Marine Actinobacteria as a source of compounds for phytopathogen control

25 Pages·2017·2.72 MB·English

RESEARCHARTICLE
Marine Actinobacteria as a source of
compounds for phytopathogen control: An
integrative metabolic-profiling / bioactivity
and taxonomical approach
LuzA.Betancur1,2,SandraJ.Naranjo-Gaybor1,3,DianaM.Vinchira-Villarraga1,Nubia
C.Moreno-Sarmiento1,LuisA.Maldonado4,ZulmaR.Suarez-Moreno5,AlejandroAcosta-
Gonza´lez6,GillermoF.Padilla-Gonzalez7,Mo´nicaPuyana8,LeonardoCastellanos1,
a1111111111 FreddyA.Ramos1*
a1111111111
1 UniversidadNacionaldeColombia,SedeBogota´,DepartamentodeQu´ımica,Carrera,EdificiodeQu´ımica
a1111111111
of427,Bogota´,Colombia,2 UniversidaddeCaldas.DepartamentodeQu´ımica.EdificioOrlandoSierra,
a1111111111
BloqueB,SedePalograndeCalle.Manizales,Caldas,Colombia,3 UniversidaddelasFuerzasArmadas,
a1111111111
ESPECarreradeIngenier´ıaAgropecuariaIASAIIAv.GeneralRumiñahuis/n,Sangolqu´ı-Ecuador,
4 UniversidadAuto´nomaMetropolitanaRector´ıa—Secretar´ıaGeneral,Prolongacio´nCanaldeMiramontes,
Col.Ex-haciendaSanJuandeDios,Tlalpan,Me´xicoDF,5 Investigacio´nyDesarrollo,EmpresaColombiana
deProductosVeterinariosVECOLS.A.,Bogota´D.C,6 UniversidaddelaSabana,FacultaddeIngenier´ıa,
AutopistaNorte,Ch´ıa,Cundinamarca,Colombia,7 UniversidadedeSãoPaulo,FaculdadedeCiências
FarmacêuticasdeRibeirãoPreto,Av.dodeSaoPaulo,FaculdadedeCiênciasFarmacêuticasdeRibeirão
OPENACCESS
Preto,Av.doCafe´,RibeirãoPreto–SP,Brazil,8 DepartamentodeCienciasBiolo´gicasyAmbientales,
Citation:BetancurLA,Naranjo-GayborSJ, ProgramadeBiolog´ıaMarina,UniversidadJorgeTadeoLozano,Carrera,Modulo,Oficina,Bogota´,Colombia
Vinchira-VillarragaDM,Moreno-SarmientoNC,
*[email protected]
MaldonadoLA,Suarez-MorenoZR,etal.(2017)
MarineActinobacteriaasasourceofcompounds
forphytopathogencontrol:Anintegrative
metabolic-profiling/bioactivityandtaxonomical Abstract
approach.PLoSONE12(2):e0170148.
doi:10.1371/journal.pone.0170148 Marinebacteriaareconsideredaspromisingsourcesforthediscoveryofnovelbiologically
Editor:Marie-JoelleVirolle,UniversiteParis-Sud, activecompounds.Inthisstudy,samplesofsediment,invertebrateandalgaewerecol-
FRANCE lectedfromtheProvidenciaandSantaCatalinacoralreef(ColombianCaribbeanSea)with
Received:September19,2016 theaimofisolatingActinobateria-likestrainabletoproduceantimicrobialandquorum
quenchingcompoundsagainstpathogens.Severalapproacheswereusedtoselectactino-
Accepted:December29,2016
bacterialisolates,obtaining203strainsfromallsamples.Accordingtotheir16SrRNAgene
Published:February22,2017
sequencing,atotalof24strainswasclassifiedwithinActinobacteriarepresentedbythree
Copyright:©2017Betancuretal.Thisisanopen
genera:Streptomyces,Micromonospora,andGordonia.Inordertoassesstheirmetabolic
accessarticledistributedunderthetermsofthe
profiles,theactinobacterialstrainsweregrowninliquidcultures,andLC-MS-basedanaly-
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and sesfromethylacetatefractionswereperformed.Basedontaxonomicalclassification,
reproductioninanymedium,providedtheoriginal screeninginformationofactivityagainstphytopathogenicstrainsandquorumquenching
authorandsourcearecredited.
activity,aswellasmetabolicprofiling,sixoutofthe24isolateswereselectedforfollow-up
DataAvailabilityStatement:Allrelevantdataare withchemicalisolationandstructureidentificationanalysesofputativemetabolitesinvolved
withinthepaperanditsSupportingInformation
inantimicrobialactivities.
files.Dataarealsoavailableatthefollowinglink:
https://osf.io/dym7w/.
Funding:LABthankstheProgramaDoctoralBecas
Colciencias(567)forthegrantedscholarship
(2012).SJNthanksSENESCYTEcuadorforthe
grantedscholarship(2012-2).AAGisrecipientof
an"EsTiempodevolver2015-2016"postdoctoral
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 1/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
fellowshipfromColcienciasandLaSabana Introduction
University(Bogota´,Colombia).Financialsupport
Duringthesecondhalfofthetwentiethcentury,oneofthemajorconcernsinagriculturewas
fromCOLCIENCIAS(grantCod.1101-659-44402.
CT.537/14),IFS(GrantF/5023-2),andUniversidad focusedonpollutionoriginatedbytheextensiveuseofhighlytoxicagrochemicalssuchaspes-
NacionaldeColombia-DIB(grant/Hermes23604). ticides[1,2].Studiessincethe1970shaveshownthat,besidestheharmfuleffectsatthepublic-
ThefunderVECOLS.A.providedsupportinthe healthlevel,theuseofpesticideshaveledtotheemergenceofphytopathogenresistancecaused
formofsalariesforauthors(ZRS),butdidnothave
bythesystematicuseofaproduct[3].Asthepresenceofpathogensincropsofglobaleco-
anyadditionalroleinthestudydesign,data
nomicimportanceispersistent,bothindustryandacademyhaveincreasedtheireffortsin
collectionandanalysis,decisiontopublish,or
findingsolutionstothisproblem.
preparationofthemanuscript.Thisdoesnotalter
ouradherencetoPLOSONEpoliciesonsharing Bacterialcoloniesofboth,pathogenicandbeneficstrainsarerecognizedassocialcommu-
dataandmaterials. nitiesthatareabletoregulatetheirgeneexpressioninadensity-dependentway,phenomenon
knownasQuorumsensing(QS)[4].QSsystemsregulatemanyprocessesrelatedtometabo-
CompetingInterests:Oneoftheauthors(ZRS)is
employedbythecommercialcompanyVECOLS.A. lism,developmentandvirulenceinbacterialcells,throughtheproductionofsmallsignaling
Therearenopatents,productsindevelopmentor molecules,thatincreaseinconcentrationwithincreasingcellnumbers[5].Oncetheconcen-
marketedproductstodeclare.Thisdoesnotalter
trationofthesemoleculesreachesaspecificthreshold,differentsignaltransductioncascades
ouradherencetoPLOSONEpoliciesonsharing
areinducedresultinginchangesingeneexpression,includingpathogeniceffects.Theexpres-
dataandmaterials.
sionofsomevirulence-relatedtraitsaredependentonQSandmanyplat-pathogenicbacteria
arereliantonthiskindofsystemstoevokediseaseinitsplanthost[6,7].Asanexample,ithas
beenwidelyrecognizedthattoxoflavinproductioninsomeBurkholderiaspecies(particularly
inB.glumae)iscontrolledbyaQSsystemandthat,theproductionofthisphytotoxinisakey
pathogenicityfactorinricerotandwilt[8].
Analternativetosolvethisproblemisthesearchofantibioticcompoundsderivedfrom
microorganisms,largelyrecognizedasthemainsourceofantimicrobialcompounds,witha
potentialuseagainstphytopathogens[9].Microorganismsfromterrestrialenvironmentshave
beenthetraditionalsourceofthesecompounds.Marinemicroorganismshavebeenrecognized
asanimportantsourceofsecondarymetaboliteswiththepotentialtocontrolthesepathogens
duringthelastyears[10,11].Furthermore,marinerepresentativesofthePhylaActinobacteria
arerecognizedasoneofthemostimportantgroupswithbiotechnologicalpotential[12],thus
contributingtoincreasingthesupplyofnewbioactivecompounds[13].Marine-derivedmetab-
olitesbecomeprototypesforthedevelopmentofnewsubstanceswithaputativeinsecticidaland
antimicrobialpotential,whichmakethemexcellentcandidatesfortheiruseasagrochemicals
[14,15].OneclearexampleisthecaseofKasugamycinTM,asystemicfungicideagainstMagna-
porthegriseaandbactericideagainstBurkholderiaglumae[16].Thisbioactivecompoundwas
isolatedforthefirsttimefromStreptomyceskasugaensis,aterrestrialActinobacteria,andlater
frommarinestrainsofStreptomycesrutgersensissubsp.gulangyunensis[17,18].
Themodeoftherapeuticefficacyofantibioticsisattributedtotheinhibitionofbacterial
growthinvivowhenantibioticconcentrationsexceedtheMIC.However,concentrations
belowtheMICcanstillattenuategrowthandtheexpressionofavarietyofbacterialvirulence
factors,compromisingtheabilityofthepathogentocausedisease.Thisactivityofantibiotics
isreferredtoassub-MICeffects,andthecompoundsthatproducetheseeffectsareknownas
quorumquenchingcompounds.
Quorumquenchingcompoundsalsohavebeenusedinordertoinhibittheexpressionof
phytopathogenvirulencefactors.Oneofthreedifferentstrategiescanachievethis:theproduc-
tionofenzymes,thatdegradatesthesignalmolecules;theinhibitionoftheenzymes(itstran-
scriptionoritsactivity)involvedinthebiosynthesisofthesignal-molecule;ortheinhibitionof
theactivationreceptorsofQS[4].Therearesomeexamplesofbacterialstrainsabletoinhibit
QSsystemsforpathogenicstrains.Forexample,theexpressionoflactonaseenzymefrom
Bacillus240B1,codifiedbytheaiiAgene,inPectobacteriumcarotovorum(formelyErwiniacar-
otovora)reducesignificantlyAHLreleaseandsoftrotdiseasesymptomsindifferentdetached
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 2/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
tissuesofpotato,eggplant,Chinesecabbage,carrotandcelery[19].Inaddition,somestrains
ofStreptomyceshavedemonstratedabilitytoinhibittheexpressionofdifferentvirulencefac-
torsregulatedbyQSinP.caratovorum,throughtheproductionofsomesecondarymetabolites
identifiedaspiericidinAandglucopiericidinA,showingthattheycouldhavepotentialuseas
controlagentsofsomeplantspathogens[20].Compoundsderivedfrommarinemicroorgan-
ismshavealsobeenevaluatedfortheirroleasquorumquenchers,suitableforactingasanti-
pathogeniccompoundsthroughinterruptionofpathogenicbacterialcommunication;this
interruptionreducesdamageinthehost[21,22].Forinstance,thelineardipeptidesPro–Gly
andN-amido-α-prolinefromActinobacteriaassociatedtoamarinespongeshowedinhibitory
activitiesagainstquorumsensing(QS)mediatedvirulencefactorsinPseudomonasaeruginosa
[23].
However,despitetheeffortstoisolatenewcompoundsfrommarineenvironments,tradi-
tionalbioprospectingapproachesincludingbioassay-guidedfractionationoftenleadtoisola-
tionofalreadyknowncompounds[24].This,inpart,isduetotheuseofclassicalapproaches
forselectingmicrobialstrainsbasedsolelyontaxonomicalorantimicrobialinformation[25].
Furthermore,studiesbasedexclusivelyonchemicaldiversityofcompoundslackdataontheir
biologicalactivity,whichlimitstheirimpactforfurtherapplications.Forthesereasons,bio-
prospectingofmicrobialstrainsforisolatingnewbioactivecompoundshasmovedtowards
integratedstrategies,whichcombinesphylogeneticdataandbioactivitytestswithdereplica-
tionapproaches,asaquickalternativeforidentifyingknownandbioactivemetabolitesina
samplemixture[26].Thesestrategies,complementedwithaccuratemultivariateanalyses
(PCA,HCA,OPLSandothers),haveshowntobeeffectiveinidentifyingnewbioactivecom-
pounds,improvingthepipelinefordrugdiscoveryprogramsusingmicroorganisms[27].
Inthisstudy,wepresentastrategytostudythemetabolicpotentialofselectedstrainsof
marineActinobacteriafromtheColombianCaribbeanSea,basedontheintegrationoftaxo-
nomicalinformation;screeningdataofantimicrobialactivitiesagainstphytopathogenicstrains
(BurkholderiaplantariiATCC43733,BurkholderiaglumaeATCC33617,Burkholderiagladioli
3704-1-FEDEARROZ-).Forantifungaltests,phytopathogenicfungiFusariumoxysporumf.
sp.dianthirace2,Colletotrichumgloeosporioides26Bandquorumquenchingactivity(Chromo-
bacteriumviolaceumATCC31532)wereused,alongwiththeirHPLC-MSmetabolicprofile.
Allthedatasetsobtainedherewereintegratedtoprioritizeselectionofisolatesforfollow-up
withchemicalisolationandstructureidentificationanalysesofactivecompounds.Adiscus-
sionontheadvantagesoftheintegrativeapproachandthepossibleoutputsofthisstrategyis
alsopresented.
Materialsandmethods
Isolationofbacterialstrains
InordertoconductthisresearchtheANLA(AutoridadNacionaldeLicenciasAmbientales)
andtheMinisteriodeAmbienteyDesarrolloSosteniblegrantedpermissiontocollectsamples
andstudytherecoveredbacteria(PermissionN˚4of10/02/2010,Anexo2,ContratodeAcceso
aRecursoGene´ticoNo108).Forbacterialstrainsisolation,smallpieces(2cm2)ofmarine
invertebrates,algaeandsedimentswerecollectedbySCUBAdivingatreefsatOldProvidence
Island(ColombianCaribbeanSea).Thegeographiccoordinatesofsamplingareawas13˚
23’0,00"Na13˚25’0.00"N81˚22’0.00"Oto81˚24’0,00".Samplesofthesamesubstrateswerecol-
lectedinduplicateatfivedifferentsitesonMarchof2013.Fragmentsofgorgoniansandalgae
werecutoffwithsharpscissors.Spongefragmentswerecutoffwithadivingknife.Sediment
samples(5cm3)werecollectedusingplasticspoons.Eachsamplewasindividuallyplacedina
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 3/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
smallziplockbagandbroughttothesurface.Samplesweregentlyrinsedwithsterilewaterand
thenplaceddirectlyonthesurfaceofsolidculturemediaplates.
TherecoveryofactinobacterialstrainswasperformedbyusingenrichedmediasuchasISP-
2agar(yeastextract4g/L,maltextract10g/L,dextrose4g/L,agar20g/L)andoatmealagar
supplementedwithsalts(oatmeal30g/L,NaCl20.8g/L,KCl0.56g/L,MgSO 4.8g/L,Rilasea
4
salt1.5g/L,glycerol1.5g/L,agar18g/L).Plateswereincubatedfortwoweeksat30˚C.
Oncebacterialgrowthwasdetected,agarwasfragmentedintopieceswithasterileneedle.
Theobtainedfragmentsweretransferredto2.0mLofsterilesalinesolution(0.85%)andthen
homogenizedinavortexandsupplementedwith50mgofCaCO inordertoimprovethe
3
growthofActinobacteriastrains.Suchsuspensionswereincubatedat26˚Cforoneweekand
thenseriallydilutedwithTween-801(0.1%).Dilutionsfrom10−1to10−5weremassiveculti-
vatedincasein-starchagar(CSA)(Casein1g/L,Starch10g/L,K HPO 0.5g/LAgar15g/L)
2 4
andincubatedat26˚Cforaperiodof4to6weeks.
Purificationofbacterialisolateswasperformedbysuccessivesub-cultureofsinglecolonies
inCSAmedium.Atotalof203isolateswasrecovered,including,162bacteriaand41fungi.
Bacterialisolateswerepreservedasfollows:i)Forthebacteriaformingmycelium(typicalof
Actinobacteria-likeorganisms),singlecoloniesweregrowninCSAplatesat4˚C(shortterm
preservation)andmyceliasuspensionswerepreservedwithglycerol(50%)at-80˚C(Long
termpreservation)[28].ii)Forthosebacteriawithnomyceliaformation,asinglecolonywas
grownintrypticsoyagar(Difco),andaloopfulofbiomasswassuspendedinTrypticSoy
Brothsupplementedwithglycerol50%,whichwerethenstoredat-80˚C.Taxonomicalidenti-
fication,biochemicalcharacterizationandmetabolicprofilingstudiesweredoneto24strains
duetotheirmicroscopicandmacroscopiccharacteristicsasActinobacteria-likestrains(vide
infra).AllthestrainswereregisteredinthecollectionofmicroorganismsoftheIBUN(Insti-
tutodeBiotecnolog´ıa—UniversidadNacionaldeColombia—SedeBogota´).
Biochemicalcharacterizationofbacterialstrains
SinglecoloniesoftheActinobacteria-likestrainswerecharacterizedbytheircolonymorphol-
ogyinnutrientagar,ISP-2,ISP-3,andISP-4(S1Fig).Catalaseandoxidase(Bactident1Oxi-
dasaMERKC)activityweretestedassuggestedforStreptomyces-likebacteria[29,30].Other
biochemicalcharacteristicssuchasfermentationoroxidationofsugars(glucose,manitol,ino-
sitol,sorbitol,rhamnose,sucrose,melibiose,amygdalinandarabinose);enzymeactivity(β-
galactosidase,arginine-dihydrolase,lysinedecarboxylase,ornithinedecarboxylase,ureasae,
tryptophandeaminase,gelatinase);citrateutilization;productionofsulfide,indoleandacet-
oin;andnitratereductionweretestedwiththeAPI20Ekit(bioMe´rieuxInc.,Durham,NC)
(S1Table).TheresultswerecomparedwiththosedescribedforotherActinobacteria-likebac-
teria[31].
ForachemotaxonomiccharacterizationoftheActinobacteriastrains,thepresenceofL,L-
diaminopimelicacid(LL-A pm)ormeso-diaminopimelicacidinthecellwalloftheisolates
2
wasdeterminedthroughtheprotocolestablishedbyStaneckandRoberts[32]withsomemod-
ifications[33].Briefly,twoorthreeloopfulsoffreshbiomass(7–14daysgrowth)weresus-
pendedin500μLofHCl6N.Suchsuspensionwashomogenizedinavortexfor10minand
thenplacedinanovenat100˚Cfor8h.Aftercentrifugation,spentsupernatantsweretrans-
ferredtoanewEppendorftube,anddriedat100˚C.500μLofsteriledistilledwaterwere
addedtoeachtube,andsampleswerecentrifugedanddriedoncemore,untilsupernatants
werecompletelydried.Thismaterialwasthesuspendedinto75μLofdistilledwater,and3μL
ofeachsampleusedforfurtherTLCanalysesand1μLdiaminopimelicacidsmixture(CHE-
M-IMPEXINT´LINC,1%indistilledwater)wereloadedoncelluloseTLCplates20x20.TLC
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 4/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
platesweredevelopedinsaturatedglasschamberscontainingmethanol-water-6NHCl-pyri-
dine(80:26:4:10,v/v).Theplatesweredried,sprayedwithafreshlypreparedninhydrin
solution(0.2%,w/vdissolvedinacetone)andheatedat100˚Cfor5min.PresenceofL,L-dia-
minopimelicacidwasdetectedinthoseregionsshowingacharacteristicolivegreencolor
(Rf=0.6).(S2Fig).
ScanningElectronMicroscope(SEM)analysis
Scanningelectronmicrographanalysisstrainsweregrowingfor8daysonISP-2agarmedium
usingfilterpaper.CulturesofstrainsarefixedusingMilloningphosphatebuffer(0.1M,pH
7.2)Glutaraldehyde(1%).Thecellswererinsedwithdistilledwatersubsequentlydehydrated
withethanol.Thefilterpaperwasfirstdried,thenfixedontostubsandcoatedwithgraphite.
ThescanningmicroscopyanalysiswasperformedusingSEM-TescanVega3SB[34,35].
Taxonomicalidentificationandaccessionnumbers
24isolateswereselectedforfurtherstudiesbasedontheirmicroscopicandmacroscopicchar-
acteristicsconsistentwithActinobacteria-likestrains.Forthetaxonomicalidentificationofthe
selectedstrains,PCRamplificationandsequencingoftheentire16SrRNAlocusweredoneby
usinguniversalprimers27Fand1492R,aspreviouslydescribed[36].Sequencesweretrimmed
andassembledwithDNABasersoftware2.9andanaccuratealignmentwasobtainedusing
theSINAonlinetool[37].Neighbor-joiningtreesofisolatesandtheirclosestrelativeswere
generatedusingtheARBsoftware[38]andtheLTPs123availableintheSILVAdatabase[39].
AllDNA16SrRNAnucleotidesequencesforthe24strainsweredepositedinGenBank/
EMBL/DDBJ,undertheaccessionnumbersKX641378toKX641401,aspresentedinTable1.
Selectedmorphotypeswereidentifiedinparallelto16SrRNAapproachbymassspectrome-
trywithMALDI-TOF(Matrix-assistedlaserdesorption/ionizationtimeofflight)Biotyper
BRUKER.Forthispurpose,strainswerere-growthinLBmediaandincubatedforeightdays
atthesametemperatures.Identificationwasdonebydirectsmearmethod,whereasinglecol-
onywaspickedandplacedontoaMALDItargetplateasathinlayerandallowedtodryat
roomtemperature.TheMALDIplatewassubsequentlyoverlaidwith1μLof70%formicacid
andthencoveredwiththematrixsolution(α-cyano-4-hydroxycinnamicacid—HCCA)and
airdried.TheprofileswerevisualizedwiththesoftwareFlexControl(version3.0)andthe
MALDIBiotyperRTC.DendogramswereobtainedusingBioTypersoftware3.0.Forcalibra-
tionandasapositivecontrol,weusedaBacterialTestStandard(BTS)Escherichiacoli(DH5α)
(BrukerDaltonikGmbH,Bremen,Germany)proteicprofile.
Bioassaysusingmarinebacteriastrains
Antimicrobialassays. Antibacterialactivityfrom24Actinobacteria-likeisolateswasfirst
testedagainst3bacterialricepathogensbyusingadirectconfrontationassay.Thebacterial
pathogensstrainsincluded:BurkholderiaplantariiATCC43733,BurkholderiaglumaeATCC
33617,andBurkholderiagladioliCIAT3704–1,kindlyprovidedbyFEDEARROZ,whichwere
isolatedfromsymptomaticriceplantsofbacterialpanicleblightandriceseedlingblight.These
bacterialstrainswererecoveredandgrownaspreviouslydescribed[40–43].Theconfrontation
assaywasperformedasfollows:singlecoloniesofeachActinobacteria-likeisolatesweremas-
sivelystreakedonMueller-Hintonagarplatescoveringhalfofthesurface,andplateswere
incubatedat28˚Cfor10days.PathogenicBurkholderiawherethenstreakedinahorizontal
line,perpendiculartotheactinobacterialgrowtharea.Plateswereincubatedat37˚Cand
growthofBurkholderiastrainswasdailymonitoredforthreedays.Actinobacteria-likeisolates
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 5/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
Table1. AffiliationofActinobacteriastrainsisolatedfrommarinesamples.
Cluster(OTU) Closestneighbors Sim% Strainnumber Strain/GenBankaccessionnumbers Source
1 Streptomycesgriseochromogenes 99.86 3 KX641378 Niphatesdigitalis(Porifera)
6 KX641383 Niphatesdigitalis(Porifera)
Streptomycesviolascens 99.86 13 KX641381 Sediment
87 KX641385 Niphatesdigitalis(Porifera)
Streptomycesresistomycificus 99.86 89.3 KX641386 Niphatesdigitalis(Porifera)
89.4 KX641387 Niphatesdigitalis(Porifera)
Streptomyceshydrogenans 99.86 161a KX641396 Bryopsissp.(Chlorophyta)
161b KX641397 Bryopsissp.(Chlorophyta)
Streptomycesalbidoflavus 99.86 184 KX641399 Euniceafusca(Gorgonacea)
194 KX641400 Sediment
208 KX641401 Euniceafusca(Gorgonacea)
2 Streptomycessp.MBRL172 99.03 182 KX641398 Amphiroasp.(Rhodophyta)
3 Streptomycessanyensis 99.72 5 KX641379 Niphatesdigitalis(Porifera)
143 KX641389 Niphatesdigitalis(Porifera)
144 KX641390 Niphatesdigitalis(Porifera)
144a KX641391 Niphatesdigitalis(Porifera)
145 KX641392 Niphatesdigitalis(Porifera)
148 KX641393 Niphatesdigitalis(Porifera)
149 KX641394 Niphatesdigitalis(Porifera)
149a KX641395 Niphatesdigitalis(Porifera)
4 Streptomycesmicroflavus 99.86 46b KX641384 Codiumsp.(Chlorophyta)
Streptomycesfulvorobeus 99.86
5 Streptomycespratensis 100 9 KX641380 Dictyotasp.(Phaeophtyta)
6 Micromonosporachalcea 99.79 102N KX641388 Dictyotasp.(Phaeophtyta)
Micromonosporamaritima 99.38
Micromonosporasediminicola 99.15
Micromonosporamarina 99.44
7 Gordoniabronchialis 98.41 25 KX641382 Xestospongiasp.(Porifera)
doi:10.1371/journal.pone.0170148.t001
abletoinducetotalorpartialgrowthinhibitionofthepathogensafter48hourswereconsid-
eredaspositiveforantibacterialactivity.
Antifungalactivitiesfromall24strainsweremeasuredasdescribedbyKaninietal.[44].
Forantifungaltests,phytopathogenicfungiFusariumoxysporumf.sp.dianthirace2,isolated
fromvascularwiltsymptomaticcarnationplants[45],andColletotrichumgloeosporioides26B,
obtainedfromyamplantswithclassicalsymptomsofanthracnose[46]weretested.Thesephy-
topathogenicfungiwerekindlyprovidedbythe“GrupodeEstudioenN˜ame”fromtheBio-
technologyInstitute,andthe“EstudiodeActividadesMetabolicasVegetales”researchgroup
oftheChemistryDepartmentfromtheUniversidadNacionaldeColombia.
Quorumquenchingassayofmarinestrains. Inordertotesttheabilityofthe24Actino-
bacteria-likeisolatestodisruptthequorumsensingsystems,Chromobacteriumviolaceum
ATCC31532wasusedasabiosensorbymeansofacrossstreakantagonisticassay[43,47,48].
Briefly,LBagarplateswereseededwitheachActinobacteria-likeisolatebystreakingthemina
horizontalline,neartooneoftheextremesoftheagarplate.Then,thebiosensorwasseeded
perpendicularlytothislineandincubatedfor48hours.ThegrowthoftheC.violaceumstrain
withoutviolaceinproduction(whitecolonies)wasregardedasaquorumquenchingpositive
result,whilethegrowthofpurplecoloniesofC.violaceumortheabsenceofgrowthofthebio-
sensorwereregardedasanegativeeffect.
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 6/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
Marinebacteriastrainscultureandextraction. Isolateswererecoveredfromtheirglyc-
erolstockintoISP-2platesincubatedat28˚C.Strainsweresubsequentlyculturedin100mL
bytriplicateinTrypticaseSoyBroth(TSBBactoTMBD)mediumin500mLconicalflasksby
shakingat130rpmat25˚Cfor2weeks.Cultureswerethencentrifugedandfiltratedby
0.22μm,andtheirspentsupernatantswereextractedthreetimeswithethylacetate(EtOAc).
Organicextractswereconcentratedundervacuumandkeptfrozenuntiltheiruseforbioactiv-
ityscreening(antibacterial,antifungalandquorumquenchingactivity)andforLC-MSanaly-
sis.AnEtOAcextractoftheculturemediaTSBwasusedasblankforbothbioassaysand
chemicalanalysis.
Bioassayusingorganicextracts. Antibacterialactivityoftheextracts:Antibacterial
activityoftheextractsagainstBurkholderiapathogensstrainswasevaluatedbyadiffusiontest
in96-wellmicrotiterplates[49].SinglecoloniesofBurkholderiaspp.weregrowninliquid
mediumKingB(KB)at30˚Cpriortouse.30μLofeachbacterialculturewerethendiluted
into200μLofKBagarmedium,in96wellmicrotiterplates.500μgofeachEtOAcextract
werethensuspendedinto30μLofDMSO5%,andaddedintoeachwell.Plateswerestatically
incubatedfor24hat37˚C,andthegrowthofeachBurkholderiaculturewasmonitoredafter
24hbyvisualinspection;thosewellsshowinginhibitionofBurkholderiagrowthwereconsid-
eredpositiveforthepresenceofantibacterialcompounds.Gentamicin0,2μg/mLwasusedas
positivecontrolforbacterialinhibition,andDMSO5%andTSBwereusedasnegative
controls.
Antifungalactivityoftheextracts:AntifungalactivitywasalsotestedusingEtOAcextracts
obtainedforeachActinobacteria-likeisolateina24-wellmicrotiterplateassay.Briefly,each
fungalstrainwasgrowninPDAplates,andaconidialsuspensionwaspreparedwith0.85%
NaClsolution.Then,thisconidialsuspensionwasusedtoinoculateeachwell,previouslyfilled
with2mLofPDA.Simultaneously,500μgofeachEtOAcextractsweredissolvedin30μL5%
DMSO,andthissolutionwasaddedtothesurfaceofeachwell,previouslyinoculatedwiththe
conidialsuspension.Theplateswerethenincubatedat26˚Cfor96h.Totalorpartialinhibi-
tionofthefungalgrowthwasregardedaspositiveforthepresenceofantifungalcompoundsin
suchanEtOAcextract.Asolutionof1%clotrimazole(5μL/well)wasusedaspositivecontrol,
and5%DMSOandTSBwereusedasnegativecontrols.
Quorumquenchingactivityoftheextracts:Organicextractsofmarinestrainswerealso
testedforthepresenceofquorumquenchingcompounds,byusingadisc-diffusionplateassay
[50].AHL-producingstrainChromobacteriumviolaceumATCC31532wasusedasabiosensor,
and5mmdiameterdiscswereloadedwith300μgofeachEtOAcfraction.Discsweresubse-
quentlyplacedonthesurfaceofagarplatespreviouslyinoculatedwithabacterialsuspension
ofC.violaceumATCC31532(OD 0.5).Plateswereincubatedfor24hat30˚C.Asnega-
600nm
tiveandpositivecontrols,300μLofDMSO(5%v/v)and200μg4-hydroxybenzaldehyde
(PHB)perdiscwereused,respectively.Presenceofquorumquenchingcompoundswasidenti-
fiedbythelackofviolaceinproductionaroundthediscs[51].Incontrast,wheneverzonesof
growthinhibitionwereobserved,thisresultwasreportedasantibacterialextract.
Metabolicprofilinganalysisofmarinestrains
SamplepreparationandHPLC-MSanalysis. Organicextractsforeachisolateand
mediumblankwerepreparedataconcentrationof1mg/mLinMeOH.Liquidchromatography-
massspectrometryexperimentswerecarriedoutinanHPLCLaChrom(VWRHITACHI-E-
LITE)coupledtoESI-ITmassspectrometerAmazonX(Bruker-Daltonics).Thechromatographic
analysisusedaXTerraC-18column(4.6×150mm,5μm)andaflowrateof1.0mL/min.The
mobilephaseconsistedof0.075%formicacid(A)andacetonitrilewith0.075%formicacid(B).
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 7/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
Thesolventprogramstartedwith10%Bfor3minandalinearincreaseto40%Bin17min,fol-
lowedbyalinearincreaseto100%Bin15min,thenfollowedby10minat100%B.Thetotal
analysistimeforeachsamplewas50min.Thesample(20μL)wasinjectedbyanautosampler.
Themassspectrometrywascarriedoutinbothpositiveandnegativeionizationmodeswitha
sprayvoltageat4kVandcapillarytemperatureat250˚C.Themassrangewassetfromm/z70–
3000.RawdatafilesgeneratedbyHPLC-MSwerefirstconvertedtonetCDFfiles.
Dataanalysis. ThenetCDFfileswerethenimportedtoMZmine2.10software[52]for
preprocessingsteps.PeakdetectioninMZminewasexecutedfollowingnoiseremoval,chro-
matogramconstruction,andpeakdeconvolutionsteps[53].First,themassvalueswere
detectedusingthecentroidmodeineachspectrumandthepeaksbelow1×105oftheheight
werediscardedasnoise.Inthesecondstep,chromatogramswereconstructedforeachofthe
massvalues,whichspanoveracertaintimerange.Theminimumtimespanoverthesameion
wassetas1minandtheerroroftheionm/zvaluewasallowedwithin100ppm.Theminimum
intensityofthehighestdatapointinthechromatogramwassetat1×105.Finally,adeconvolu-
tionalgorithmwasappliedtoeachconstructedchromatogramofeachmassiontorecognize
theactualchromatographicpeaks.The“baselinecutoff”algorithm,whichsearchesforlocal
minimainthechromatogramandseparatesindividualpeaksatminimalpointswasused.The
separatedpeakswerethendeisotopedusingthefunctionofisotopicpeaksgrouperinwhich
wesetm/ztoleranceat1m/z;retentiontimetoleranceat1(min);maximumchargeof3;and
representativeisotopebeingthemostintense.Theretentiontimenormalizerwasalsoused
afterdeisotopingtoreduceinter-batchvariationbysettingm/ztoleranceat1m/z;retention
timetoleranceat1absolute(min),andminimumstandardintensityat1.1×105.Selected
MZmineparameterswerechosenaccordingtothecharacteristicsoftheLC-MSsystem(e.g.
resolution,etc)andexperimentalresults(retentiontimeandm/zdeviation).Theremaining
peaksindifferentsampleswerealignedbasedonthemassandretentiontimeofeachpeak.The
ionm/ztoleranceforalignmentwas1.0or100ppm,retentiontimewas1absolute(min),and
weightform/zwas20andRtwas15respectively.Followingalignment,theresultingpeaklist
wasgap-filledwithmissingpeaksusingtheintensitytoleranceof20%andretentiontimetoler-
anceof1min.Thealignedpeaklistwasthenexportedasacommaseparatedvalue(.CSV)file.
DatawereimportedtoSIMCA-P14(Umetrics,Sweden)forfurthermultivariatedataanalysis
[54].BarcodingwasgeneratedinMicrosoftExcelusingtheIFfunction(=IF(cell>0,1,0)).This
presence-absencecreatedabinarydatasetforeachbacterialextractshowingtheoverallchemi-
caldiversity.Valuesof“1”areshownasblack,whilevaluesof“0”arewhite[27].Additionally,
anin-housedevelopedExceltoolwasusedtosortandremovefeatures(pairofm/zratiosand
retentiontimes)associatedwithculturedmediablank(TSB).Dereplicationprocesswasdone
usingAntiMarinnaturalproductsdatabase,andisotopicabundancewasconfirmedin
HPLC-MSrawdata[54].
Resultsanddiscussions
Marinebacteriaisolationandcharacterization
EnvironmentalsamplescollectedfromthecoralreefofProvidencia(Colombia,South-west
CaribbeanSea)werestudiedtorecoverassociatedmicroorganisms.Environmentalsamples
wereprocessedbyusingclassicalenrichmentprotocolsforisolationofculturableActinobac-
teria,suchastheuseofISP-2andoatmealagarculturemedia.[55].Oneweeklater,oncethe
bacterialgrowthwasdetected,bothagarswerefragmentedintopiecesandtransferredtosaline
solutionsupplementedwithCaCO ,inordertoimprovethegrowthofActinobacteriastrains
3
[56].Purificationofbacterialisolateswasperformedbysuccessivesub-cultureofsinglecolo-
niesinCSAmedium,recovering203isolates:162weredescribedasbacteriawhile41asfungi.
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 8/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
InordertoidentifyActinobacteriastrains,thebacteriaweremorphologicallycharacterizedin
fourculturemedia(ISP-2,ISP-3,ISP-4,nutrientagar),andbiochemicaltestsforsugarfermen-
tationandoxidationaswellasforotherenzymeactivitieswerealsoperformed.Determination
ofL,L-diaminopimelicacids(L,L-A pm)aschemotaxonomicmarkerforStreptomyceswas
2
alsocarriedout[57].Acompletedescriptionoftheobtainedresultsforallthesebiochemical
traitsispresentedinTableandFig(S1TableandS1Fig).Morphologicalandbiochemicaldata
showedthat22isolatesbelongtothegenusStreptomycesastheycontainedwhole-organism
hydrolysatesrichinL,L-A pm,andtheyformedextensivelybranchedsubstratemyceliathat
2
carriedabundantaerialhyphaeabletodifferentiateintochainsofspores.Thissetofisolates
wasofparticularinterestbecauseStreptomycetesareknowntobeoneofthemostprolific
sourcesofsecondarymetabolites[58,59].Theothertwoisolates(25and102N)showedto
containmeso-A pm(S2Fig),whichindicatethattheybelongtodifferentgenera,anditneeds
2
the16SrRNAtobeestablished.Overall,resultsofbiochemicalprofilesobtainedbyAPI20E
testinall24strainsexhibiteddifferences,whichcouldindicatethatisolatesarenotredundant
(S1Table).Forexample,althoughstrains9and5showedthesamebiochemicaltestsprofile,
strain9wasconsidereddifferentfrom5sinceitsmorphologyinfourdifferentculturemedia
(ISP-2,ISP-3,ISP-4,nutrientagar)wasdifferentfromthatpresentedbystrains5.Inthesame
sense,itwasfoundthatisolates89.3and89.4aresimilarintheirbiochemicalprofile,andthey
onlydifferintheiroxidasephenotype.Regardingthecatalasetest,allstrainswerecatalaseposi-
tive,exceptthatof161b.AdditionallytheSEMofthestrains(S3Fig)showthespore-surface
morphologyandthecharacteristicgrowthofActinobacteria.Theseproceduresallowedusto
recover24Actinobacteriaisolatesoutofthe162bacterialisolates(c.a.15%)obtainedfrom
marinesources.
16SrRNAanalysespresentedinTable1andFig1(each>1400bpinlength)suggestedthat
all24isolateswerephylogeneticallyaffiliatedtophylaActinobacteria,representingthreedifferent
generanamelyStreptomyces(22strains),Gordonia(onestrain,number25)andMicromonospora
(onestrain,number102N).Theseresultsareinagreementwiththebiochemicalprofilesobtained
fromAPI20-E,whichsuggestthattheirphenotypeisconsistentwiththeir16SrRNAgenotype.
Furtherstudieswillbecarriedoutinordertodeterminetheprecisetaxonomicalidentificationto
thespecieslevel.ThemajorityoftheActinobacteriaarefree-livingorganismsthatarewidelydis-
tributedinbothterrestrialandmarineecosystems,thegeneraofthisphylumexhibitenormous
Fig1.Comparisonofaphylogeneticreconstructionbasedon16SrRNAgenesoftheactinobacterial
isolatesandMALDI-TOFprofilesclustering.ANeighborJoiningphylogenetictreebasedon16SrRNA
genesoftheactinobacterialisolatesandtheirclosestrelatives(cultivableanduncultivable)wasdone(left
side).Bootstrapcalculationwasdone(1000repetitions)andisshowforthemainbranchesinthetree.A
dendrogrambasedonMALDI-TOFprofilesofeachisolate(rightside)wascalculatedtocomparetheobtained
resultsbythesetwomethodologiesofclassification.
doi:10.1371/journal.pone.0170148.g001
PLOSONE|DOI:10.1371/journal.pone.0170148 February22,2017 9/25
MarineActinobacteriaasasourceofcompoundsforphytopathogencontrol
diversityintermsoftheirmorphology,physiology,andmetaboliccapabilities.Theproducing
capacityofindividualActinobacteriacanalsovaryenormously.SomeStreptomycesspeciespro-
duceasingleantibiotic,whileothersproducearangeofdifferentcompoundsandcompound
classes[60].
TheclosestneighborsandsimilaritypercentageforeachstrainarepresentedinTable1.
Thephylogenetictreebasedon16SrRNAlocusforthe24Actinobacteriaisolatespresentedin
Fig1showssevenoperationaltaxonomicalunits(OTUs).AtotalofelevenStreptomyceswas
formingthefirstclade(OTU1),composedmainlyofrelatedstrainsrecoveredfromfivediffer-
entsources(Table1),suggestingtheubiquityofthecluster,whichisnotconsideredasa
redundantparameter.Itsclosestneighborshavebeenrecoveredmainlyfromsoil.OnlyS.albi-
doflavus,recoveredmainlyfromseaandalsosoilexhibitsboth,antifungalandantibacterial
activities[61,62].Twocloseneighbors,S.violacensandS.resistomycifushaveshownantibacte-
rialactivity.SrainsofS.violascenshavebeenreportedtoproduceantibioticssuchasactinomy-
cin[63].TheotherneighborsS.griseochromogensandS.hydrogenanshaveshownantifungal
activity[64,65]
Strains5,143,144,144a,145,148,149and149aweregroupedwithinthesamecluster
(OTU5).TheclosestneighbortothisgroupwasStreptomycessanyensis.Thisstrainhasbeen
recoveredfrommangrovesedimentsandhasthisisitsfirstreportofitsantimicrobialactivity
[66,67].AlltheseeightstrainswererecoveredfromthesamespongeNiphatesdigitalis.How-
ever,severaldifferencesobservedbetweentheirbiochemicalprofilesandtheirmorphologies
indifferentialISPmediastronglyindicatesthattheyshouldbeconsideredasdifferentisolates.
Otherfiveisolatesweregroupedinfivesingleclusters(i)Strain182Streptomycessp.(OTU2),
whichiscloselyrelatedtoStreptomycescanchipurensis[68];(ii)Strain9(OTU3),whichgroups
togetherwithStreptomycespratensis,thisstrainhasbeenrecoveredfromterrestrialenviron-
mentsandtherearenotreportsforitsbiologicalactivity[69];(iii);Strain46b(OTU4),which
iscloselyrelatedtoStreptomycesmicroflavusandStreptomycesfulvorobeus.S.microflavushas
beenreportedfromseaandsoils,whileS.fulvorubeushasbeenreportedonlyfromthesea
[70].Neitherofthesetwostrainshaveantimicrobialactivityreports(iv)Strain102N(OTU6)
filiatedtoMicromonospora sp.astheclosestneighbor;Micromonospora speciesarewidelydis-
tributedinnature,livingindifferentenvironments.Theyhavelongbeenknownasasignifi-
cantsourceofsecondarymetabolitesanditwasrecentlydemonstratedthatMicromonospora
speciesmayalsoinfluenceplantgrowthanddevelopment.Thegenushasalsobeenreportedto
producealargenumberofantibioticsandissecondonlyafterStreptomycesinthisrespect,syn-
thesizingupto500differentmoleculeswithdiversestructuresandproperties.Micromonos-
poraspeciescanalsoproducehydrolyticenzymes,whichallowsthemtoplayanactiverolein
thedegradationoforganicmatterintheirnaturalhabitats[71].and(v)strain25(OTU7)with
Gordoniasp.astheclosestneighbor.MembersofthegenusGordoniawereoriginallyisolated
asopportunisticpathogensinhumans.However,inrecentyears,mostnovelmembersofthe
genushavebeenisolatedfromterrestrialandmarinesources[72]Severalmembersofthis
taxonplayimportantrolesinbiodegradationandbioremediation.Theproductionofsurface-
activecompoundsbyGordoniasp.hasbeenassociatedwiththeirabilitytodegradethehydro-
phobiccompounds[73].Importantly,accordingtothecurrentlimitofcircumscriptionofa
prokaryoticspeciesbasedonthe16SrDNAsequence[74],theisolate25couldbeaputative
newspeciesbecausepercentageof16SrDNAsimilarityagainstitsclosestrelativeisbelow
98.7%.Ingeneral,wedidnotfindanysignificantcorrelationbetweenisolationsourcesand
bacterialspecieshererecovered,whichindicatesthatallsourcesharbordifferentActinobac-
teriaspecies,withoutapparentrecruitmentofaparticularOTUsbyanyspecificsource.
Recently,matrixassistedlaserdesorption/ionizationtimeofflightmassspectrometry
(MALDI-TOFMS)hasbeenusedforbacterialdereplicationandpreliminaryidentificationof
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