Potential for biocontrol of melanized fungi by actinobacteria isolated from intertidal region of Ilha Do Mel, Paraná, Brazil

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

Environmental

Microbiology

Potential

for

biocontrol

of

melanized

fungi

by

actinobacteria

isolated

from

intertidal

region

of

Ilha

Do

Mel,

Paraná,

Brazil

Camila

de

Araújo

Dalitz

,

Mariana

Vieira

Porsani,

Izabel

Cristina

Figel,

Ida

C.

Pimentel,

Patrícia

R.

Dalzoto

UniversidadeFederaldoParaná,DepartamentodePatologiaBásica,Curitiba,PR,Brazil

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t

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f

o

Received27November2014 Accepted20June2016

Availableonline11October2016 AssociateEditor:CarlosPelleschi Taborda Keywords: Actinobacteria Melanizedfungi Antimicrobialactivity 16SrDNA

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b

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t

Actinobacteriaoccurinmanyenvironmentsandhavethecapacitytoproducesecondary metaboliteswithantibioticpotential.Identificationandtaxonomyofactinobacteriathat produceantimicrobialsubstancesisessentialforthescreeningofnewcompounds,and sequencingofthe16SregionofribosomalDNA(rDNA),whichisconservedandpresentinall bacteria,isanimportantmethodofidentification.Melanizedfungiarefree-livingorganisms, whichcanalsobepathogensofclinicalimportance.Thisworkaimedtoevaluategrowth inhibitionofmelanizedfungibyactinobacteriaandtoidentifythelattertothespecieslevel. Inthisstudy,antimicrobialactivityof13actinobacterialisolatesfromthegenusStreptomyces wasevaluatedagainstsevenmelanizedfungiofthegeneraExophiala,Cladosporium,and Rhinocladiella.Inalltests,allactinobacterialisolatesshowedinhibitoryactivityagainstall isolatesofmelanizedfungi,andonlyoneactinobacterialisolatehadlessefficientinhibitory activity.The16SrDNAregionoffivepreviouslyunidentifiedactinobacterialisolatesfromIlha doMel,Paraná,Brazil,wassequenced;fouroftheisolateswereidentifiedasStreptomyces globisporussubsp.globisporus,andoneisolatewasidentifiedasStreptomycesaureus.This workhighlightsthepotentialofactinobacteriawithantifungalactivityandtheirroleinthe pursuitofnovelantimicrobialsubstances.

©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.Thisis anopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/

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

Introduction

Actinobacteria occur in various environments1 _{and have}

the capacity to produce extracellular enzymes and sec-ondary metabolites withantibiotic properties,2 _thus

show-ing significant biotechnological and therapeuticpotential.3

∗ _{Correspondingauthor.}

E-mail:camidalitz@gmail.com(C.A.Dalitz).

Actinobacteria from intertidal regions produce unique metabolitesandcarryoutuniquephysiologicalprocessesdue toextremeenvironmentalconditions,suchassalinity, tem-perature, and humidity.4 _{Actinobacteria from theintertidal}

region of Ilha do Mel, Paraná, Brazil, have already shown promisingresultsininhibitionofpathogenicorganismsand productionofsubstanceswithantimicrobialpotential.5_Other

http://dx.doi.org/10.1016/j.bjm.2016.09.010

1517-8382/©2016SociedadeBrasileiradeMicrobiologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

studieshavefoundantibacterialandantifungalpropertiesin extractsfromactinobacteriaisolatedfromforestsoil,6

high-lightingtheimportanceofstudyingantimicrobialpotentialof secondarymetabolitesproducedbyactinobacteria.

Melanizedfungi, whichare dark-colored organismsdue tothepresenceofmelaninintheircellwall,7_canbe

sapro-trophic or pathogenic to humans, vertebrates, or plants. Diseasescausedbymelanizedfungiareknownas eumyce-toma,chromoblastomycosis,andphaeohyphomycosis.These fungiusuallybelongtothegeneraExophiala,Cladosporium,and Rhinocladiella.Thetreatmentisusuallyclinicalorsurgical,and thedrugsitraconazoleandketoconazolearefrequentlyused totreattheseinfections.8

Identification and taxonomy of actinobacteria are very importantfortheresearchofnewcompounds,providing infor-mationabouttherelationsbetweenorganismsandabouttheir potentialsecondarymetabolites.9_{Animportantmethodfor}

identificationofactinobacteriaisthesequencingofthe16S region oftheir ribosomal DNA (rDNA). Thisregion is con-servedandpresentinallbacteria.10_{Comparisonofsequences}

fromunidentifiedisolateswiththosealreadyknownallows theconstruction ofphylogenetictrees andidentification of organisms.11_{Theaimofthisworkwastoevaluateinhibitory}

activityofactinobacteriaagainstmelanizedfungiandidentify theactinobacterialisolatestothespecieslevel.

Materials

and

methods

Microbialstrains

Inthisstudy,13actinobacterialstrains(Table1)previously iso-latedfrommarinesediments5_{andsevenstrainsofmelanized}

fungi(Table1)previouslyisolatedfromdialysiswaterof vari-ousdialysisunitsinCuritiba,Brazil,12_{wereusedininhibitory}

activitytests.

Allorganisms werestored inthebiologicalcollection of LabMicro,UniversidadeFederaldoParaná,Curitiba,Brazil.

Inhibitiontests

InhibitoryactivityofStreptomycesspp.againstthemelanized fungibelongingtothegeneraExophiala,Rhinocladiella,and Cla-dosporiumwasevaluatedusinginhibitiontests.13

Theinhibitiontestsconsistedofspreadingasaline solu-tionwith3×108_{actinobacterialcellspermilliliter,according}

totheMcFarlandturbidityscale,onSabouraudagarmedium inaPetridishusingaDrigalskispatula.Asmallblockwitha diameterof6mmwasremovedfromthecenterofthedishand replacedwithanotheronecontainingafungalculturegrown for10daysat27◦ConSabouraudagar.Thecontrolconsisted ofaPetridishcontainingthefungalculturealone.Alltests wereperformedintriplicate.

Thegrowthdiameterofthefungalisolateswasmeasured after7and14daysofincubationat27◦ConSabouraudagar. ThegrowthofthefungusinPetridishesthatcontained acti-nobacteriawasthencomparedtothegrowthofthecontrol samplesusingstatisticalanalysis.

The data were transformed using log (x+2) and ana-lyzed using analysis of variance and Tukey’s test at 5%

Table1–Actinobacteriaisolatedfromtheintertidal

regionofIlhaDoMel,Parana,Brazil5_andthefungal

isolatesfromdialysisunits.12

Isolate Molecularidentification Genbank access ADG2712B83 Streptomycesparvus JX997139 ASG315A43 Streptomycesbacillaris JX997140 ADG3211A60 Streptomycesseoulensis JX997141 AD3B17 Streptomyceslongwoodensis JX997148 ASG353A43 Streptomycescavourensis JX997146 AD11B76 Streptomycescavourensis JX997147 AS3A26 Streptomycescavourensis JX997143 ADG3412B82 Streptomycesmalachitospinus JX997142 ADG353A40a _{Streptomycesglobisporus} globisporus KJ155504 ADG353B14a _{Streptomycesglobisporus} globisporus KJ155505 ADG353A29a _{Streptomycesglobisporus} globisporus KJ155506 AD3A26a _{Streptomycesaureus KJ155507} ADG313A69a _{Streptomycesglobisporus} globisporus KJ155508 03/830-09A3 Cladophialophorachaetospira

Cladosporiumsp.

JN650527 09/833-09B3 Exophialapisciphila JN650528 20/832-09B2 Exophialapisciphila JN650529 40/952-09B3 Exophialapisciphila JN650530 53/960-09E2 Exophialapisciphila JN650532 160/137-10D2 Exophialapisciphila Rhinocladiellasimilis JN650534 168/226-10A2 Pseudocladosporiumsp. Exophialapisciphila JN650535

a _{-Strainsthathavebeenidentifiedinthepresentwork.}

probability. In addition, a factorial experiment (1×1) was performed.Allstatisticaltestswereperformedusingthe ASSI-STAT7.6software.14

16SrDNAsequencing

All organisms havebeen previouslyidentified morphologi-cally asbelonging tothe genusStreptomyces,and eightout ofthe13strainstestedhavebeenpreviouslyidentifiedusing molecularmethods(Table1).5_{Theremainingstrains,ADG35}

3A29,ADG353A40,ADG353B14,AD3A26,andADG31 13A69,wereidentifiedinthisworkusing16SrDNA sequenc-ing. Actinobacterial isolates were grown for three days at 27◦C in Czapek–Dox medium. DNA was extracted as pre-viously described16 _{and amplifiedusing the primers 9F (5}

GAGTTTGATCCTGGCTCAG 3)and Sm5R (5 GAACTGAGAC-CGGCTTTTTGA 3). DenaturationofDNA wasperformedat 95◦C for 5min, followed by 30 cycles of45s at94◦C, 45s at65◦C,and1minat72◦C,andafinalextensionof10min at 72◦C.15 _{Polymerase chain reaction products were}

puri-fied and sequenced using an ABI 3130sequencer (Applied Biosystems).16

Thesequenceswerethen analyzedusingthe Staden1.6 software17 _{andaligned usingtheMEGA 4.0software.}18 _The

sequences were then compared to those deposited to the NationalCenterforBiotechnologyInformationdatabaseusing theBLASTalgorithm.19

Isolate AD G35 3A 29 Isolate AD G35 3A 40 Isolate AD G35 3B 14 Isolate AD G31 13A 69

Isolate AD 3A 26

Streptomyces globisporus globisporus H83-3 Streptomyces globisporus globisporus 1366 9A Streptomyces globisporus globisporus NBRC12208 (T) Streptomyces globisporus globisporus 13638A

Streptomyces badius NRRL B-2567 (T) Streptomyces globisporus strain FX-1

Streptomyces badius OACT41O Streptomyces bacillaris strain BZ10-33 Streptomyces bacillaris S4BW2 Streptomyces bacillaris CFCC3101 Streptomyces bacillaris CFCC3171 Streptomyces bacillaris NBRC13487 (T) Streptomyces kanamyceticus NBRC100912 (T) Streptomyces kanamyceticus KCC S-0433 Streptomyces aureus NBRC100912 (T) Streptomyces olivochromogenes NBRC3178 (T) Streptomyces olivochromogenes NBRC13067 Streptomyces olivochromogenes NBRC3561 Streptomyces olivochromogenes GMC105 Streptomyces aureus 173978 Streptomyces aureus 174462 Streptomyces aureus 173412 Streptomyces aureus 173414 Streptomyces aureus 173862 Microbiospora mesophila jCM3151 (T) 001 99 78 46 92 100 71 97 92 95 47 88 100 95

Fig.1–Dendrogramofthesequencesofthe16SregionoftherDNAofStreptomycessp.comparedtootherstrainsfrom GenBank.(T)indicatestypestrains.

Results

and

discussion

Inhibitiontests

The13 actinobacterialisolateswerescreenedforinhibitory activityagainstthesevenisolatesofmelanizedfungi(Table1). In all tests, statistically significant inhibitory activity was observed,andtheF-valuewasalsohighlysignificant(p<0.01). To identify the actinobacterial isolate with the great-est inhibition potential, a factorial experiment (1×1) was performed. The F-value was highly significant (p<0.01), demonstratingasimilarinhibitionpotentialof12outofthe13 isolatestested.Theonlyisolatethatshowedalowerinhibitory efficiencywasADG3113A69(Streptomycesglobisporussubsp. globisporus). Fungal strains 03/830-09A3 (Cladosporium sp.) and 40/952-09B3 (Exophiala pisciphila) were more efficiently inhibitedbytheactinobacteriaassayedthantheotherfungal isolates.

The 13 actinobacterial isolates tested in the present work had already shown inhibitory activity against other pathogenic microorganisms in a previous study.5 _Among

the 116 actinobacteria isolated by the author from inter-tidal regions of Ilha do Mel, Paraná, Brazil, 68% showed activityagainstthe pathogenicstrainsStaphylococcus aureus ATCC25923,CandidaalbicansATCC10231,EscherichiacoliATCC

25922, and Pseudomonas aeruginosa ATCC 27853.This study highlighted the inhibitory activity of these actinobacteria and theirpotentialtoproduceantimicrobialandantifungal compounds.

Other authors have also demonstrated inhibitory activ-ity of actinobacteria from the genus Streptomyces against other microorganisms. Strains of Streptomyces spp. were tested againstthe phytopathogenicfungiPhytophthora para-sitica, Guignardia citricarpa, Rhizoctonia solani, Colletotrichum sublineolum, Pythium sp., and Fusarium oxysporum. Most of theactinobacterialstrainsshowedinhibitoryactivityagainst the fungi. This activity was often species-related,showing the importanceofidentifyingorganismswithantimicrobial potential.Theresultsofthisstudyhavealsorevealeda corre-lationoffungalinhibitionwithchitinolyticactivity,indicating aroleofcompoundsproducedbyactinobacteriain antimicro-bialactivity.20

Inanotherstudy,inhibitoryactivityhasalsobeendetected in actinobacterial isolates from the genera Streptomyces, Nocardia,Kitasatospora,Amycolatopsis,Rhodococcus,and Gordo-nia against Bacillus subtilis, E. coli, C. albicans, Xanthomonas campestris,andMucorracemosus.Theresultsofthisstudy indi-catedtheinhibitionpotentialofactinobacteriafromthegenus Streptomycesagainstdiversemicroorganisms.21

Theseresultshighlighttherelevanceofactinobacteriato thescreeningfornewcompoundswithantimicrobialactivity

andtheirpotentialtoproduceantimicrobialand antifungal compounds.

16SrDNAsequencing

The16SrDNAsequenceswereeditedusingtheStaden soft-ware,andtheBLASTalgorithm(http://blast.ncbi.nlm.nih.gov) wasusedtocomparethesequenceswithothersequences pre-viouslydepositedinthe GenBankdatabase.Thesequences werealignedtothoseoftypestrainsusingtheMEGA5.2 soft-ware.Followingthealignment,adendrogramwasconstructed usingtheneighbor-joiningmethodwith1000bootstrap repli-cates,theTamurathree-parametermodel,andtheGamma(G) distribution(Fig.1)inthesamesoftware.

16SrDNAoftheactinobacterialisolatesADG353A29,AD G353A40,ADG353B14,AD3A26,andADG3113A69was sequenced.FouroftheseisolateswereidentifiedasS. globis-porussubsp.globisporus,andonewasidentifiedasS.aureus.

OtherauthorshavepreviouslyisolatedS.globisporusfrom diverseenvironments,suchassoil22_{andmangroves.}23

S.globisporus has been isolated from soilsamples from China, and its secondary metabolites were isolated and evaluated. Compound C-1027 was tested for antimicrobial andantitumoractivitiesandshowedantimicrobialactivities againstmostgram-positivebacteria;however,noactivitywas observedagainstMycobacteriumsp.orgram-negativebacteria. Thiscompound wasalsotested againstKBcarcinomacells invitro,andapotentcytotoxicityeffectwasobserved,aswell astheabilitytoinhibittransplantabletumorsinmice.22

S.aureushasbeenfoundinThailandsoils21_andmangroves

inIndia.23_{TheantifungalactivityofS.aureushasbeentested}

againstthephytopathogenicfungusValsapaulowniae,24 _and

inhibitionofthefungalgrowthwasobserved.

Besides antimicrobial activity, S. aureus can also pro-duce metabolites able to degrade pollutants. Strain HP-S-01 was isolated from activated sludge; its capability of degrading soil pollutants was measured, and its metabo-lite 3-phenoxybenzaldehyde was analyzed. The results showed that 47.9% of ␤-cypermethrin and 67.0% of 3-phenoxybenzaldehyde were degraded in the absence of additionalcarbonsources.Thestudysuggestedthatthe HP-S-01strainofS.aureuscanbeusedforbioremediation;however, more research is needed before its application at a large scale.25

Theresultsofthisstudyledustoconcludethatthe iso-lates ofStreptomyces spp. from Ilha do Mel,Paraná, Brazil, have a statistically significant potential to inhibit growth ofmelanizedfungifromthegeneraExophiala,Cladosporium, andRhinocladiella.Amongthefivepreviouslyunidentified iso-latestestedinthe present study,fourwere identifiedas S. globisporus subsp. globisporus, and one was identified as S. aureus.Furtherresearchisnecessarytobetterunderstandthis inhibitionpotentialandits mechanisms.Theseresultsalso highlightedtheimportanceofactinobacteriainthepursuitof newcompoundswithantimicrobialpotential.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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