The potential of compounds isolated from Xylaria spp. as antifungal agents against anthracnose

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

Biotechnology

and

Industrial

Microbiology

The

potential

of

compounds

isolated

from

Xylaria

spp.

as

antifungal

agents

against

anthracnose

Luciana

M.

Elias

_,

_Diana

_Fortkamp

_,

_Sérgio

_B.

_Sartori

_,

_Marília

_C.

_Ferreira

_,

Luiz

H.

Gomes

_,

_João

_L.

_Azevedo

_,

_Quimi

_V.

_Montoya

_,

_André

_Rodrigues

_,

Antonio

G.

Ferreira

_,

_Simone

_P.

_Lira

a_{UniversidadedeSãoPaulo,EscolaSuperiordeAgricultura“LuizdeQueiroz”,DepartamentodeCiênciasExatas,Piracicaba,SP,Brazil} b_{UniversidadedeSãoPaulo,EscolaSuperiordeAgricultura“LuizdeQueiroz”,DepartamentodeGenética,Piracicaba,SP,Brazil} c_{UniversidadeEstadualPaulista“JúliodeMesquitaFilho”,InstitutodeBiociências,DepartamentodeBioquímicaeMicrobiologia,Rio}

Claro,SP,Brazil

d_{UniversidadeFederaldeSãoCarlos,DepartamentodeQuímica,SãoCarlos,SP,Brazil}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received6March2017 Accepted9March2018 Availableonline31March2018 AssociateEditor:LuisHenrique Guimarães Keywords: 2-Hexylidene-3-methylbutanedioic acid Amazon Anthracnose Plantpathogen

a

b

s

t

r

a

c

t

AnthracnoseisacropdiseaseusuallycausedbyfungiinthegenusColletotrichumor Gloeospo-rium.Theseareconsideredoneofthemainpathogens,causingsignificanteconomiclosses, suchasinpeppersandguarana.Thecurrentformsofcontrolincludetheuseofresistant cultivars,sanitarypruningandfungicides.However,evenwiththeuseofsomemethods ofcontrollingthesecultures,thecropsarenotfreeofanthracnose.Additionally,excessive applicationoffungicidesincreasestheresistanceofpathogenstoagrochemicalsandcause harmtohumanhealth andtheenvironment.Inordertofindnaturalantifungalagents againstguaranaanthracnose,endophyticfungiwereisolatedfromAmazonguarana.The compoundspiliformicacidandcytochalasinDwereisolatedbychromatographictechniques fromtwoXylariaspp.,guidedbyassayswithColletotrichumgloeosporioides.Theisolated com-poundswereidentifiedbyspectrometrictechniques,asNMRandmassspectrometry.This isthefirstreportthatpiliformicacidandcytochalasinDhaveantifungalactivityagainst C.gloeosporioideswithMIC2.92and2.46␮molmL−1respectively.Captanand difenocona-zolewereincludedaspositivecontrols(MIC16.63and0.02␮molmL−1_{,respectively).Thus,}

Xylariaspeciespresentedabiotechnologicalpotentialandproductionofdifferentactive compoundswhichmightbepromisingagainstanthracnosedisease.

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

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

∗ _{Correspondingauthor.}

E-mail:splira@usp.br(S.P.Lira).

https://doi.org/10.1016/j.bjm.2018.03.003

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

Introduction

Anthracnoseisoneofthemostseriousplantdiseases affect-ing different cultures worldwide. This disease is usually caused byfilamentous fungi in the genus Colletotrichumor Gloeosporium, and these pathogens cause great losses and hence reduction in the quality and quantity of fruits and vegetables.1–4_{Anthracnoseisconsideredthemaindiseaseof}

guarana(Paullinia cupana ‘sorbilis’ Mart., Sapindaceae). The pathogeninfectsleavesandstems,causingdeformationand necroticlesions.5,6 _{Thecurrentformsofcontrolincludethe}

useofresistant cultivars,sanitarypruningand fungicides.7

However, even with the use ofsome methods of control-ling these cultures, they are not are free of anthracnose. Additionally,excessiveapplicationoffungicidesincreasesthe resistanceofpathogenstoagrochemicalsandcauseharmto humanhealthandtheenvironment.8

Therefore,thesearchfornaturalproductswithantifungal activityofagronomicrelevanceisanimportantalternativefor thecontrolofdiseasessuchasanthracnose.9_{Inthesearchfor}

bioactivecompounds,onlyasmallfractionoftheestimated fungalbiodiversity hasbeenchemicallyinvestigated,10 _and

onlyasmallnumberofthesehavealreadybeenculturedand selectedfordrugproduction.11,12_{Fungihavespecialinterest}

becausetheyareknowntoproduceseveralsecondary metabo-liteswithawiderangeofapplications.13_{Thefungiofthegenus}

Xylariastandoutintheproductionofsecondarymetabolites, mainlyofthe classofcytochalasins,isocumarins,lactones, sesquiterpenos,triterpenosandxanthones,andwiththemost diversebiologicalactivities,suchasanticancer,antioxidant, antimicrobial, antiinflammatory, antiviral, inhibiting HIV-1 protease,antibioticandantitumor,14,15amongothers.

Inapreviousstudy,ascreeningwasdonewiththefractions of16endophyticfungiisolatedfromGuaranaplantfrom Ama-zonia,andtwoXylariaspp.wereselectedbecauseitsactivity againstthephytopathogenColletotrichumgloeosporioides.Thus, thisresearchaimedtoevaluatetheantifungalpotentialof sec-ondarymetabolitesproducedbythistwoendophyticXylaria spp.isolatedfromguarana(P.cupana).

Experimental

Generalprocedures

Optical rotations were taken on a Polartronic H Schmidt & Haensch digital polarimeter (1dm cell) at23◦C. The1_H

nuclearmagneticresonance(NMR)wasrecordedonaBruker 14.1Tesla, AVANCE III model spectrometer (operating at 600.13MHz for hydrogen frequency) with crioprobeTM_{. The}

chemicalshiftsaregivenonaı(ppm)scaleandreferenced totetramethylsilane(TMS).High-resolutionelectrospray ion-izationmassspectrometry(HR-ESIMS)wasacquiredbydirect infusiononaQ-TOFMaxisImpactinthefollowingconditions: capillaryvoltage, 4.50kV; operating inelectrospraypositive mode;Theionsourcewassettoanebulizerpressureof0.3bar, adrygasflowrateof4Lmin−1,andadrytemperatureof180◦C; detectionrange: 100–700Dawithtotal ioncountextracting acquisition.Data acquisitionwasperformedusingaBruker CompassDataAnalysis4.2.

High-performance liquid chromatography (HPLC) was conducted using a Agilent 1100Series UV/Viswith a qua-ternary pump, coupled to a UV detector MWD (Multiple Wavelength Detector), using a reversed phase column C18

(250mm×4.60mm, 5␮m; Phenomenex Kinetex), with a mobile phase H2O/MeOH (80:20, v/v), flow rate 1mLmin−1

and =254nm. HPLC-grade solvents were utilized. Solid-phase extractionwas carriedout usingsilica gelcartridges ofdifferentdimensions(Phenomenex).Gelpermeation chro-matography was performed in a glass column filled with Sephadex LH-20(Pharmacia). Silica gel254 (Macherey-Nagel)

were usedforthin-layerchromatography(TLC).Spotswere detectedunderUVlight(254and365nm).

Fungalisolation

Theendophyticfungi(isolates249and214)wereisolatedfrom leaves ofguaranaplant(P.cupana)withanthracnose symp-toms(necroticlesions),collectedatManaus-AM(03◦225),at the Experimental Farm of the Federal University of Ama-zonas (UFAM), and Maués-AM (57◦420), at Fazenda Santa Helena (Ambev– AmericanBeverageCompany), in Novem-ber 2010.The collected sampleswere leavesand branches ofatotal of20adultplants(10ofeach locality).Complete intactleaveswererinsedwithrunningwaterandthenwith distilledwater.Thecleanedleavesweresterilizedby consecu-tivewashesin75%EtOH(1min),3%NaOCl(3min)and75% EtOH (30s),and theywere rinsedwithsteriledistilled H2O

twotimes.16,17_{Thesterilizedmaterialwascutinto8×12mm}

and 3–4 pieces were deposited on a Petri dish containing potatodextroseagar(PDA,KasviTM_{,Brazil),chloramphenicol}

and streptomycin(100␮gmL−1 ofeach) topreventbacterial growth. Plates were incubated at 27◦C. Petri dishes were observeddaily,andtheindividualhyphatipsoftheemerging colonieswerere-inoculatedinnewPDAplatesuntilpure cul-tureswereobtained.Pureculturesoftheendophytes(isolates 249and214)werepreservedbytheCastellanimethod.18

Fungalidentification

Purecultureofisolates249and214werefirstgrowninmalt agar 2%(in gL−1: 20 malt extract, and 15 agar, Acumedia, NeogenTM_)at25◦_{Cforsevendays.Then,freshmyceliawere}

harvested from and usedforDNA extractionbased on the CTAB method (cetyltrimethylammonium bromide)usedin Montoyaetal.19_{WeamplifiedtheITSbarcodingregionusing}

ITS4andITS5primers.Ampliconswerecleanedupand sub-mittedtocycle-sequencingreactionsusingBigDyeTerminator v.3.1Kit(ThermoFischerScientific).Bidirectionalsequences weregeneratedinABI3530(ThermoFischerScientific)using thesameprimerpair.

Forwardandreversesequenceswereassembledincontigs inBioedit.20_{Then,contigswerecomparedwithhomologous}

sequencesdepositedintheNCBI-Genbank.

To refinethe taxonomicassignment ofisolates249 and 214,wecarriedoutaphylogeneticanalysisinMEGAv.6.0.21

For this analysis, weretrieved sequences from closet rela-tivetaxadepositedinthedatabase.Sequenceswerealigned in MAFFT,22 _{and this dataset was used to infer a}

the Kimura 2-parameters as the nucleotide substitution model.Branchsupportwascalculatedusing1000bootstrap pseudoreplicates.

Isolationoftheactivecompounds

Theendophyticfungi249and214wereseparatelyfermented in malt extract broth (in gL−1: 20 malt extract, KasviTM_,

Brazil) at27◦C under agitation (150rpm) forfive days (5L; 20mL×250mLbatchesin500mLErlenmeyerflasks,foreach fungalstrain).Themyceliumwasseparatedfromtheliquid extractusingvacuumfiltration(filterMachereyNagelTM_,MN

640m,7cm)andtheliquidextractwasextractedthreetimes withEtOAc(100mLoftheliquidextractto150mLofEtOAc). Evaporationofthesolventfromtheextractinvacuogavean EtOAcextract(306.4mgforthestrain249and700.0mgforthe strain214).

The EtOAc extract from the strain 249 was chro-matographedonaSep-Paksilicagelcolumn(5g,WatersTM_).

ThesamplewassolubilizedinamixtureofhexaneandCH2Cl2

(100␮L)andappliedtothetopofthecolumn,whichwas elu-tingsuccessivelyhexane/CH2Cl2/MeOH,startingwith100mL

of100%hexaneandincreasingthegradientofCH2Cl2 until

100%CH2Cl2,andthenincreasingthegradientofMeOHuntil

100%. Based on the TLC monitoring, all the fractions col-lected were combined insixmainfractions (a–f).Bioactive fractiond(221.4mg)wassubjectedtofurtherCCfractionation oversilicagel(5g)elutedwithhexane/CH2Cl2/MeOHtogive

fivefractions(d1–d5).Bioactivefractiond1(105mg)was frac-tionedbygel filtrationoverSephadexLH-20 columneluted with100% MeOH to givefive fractions(d1a–d1e), collected basedon the retention time.Bioactive fractiond1b (44mg) waspurifiedonreverse phaseHPLCusing anisocratic elu-tion H2O/MeOH(80:20) during25minto givefour fractions

(d1b1–d1b4),beingtheactived1b4(retentiontime:11.22min) with6.8mg.

The EtOAc extract from the strain 214 was chro-matographedonaSep-Paksilicagelcolumn(10g,Waters®) elutingsuccessivelyhexane/CH2Cl2/EtOAc/MeOHtogivesix

fractions: 50mL hexane: 50mLCH2Cl2 (fraction a), 100mL

CH2Cl2 (fractionb), 50mLCH2Cl2: 50mLEtOAc (fractionc),

100mLEtOAc(fractiond),50mLEtOAc:50mLMeOH(fraction e)and100mLMeOH(fractionf).Bioactivefractiond(375mg), wassubjectedtofurtherCCfractionationoversilicagel(5g) thesamewaydescribedabovetogivesixfractions(d1–d6). Bioactive fraction d3 (50mL CH2Cl2: 50mL EtOAc, 171mg)

waspurifiedonreversephaseHPLCduring15minusingan isocratic elution H2O/MeOH (80:20) to give three fractions

(d3a–d3c),beingtheactived3cwithretentiontime5.92min (9.6mg).

Antifungalbioassays

Disk-diffusionassay

Thedisk-diffusionassay(paperdiskmethod)23_wasusedto

determinetheantifungalactivityduringtheseparationand purificationprocedures.Eachfractionobtainedfromdifferent chromatographswassolubilizedinMeOHandapplied(10␮L) to6mmdiametersterilepaperdisks(with2mgofeachEtOAc extract,1mgofeachfractionor0.5mgofeachpurecompound)

and placedat oneedgeofthe 60mm PDAPetri dish plate (KasviTM_{,Brazil).Aninoculumofthepathogen(a7mm}

diam-eter disc removedfrom a newpeaked plate)was addedto anotheredgeofthePDAplate.Theclearinhibitionzonesof mycelialgrowtharoundthepaperdisksweremeasuredafter incubationfor7daysat27◦C.Afterthisperiod,theinhibition percentageswere calculatedcomparedtothecontrolusing theanalysisprogramImageJ(ImageProcessingandAnalysisin Java).Thisexperimentwasconductedwithonerepeat.Forthe negativecontrol,10␮LofMeOHwasappliedinasterilepaper disk.

In order to know the fungicidalor fungistaticactionof the isolated compounds, asmall part of the funginext to the paper disk with each compound was transferred to a newplate.Theactionwascharacterizedbyfungicideifthe fungus does not grow in the new plate, and fungistatic if growth. Colletotrichumgloeosporioidesusedinthis study was cededbytheUFAM,Amazonas-AM, Brazil.This phytopato-genicfungalstrainwasisolatedfromGuaranaleavesinfected withanthracnoseandisdepositedinthe Centralde Recur-sosMicrobianosdaUNESP(CRM-UNESP)underthecodeCRM 1352.

Microdilutionassays

Broth microdilution techniques were performed in 96-well microplatesaccordingtotheguidelinesoftheNational Com-mitteeforClinicalLaboratoryStandards24withmodifications.For the assay,compoundtestwells(CTWs)were preparedwith stock solutionsofeach compound inDMSO (250mgmL−1), dilutedwithmalt2%medium(ingL−1:20maltextract,Kasvi, Brazil) tofinalof250, 125, 62.5,31.25,15.6,7.81,3.90,1.95, 0.77,0.48and0.24␮gperwell.AninoculumsuspensionofC. gloeosporioidessporeswasobtainedfroma14days-oldculture andwasaddedintoliquidmalt2%medium(105_sporesmL−1_,

60␮L) toeach well (final volumein the well=100␮L, com-pletedwithliquidmalt 2%medium).Spores wereobtained through the fungalculture onsolid malt2%mediumafter 14 days. The plate was scraped,and steriledistilled water was added. This solution was filteredin sterileglass wool toseparatethespores,andthe countingofthe sporeswas doneinNeubauerchamber.Agrowthcontrolwell(containing medium,inoculum,andthehighestconcentrationofDMSO usedinaCTW,butcompound-free)wasincluded.Microplates wereincubatedinB.O.D.at28◦CandwerereadinaTECAN microplatereader,SUNRISEmodel,operatedbythesoftware Magellan v7.1, at 620nm, in intervals of 12h, for 144h. The active ingredientsof commercial fungicides CaptanTM

(N-(trichloromethylthio)ciclohex-4-ene-1,2-dicarboximide) andScoreTM_{(difenoconazole,}

cis-trans-3-chloro-4-[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl 4-chlorophenyl ether) were used as positivecontrol in the same concentrations oftheisolated compounds. TheMICs were determined spectrophotometrically after the above mentioned incubation periods, according to the NCCLS guidelines,24 _{withmodifications.For commercialfungicides}

and compound, the MICs were determined as the lowest concentrationshowingabsenceorequaltotheinitialgrowth compared with the growth (until 12h) in the drug-free well.

Xylaria cubensis BCC 1144 Xylaria cubensis BCC 1219 Xylaria cubensis BCC 1027 Xylaria cubensis BCC 18872 Xylaria cubensis BCC 1303 Xylaria cubensis FL1034 Xylaria cubensis FL0621 Xylaria cubensis FL1044 Xylaria cubensis FL1386 Xylaria hypoxylon CBS 590.72 Xylaria hypoxylon CBS 868.72

Xylaria castorea ATCC 76020 Xylaria longipes SFC 960725-6 Xylaria longipes CBS 148.73 Xylaria enteroleuca CBS 651.89 Xylaria fioriana CBS 486.61 Xylaria cornudamae CBS 724.69 Xylaria apiculata CBS 365.81 Xylaria mali CBS 385.35 Xylaria arbuscula CBS 454.63 Xylaria arbuscula CBS 452.63

Hypoxylon fragiforme YMJ 383 Hypoxylon fragiforme olrim 777

Nectria cinnabarina CBS 279.48 Daldinia eschscholzii AB284189

Daldinia concentrica CBS 139.73 Daldinia vernicosa CBS 157.32 Diatrype disciformis D8M

Xylaria acute ATCC 56487 Xylaria sp. 214 Xylaria sp. 249 0.05 96 100 100 100 93 76 91 51 59 66 79 64 100 67 99 82 100 99 66

Fig.1–PhylogenetictreeofXylariaspp.249and214.

Results

Isolationandidentificationoftheendophytes

Thestrains249and214wereisolatedfromtheguaranaleaves intheAmazonintwodifferentplaces.Afterpurificationofthe strainsandbasedonthephylogeneticanalysis,thesequences ofisolates249and214clusteredwithsequencesfromother Xylariaspecieswithhighbootstrapsupport.Thus,herewe ten-tativelyidentifiedthesefungiasXylariasp.(Fig.1).Thefungal strainsaredepositedattheCentraldeRecursosMicrobianos daUNESP(CRM-UNESP)undercodesCRM1915(Xylariasp.214) andCRM1916(Xylariasp.249).

Bioguidedisolationoftheactivecompound

TheEtOAcextract(306mg)fromthestrain249showed60.39% inhibitiontoC.gloeosporioides.Thisextractwasfractionated bychromatographyonasilica-gelcolumn,permeation chro-matographyonSephadexLH-20gelandreversephaseHPLC. The active compound showed 51.33% activity (code d1b4, 6.8mg).

TheEtOAc extract (700mg) from the strain 214 showed 30.76%inhibitiontoC.gloeosporioides.Thisextractwas frac-tionated by chromatography on a silica-gel column and

reversephaseHPLC.Theactivecompound(coded3c,9.6mg) showed38.76%inhibitiontoC.gloeosporioides.

Thesecompoundswereidentifiedbyanalysisof spectro-scopicdata(MSandNMR)andcomparedwithcompoundsin theDictionaryofNaturalProductsdatabase.27

Identificationofthebioactivecompounds

Compoundd1b4

Thecompoundd1b4(6.8mg)presentsanUVmaxat227.2nm.

Inthehigh-resolutionmassspectrum,molecularionswere observedatm/z215.1268[M+H]+_{,m/z237.1103[M+Na]}+ _and

m/z451.2304[2M+Na]+_{,whichwasappropriateforthe}

molec-ularformulaC11H18O4(214.1189Da).Withthedataobtainedin

the1_{HNMRspectrum,itwaspossibletocorrelatethe}

hydro-gensshifts(ıH)ofd1b4withthecompoundpiliformicacid.26,27

Theexperimentofopticalrotationprovided[˛]D23−0.0375

(c,1mgmL−1inMeOH),allowingidentifyitas(−)-piliformic acid. Piliformic acid (Fig. 2-A), exists in levo, dextro and racemicform,althoughtheabsolutestereochemistryhasnot beendetermined.27

Compoundd3c

Thecompound d3c presents an UVmax at217 and 286nm.

Fig.2–Structuresofthepiliformicacid(C11H18O4)(A)andcytochalasinD(C30H37NO6)(B)producedbyXylariasp.249and

Xylariasp.214,respectively,isolatedfromguaranaplant. 1,4 1,2 1 0,8 0,6 0,4 0,2 0 1,4 1,2 1 0,8 0,6 0,4 0,2 0 0 12 24 36 48 60 72 84 96 108120132144 0 12 24 36 48 60 72 84 96 108120 132 144 Hours Hours Absorbance 620 nm Absorbance 620 nm Piliformic acid [umol mL-1] Cytochalasin D [umol mL-1] 5.83 2.46 1.23 0.30 0.076 0.015 Contorol 2.91 1.45 0.18 0.035 Control

A

B

Fig.3–Dataobtainedinmicrodilutionassayoftheisolatedcompoundpiliformicacid(A)andcytochalasinD(B)against sporesofC.gloeosporioides.

observed atm/z 508.268 [M+H]+_{, m/z 530.251 [M+Na]}+_{, m/z}

1037.513 [2M+Na]+_{, m/z 430.236 [M-OCOCH}

3 H2O]+, m/z

1015.5310[2M+H]+_{,whichwasappropriateforthemolecular}

formulaC30H37NO6(507.260Da).Withthedataobtainedinthe 1_{HNMRspectrum,itwaspossibletocorrelatethehydrogens}

shifts(ıH)ofd3cwiththecompoundcytochalasinD.28

Theexperimentofopticalrotationprovided[␣]D230.0192(c,

1mgmL−1inMeOH),consistentwiththeliterature,allowing identifyitas(+)-cytochalasinD(Fig.2-B).

Evaluationoftheactivecompounds

Theisolatedcompoundspiliformicacid andcytochalasinD havefungistaticactivityagainstC.gloeosporioides.Piliformic acidinhibited51.33%ofthemycelialgrowthofC. gloeospori-oidesbythediskdiffusionmethod,andcytochalasinD38.76%, bothinaconcentrationof500␮gdisk−1.Inthemicrodilution assay(Fig.3)allthecompoundsshowedantifungalactivity. Captananddifenoconazolewereincludedaspositivecontrols (MICof16.63and0.02␮molmL−1,respectively).Piliformicacid andcytochalasinDwereactiveagainstC.gloeosporioides,with aMICof2.92and 2.46␮molmL−1,respectively. Bothhavea higherMICthantheactiveingredientdifenoconazole,buta lowerMICthancaptan.Commercialfungicideswerechosen because theyare from different chemical classes and pre-senteddistinctmechanismsofaction.

Discussion

The piliformic acid (CAS 98985-75-2), also known as 2-hexylidene-3-methylbutanedioic acid,

2-cyclohexylidene-3-methylsuccinic acid or 3-Nonene-2,3-dicarboxylic acid25

belongstothechemicalclassofthepolyketides.29_The

biosyn-thesisofpiliformicacidhasbeenstudied.Themetabolitecan beretro-biosyntheticallycleavedtogenerateaC8andaC3 moiety, and the C8 unitis deriveddirectly from octanoate thatoriginatesfromafattyacidsynthase(FAS)ratherthan fromapolyketidesynthase(PKS).26_{Thesynthesiswasalready}

studiedbyMangaleswaranandArgade,27_{anditwasaneasy,}

four-step.

Piliformicacidwas identifiedthefirsttimebyAnderson etal.30_{asametaboliteproducedbyfungiofthefamily}

Xylari-aceae:Hypoxylon(H.deustum),Poronia(P.piliformis)andXylaria (X. polymorpha, X.longipes, X.Maliand X.hypoxylon).26 _This

compound iscommonlyfoundinendophyticfungi belong-ingtotheXylariaceae,especiallyinthegenusXylaria.Itwas alreadyisolatedfrom themarine-derivedfungusXylariasp. PSU-F10031_{andalsofromanendophyticXylariasp.ofPinus}

strobus.32_{Thismetaboliteisalsoproducedbyothersfungi,as}

several endophyticfungiisolated from themangroveplant Avicennia marina33 _{and from an endophytic Aspergillus}

alla-habadiiBCC4533534_amongothers.

The cytochalasin D belongs to the chemical class of the N-containing compounds and was first reported by AldridgeandTurner,35_{isolatedfromthefungusMetarhizium}

anisopliae. Subsequently this compound was isolated from other fungi, such as Hypoxylon terricola36 _{and Engleromyces}

goetzii.37

Thereareover60knowncytochalasins,wichwereisolated fromdifferentgeneraoffungi,suchasPhomasp.,Daldiniasp. Chalarasp.,Hypoxylonspp.,amongothers.Theproductionof cytochalasinDhasbeenreportedbyfungiinthegenusXylaria, as X. hypoxylon,15 X. mellisii,38 Xylaria sp.,39 X. carpophila,40

X.cubensis,41_X.arbuscula42_{andXylariasp.NC1214,afungal}

endophyteofthemossHypnumsp.43

Inourwork,wediscoveredthefungistaticactivityof pil-iformicacidandcytochalasinDagainsttheplantpathogen C.gloeosporioides.Studiessuggestthatfungistaticagentsare unabletoinhibitingthe morphogenetictransformationand aremorelikelytoblockgrowthbybudding.44

Inliterature,fewbiologicalactivitieshavebeenrelatedfor thepiliformicacid.Thiscompoundhasmoderate cytotoxic-ityagainstcancercellsKBeBC-145_{andantifungalactivityin}

yeastgeneraNadsonia,Nematospora, Rhodotorulaand Saccha-romycestoaconcentration>100mgL−1intestsperformedby thediskdiffusionmethod.46_{Piliformicacidalsoshowedmild}

antibacterial activity against standard Staphylococcus aureus ATCC 25923 and methicillin-resistant strain.47 _The

activ-ity againstthe plantpathogens Cladosporium cladosporioides andC.sphaerospermuminbioautographyassay(with25and 10␮gmL−1)wasalsodescribed.48

Thebiologicalactivitiesofcytochalasinsincludeglucose transport, inhibition ofthe actin polymerization,secretion ofhormones,immunosuppressiveactivity,antibiotic, antivi-ral, herbicidal and antifungal activity.49–53 _{The activity of}

cytochalasinDagainstfivetumorcelllineshavebeentested, and the IC50 ranged from 0.22 to 1.44␮M.43 In agronomy,

asan antifungalagent,cytochalasin Dhas activity against theplantpathogenBotrytis cinerea,thecausalagentofgray moldonplants,ataconcentrationof25␮gdisc−1,inadisk diffusion method bioassay.49 _{The cytochalasin D was also}

testedagainst C. cladosporioidesand C. sphaerospermum,the causalagentsofverrucoseinplants,bythebybioautography method.Itinhibitedtheplantpathogensinaconcentration of10 and 25␮gmL−1, respectively.51 _{Inour study,}

cytocha-lasin D was active against C. gloeosporioides with a MIC of 2.46␮molmL−1.Intheliterature,thereareotherscompounds withactivityagainstthisplantpathogen,aspreussomerinEG1 and its derivatives monoacetylpreussomerin and diacetyl-preussomerinEG1,withMICat100␮gmL−1(0.28␮molmL−1) by dilution in agar (radial growth-inhibitory activity).54 _In

anotherstudy,thecompoundscyclotryprostatinAand trypto-quivalineOweretestedagainstC.gloeosporioidesby microdilu-tiontestandshowedMICof50␮gmL−1(0.12␮molmL−1)and 100␮gmL−1 (0.18␮molmL−1),respectively.55 _Thecompound

dehydrozaluzaninexhibitedactivityat30mM.56_Theisolated

compoundspiliformicacidandcytochalasinDhaveMIC val-uesof2.92and2.46␮molmL−1.

Themechanismofactionofcytochalasin Dinvolves its bindingcapacity to actin filaments and therefore prevents polymerizationandelongation.Thereforeaffectcell morphol-ogyandinhibitcellularprocessessuchascelldivision,which becomessomecasesapoptosis.57 Moreover,cytochalasinD inhibitsproteinsynthesis.58

Theisolatedcompoundsacidpiliformicandcytochalasin Dwe are founded topresent promising antifungalactivity againstC.gloeosporioides(fromguaranaplant),whichcauses theanthracnosedisease.Thesecompoundswereproducedby endophyticXylariaspp.(fromManausandMaués),inthe Ama-zoniaForest.Thus,itwaspossibletoverifythatbothXylaria speciespresentedabiotechnologicalpotentialandproduction ofdifferentactivecompounds.Thecompoundsisolatedinthe presentworkareherereportedforthefirsttimeas

antifun-galagentsagainstC.gloeosporioides,arelevantplantpathogen worldwide.However,toxicitytestsandinvivobioassaysneed tobeperformed.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

FinancialsupportwasprovidedbyFAPESPgrant(2014/15760-3) andBIOTA/BIOprospecTAFAPESPgrant(2013/50228-8). Schol-arshipstoL.M.Elias.(FAPESP:2012/02000-5),M.C.Ferreira. (FAPESP:2014/05940-4),fromCNPq(142079/2016-2)andCAPES arealsogratefullyacknowledged.WethankUFAM,T.E. Bez-erra,Dr.R.G.deS.Berlinck,Dr.M.W.PaixãoandDr.N.S.M. Junior.

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