics

MatoGrossostate SantoAntôniodeLeverger S15◦5931.8W55◦4857.4 RiverAricáfloodplain Lenticenvironment,lowdepth SãoPaulostate MogidasCruzes S23◦3158.5W46◦0838.3 RiverTietêfloodplain Lenticenvironment,mediumdepth

diversity,sinceitcanrepresentapproximately58%ofthetotal

viable microbial biomass,17 _{playing a key role in the trap.}

Caravieriet al.18 _{were the firstto reportthe totalbacterial}

diversityfromUtriculariahydrocarpaandGenliseafiliformistraps usinganon-cultivableapproach,showingthatonly1.2%of theobservedoperationaltaxonomicunits(OTU)wereshared bybothanalyzedplants(U.hydrocarpaandG.filiformis). More-over,inadditiontoplantgenotype,trapagecanalsoinfluence themicrobialcommunitiesinsidethevesicles.19

Thepresentstudyaimstoisolatethebacterialcommunity from differentorgans(stolonsand utricles)ofU.breviscapa,

whichmayhavegreatbiotechnologicalpotential,inorderto buildthefirstbacteriumcollectionforU.breviscapaandstudy thediversityofcultivablebacteria,aimingtounderstandtheir functioninthisuniqueenvironment.

Materials

and

methods

Plantsampling

ThesamplesofU.breviscapaplantswerecollectedfromtwo differentfloodplainsinBrazil:(1)SantoAntôniodeLeverger municipality,MatoGrossostate(MT)and(2)RioTietêflood plain, Mogi das Cruzes municipality, São Paulo state (SP)

(Table1andFig.1).Thesampleplantswerestoredinplastic

bagsdulyidentifiedwithsamplenumberandthepointfrom whichtheywerecollected,maintainedinfreshwaterat envi-ronmental temperature. Aftercollection, the sampleswere transportedtotheLaboratoryofMolecularBiologyand Micro-bialEcologywheretheisolationwasimmediatelycarriedout. VouchersaredepositedinHerbariumHUMC.

Bacterialisolation

ThemacerationmethodwasadaptedfromKuklinsky-Sobral et al.20 _{for the bacterial isolation from weighed stolons}

and utricles of U. breviscapa previously washed in ster-ilized water. Bacteria were isolated by soaking 5 stolon fragments (approximately 5mm per fragment) or 10 utri-clesinphosphate-bufferedsaline(PBS)containingNa2HPO4

(1.44gL−1), KH2PO4 (0.24gL−1), KCl (0.20gL−1), and NaCl

(8.00gL−1),adjustingthepHto7.4,andmacerating.After mac-eration,thesamplevolumewasadjustedto1mL,appropriate dilutionswerecarriedoutandplatedonto10%trypticasesoy agar (TSA) supplementedwith50mgmL−1 ofthe fungicide Imazalil(Magnate500CE,Agricur),andtheplateswere incu-batedat28◦Cfor2–15days.Thecolonieswereremovedfrom theplates,inoculatedonto10%TSAagarmedium,incubated at28◦Cfor2–10days,andtheneachisolatewassuspendedin a20%glycerolsolutionandstoredat−70◦_C.

Fig.1–HabitofUtriculariabreviscapawithinflorescence (bar=10mm).Thedetailshowsastolonwithutricles (arrowindicatesanutricule;bar=5mm).

Statisticalanalyseswere carriedoutwithbiological trip-licatesforeachtreatmentforbacterialquantificationinthe isolationexperiments,whichwereperformedinacompletely randomdesign.Thesignificanceoftheobserveddifferences wasverifiedusinganewanalysisofvariance(p=0.05). Analy-seswereconductedusingRsoftwareversion3.0.1.

Polymerasechainreaction(PCR)amplificationand16S rDNAsequencing

Aftercultivation,thebacterialDNAwasextractedaccording toAraújoetal.21_{.Apartialsequenceofthe16SrRNAgene}

wasamplifiedusingthepairofprimersR137822_andP027F.23

PCRswereperformedaccordingtoDouradoet al.24_{All PCR}

amplificationwascheckedthroughelectrophoresisonagarose gel (1.5%, w/v agarose) and UV visualization of the ethid-iumbromide-stainedgels,afterwhichthePCRproductswere purifiedusingaGFXPCRDNAandgelbandpurificationkit (AmershamBiosciences)andsequencedbySanger Sequenc-ingTechnology25_{usingtheprimer1378R.}

Analysisofthe16SrRNAgenesequence

Sequences were obtained from the bacteria isolated from

U. breviscapa stolonsandutriclesfrom SãoPaulo andMato

Grossostate.Priortothe analysis,all chromatogramswere trimmed with Phred-Phrap (http://www.phrap.com/phred/). Thesequenceswereclusteredasoperationaltaxonomicunits (OTU) using MOTHUR26 _{and a cut-off of 97% identity and}

further examined using rarefaction analysis and Libshuff, and dendrograms were constructed. Furthermore, richness and diversity were also calculated using MOTHUR based on non-parametric richness (Ace and Chao1)and diversity (Shannon-Weaver and Simpson) indexes with cut-offs for similarity at97% (0.03), 95% (0.05) and 91% (0.09) identity. TaxonomicclassificationswereperformedusingRDPQuery

(https://rdp.cme.msu.edu)asdatabaseparameters.For

phe-neticanalysis,thesequenceswerealignedusingClustalW,27

and the distance was calculatedusing the Jukes and Can-tor model28 _{using Neighbor Joining method in MEGA 5}

software.29 _{Thebranches weretestedwithbootstrap}

analy-ses(1000replications),andthelayoutoftreeswasdesigned usingtheonlineapplication“InteractiveTreeOfLife”(iTOL)

(http://itol.embl.de/).30

Atotalof200DNA sequencesofpartial16SrRNAgenes were deposited in the GenBank database under accession numbersKY453794toKY453980.

Results

and

discussion

A total of200 sequences were obtainedfrom bacteria iso-lated from U. breviscapa. Higher isolated bacterial density was observedinassociation withplantsfrom MatoGrosso (MT)(Fig.2).ThebacterialdensityaveragesfromMTsamples were 4.9×105_CFUutricle−1 _{and 3.6×10}7_{CFUg of stolon}−1_,

whilethesamplesfromSãoPaulo(SP) hadbacterial densi-ties of6.0×104_CFUutricle−1 _{and 1.2×10}7_CFUgofstolon−1

(Fig.2).

TherichnessestimatorofOTUsperformedwiththeChao1 and Aceindexes at97% (Table 2)showed a greater bacte-rial richness inSP plants than in MTplants. According to theChao1parameter,wecanobservethehighestrichnessin utriclesfollowedbystolonsinSPplants.However,basedon theAceestimator,theoppositewasobserved,inwhichthe Aceindexshowedthehighestrichnessinstolonsfollowedby

0,0000# 1,0000# 2,0000# 3,0000# 4,0000# 5,0000# 6,0000# 7,0000#

Mato#Grosso# #São#Paulo# Mato#Grosso# #São#Paulo#

Log10&UFC.unit/1&&

A

B

Fig.2–Bacterialdensityin(A)utriclesand(B)stolonsofU.breviscapafromMatoGrosso(MT)andSãoPaulo(SP). Statisticallydifferentatp<0.05.

Table2–DiversityindexandrichnessestimationofanalyzedOTUs.

Library Similarity OTUs Chao-1 Ace Shannon(H) Simpson(1-D)

Richnessestimator Diversityindexes

utricSP 97% 37 107(62–236) 104(63–211) 3.2(3.0–3.5) 0.95(0.98–0.93)

utricMT 97% 25 56(34–124) 66(38–152) 2.5(2.1–2.9) 0.85(0.94–0.76)

stolSP 97% 34 93(55–196) 228(144–373) 3.3(3.1–3.6) 0.97(0.99–0.95)

stolMT 97% 17 56(27–168) 82(46–163) 2.64(2.3–2.9) 0.94(0.99–0.90)

utricles.IntermsofbacterialrichnessinMTplants, Chao1

estimatesastatisticallyequalrichnessinbothutriclesand

stolons(Table2).

Itis common knowledge that the performance of non-parametric estimators depends on the species abundance distributioninthesample,andthepreferenceforoneover anotherisadifficultissue.Basualdo31observedinhiswork thatthe Aceindexwas betterwhenthe observedrichness waslow andChao1when theobserved richnesswas high. Inanotherstudy,Hortaletal.32_{mentionedthat}

abundance-basednon-parametricestimators(AceandChao1)aremore precisecomparedwithother richness estimators;however, theirprecisiondiminishesatlowersamplingintensities, pro-ducinglessconsistentresults.Thissuggeststhatthechoice among richness estimator parameters can vary between samplingcharacteristics,eveninthesamestudy.

AccordingtotheSimpsonindexdiversityat97%,ahigher diversityofOTUswasfoundinlibrariesconstructedfromSP plants,withthehighestdiversityinstolons(stolSP)followed byutricles(utricSP).SimilartoSãoPaulo,thebacterial diver-sityindexinMTplantswasalsohigherinstolons(stolMT) followedbyutricles(utricMT).TheShannondiversityindexat 97%showedthesameresult.

Therefore,forallindexesused(Chao1,Ace,Simpsonand Shannon),higherdiversityrateswereobservedinplantsfrom SãoPaulo.ThereisnoapparentreasonwhySãoPaulosamples presentahigherdiversitywhencomparedwithMatoGrosso samples,but itisknownthattherearevarious characteris-ticsthatcouldbeconsideredtocontributetothisvariation, ofwhichthetrapagehasbeenthemostcitedinthe litera-ture,but thequalityofwatercanalsoimpactthemicrobial diversity. Plachno et al.19 _{found that old traps were}

colo-nizedbyattachedbacteria,buttheycanalsobefoundfreely suspendedin trap fluid, as shown in Sirová et al.17 How-ever, determining utricle dynamics is further complicated by the rapid aging of traps (or pitcher), as their life cycle iscompleted over approximately 30 days, and many envi-ronmentalchangesoccurduringthistime.33_{Althoughtheir}

lifecycle isshort,theinteriorofutriclespresentslow con-centrationsofoxygen,and it hasbeen postulated thatthe organisms trapped byUtricularia die as a resultof oxygen

Table3–Libshuffanalysiscomparingcoveragebetween libraries.

Comparison Score Significance

stolMT-stolSP 0.006 0.0044 stolSP-stolMT 0.025 <0.0001 stolMT-utricMT 0.010 0.0015 utricMT-stolMT 0.053 <0.0001 stolMT-utricSP 0.011 0.0037 utricSP-stolMT 0.069 <0.0001 stolSP-utricMT 0.029 <0.0001 utricMT-stolSP 0.009 <0.0001 stolSP-utricSP 0.001 0.5787 utricSP-stolSP 0.006 0.0009 utricMT-utricSP 0.005 0.0001 utricSP-utricMT 0.048 <0.0001

depletion.34_{Thisanoxicenvironmentisreflectedinits}

com-mensalcommunities,selectingforanaerobicandfacultatively aerobicbacteria.35_Pitschetal.36_{reportedaciliatecommensal}

thatusesalgaetocircumventthelowconcentrationof oxy-geninsidetheutricles.Furthermore,someresearchershave assumedthatthishighabundanceofcommensalorganisms alsooccursinemptytraps;inthesameway,these microorgan-ismsarenotspecializedforlivinginsidethetraps,andthey canthereforeliveeitherontheexternalsurfaceorfreelyas planktonintheambientwater.37

Another factor that could influence the microbial com-munity inside the traps is the composition of the water surroundingthetrapthatwillbesuckedintogetherwiththe preyorevenattachedtotheprey.Thus,thebacterial commu-nityisselectedaccordingtothephysical–chemicalconditions inside the trap. Itis importanttoemphasize that the two sampled siteswerelocatedinfloodplainareas.River flood-plainsystemsareknownfortheirheterogeneityinhabitats andhydrologicalpulses,presentinggreattemporaland spa-tialvariationintheirlimnologicalconditions,which,inturn, shouldinfluencethebacterialcommunitycomposition.38

TheLibshuffsignificancetestindicatedthatthoselibraries belongingtothesame partoftheplant(stolSP/stolMT and utricSP/utricMT) (Table 3) differsignificantly foreach sam-pled site (São Paulo and MatoGrosso). Moreover,although

utricMT

stolMT

stolSP

utricSP

0.1

Fig.4–PhenetictreesrepresentingthecultivablebacterialfoundinutriclesandstolonsfromU.breviscapawerebuiltusing NeighborJoiningmethodinMEGA5software.(A)IsolatesfromMatoGrossoand(B)fromSãoPaulo.Thesolidgraycircles nexttothetreebranchescorrespondtobootstrapvalueshigherthan50%.Brachyspirainnocens(S000437178);Brachyspira

hyodysenteriae(S000437188),Brachyspirahyodysenteriae(S000437188)andBrachyspirahyodysenteriae(S000437188)wasused

theyareassociatedwiththesamesite,thesignificance analy-sisforutricleMTandstolonMTshowsasignificantdifference betweentheseparts ofthe hostplant.On the other hand, whencomparingutricleSPandstolonSP,whichpresenthigher richnessanddiversityintheir16SrDNAlibraries,donotvary, indicatingthatonlyinless diverseenvironments are there differentcolonizationpatternsbetweenstolonsandutricles.

Adendrogramofbacterialcommunitiesshowed similar-itiesbetweenthe samesite libraries(Fig.3).Agreeing with therichnessanddiversityindexes,thelibrariesweregrouped accordingtothesampledsite,groupingutriclesandstolons fromSãoPaulo(utricSPandstolSP)inthesamebranch(with presents higher diversity), while utricles and stolons from MatoGrosso(utricMTandstolMT)weregroupedinanother branch(whichpresentslower diversity).Thisindicatesthat thereisgreaterbacterialcommunitysimilaritybetween dif-ferentpartsofthesamehostplantcollectedfromthesame sampledsite (SãoPauloor MatoGrosso),corroborating the hypothesis that the place of origin plays the mainrole in bacteria-plantinteractions,sincethe environmentsupplies themicrobialdiversitytotheplanthost.

Inlookingatthe200isolatesfromSPandMT,respectively, the16SrRNAanalysisofplantisolatesfromMTshowed22 bac-terialgenera,amongthem Sphingomonas(36.71%),Aquitalea

(18.99%)andBacillus(6.33%),whileintheplantisolatesfrom SãoPaulo,wefound 31bacterialgenera, ofwhich the pre-dominantgenerawereMicrobacterium(20.66%),Sphingomonas

(14.05%)andPelomonas(9.09%)(Fig.4).Caravierietal.18 also foundSphingomonasandMicrobacteriumgenerainassociation withU.hydrocarpa,buttheculture-independentmethodsused intheirworkfoundtheseOTUsamongthelessabundant gen-era.

Sixgenerawerecommontobothsampledsites(SPandMT):

Aquitalea,Azospirillum,Bacillus,Chromobacterium, Novosphingo-biumandSphingomonas(Figs.4and5).Asshownforbacterial density,itwasalsoverifiedthattheabundanceofcultivable bacterialspeciesvariedaccordingtothesampledsites, sug-gesting that the plant selects the bacterial community in itstissues,andthisselectionmustoccurfromthediversity presentinitssurroundingenvironment.Moreover,Koopman etal.39_{observedthatthebacterialcommunitywithinthetraps}

ofSarraceniaalataplantswassignificantlydifferentfromthe soilsurroundingtheplantandvariedovertime,suggesting thatthe communityassociatedwiththe plantsshould not occurrandomlybutshouldbedependentontheinteraction betweenthe plantgenotype and the environmental condi-tions.

The Utricularia samples were taken from muddy and

dirtywater,wherethemostabundantgeneraofboth stud-ied sites belonged to the Sphingomonas genus, which has beenpreviouslyreportedtocolonizedifferentpolluted envi-ronments, both aquatic and terrestrial, being able to use polycyclicaromatic hydrocarbons as its sole sourceof car-bon and energy.40 _{Moreover,this genus has been reported}

tohaveabeneficial interactionwithplants,producing phy-tohormones such as gibberellins and indole acetic acid, promotingthegrowthofSolanumlycopersicum,41_and

produc-ing exopolysaccharides,42,43 _{which havealso beenreported}

tobeafactorinplant–bacteriuminteractionsforPaenibacillus polymyxa.44 3 3 1 1 1 1 1 utricMT stolMT

12

16

14

9

Fig.5–VenndiagramrepresentedbysharedUTO’sof stolons(stol)andutricules(utric)ofU.breviscapafromMato Grosso(MT)andSãoPaulo(SP)(dissimilarityof0.09). Therefore:utricMT:utriculesfromMatoGrosso,stolSP: stolonsfromSãoPaulo,utricSP:utriculesfromSãoPaulo andstolMT:stolonsfromMatoGrosso.

Inthepresent work,thegenusAquitaleawasoneofthe mostabundantgeneraassociatedwithutriclesfromsampled

SPandMTUtricularia.ThegenusAquitaleawasdescribedin

2006byLauetal.,45 _{fromafacultativeanaerobicbacterium}

strainlivinginlakes.Thisgenusismostcommonlyfoundin humicandoligotrophiclakesand inother watersources.46

Wooetal.47_{showedthatthegenusAquitaleawasoneofthe}

mostabundantgeneraisolatedfromwettropicalforestsoils, an environmentrich inorganic matterto decompose, pre-senting high levels of enzymeactivities. Although little is knownaboutthemechanismofdigestioninUtricularia, evi-dencehasbeenraisedforthepresenceofenzymaticactivity insidethe trap,anda considerableproportionofthe enzy-maticactivityinthetrapfluidisassumedtobederivedfrom thebacterialcommunity.48

Other abundantgenera associated withUtricularia from

SPwerePelomonasandMicrobacterium,whichhavealsobeen

reportedasbeneficialbacteriatohostplants.Pelomonasspp. isolatedfromindustrialwaterorfromhumicandoligotrophic lakesareabletofixnitrogenandreducenitrate45,49_;theycan

alsobeinvolvedinthebioremediationprocessbecausethey degradearomaticcompounds. Microbacteriumisolated endo-phytically and from the plant rhizoplane are also able to fixnitrogen,50_{solubilizephosphate,oxidizesulphur,reduce}

nitrate and produce bothindoleacetic acid (IAA) and ACC deaminase(associatedwithstressreductioninplantsbythe inhibitionofethylenesynthesis),showingtheirpotentialto promoteplantgrowth.51

TheBacillusspp.foundinbothlibraries(SPandMT)also haveplantbeneficialcharacteristics,suchasthepresenceof thenitratereductaseenzymeandthepotentialtoinhibit sev-eralphytopathogenicfungi(forexample,Alternariaalternata,

Fig.6–PhenetictreerepresentingthecultivableAcidobacteriafoundinassociationwithutriclesofU.breviscapafromSP usingNeighborJoiningmethodinMEGA5software.Numbersabovethebranchesindicatesbootstrapvalues.Brachyspira

specieswereusedasoutgroup.

capsiciandRhizoctoniasolani),demonstratingtheirimportant roleinhostplantdefense.52

Furthermore,SPplantspresentedexclusivegroups,such astheActinobacteriathatwereisolatedfromstolonsand utri-cles,confirmingthespecificitydependentonthegeographic location from which the plants were collected. However, despitepresentingsimilarlibrarieswithnodifferenceinthe Libshuffsignificancetest,plantorganscanalsoselecta bacte-rialgroup,suchasthephylaAcidobacteriaandBacteroidetes, whichwere foundonlyin utriclesfrom plantscollected in

SãoPauloandMatoGrosso,respectively(Fig.4).Theseresults supportthehypothesisthatplantsselectthebacterial com-munities associatedwithstolonsandutriclesbased onthe availablecommunity present intheirsurrounding environ-ment.

Anotherimportantpointweneedtoaddressisthe utri-cleisolatesthatbelongtotheAcidobacteriagroup,identified as thegenus Terriglobusalbidus (99%similarity), agenusof fastidiousgrowth(Fig.6).PhylumAcidobacteriaisoneofthe mostabundantinsoils,anditsmembershavebeenfoundby

cultivationindependentmethodsinmanytypesof environ-ments.Acidobacteriaisknowntobeabundantintrapfluid, andsomespeciesareknowntoliveinacidicenvironments.53

Sirováetal.17_{showedthatthetrapfluidpHislowinU.} vul-garis,rangingfrompH4.2to5.1accordingtothetrapage,and thiscouldbecorrelatedwiththephosphataseactivityinside thetrap.

However,membersoftheAcidobacteriagrouphaveproven difficulttoisolateandcultivateunderlaboratoryconditions, andtherearestillfewspecieswithvalidnames,andfeware welldefined;amongthemaremembersofthegenera Acidobac-terium,Acanthopleuribacter, Bryobacter, Edaphobacter,Geothrix, Granulicella,HolophagaandTerriglobus.54,55_{Terriglobussp.was}

previouslyisolatedfromwater56_{andfromspecificsoil}

con-ditions,suchasdesertsoils,57 _{arctictundrasoils}58 _andthe

rhizosphereofamedicinalplant.59 _{T.albiduswasdescribed}

in201560_{isolatedfromasemiaridsavannahsoilcollectedin}

northernNamibia,howeverthepresentworkpresentthefirst reportofcultivablebacteriabelongingtothisgenusisolated inassociationwithacarnivorousplant.

Caravieri et al.,18 _{using culture-independent methods,}

reportedthatAcidobacteriumwasoneofthedominant gen-erainassociationwithU.hydrocarpa.Thereareadvantagesin retrievinganAcidobacteriumisolateduetoitsbiotechnological applications,suchastheproductionofanextracellularmatrix thatapparently facilitatesthe flocculationofcellsinliquid media.61 _{Therefore,ourresearchgroup iscurrently}

investi-gatingthebiotechnologicalpotentialoftheseAcidobacterium

isolates.

Conclusions

TheresultsshowthatthebacterialdiversityofU.breviscapa

variedaccordingtotheisolationlocation(SãoPauloandMato Grosso)ratherthanorgans.Thisisalsothefirstreportofan Acidobacteria(Terriglobusgenus)isolatedinassociationwith utriclesofU.breviscapa.Theroleofthesebacteriainsidethe utriclesandstolonsmustbefurtherinvestigatedinorderto understandtheirpopulationdynamicswithinthehostplant.

Conflicts

of

interest

Declarationsofinterest:none

r

e

f

e

r

e

n

c

e

s

1.KrólE,PłachnoBJ,AdamecL,StolarzM,DziubinskaH,

TrebaczK.Quiteafewreasonsforcallingcarnivores‘the

mostwonderfulplantsintheworld’.AnnBot.2011;109:47–64.

2.EllisonAM,GotelliNJ.Energeticsandtheevolutionof

carnivorousplants-Darwin’s‘mostwonderfulplantsinthe

world’.JExpBot.2009;60:19–42.

3.ReifenrathK,TheisenI,SchnitzlerJ,PorembskiS,Barthlott

W.TraparchitectureincarnivorousUtricularia

(Lentibulariaceae).Flora.2006;201:597–605.

4.GivnishTJ.Newevidenceontheoriginofcarnivorousplants.

ProcNatlAcadSciUSA.2015;112:10–11.

5.AdamecL.Mineralnutritionofcarnivorousplants.BotRev.

1997;63:273–299.

6.EllisonAM,GotelliNJ.Evolutionaryecologyofcarnivorous

plants.TrendsEcolEvol.2001;16:623–629.

7.TaylorP.ThegenusUtricularia.In:Ataxonomicmonograph.

KewbulletinadditionalseriesXIV.RoyalBotanicGardens.

Londres:Kew;1989:668–671.

8.PoppingaS,DaberLE,WestermeierAS,etal.Biomechanical

analysisofpreycaptureinthecarnivorousSouthern

bladderwort(Utriculariaaustralis).SciRep.2017;7:1776.

9.PłachnoBJ,AdamecLL,LichtscheidlIK,PeroutkaM,

AdlassnigW,VrbaJ.Fluorescencelabellingofphosphatase

activityindigestiveglandsofcarnivorousplants.PlantBiol.

2006;8:813–820.

10.ChaseMW,ChristenhuszMJM,SandersD,FayMF.Murderous

plants:VictorianGothic,Darwinandmoderninsightsinto

vegetablecarnivory.BotJLinnSoc.2009;161:329–356.

11.AdlassnigW,PeroutkaM,LendlT.Trapsofcarnivorous

pitcherplantsasahabitat:compositionofthefluid,

biodiversityandmutualisticactivities.AnnBot.

2011;107:181–194.

12.BordiecS,PaquisS,LacroixH,etal.Comparativeanalysisof

defenceresponsesinducedbytheendophyticplant

growth-promotingrhizobacteriumBurkholderiaphytofirmans

strainPsJNandthenon-hostbacteriumPseudomonassyringae

pv.pisiingrapevinecellsuspensions.JExpBot.

2011;62:595–603.

13.McinroyJA,KloepperJW.Surveyofindigenousbacterial

endophytesfromcottonandsweetcorn.PlantSoil.

1995;173:337–342.

14.McinroyJA,KloepperJW.Populationdynamicsofendophytic

bacteriainfield-grownsweetcornandcotton.CanJMicrobiol.

1995;41:895–901.

15.RyanRP,GermaineK,FranksA,RyanDJ,DowlingDN.

Bacterialendophytes:recentdevelopmentsand

applications.FEMSMicrobiolLett.2008;278:1–9.

16.SessitschA,KuffnerM,KiddP,etal.Theroleof

plant-associatedbacteriainthemobilizationand

phytoextractionoftraceelementsincontaminatedsoils.Soil

BiolBiochem.2013;60:182–194.

17.SirováD,BorovecJ,CernaB,RejmánkováE,AdamecL.

Microbialcommunitydevelopmentinthetrapsofaquatic

Utriculariaspecies.AquatBot.2009;90:129–136.

18.CaravieriFA,FerreiraAJ,FerreiraA,ClivatiD,MirandaVFO,

AraújoWL.Bacterialcommunityassociatedwithtrapsofthe

carnivoralplantsUtriculariahydrocarpaandGenliseafiliformis.

AquatBot.2014;116:8–12.

19.PłachnoB,ŁukaszekM,WołowskiK,AdamecL,StolarczykP.

AgingofUtriculariatrapsandvariabilityofmicroorganisms

associatedwiththatmicro-habitat.AquatBot.2012;97:44–48.

20.Kuklinsky-SobralJ,AraújoWL,MendesR,Pizzirani-Kleiner

AA,AzevedoJL.Isolationandcharacterizationofendophytic

bacteriafromsoybean(Glycinemax)growninsoiltreated

withglyphosateherbicide.PlantSoil.2005;273:91–99.

21.AraújoWL,MarconJ,MaccheroniWJr,vanElsasJD,van

VuurdeJW,AzevedoJL.Diversityofendophyticbacterial

populationsandtheirinteractionwithXylellafastidiosain

citrusplants.ApplEnvironMicrobiol.2002;68:4906–4914.

22.HeuerH,KrsekM,BakerP,SmallaK,WellingtonEMH.

Analysisofactinomycetecommunitiesbyspecific

amplificationofgenesencoding16SrRNAand

gel-electrophoreticseparationindenaturinggradients.Appl

EnvironMicrobiol.1997;63:3233–3241.

23.LaneDJ,PaceB,OlsenGJ,StahlDA,SoginML,PaceNR.

Rapid-determinationof16SribosomalRNAsequencesfor

phylogeneticanalyses.ProcNatlAcadSciUSA.

1985;82:6955–6959.

24.DouradoMN,AndreoteFD,Dini-AndreoteF,ContiR,Araújo

JM,AraújoWL.Analysisof16SrRNAandmxaFgenes

revealinginsightsintoMethylobacteriumniche-specificplant

25.SangerF,NicklenS,CoulsonAR.DNAsequencingwith

chain-terminatinginhibitors.ProcNatlAcadSciUSA.

1977;74:5463–5467.

26.SchlossPD,WestcottSL,RyabinT,etal.Introducingmothur:

open-source,platform-independent,community-supported

softwarefordescribingandcomparingmicrobial

communities.ApplEnvironMicrobiol.2009;75:7537–7541.

27.ThompsonJD,HigginsDG,GibsonTJ.ClustalW:improving

thesensitivityofprogressivemultiplesequencealignment

throughsequenceweighting,position-specificgappenalties

andweightmatrixchoice.NuclAcidRes.1994;22:4673–4680.

28.JukesTH,CantorCR.Evolutionofproteinmolecules.In:

MunroHN,ed.Mammalianproteinmetabolism.NewYork,USA:

AcademicPress;1969:21–132.

29.TamuraK,PetersonD,PetersonN,StecherG,NeiM,Kumar

S.MEGA5:molecularevolutionarygeneticsanalysisusing

maximumlikelihood,evolutionarydistance,andmaximum

parsimonymethods.MolBiolEvol.2011;28:2731–2739.

30.LetunicI,BorkP.InteractiveTreeOfLife(iTOL):anonlinetool

forphylogenetictreedisplayandannotation.Bioinformatics.

2007;23:127–128.

31.BasualdoCV.Choosingthebestnon-parametricrichness

estimatorforbenthicmacroinvertebratesdatabases.RevSoc

EntomolArgent.2011;70:27–38.

32.HortalJ,BorgesPA,GasparC.Evaluatingtheperformanceof

speciesrichnessestimators:sensitivitytosamplegrainsize.

JAnimEcol.2006;75:274–287.

33.FridayLE.RapidturnoveroftrapsinUtriculariavulgarisL.

Oecologia.1989;80:272–277.

34.PlachnoBJ.ThecarnivorousplantsandCharlesDarwin–a

storyaboutbeautifulfascination.In:ZarzyckiK,MirekZ,

KorzeniakU,eds.KarolDarwinwoczachpolskichbotaników

XIX–XXIw.PANIBKraków;2010.

35.AlcarazLD,Martínez-SánchezS,TorresI,Ibarra-LacietteE,

Herrera-EstrellaL.ThemetagenomeofUltriculariagibba’s

traps:intothemicrobialinputtoacarnivorousplant.PLOS

ONE.2016;11:1–21.

36.PitschG,AdamecL,DirrenS,etal.ThegreenTetrahymena

utriculariaen.sp.(Ciliophora,Oligohymenophorea)withits

endosymbioticalgae(Micractiniumsp.),livingintrapsofa

carnivorousaquaticplant.JEukaryotMicrobiol.

2017;64:322–335.

37.AdamecL.Thesmallestbutfastest.Ecophysiological

characteristicsoftrapsofaquaticcarnivorousUltricularia.

PlantSignalBehav.2011;6:640–646.

38.LaqueT,FarjallaVF,RosadoAS,EstevesFA.Spatiotemporal

variationofbacterialcommunitycompositionandpositive

controllingfactorsintropicalshallowlagoons.MicrobEcol.

2010;59:819–829.

39.KoopmanMM,FuselierDM,HirdS,CarstensBC.The

carnivorouspalepitcherplantharborsdiverse,distinct,and

time-dependentbacterialcommunities.ApplEnviron

Microbiol.2010;76:1851–1860.

40.LeysNMEJ,RyngaertA,BastiaensL,VerstraeteW,TopEM,

SpringaelD.Occurrenceandphylogeneticdiversityof

Sphingomonasstrainsinsoilscontaminatedwithpolycyclic

aromatichydrocarbons.ApplEnvironMicrobiol.

2004;70:1944–1955.

41.KhanAL,WaqasM,KangS,etal.Bacterialendophyte

Sphingomonassp.LK11producesgibberellinsandIAAand

promotestomatoplantgrowth.JMicrobiol.2014;52:689–695.

42.PrajapatiVD,JaniGK,ZalaBS,KhutliwalaTA.Aninsightinto

theemergingexopolysaccharidegellangumasanovel

polymer.CarbohydPolym.2013;93:670–678.

43.SchultheisE,DregerMA,NimtzM,WrayV,HempelDC,

NörtemannB.Structuralcharacterizationofthe

exopolysaccharidePS-EDIVfromSphingomonaspituitosas

trainDSM13101.ApplMicrobiolBiotechnol.2008;78:1017–1024.

44.YegorenkovaIV,TregubovaKV,IgnatovVV.Paenibacillus

polymyxaRhizobacteriaandtheirsynthesizedexoglycansin

interactionwithwheatrootscolonization.CurrMicrobiol.

2013;66:481–486.

45.LauH,FarynaJ,TriplettEW.Aquitaleamagnusoniigen.nov.,

sp.nov.,anovelGram-negativebacteriumisolatedfroma

humiclake.IntJSystEvolMicrobiol.2006;56:867–871.

46.SedlacekJ,KwonSW,SvecP,etal.Aquitaleapelogenessp.nov.,

isolatedfrommineralpeloid.IntJSystEvolMicrobiol.

2015;66:962–967.

47.WooHL,HazenTC,SimmonsBA,DeAngelisKM.Enzyme

activitiesofanaerobiclignocellulolyticbacteriaisolatedfrom

wettropicalforestsoils.SystApplMicrobiol.2010;37:60–67.

48.SirováD,AdamecL,VrbaJ.Enzymaticactivitiesintrapsof

fouraquaticspeciesofthecarnivorousgenusUtricularia.

NewPhytol.2003;159:669–675.

49.GomilaM,BowienB,FalsenE,MooreERB,LalucatJ.

DescriptionofPelomonasaquaticasp.nov.andPelomonas

puraquaesp.nov.,isolatedfromindustrialandhaemodialysis

water.IntJSystEvolMicrobiol.2007;57:2629–2635.

50.ZakhiaF,JederH,WillemsA,GillisM,DreyfusB,LajudieP.

Diversebacteriaassociatedwithrootnodulesof

spontaneouslegumesinTunisiaandfirstreportfornifH-like

genewithinthegeneraMicrobacteriumandStarkeya.Microb

Ecol.2006;51:375–393.

51.MadhaiyanM,PoonguzhaliS,LeeJ,LeeK,SaravananVS,

SanthanakrishnanP.Microbacteriumazadirachtaesp.nov.,a

plantgrowth-promotingActinobacteriumisolatedfromthe

rhizoplaneofneemseedlings.IntJSystEvolMicrobiol.

2010;60:1687–1692.

52.ZhaoZ,WangaQ,WangK,BrianK,LiuC,GuY.Studyofthe

antifungalactivityofBacillusvallismortisZZ185invitroand

identificationofitsantifungalcomponents.BioresourTechnol.

2010;101:292–297.

53.TakeuchiY,ChaffronS,SalcherMM,etal.Bacterialdiversity

andcompositionintheluidofpitcherplantsofthegenus

Nepenthes.SystApplMicrobiol.2015;38:330–339.

54.EichorstSA,KuskeCR,SchmidtTM.Influenceofplant

polymersonthedistributionandcultivationofbacteriain

thephylumAcidobacteria.ApplEnvironMicrobiol.

2010;77:586–596.

55.OkamuraK,KawaiA,YamadaT,HiraishiA.Acidipilarosea

gen.nov.,sp.nov.,anacidophilicchemoorganotrophic

bacteriumbelongingtothephylumAcidobacteria.FEMS

MicrobiolLett.2011;317:138–142.

56.BaikKS,ChoiJS,KwonJ,etal.Terriglobusaquaticussp.nov.,

isolatedfromanartificialreservoir.IntJSystEvolMicrobiol.

2013;63:4744–4749.

57.AbedRM,Al-KindiS,Al-KharusiS.Diversityofbacterial

communitiesalongapetroleumcontaminationgradientin

desertsoils.MicrobEcol.2015;69:95–105.

58.RawatSR,MännistöMK,BrombergY,HäggblomMM.

Comparativegenomicandphysiologicalanalysisprovides

insightsintotheroleofAcidobacteriainorganiccarbon

utilizationinArctictundrasoils.FEMSMicrobiolEcol.

2012;82:341–355.

59.WhangKS,LeeJC,LeeHR,HanSI,ChungSH.Terriglobustenax

sp.nov.,anexopolysaccharide-producingAcidobacterium

isolatedfromrhizospheresoilofamedicinalplant.IntJSyst

EvolMicrobiol.2014;64:431–437.

60.PascualJ,WustPK,GeppertA,FoeselBU,HuberKJ,

OvermannJ.Terriglobusalbidussp.nov.,amemberofthe

familyAcidobacteriaceaeisolatedfromNamibiansemiarid

savannahsoil.IntJSystEvolMicrobiol.2015;65:3297–3304.

61.EichorstSA,BreznakJA,SchmidtTM.Isolationand

characterizationofsoilbacteriathatdefineTerriglobusgen.

nov.,inthephylumacidobacteria.ApplEnvironMicrobiol.