Antagonistic activity of fungi of Olea europaea L. against Colletotrichum acutatum

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Contents lists available atScienceDirect

Microbiological

Research

j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / m i c r e s

Antagonistic

activity

of

fungi

of

Olea

europaea

L. against

Colletotrichum

acutatum

Miguel

C. Landum

a

_,

_Maria

_do

_Rosário

_Félix

b

_,

_Joana

_Alho

a

_,

_Raquel

_Garcia

a

_,

Maria

João

Cabrita

b

_,

_Fernando

_Rei

b

_,

_Carla

_M.R.

_Varanda

a,∗

a_Instituto_de_Ciências_Agrárias_e_Ambientais_{Mediterrânicas,}_Instituto_de_Investigaç_ão_e_Formaç_ão_Avanç_ada,_Universidade_de_Évora,_Núcleo_da_Mitra,_Ap. 94,7006-554Évora,Portugal

b_Departamento_de_Fitotecnia,_Escola_de_Ciências_e_Tecnologia,_Instituto_de_Ciências_Agrárias_e_Ambientais_{Mediterrânicas,}_Universidade_de_Évora,_Núcleo daMitra,Ap.94,7006-554Évora,Portugal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received29October2015

Receivedinrevisedform1December2015 Accepted4December2015

Availableonline12December2015 Keywords: Anthracnosis Biocontrol Olive Volatiles

a

b

s

t

r

a

c

t

Funginaturallypresentinolivetreeswereidentiﬁedandtestedfortheirantagonisticpotentialagainst

Colletotrichumacutatum.Atotalof14isolateswereidentiﬁed,12belongedtogeneraAlternaria,

Epicoc-cum,Fusarium,Aspergillus,Anthrinium,Chaetomium,Diaporthe,Nigrospora,onetofamilyXylariaceaeand

onewasunclassiﬁed.AllfungalisolatesshowedsomeinhibitoryactionoverthegrowthofC.acutatum

duringdualculturegrowth,however,whenagar-diffusibletestswereperformedonlyﬁvefungalisolates

causedC.acutatumgrowthinhibition:Alternariasp.isolate2(26.8%),thefungusfromXylariaceaefamily

(14.3%),Alternariasp.isolate1(10.7%);Diaporthesp.(10.7%),Nigrosporaoryzae(3.5%).Volatilesubstances

producedbytheseisolateswereidentiﬁedthroughgas-chromatographytechniques,asphenylethyl

alco-hol,4-methylquinazoline,benzothiazole,benzylalcohol,lilial,galaxolide,amongothers.Theseinhibitory

volatilescouldplayasigniﬁcantroleinreductionofC.acutatumexpansioninoliveandtheirstudyas

potentialbiocontrolagentsshouldbefurtherexplored.

1. Introduction

ColletotrichumisagenusofAscomycotafungithatcontainsome

ofthemostsuccessfulplantpathogenicfungalspeciescausinghigh

economiclossestoawiderangeofwoodyandherbaceouscrops,

especiallyfruits,vegetablesandornamentalsbothintropicaland

temperateregionsworldwide(Baileyetal.,1992;Zivkovicetal.,

2010;Baroncellietal.,2014).

Duetotheirscientiﬁcandeconomicimportance,speciesfrom

thegenusColletotrichumhaverecentlybeenrankedinthetopten

fungalpathogens(Deanetal.,2012).Oneofthemostpathogenic

speciesofthisgenusisColletotrichumacutatumSimmonds,which

causesanthracnose andblightinimportanthosts suchasolive,

almond, peach, citrus, strawberry, among others (Förster and

Adaskaveg,1999;MartínandGarcía-Figueres,1999;Timmerand Brown,2000;Zaitlinetal.,2000;Curryetal.,2002).Colletotrichum

acutatumcanaffectmostpartsoftheplantandsymptomsrange

fromshoot and leaf spots to fruitrot. Symptoms onfruits are

extremelyimportantduetotheeconomiclossestheycause,not

∗ Correspondingauthor.Fax:+351266760822.

E-mailaddress:carlavaranda@uevora.pt(C.M.R.Varanda).

onlyintheﬁeld(pre-harvest)butalsoduringstorage(post-harvest)

(Baileyetal.,1992;Xiaetal.,2011;Menezesetal.,2014;Zhong etal.,2014).

At present most effective control measures against

Col-letotrichum diseases rely on cultural control, using resistant

cultivars,chemicalcontroland/orbiologicalcontrolusing

antago-nisticorganisms.Measuresofculturalcontrolareextremelyhelpful

todecreaseinoculalevelsandtoworsenfungioptimal

develop-mentconditions;however,theyarenotsufﬁcientandtocombatC.

acutatumtheyshouldbeusedaspartofanintegratedcontrol.In

addition,resistant/tolerantvarietiesarenotavailableforallcrops,

costsassociatedwithreplacinganestablishedcropwitha

resis-tantareveryhighandproducersusuallyselectcultivarsbasedon

othercriteriathandiseaseresistance.Duetothis,themostcommon

strategytocontrolColletotrichumistheuseofchemicalfungicides.

Althoughfungicidesreducetheseverityofthedisease,eradication

isdifﬁculttoattainandrepeatedapplicationsareoftennecessary

tomaintainprotection.Inaddition,theneedtorespectfungicide

securityperiodsbeforeharvesting mayresultin an incomplete

protectionofthefruitsinadevelopmentalstageofveryhigh

sus-ceptibilityindiseasesasoliveanthracnose(Cacciolaetal.,2012).

Negativeeffectsoftheuseoffungicidesareevident;theabusein

theemploymentofchemicalcompoundshasfavoredthe

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orationofhumanhealth,environmentalcontaminationandthe

developmentofpathogensresistanttofungicides(Prapagdeeetal.,

2008).

Biologicalcontrolassumesthereforeagreatimportance.Inthe

lastthreedecadestherehasbeenanincreasedinterestintheuse

ofbiologicalagentsforfungalplantpathogenscontroland

numer-ousmicroorganismshavebeenidentiﬁedaspotentialbio-control

agents(Alabouvetteetal.,2006).Planthostshavebeenthebest

placestoobtaingoodantagonistsagainstpathogensattackingthose

hosts.Plantsusuallyhaveseveralfungipresenteitherontheir

sur-faces(epiphytes)orinsidetissues(endophytes)thatdonotcause

anyvisibledamage. Thesefungiarevery diverseand theirrole

lacksstudy.Onthecategoryofpotentialbio-controlagents,

endo-phytic microorganismshave gained a considerableimportance.

Somestudies have shown thatendophytic fungiproduce

com-poundsthatinhibitthegrowthofotherfungibothintheﬁeldandin

storage(MercierandJiménez,2004;Rodriguezetal.,2009;Wang

etal.,2013).

The aim of this study focuses on assessing the ability of

severalfungiisolatedfromolivetoinhibitthegrowthofthe

phy-topathogenicfungusC.acutatumthroughtheirantagonisticactivity

andeffectofsecondarymetabolites.

2. Materialsandmethods

2.1. Isolationofpathogen

Thephytopathogenic fungusC.acutatumbelongs tothe

col-lection of the Mycology Laboratory, Institute of Mediterranean

Agricultural andEnvironmental Sciences(ICAAM), University of

Évora,Portugal.Itwasoriginallyisolatedfromfruitsfromanolive

treecv.GalegavulgargrowinginElvas,Portugal.StocksofC.

acu-tatumisolatesweregrownonpotatodextroseagar(PDA)(Oxoid)

plates,atroomtemperature(25–28◦C)andstoredat4◦Cforlater

use.

2.2. Samplecollectionandisolationoffungalisolates

Samplesofsymptomlessleaveswerecollectedfrom50

asymp-tomaticolivetreesfromcv.Galegavulgarfromanorchardlocated

intheSouthofPortugal(Évora).Tosearchforepiphytesand

endo-phytes,halfof thecollectedleaveswasnot disinfectedandthe

otherhalfwassurfacesterilizedbytreatmentwithethanol70%

(v/v)for2min,3%sodiumhypochloritefor3min,ethanol70%(v/v)

for1min,andaﬁnalwashrepeatedthreetimesinsteriledistilled

waterfor1mineach(Vermaetal.,2007).Treatedanduntreated

leaveswereplacedonPetridishes(9cm)containingPDA.Plates

wereincubatedforsevendaysatroomtemperature(25±3◦C).

Fungiwerefurtherpuriﬁedbytransferringthemyceliafromthe

marginofthegrowingfungalcoloniestoindividualPetridishes

(5cm)containingfreshPDAandincubatedatroomtemperature

(25±3◦C)forsevendays.

2.3. Fungalidentiﬁcation

DNAwasextractedfromfungalculturesgrowinginPDAplates

forsevendays,usingtheDNeasyPlantMiniKit(Qiagen),in

accor-dance to manufacturer’s instructions. The internal transcribed

spacer (ITS) region of nuclear rDNA was PCR ampliﬁed from

genomicDNAbyusingITS1andITS4primers(Whiteetal.,1990).

The PCR reactions consisted of 30–80ng of genomic DNA,

10mMTris–HCl(pH8.6),50mMKCl,1.5mMMgCl2,0.2mMdNTPs

(Fermentas),1_␮Mofeachprimerand2.5UofDreamTaqDNA

poly-merase(Fermentas)inatotalvolumeof50␮L.Ampliﬁcationwas

carriedoutinaThermalCycler(Bio-Rad)at95◦Cfor2minfollowed

by40cyclesof95◦Cfor30s,50◦Cfor50s,and72◦Cfor60sanda

ﬁnalextensionat72◦Cfor10min.

PCRproductswerepuriﬁedusingGFXGelDNAPuriﬁcationKit

(GEHealthcareBiosciences)andsequencedinforwardandreverse

directionsbyMacrogen(TheNetherlands).Sequenceanalysisofthe

ITSsequenceswascarriedoutusingBioEditSequenceAlignment

Editorv.7.2.3(Hall,1999).Thesearchforhomologoussequences

wasdoneusingBasicLocalAlignmentSearchToolsattheNational

CenterforBiotechnologyInformationandonFungalBarcode

web-site(http://www.fungalbarcoding.org).

2.4. Antagonistictests

2.4.1. Directinhibitiontest

Fungalisolatesweretestedinvitrofortheirantagonisticactivity

againstC.acutatumusingthedirectoppositionmethod(Dennisand

Webster,1971).Brieﬂy,a5mmmyceliadiscfromthemarginof

activelygrowingcolonyofC.acutatumwasplacedatabout1cm

fromthewallofa9cmPDAplateandattheoppositeside,asimilar

sizeddiscofthefungalisolatewasplaced.Plateswereincubated

at25±2◦Candthreereplicateswereusedforeachfungustested.

ControltestswerealsocarriedoutusingC.acutatumalone.Forthe

estimationofthegrowthinhibitionpercentage,theradialgrowth

ofC.acutatumwitheachoftheisolatedfungiandcontrolplateswas

measuredwithavernercaliperandrecordedconsecutivelyforﬁve

daysandonthedayseven,tenand15.Theinhibitionpercentage

wascalculatedusingthefollowingformula(RoyseandRies,1977):

I[inhibitionpercentage] =

R1[colonyradiusincontrol]−R2[colonyradiusintest] R1

×100

Theinteractiontypebetweenthetwofungiwasassessedusing

a scale from Ato D(Dharmaputra, 2003; Demici et al., 2012):

A=growthinhibitionofC.acutatumoncontactwithinteracting

fun-gus;B=mutualinterminglingofC.acutatumandinteractingfungus

butbothgrowslowlyandatdifferentrate;C=mutualinhibition

withaspacedistance<0.2cm;D=mutualinhibitionat distance

>0.2cm.

Microscopic observations were made to the margins of C.

acutatumexposedtofungalisolates.Theseweremountedon

micro-scopicslides andstainedwithlactophenolblue.A non-exposed

C.acutatumwasusedascontrol.Slides wereexaminedundera

microscope(OlympusBX41).

2.4.2. Volatilecompoundstest

Toevaluatethepossibleproductionofvolatilecompoundsthat

couldhavesomeactivityonthegrowthofC.acutatum,asimple

trialwasconducted(RahmansyahandRahmansyah,2013).

IsolatedfungiwereincubatedonPDAPetridishes(9cm)for

ﬁvedays.Insmall(5cm)PDAplatesC.acutatumincubatedfor48h.

Afterthoseperiods,thesmallplatescontainingthephytopatogenic

funguswereplacedinvertedontopofeachoftheisolatedfungus.

Thetopwassealedwithparaﬁlmand adhesivetapetoprevent

diffusionofvolatiles.ThegrowthofC.acutatumineachoftheplates

containingadifferentfunguswascomparedwithacontrolplaced

invertedinaplatecontainingonlyPDAmedium.Threereplicates

wereused.

Afterﬁvedaysofincubationat25±3◦C,thediametersofthe

pathogencoloniesweremeasuredandthepercentageofinhibition

wascalculatedusingthesameformulaaspreviouslydescribed.

2.4.3. Non-volatilecompoundstest

Erlenmeyer’sﬂaskseach containing15mlofpotato dextrose

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themarginsofactivelygrowingcoloniesoffungalisolatesand

incu-batedfor 12 daysat 25±2◦C. Afterthat periodsolutions were

ﬁlteredusingﬁlterpapers150mm(Whatman)toremovespores

andotherfungalstructures.

75␮l,100␮land200␮lofthedifferentcultureﬁltrateswere

placedina1cmcircularholemadedirectlyinthemediuminthe

oppositesidewhereC.acutatumwasgrowingfortwodaysina9cm

PDAplate.Threereplicatesforeachﬁlteredsolutionwereusedas

wellasacontrolwheretheholewasﬁlledwith75_␮lofPDBalone.

2.5. Extractionandchromatographicanalysisofvolatiles

Extractionswereperformedasdescribedpreviously(Vilanova

etal.,2012).Brieﬂy,toa10mltube,8mlofPDBwhereineach

fun-gusgrewandamagneticstirbar(22.2mm×4.8mm)wereadded.

3-Octanolwasusedasinternalstandard.Extractionwasdoneby

stirringthesamplewith500␮lofdichloromethane(Merck,

Darm-stadt,Germany) for15min.Aftercoolingat0◦Cfor10min,the

magneticstirbarwasremovedandtheorganicphasewasobtained

Fig.1.Illustrationofsomeofthefungiusedinthedirectinhibitiontestandthedifferenttypeofinteractionsobserved7daysafterinoculation.Topleft:C.acutatum(control plate);topright:interactiontypeCshownbetweenNigrosporaoryzaeandC.acutatum;bottomleft:interactiontypeAshownbyAspergillusnigerandC.acutatum;bottom right:interactiontypeDshownbyAlternariasp.isolate2andC.acutatum.

Fig.2. Colletotrichumacutatumradialmycelialgrowth(cm)overtimeregisteredindirectinhibitiontestsusingfungiisolatedfrom(A)non-disinfectedand(B)disinfected leaves.

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Fig.3. Inhibitionpercentagevaluesobserved7daysafterinoculationofbothfungiinPDAplatesduringdirectinhibitiontest.

Fig.4. Inhibitionpercentagevaluesobserved7daysafterinoculationofbothfungiduringvolatilecompoundtest.

Table1

Classiﬁcation oftheinteraction typebetweenthe antagonisticfungiand Col-letotrichumacutatum.

Fungus Typeofinteraction

Aspergillusniger;unclassiﬁedendophytic fungus

A

Nigrosporaoryzae C

Alternariasp.isolate1and2;Arthriniumsp.;Chaetomium sp.;Diaporthesp.;Epicoccumnigrumisolate1,2and3; Fusariumsp.isolate1and2;Xylariaceae

D

bycentrifugation (2948×g, 5min,4◦C),withthe extractbeing recovered in a vial using a Pasteur pipette. Then thearomatic extractwasdriedwithanhydroussodiumsulfate(Merck, Darm-stadt,Germany)andcollectedagaininanewvial.

AThermoFinniganTraceGCgaschromatograph(Thermo Finni-gan, Austin, TX), equipped with a Thermo Finnigan Polaris Q

mass selectivedetectorwasused. Samples(1_␮l)wereinjected inthesplitlessmodeandvolatileswereseparatedusingafused silica capillarycolumn, ZB-Wax30m, 0.25mm i.dand 0.25␮m ﬁlmthickness (ZebronCapillaryGC column;PhenomenexUSA) under the following column temperature program:40◦C (held 1min)heatedat4◦C/minto100◦Candthenheatedat8◦C/minto 250◦C(heldfor10min);andaRtx-5(Crossbond5%diphenil—95% dimetilpolysiloxane)30m,0.25mmi.dand0.25␮mﬁlmthickness (RESTEK,USA)usingthefollowingcolumntemperatureprogram: 40◦C(held1min)heatedat5◦C/minto250◦C(held5min)and thenheatedat5◦C/minto300◦C(held1min).

Carriergaswasgradeheliumataflowrate(constantflow)of 1.0ml/min;theinjectiontemperaturewas250◦C,insplitlessmode withasplitflowof50ml/min.Detectionwascarriedoutbypositive ionelectronimpact(EI)massspectrometryinthefullscanmode, usinganionizationenergyof70eVandatransferlinetemperature of250◦C.

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Fig.5.Inhibitionpercentagevaluesobserved7daysafterinoculationduringnon-volatilecompoundstest.

Table2

Compoundsidentiﬁedinatleastonesample,andsomecompoundsbelongingtodifferentchemicalfamilies.

Compounds MW MSions N.oryzae Alternariasp.isolate2 Alternariasp.isolate1 Xylariaceae Diaporthesp.

Phenylethylalcohol 122 91,92,122,65 x x – x x 4-Methylquinazoline 144 144,103,117,129 – – – x – Benzothiazole 135 135,108,69,82 – – – x – Benzylalcohol 108 79,108,107,77 – x – – – Lilial 204 189,147,131,117 x x – x x Galaxolide 258 243,213,244,258 x x – x x Chemicalfamilies

Propanoicacidderivatives 71,43,89 x x x x x

Pyrazinecompounds 154,85 x x x x x Benzaldehydederivatives 92 x – – – – Ketonescompounds 55,43,83,111,126 x – – – – Aminesderivatives 72,44 x x – x x Phenol,bis-dimethylethyl 191,57,206 x – – x x Table3

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Themassacquisitionrangewasm/z30–450andthescanning rate3scans−1.Chromatographicpeakswereidentiﬁedby com-paringtheirmassspectrawiththosereportedintheliteratureand incommerciallibrariesNISTMSSearch2.0andWiley7.Thelinear retentionindexvalueswerecalculatedinbothcolumnsdescribed. Foreachcompound,thelinearretentionindexescalculatedwere comparedwiththesereportedbyotherauthorsandNISTlibrary inordertohaveanothertooltohelpontheidentiﬁcationof com-pounds.Allsampleswereanalyzedinduplicate.

2.6. Statisticalanalysis

Analysisof variance (ANOVA) was conductedregarding dif-ferencesamongantagonisticfungiineachperiod,usingtheIBM SPSSstatistical package v.20. Multiplemean comparisonswere madeusingTukeyHSDtestwhenstatisticaldifferenceswerefound betweendatasets(P<0.05).

3. Results

Atotalof14differentfungiisolateswererecoveredfrom disin-fectedandnon-disinfectedleavesof50treesofOleaeuropaeacv. GalegavulgarfromanorchardlocatedinÉvora(Portugal).

Sequencingresultsconﬁrmed thepresence of sevendistinct fungiinnon-disinfectedleaves:Alternariasp.isolate1;Aspergillus niger;Epicoccumnigrumisolate1andisolate2;Fusariumsp.isolate 1;Anthriniumsp.andafungusoftheXylariaceaefamilywhichwas notpossibletodeterminatethespecies;andsevenspeciesin dis-infectedleaves:Alternariasp.isolate2;Chaetomiumsp.;Diaporthe sp.;E.nigrumisolate3;Fusariumsp.isolate2;Nigrosporaoryzaeand anundeterminatedendophyticfungus.

Mostpredominantspecies wereE.nigrum;Alternariasp.and Fusariumsp.,totallingsevenofthe14fungalspeciesdetected.

All14fungalisolateswerescreenedfortheirantagonistic activ-ityagainstthephytopatogenicfungusC.acutatum.

C.acutatumthroughout thedirect inhibition testswas char-acterizedbyacontinuousincreasinggrowth(Table4)exceptin

thepresenceofA.niger,N.oryzaeandtheunclassiﬁedendophytic

fungus(Fig.2AandB).A.niger(Fig.1bottomleft)andthe

unclassi-ﬁedendophyticfungiinhibitedthegrowthofC.acutatumthrough

contact(interaction typeA)whereasN.oryzae(Fig.1 topright)

inhibitedthegrowthofC.acutatumwithaspacedistanceofless

than0.2cm(interactiontypeC)(Table1).Alltheotherfungal

iso-latesshowedsomemutualinhibitionandtheirgrowthpresented

distancesofmorethan0.2cmbetweenantagonistandpathogenic

fungi(interactiontypeD)(Fig.1bottomright).

Theinhibitionpercentagescalculatedforfungalisolatesranged

from11.8%to86.3%(Fig.3),showingthatallfungalisolateshad

someinhibitoryactionoverthegrowthofC.acutatum.Asexpected

fromtheinteractiontypesfound,A.niger,N.oryzaeandthe

unclas-siﬁedendophytewerethosethatdemonstratedgreaterabilityto

inhibitthegrowthofC.acutatuminthedirectinhibitiontest,with

percentagesof86.3%,66.7%and68.6%,respectively.

MicroscopicobservationsoftheinteractionsbetweenC.

acuta-tumandtheantagonisticfungididnotshowanyalterationsinthe

pathogenhyphae(datanotshown).

Allthe14fungalisolatestestedproducedvolatilesthatcaused

someinhibition onthegrowthof C. acutatum.Although

differ-encesbetweenisolateswerenotsigniﬁcant,themosteffectivewere

Alternariasp.isolate2,E.nigrumisolate3andFusariumsp.isolate

2withaninhibitionofgrowthof48%andthelesseffectivewere

Diaporthesp.andN.oryzaepresenting28%(Fig.4;Table5).

Asfortheagardiffusibletests,onlyﬁvefungalisolates,fourof

theseisolatedfromdisinfectedleaves,producedsubstances

capa-ble of diminishingthe radialgrowth of C.acutatum presenting Table

4 Mean (± Std. error) of radial mycelial growth (cm) of Colletotrichum acutatum over time during the direct inhibition test. Day Control Alternaria sp. isolate 1 Alternaria sp. isolate 2 Arthrinium sp. Aspergillus Chaetomium sp. Diaporthe sp. Epicoccum nigrum isolate 1 Epicoccum isolate 2 Epicoccum isolate 3 Fusarium sp. isolate 1 Fusarium sp. isolate 2 Nigrospora

Unclassiﬁed endophytic fungus

Xylariaceae 1 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 2 0.47 ± 0.03 0.37 ± 0.09 0.33 ± 0.07 0.20 ± 0.06 0.20 ± 0.00 0.30 ± 0.00 0.23 ± 0.07 0.23 ± 0.03 0.40 ± 0.00 0.23 ± 0.03 0.33 ± 0.07 0.33 ± 0.07 0.37 ± 0.03 0.33 ± 0.03 0.27 ± 0.03 3 0.53 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.20 ± 0.06 0.23 ± 0.03 0.33 ± 0.07 0.40 ± 0.00 0.33 ± 0.03 0.43 ± 0.03 0.33 ± 0.03 0.47 ± 0.03 0.47 ± 0.03 0.37 ± 0.03 0.37 ± 0.07 0.40 ± 0.06 4 0.57 ± 0.03 0.57 ± 0.03 0.57 ± 0.03 0.20 ± 0.06 0.23 ± 0.03 0.40 ± 0.00 0.37 ± 0.09 0.40 ± 0.00 0.43 ± 0.03 0.33 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.40 ± 0.00 0.37 ± 0.07 0.47 ± 0.03 5 0.67 ± 0.03 0.57 ± 0.03 0.57 ± 0.03 0.43 ± 0.03 0.23 ± 0.03 0.43 ± 0.03 0.50 ± 0.00 0.57 ± 0.03 0.50 ± 0.00 0.50 ± 0.00 0.57 ± 0.03 0.53 ± 0.03 0.47 ± 0.03 0.37 ± 0.07 0.53 ± 0.03 7 1.70 ± 0.00 1.07 ± 0.07 1.23 ± 0.09 1.27 ± 0.09 0.23 ± 0.03 1.33 ± 0.07 1.50 ± 0.06 1.43 ± 0.03 1.30 ± 0.15 1.27 ± 0.03 1.17 ± 0.12 1.43 ± 0.03 0.57 ± 0.03 0.37 ± 0.07 1.13 ± 0.03 10 2,07 ± 0.03 1.63 ± 0.09 1.50 ± 0.15 1.83 ± 0.09 0.23 ± 0.03 1.77 ± 0.18 1.67 ± 0.12 1.90 ± 0.10 1.77 ± 0.09 1.53 ± 0.03 1.90 ± 0.06 2,03 ± 0.07 0.57 ± 0.03 0.53 ± 0.03 1.93 ± 0.12 15 3,97 ± 0.03 1.63 ± 0.09 1.50 ± 0.15 1.83 ± 0.09 0.23 ± 0.03 1.77 ± 0.18 1.67 ± 0.12 1.90 ± 0.10 1.77 ± 0.09 1.53 ± 0.03 1. 90 ± 0.06 2,03 ± 0.07 0.57 ± 0.03 0.53 ± 0.03 1.93 ± 0.12

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Table 5 Mean (± Std. error) of radial mycelial growth (cm) of Colletotrichum acutatum over time during the volatile test. Day Control Alternaria sp. isolate 1 Alternaria sp. isolate 2 Arthrinium sp. Aspergillus niger Chaetomium sp. Diaporthe sp. Epicoccum Epicoccum isolate 2 Epicoccum isolate 3 Fusarium sp. isolate 1 Fusarium sp. isolate 2 Nigrospora oryzae Unclassiﬁed endophytic fungus

Xylariaceae 1 0.50 ± 0.00 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.40 ± 0.00 0.43 ± 0.03 0.43 ± 0.03 0.40 ± 0.00 0.43 ± 0.03 0.50 ± 0.00 0.50 ± 0.00 2 0.63 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.03 0.40 ± 0.00 0.43 ± 0.03 0.43 ± 0.03 0.40 ± 0.00 0.47 ± 0.00 0.50 ± 0.00 0.50 ± 0.00 3 0.67 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.03 0.40 ± 0.00 0.43 ± 0.03 0.43 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.50 ± 0.00 4 0.67 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.03 0.40 ± 0.00 0.43 ± 0.03 0.43 ± 0.03 0.43 ± 0.03 0.50 ± 0.03 0.50 ± 0.00 0.50 ± 0.00 5 0.73 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.50 ± 0.00 0.43 ± 0.03 0.60 ± 0.00 0.50 ± 0.00 0.53 ± 0.03 6 0.80 ± 0.00 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.50 ± 0.00 0.43 ± 0.03 0.60 ± 0.00 0.50 ± 0.00 0.53 ± 0.03 7 0.83 ± 0.03 0.47 ± 0.03 0.43 ± 0.03 0.47 ± 0.03 0.50 ± 0.00 0.47 ± 0.03 0.60 ± 0.00 0.47 ± 0.0.03 0.47 ± 0.03 0.43 ± 0.03 0.50 ± 0.00 0.43 ± 0.03 0.60 ± 0.00 0.50 ± 0.00 0.53 ± 0.03

someinhibitionvalues:Alternariasp.isolate2 (26.8%),the fun-gusfromXylariaceaefamily(14.3%),Alternariasp.isolate1(10.7%); Diaporthesp.(10.7%),N.oryzae(3.5%)(Fig.5).

Althoughonlya fewvolatilecompoundswereidentiﬁed,the

ﬁvesamplesunderstudypresentedsomedifferentcharacteristics

(Table2).Remarkably,thesampleofAlternariasp.isolate1was

thepoorestoneintermsofvolatilecompounds.Somecompounds

seemtobecharacteristicofsomesamples,namelyXylariaceae

(4-methylquinazolineand benzothiazole) andAlternaria sp. isolate

2(benzylalcohol).Phenylethylalcoholispresentinallsamples,

exceptinsampleofAlternariasp.isolate1,althoughindifferent

concentrations.InsamplesDiaporthesp.andN.oryzae,itwasthe

largestpeakwhileinXylariaceaesamplethepeakareaisverysmall.

DespiteAlternariasp.isolate1peakhasasimilarrelativeareatothat

presentedinDiaporthesp.andN.oryzae,thereisahugepeakthat

showsafragmentationpatternthatcanbeascribedasanesterof

anorganicacid,probablycontainingaﬂuoratom.

Pyrazinecompoundsandpropanoicacidderivatives,two

chem-ical families, were detected in all the fungal samples tested

(Table 3). Compoundspresenting fragmentation ionstypical of

pyrazinecompounds,m/z154andm/z85,appearseveraltimesin

thechromatogram,whichseemstoindicatethepresenceofatleast

fourpyrazinederivatives.Regardingpropanoicacidderivativesall

samplesseemstohavetwodifferentcompoundsbelongingtothis

family.

4. Discussion

Plantsare often colonizedby many fungithat do not cause

anydiseasesymptoms.Manyofthesemayhavebeneﬁcialeffects

on plant growth by providing essential nutrients to the plant

(Harrison,2005),indirectlymakingitlesssusceptibletopathogens,

or directly protecting them through an antagonistic effect on

pathogens(Wangetal.,2013).

Inthisstudy,14fungalisolateswereobtainedfrom50olive

trees.Fiveoftheseisolateswereidentiﬁedtospecieslevel,seven

togenuslevel,oneatfamilylevelandonewasunidentiﬁed.Most

predominantfungiwereisolatesofE.nigrum,Alternariasp.and

Fusariumsp.totallinghalfofthefungiisolated.Isolatesbelonging

tothesespecies/generawerefoundinbothdisinfectedand

non-disinfectedleaves.Fungalisolatesobtainedfromdisinfectedleaves

are endophytesand fungal isolates obtainedin non-disinfected

leavesmaybeeitherendophytesorepiphytes.However,allfungal

isolatesobtainedinthisstudyarecommonlyfoundasendophytes

inawiderangeofplantsandnotexclusiveofolive.

Resultsofdirectinhibitiontestsrevealedthatallfungalisolates

inhibitedmycelial growthof C.acutatum.Thehighestpathogen

growthinhibitionpercentages86.3%,66.7%and68.6%,were

pro-ducedbyA.niger,N.oryzaeandanunclassiﬁedendophytefungus,

respectively.

Althoughinitiallyallfungiwereplacedonoppositesidesofthe

Petridishes,fromdaytenforward,antagonistmycelialgrowthled

toadirectcontactofthepathogenwithA.nigerortoadistance

oflessthan0.2cmwithN.oryzaeandtheunidentiﬁedendophyte

fungus.This couldbe anindication of theimportance thatthe

physicalproximitybetweenfungicouldhaveontheirantagonistic

capability,sincecompetitionforspaceandfoodcanbean

impor-tantlimitingfactorfortheestablishmentofanyfungus,factwell

establishedforotherantagonists(Alabouvetteetal.,2006;Howell,

2003).

On the otherhand, thepathogen growth inhibition starting

fromdaytwoinsomeisolates,especiallyAnthriniumsp.,A.niger,

Diaporthe sp.; E. nigrum isolates 1 and 3 and the unidentiﬁed

Xylariaceaemember,wouldsuggestthattheymightreduceC.

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competitionorparasitism.Infact,theantagonisticpropertiesof

microorganismsthatactasbiocontrolagentsarebasedonthe

acti-vation ofmultiple mechanisms.Theycan controlpathogens by

competingfornutrientsandspace,bymycoparasitism,antibiosis

ormetaboliteproduction(HeydariandPessarakli,2010)eitherby

affectingthepathogenofbypromotingplantgrowthanddefence

mechanisms.

All fungal isolates identiﬁed in this study are ascomycetes,

which areknown tobeactiveproducers ofantimicrobial

com-pounds (Zhi-Lin et al., 2012). Some species of Aspergillus and

Fusarium,havealreadybeenproﬁledfortheirorganiccompounds

and some antifungal components were isolated (Morath et al.,

2012; Siddiqueeet al.,2015).Alternaria sp.,A. niger, Epicoccum

sp.and membersof theXylariaceaefamily have alsoshownto

producecompoundsmostlyunidentiﬁed(Newcombeetal.,2009;

Wanietal.,2010;Zhi-Linet al.,2012).Thevolatilecompounds

testshowedthatallisolatesproducedvolatilesthat,atsomelevel,

diminishedthegrowthofC.acutatum.Differencesingrowthwere

signiﬁcantfromdayﬁveonwardsforallfungalisolatesandslightly

moreaccentuatedinAlternariasp.isolate2,E.nigrumisolate3,

Fusariumsp.isolate2(Table5).

Asfortheagardiffusiblecompoundstest,greaterdifferences

wereobserved,fromthe14isolatestested,onlyﬁveisolates

pro-ducedcompoundscapableofinhibitingthegrowthofC.acutatum.

AmongthesearethetwoisolatesofAlternariasp.thatreached

inhi-bitionpercentagesof26.8%and10.7%whenthehighestsolution

volume(200␮l)wasused.Thisobservationisinlinewithrecent

studiesthathighlighttheabilityofsomeAlternariasp.isolatesto

producebioactivemetaboliteswithantifungalactivities(Louetal.,

2013).Otherisolatesthatproducedcompoundscapableof

reduc-ingC.acutatumgrowth,includedDiaporthesp.,N.oryzaeandthe

unidentiﬁedXylariaceae.Thelackofresultsontherestofthe

iso-latestestedmaybeduetolackofproductionofsubstanceswith

antifungalactivityingeneralorinparticularagainstC.acutatum

orininsufﬁcientconcentrationstoproduceapositiveinhibitory

result.Agardiffusiblesubstancesproducedbytheseisolateswere

identiﬁed throughgaschromatography techniques.The highest

varietyofcompounds(nine)wasdetectedinN.oryzaeandinthe

Xylariaceaememberandthelowest(two)inAlternariasp.isolate

1.

Allcompoundsdetectedareknowntohavesomeantifungal

properties(Carter etal.,1958;Morath etal.,2012;Yadavetal.,

2011).Thebenzylalcohol,detectedonlyinAlternariasp.isolate2,

mayberesponsibleforthehighestinhibitionofC.acutatumgrowth

causedbythisisolate,eitherbyitselforinassociationwiththeother

compounds.Thecompounds4-methylquinazolineand

benzothia-zole,onlyfoundintheXylariaceaemember,mayberesponsiblefor

thesecondhighestinhibitionofC.acutatum.

Twofamiliesofcompounds,pyrazineandpropanoicacid

deriva-tives, already known as substances with antifungal properties

(Morathetal.,2012),weredetectedinallthefungitested.

IdenticalcompoundsweredetectedinsamplesfromN.oryzae

andDiaporthesp.withtheexceptionofbenzaldehydederivatives

andketonescompoundsthatwereonlydetectedinN.oryzae.These

benzaldehydederivativesandketonescompoundshoweverdonot

seemtoenhanceN.oryzaeantifungalactivityagainstC.acutatum.

Itisofgreatinteresttotestthesefungalcompounds

individu-ally,namelybenzylalcoholproducedbyAlternariasp.isolate2,or

combinedandathigherdosestodeterminetheirpossibleuseas

controlsubstancesofC.acutatum.

Interestingly, fungi that produced more antagonistic

com-poundsagainstC.acutatumusuallypresentedthelowestinhibition

percentage values in the direct inhibition test, suggesting that,

althoughfungimayhavedifferentcompetitionstrategiestheymay

havea predominant one,which maydependontheconditions

and/orpathogen.Forexample,someisolatesofA.nigerhaveshown

toproduceinhibitorycompoundsagainstavarietyoffungi(

Zhi-Lin etal.,2012),howevertheisolateusedinthis studydidnot

standoutintheproduction ofcompoundscapableofinhibiting

C.acutatumgrowth,andA.nigerwasmoresuccessfulininhibiting

C.acutatumgrowththrougharapidgrowthandcompetitionfor

space/nutrients.

Inagriculture,thereisanincreasedinterestintheuseof

biolog-icalcontrolagents.Manyspeciesoforganismshavebeenstudied

asantagonistsasawiderangeofpathogens,howeverthestudy

ofantagonistsagainstC.acutatum,islimited.Theoutcomeofthis

studyisparticularlyusefulintheidentiﬁcationoflikelyfungal

iso-lates,orsubstances,candidatesforC.acutatumbiocontrolaswell

in understandingthemechanisms theyusetoreducepathogen

growth.

Thisstudyincreasestheknowledgeoffungalcommunitiesand

thesubstancestheyproducethatmaybeusedinbiological

con-trolofC.acutatum,thecausalagentofthemostimportantfungal

diseaseofolivefruitsworldwide,oliveanthracnose.Current

con-trolmeasuresarebasedonpreventiveuseofchemicaltreatments,

selectionofresistantcultivarsandearlyharvesting(Cacciolaetal.,

2012).Thisstudycontributestothedevelopmentand

implementa-tionofenvironmentalfriendlymanagementstrategiesbyreducing

theexcessoffungicideoncropplants,whichisoneofthemain

concernsofpublichealth.

Acknowledgments

This work was supported by Inalentejo

ALENT-07-0324-FEDER-001747, OPERAC¸ÃO: Gestão Integrada da Protec¸ão do

Olival Alentejano. Contributos para o seu desenvolvimento e

implementac¸ão. Carla Marisa R. Varanda is recipient of a PhD

fellowship from Fundac¸ão para a Ciência ea Tecnologia (FCT),

SFRH/BPD/76194/2011, ﬁnanced by QREN – POPH – Typology

4.1—co-ﬁnancedbyMESnationalfundingandtheEuropeanSocial

Fund.ThisworkhasbeensupportedbyFEDERandNationalfunds,

through the Programa Operacional Regional do Alentejo

(InA-lentejo)OperationALENT-07-0262-FEDER-001871/Laboratóriode

BiotecnologiaAplicadaeTecnologiasAgro-Ambientais.

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