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BASE Biotechnol. Agron. Soc. Environ.201216(3),325-336

DevelopmentandapplicationofamicroplatemethodtoevaluatetheefficacyofessentialoilsagainstPenicillium italicumWehmer,Penicillium digitatumSacc.andColletotrichum musea(Berk.&M.A.Curtis)Arx,threepostharvestfungalpathogensoffruitsHuguesSosthèneKouassi(1,2),MohammedBajji(3),YvesBrostaux(4),AbdesselamZhiri(5),AbdoulayeSamb(2),PhilippeLepoivre(1),MohammedHaïssamJijakli(1)(1)Univ.Liege-GemblouxAgro-BioTech.PlantPathologyUnit.PassagedesDéportés,2.B-5030Gembloux(Belgium).E-mail:[email protected](2)CheikhAntaDiopUniversity.FacultyofScienceandTechnology.LaboratoryofChemistryandBiochemistryofNaturalProducts.BP5005.Dakar-Fann(Senegal).(3)WalloonAgriculturalResearchCentre(CRA-W).LifeSciencesDepartment.BioengineeringUnit.ChausséedeCharleroi,234.B-5030Gembloux(Belgium).(4)Univ.Liege-GemblouxAgro-BioTech.AppliedStatistics,ComputerScienceandMathematics.PassagedesDéportés,2.B-5030Gembloux(Belgium).(5)PranarômInternationalS.A.AvenuedesArtisans,37.B-7822Ghislenghien(Belgium).

ReceivedonMarch5,2010;acceptedonAugust24,2010.

Amicrobioassaywasdevelopedforevaluatingthein vitroantifungalactivityof30preselectedessentialoils.Atemplatebasedon10serialdilutionswitheight replicatesperdilutionarrangedon two96-wellELISAplateswasusedasa reproducibleand standardized design to identify the in vitro effectiveness of these essential oils againstPenicillium italicumWehmer,Penicillium digitatumSacc.andColletotrichum musea (Berk.&M.A.Curtis)Arx, threepostharvestfungalpathogens,onfruits.Growthofmyceliumwasmonitoredbymeasuringopticaldensity(492nm).Cinnamomumzeylanicum,Cinnamomum verumandEugenia caryophylluswerefoundtobestillactiveagainstallthethreepathogensevenat100ppm.Comparedtoothermethods,thismicrobioassayprovedtobearapid,reproducible,andefficientmethodfortestingtheefficacyofessentialoilsthatinhibitsporegerminationinP. italicum,P. digitatumandC. musea.Theassayrequiresrelativelysmallamountsofessentialoils.Keywords.Essentialoils,antifungalproperties,fungaldiseases,postharvestdiseases,postharvestdecay,moulds,bioassays,in vitro.

Développement et application d’une méthode de microplaques pour évaluer l’efficacité d’huiles essentielles contre Penicillium italicum Wehmer, Penicillium digitatum Sacc. et Colletotrichum musea (Berk. & M.A. Curtis) Arx, trois champignons pathogènes de post-récolte sur fruits.Untestmicrobiologiqueaétédéveloppépourl’évaluationin vitrodel’activitéantifongiquede30huilesessentiellesprésélectionnées.Unmodèle,reproductibleetstandardisé,basésur10sériesdedilutionsavechuitrépétitionsparsérierepartiessurdeuxplaquesELISAde96puitschacune,aétéutilisépouridentifierl’efficacité de ces huiles essentielles contrePenicillium italicumWehmer,Penicillium digitatum Sacc. etColletotrichum musea(Berk.&M.A.Curtis)Arx,troischampignonspathogènesdepost-récoltesurfruits.Lacroissancemycélienneaétésuivie enmesurant ladensitéoptiqueà492nm.Cinnamomum zeylanicum,Cinnamomum verum etEugenia caryophyllusétaientencoreactivescontrelestroispathogènes,mêmeà100ppm.Comparéauxautresméthodes,cetestmicrobiologiquedéveloppé s’est avéréêtreuneméthode rapide, reproductible et efficacepour tester l’efficacitédeshuiles essentiellesquiinhibentlagerminationdessporesdeP. italicum,P. digitatumetC. musea.Cetestnécessiterelativementunefaiblequantitéd’huileessentiellepourlesessais.Mots-clés. Huile essentielle, propriété antifongique, maladie fongique, maladie postrécolte, moisissure, test biologique,in vitro.

326 Biotechnol. Agron. Soc. Environ. 201216(3),325-336 KouassiH.S.,BajjiM.,BrostauxY.etal.

1. INTRODUCTION

The banana and citrus fruit sector is still facingseriouseconomiclossesdueinparttopreharvestandpostharvestfungaldiseases.Inthepostharvestcontext,anthracnose(causedbyColletotrichum musea[Berk.&M.A.Curtis]Arx)isthemostcommonandimportantdiseaseofbanana(Muirheadetal.,2000),whilegreenmould (caused byPenicillium digitatum Sacc.) andbluemould(causedbyPenicillium italicumWehmer)arethemostcommonlyencountereddiseasesincitrus(Timmeretal.,2003).

Tocopewiththesediseases,fungicidaltreatmenthas long been used. Currently, this approach to thetreatment of fungal diseases is being challengedbecauseof:– thepresenceandpersistenceofsignificantlevelsof fungicidal residues in fruits and vegetables (Dogheimetal.,2002;Blascoetal.,2006);– chemical fungicide toxicity and the consequent risks to consumer health and to the environment (Unnikrishnanetal.,2002);– development of resistant pathogen populations (deLapeyredeBellaireetal.,1997;Ghosophaetal., 2007);– limitationson thenumberof chemical fungicides used for postharvest application (Aubertot et al., 2005).

Therefore, the fruit sector urgently needs todevelop alternative postharvest treatments that areacceptable to consumers, and thathave anegligiblerisk to human health and the environment (i.e.the so-called “natural” fungicides). Given theirtraditionally known antifungal activities, essentialoils (liquid or vapor) have been reported to inhibitpostharvest fungal growth both in vitro and in vivo(Bakkalietal.,2008).Todate,noparticularresistanceto essential oils has been reported and the risk ofdevelopmentofresistantracesoffungiisnegligible.Thismay be due to themixture of oil componentswith apparently different modes of action affectingseveraltargetsatthesametime.Moreover,essentialoils arebiodegradable,non-persistent andnon-toxicforhumanconsumptionatlowdoses(Isman,2000).Fortheabove-mentionedreasons,essentialoilsmighteffectively replace chemical fungicides for somespecificapplications.

Despite progress in this domain, biofungicidesbasedonessentialoilsarenotyetwellestablishedinthemarket and remain largely awish rather than arealisticalternative.Severalfactorsexplainthelargedisparity that persists between the accessibility ofsuch biofungicides and the disavowal of chemicalfungicides. Some of the most commonly citeddisadvantagesofusingbiofungicidesareasfollows:

– difficulties involved in setting up commercial production;– problemswithformulation;– doubtsovertheireffectiveness;– difficultyinobtainingresearchfunding;– costofproduction;– the necessity of registering essential oil-based biofungicides.

In order that such a biocontrol strategy forpostharvest treatment becomes acceptable, it seemsimportant to adopt an initiative to increase thechances of success in marketing biofungicides. TherecommendationsprovidedbyCosetal.(2006)forthescreeningofnaturalproductseffectiveagainsthumandiseasescouldbeadaptedtothesearchforantifungalessentialoilsforuseagainstplantdiseases.Inordertobeeffective,suchanapproachwouldneedtotakeintoaccounttheselectionofappropriateessentialoilsandtheuseofanappropriatebioassay.

Essentialoilsmustbeselectedaccordingtocriteriathat will promote their practical use. These criteriamay include toxicity, cost, yield and availability oftheessentialoils.Regardingtheappropriatebioassay,Datry et al. (1995) have critically reviewedmethodsusedfortestingtheefficacyofantifungalcompounds.Most of these are labor-intensive, time-consuming,expensiveandoftenunreliable.Forexample,methods(Disk-diffusion,E-test)usingasolidculturemedium(PDA,water-agar) on Petri dishes are unsuitable forscreening a large number of essential oils becausethey are time-consuming. Moreover, the MinimumInhibitory Concentration (MIC) obtained by thesemethods is higher than that obtained using a liquidculturemedium(Fernández-Torresetal.,2002).Thiscanbeexplainedbythefactthatantifungalsubstances(especiallyessentialoils)diffuselessinasolidthaninaliquidmediumandthesurfaceofcontactbetweenthemicroorganismandactivesubstancesisgreaterintheliquid than in the solidmedium.The poor solubilityofessentialoils insolidmediacanalsoexplain theirhighMICsintheseconditions.Furthermore,methodsfor determining theMIC of essential oils, i.e. usingthe counting of spore germination under an opticalor binocular microscope, measuring the length ofmycelialgrowthorcolonydiameterofthefungus,andestimatingthevisualturbidityofthesolution,aremorecumbersome and less accurate than those using theestimationofthefungusbiomassbyspectrophotometry(Banerjeeetal.,1993;Arthington-Skaggsetal.,2002).Fortheabove-mentionedreasons,themicrotiterELISAmicroplatemethod, which has proved useful for thepharmaceutical industry, is more appropriate for therapidscreeningofessentialoils.Usingsuchamethod,Wilson et al. (1997) andKuhajek et al. (2003) havedevelopedoperatingconditions for rapidquantitative

Antifungalactivityofessentialoilsagainstpostharvestfungi 327

andqualitativemicroscalebioassaysforthediscoveryofnovel compounds for the controlofPhytophthoraspp. and Botrytis cinerea, respectively. The in vitroassessment ofP. italicum,P. digitatum andC. museapresents a challenge because these microorganismsmay differentially be sensitive to the operatingconditionsdefinedbytheseauthors.Thedevelopmentofanefficientandreproduciblemethodforthein vitroevaluation of essential oils is thus needed for eachstudied fungus.The specific aims of this studyweretherefore:– to standardize the in vitro growth of P. italicum, P. digitatumandC. museaonELISAmicroplates;– toselectinterestingessentialoilssuitableforusein biofungicides;– toevaluate theirantifungal in vitroactivityagainst thethreepostharvestphytopathogens.

2. MATERIALS AND METHODS

2.1. Fungal strains and production of conidia

Penicillium italicum (strain PIRBM1), P. digitatum(strainPDRBM1)andC. musea(strainCO-CMR-65)wereobtainedfromthePlantPathologyUnitcollection(GemblouxAgro-BioTech,Belgium).ThePenicilliumstrains were isolated from citrus in Morocco (ENAMeknes) and the C. musea strain from bananas inCameroon (CARBAP). All fungal species weremaintained in 25% glycerol at -80°C. For conidiaproduction, small agar blocks containing myceliumwere cut from the fungus culture and transferred toa fresh Potato Dextrose Agar (PDA) medium forPenicillium and to a modified Mathur’s medium(MM) forC. musea.Replicate plateswere incubatedindarknessat23°Ctoallowsporangiumproduction.

2.2. Inoculum preparation

Theinoculumwaspreparedunderasepticconditions.Tenml of 0.05%Tween 20 in sterile distilled water(SDW) were added to a Petri dish on a 14-day-oldfungalcolony.Conidiaweredislodgedbyrubbingthesurfaceareawithaglassrod.Thewashingswerethenfilteredthroughadoublesterilizedlayeroffineclothtoremovemycelialfragments.Thenumberofconidiawas determinedwith a hemocytometer (Bürker) andadjustedtothedesiredconidiaconcentration.

2.3. Culture media preparation

Concentrated orange (Penicillium) or banana(C. musea) juices, previously filtered through amembranefiltertoremoveparticlesthatcouldinterferewith photometric evaluation of the fungus growth,

wereautoclavedat110°Cfor1handthendilutedwithSDWtoobtainthedesiredjuiceconcentration.

2.4. ELISA microplates, sealant and microplate photometer

ELISAmicroplates(96well)withflat-bottom/standard(Nunc MicroWell [untreated]; Greiner Bio-One)were used to estimate the in vitro growth of fungalstrains. These microplates are well suited for theirmicroscopic applications aswell as for the precisionoftheiropticalmeasurements(flatbottomofthewells,100%transmittanceat≥490nm).Themicroplatesaremanufactured in a polystyrene that is characterizedby its high clarity and resistance to many standardlaboratorychemicals.

Asealant(EASYsealTM2)coatedwithanacrylateadhesivewaschosentosealthemicroplates.Itisnon-cytotoxic,easytouseandcharacterizedbyitscapacityto protect against evaporation and contamination.Above330nm,thepercentageoflightabsorbedbythesealant isalmostnegligibleand thusappearssuitableforopticalmeasurements.

The 96-well microplate reader (MultiskanRC, Labystems; Thermo Scientific) was used as aphotometer,which is speciallyused forELISA.ThisdeviceiscontrolledbyPCsoftwareandcanshaketheplate to homogenate the solution before reading thewholeplateinonly5seconds.

The microplates, the sealant and the photometerwerecarefullyselectedinordertominimizetheeffectofthematerialonfungalgrowth.

2.5. Essential oils

Thirtyessentialoilswereselectedaccordingtointrinsic(yield,toxicity)andextrinsic(availability,cost)criteria(PranarômInternationalS.A,unpublisheddata); theirclassification according to their main components isshown in table 1.TheseessentialoilswereprovidedbyPranarômS.A.(essentialoilsproductioncompany).The oilswere previously diluted inmethanol beforetestingtheirantifungalactivity.

2.6. Experiment 1: in vitro growth of fungal strains

This experimentwas carried out in order to identifyappropriate conditions for optimal fungal growth onELISA microplates. To this end, the in vitro growthof each fungal strain was monitored using threeconidiaconcentrations(104,105and106conidia.ml-1)and three culture media, corresponding to differentdilutionsoforangeorbanana juice (3x 10-2,3x 10-3and 3x 10-4v/v). The effect of each combinationon the growth of each pathogen was assessed usingdifferentnumbers(3 to16)ofreplicates inavolume


of 200µl using sealed 96-well ELISA microplates.Given the number of replicates, all replicates ofthe same combination were pipetted into wellsof three 96-well ELISA microplates, following amodification of the procedure reported byCos et al.(2006).The control for eachgroupof replicateswasa well containing non-infected medium. Myceliumgrowthwasmonitored photometrically at 492nmby

measuring the optical density (OD) of eachwell for196h (at 2-h intervals during the first 12h and thenat 24-h intervals) of incubation at 23°C in the dark.Before each measurement, microplates were shakenfor10s.ODduetopathogengrowthwasobtainedbysubtracting the OD values of inoculated wells fromthose of uninoculatedwells from the same group ofrepetitions.

Table 1.Essentialoilstested—Les huiles essentielles testées.Essential oil * Distilled plant part Main constituents of essential oils

(terpenes and aromatic compounds)Citrus limonum Zest Non-oxygenatedcompoundsCitrus sinensis ZestMyristica fragrans FruitZinziber officinalis RhizomeMentha piperita Aerialpart AlcoholsMelaleuca alternifolia LeafOriganum majorana FloweringtopOrmenis mixta LeafCymbopogon martinii AerialpartMonarda fistulosa AerialpartCymbopogon nardus AerialpartThymus satureioides FloweringtopCymbopogon citratus Aerialpart AldehydesCinnamomum zeylanicum BarkCinnamomum verum FE BarkMentha pulegium Aerialpart KetonesMentha spicata FloweringtopThymus vulgaris Floweringtop PhenolsTrachyspermum ammi FruitEugenia caryophyllus BudCoridothymus capitatus FloweringtopOriganum compactum FloweringtopOriganum heracleoticum FloweringtopLavandula angustifilia Floweringtop EstersPimpinella anisum Fruit EtheroxidesFoeniculum vulgare AerialpartOcimum basilicum FloweringtopEucalyptus globulus LeafCinnamosma fragrans LeafMelaleuca cajuputii Leaf*Essentialoilsinboldwerethemosteffectiveoilsagainstthethreepathogensat1,000ppm—Les huiles essentielles en gras sont les huiles les plus efficaces contre les trois pathogènes à 1 000 ppm.


2.7. Experiment 2: evaluation of the effect of methanol on the in vitro growth of fungal strains

This second experiment was conducted in order toidentifythehighestmethanolconcentrationthatcouldbe appliedwithout a significant effect on thegrowthof the three studied fungi. Seven different methanolconcentrations (0, 0.5, 1, 5, 10, 17 and 33%, v/v)were tested in a volume of 200µl of diluted fruitjuice (3x 10-2, v/v) containing the fungal inoculum(104conidia.ml-1) and using eight replicates permethanolconcentrationandperfungus.Growth(ODat490nm)wasassessedusingsealedmicroplatesat24-hintervalsduring192hofincubationat23°C.Inorderto control the heterogeneity within and between theplatesobservedinExperiment1,ablockedrandomizeddesignwasused(Figure 1).

2.8. Experiment 3: screening of essential oils

The present experiment was carried out to selectinterestingessentialoilsthatcouldbeusedasabiologicalcontrol for postharvest diseases (anthracnose, greenandbluemoulds) inbananaandcitrus fruits.To thisend, the antifungal activity of selected essential oilswasevaluatedusingELISAmicroplateswithablockedrandomized design, as described previously. Thegrowthofeachpathogenwasmonitoredinavolumeof200µlcontainingdilutedfruitjuice(3x 10-2,v/v),theinoculum(104conidia.ml-1),methanol(0.5%,v/v)andtheessentialoil(1,000;500and100ppm).Incubation

and OD measurements were carried out as for thesecondexperiment.

3. STATISTICAL ANALYSIS

StatisticalanalyseswereconductedwiththeRstatisticalsoftware2.7.1.(RDevelopmentCoreTeam,2008).


Theoptimalnumberofreplicateswasfixedusingthestandarderrorofthemeanasaselectioncriterion.Inperfectspatialhomogeneitystate,thestandarderrorofthemeanisrepresentedinformula(1):

(1)

whereσmeansstandarddeviationandnisthenumberofobservations.

Theaverage standard errorof themeanwas thencalculated for each conidia concentration and fruitjuicedilutioncombinationandforeachreplicatesize.The selected number of replicateswas theminimumnumber of replicates for which an increase gave nofurther significant improvement to the standard errorofthemean.

Theobservedvaluesofthestandarderrorforeachreplicate size were also compared to the theoreticalstandard error, calculated on all the replicates fora combination of conidia concentration and juicedilution with formula (1). Dissimilarity of the twovaluescangiveaninsightintotheheterogeneityoftheexperimentalspace(ELISAplates).

The effects of conidia concentration and juicedilution on fungal growth were analyzed using atwo-wayANOVAforeachfungus,using theaverageobservedODattributedtothefungusat168-192hasa response,with thenumberof replicates selected inthefirststep.AmultiplecomparisonofthemeanswasconductedusingtheTukeytest.


Theeffectofmethanolconcentrationonfungalgrowthwasanalyzedusingatwo-waymixedmodelforeachfungus (methanol concentrations and blocks), usingthe average observedOD attributed to the fungus at72-96h (active growth) and 168-192h (plateau) asa response.Amultiplecomparisonof themeanswasconductedbyacontrasttest,taking0%methanolasareference(Dunnett’stest).

σ-=σx

xn

Figure 1.Templateforin vitroscreeningofessentialoilsonElisaplate.Templatefor tenessentialoils(EO)witheightreplicates shared on two Elisa plates —Modèle pour la sélection invitrod’huiles essentielles (HE) sur plaque Elisa. Modèle pour dix huiles essentielles avec huit répétitions partagées sur deux plaques Elisa.Xn:solutionwithEOwithpathogen—solution HE avec pathogène ;Tn:solutionwithEOwithoutpathogen—solution HE sans pathogène;X’:solutionwithoutEOwithpathogen—solution sans HE avec pathogène;T’:solutionwithoutEOwithoutpathogen—solution sans HE sans agent pathogène;n:numberofEO—nombre d’HE=1,2…10.

Block 1Plate 1 Plate 2

Block 2 Block 3

Block 4



The effect of essential oils on fungal growth wasadjustedbyatwo-way(oilsandblocks)mixedmodelfor each fungus and each oil concentration, usingthe average observedOD attributed to the fungus at168-192h(plateau)asa response.Thepercentageofinhibitionofessentialoilswithrespect to thecontrolwascalculatedwiththefollowingformula(2):

%inhibition=ODControl-ODTreatmentx100 (2)

ODControl

where ODControl = [OD (X’) - OD (T’)], ODTreatment =[OD(Xn)-OD(Tn)](Figure 1).

Standarderrorsoftheseparameterswerecalculatedfrom the mixed model. Data presented were pooledfromtwoindependentexperiments.

4. RESULTS AND DISCUSSION


Observedandtheoreticalstandarderrorsofthemeansof each conidia concentration, juice dilution andreplicate size combinations are shown in figure 2a(P. italicum), figure 2b (P. digitatum) and figure 2c(C. musea).Astheoreticalstandarderrorsaregenerallyhigherthanthoseobserved,weexpectedheterogeneityin the responsebetweenand/orwithinELISAplates,which would need to be taken into account in thenextexperimentaldesigns.Asexpected, the standarderrors decreased with the increase in the number ofreplicates.However,thedecreaseinthestandarderrorsbecamepracticallynegligiblebeyondeightreplicates.Consequently, the number of replicates for furtherexperimentswasfixedtoeight,followingadesignthatcontrolsinter-andintra-plateheterogeneity(2platesx2blocksx2wells=8replicates)(Figure 1).Toavoidany possibility of contamination, uninoculated wells(withoutpathogen)wereseparatedfromtheinoculatedwells (with pathogen). Unlike in the experiments ofCosetal. (2006), thewellssituatedat thecornersoftheplatewereusedhere,asweobservednoproblemofevaporationfromthesewells.Thedesignreportedhere allowsmore serial testing of essential oils thanthedesignproposedbyCosetal.(2006)butwithlessrepetition per treatment. This design saves time byreducing thenumberof repetitionsandby increasingthenumberofessentialoilsthatcanbetested.

In order to identify the appropriate juice dilutionandconidiaconcentrationfortheassay,growthofeach

pathogen was evaluated for three different conidiaconcentrations and three juice dilutions (Figure 3).Growth was not photometrically quantifiable before24hofincubation,andtheamountofgrowthdiffereddepending on themedium. For the three fungi, non-significant interaction between conidia concentrationand juice dilution was detected (Fmax= F4,63= 2.09,p-value=0.093)andjuicedilutions3x 10-3and3x 10-4v/v showed a significantly lower growth comparedto 3x 10-2v/v (Fmin= F2,42= 55.19, p-value<0.001)(Table 2).Atthisdilution(3x 10-2v/v),growthstartedtoincreaseat24handreachedaplateauatbetween168and192h,whatevertheconidiaconcentrationandthepathogentested.By168-192h,growthleveledoffforallconidiaconcentrationsandC. museashowedlowergrowth (OD=0.174) than P. italicum (OD=0.601)andP. digitatum(OD=0.577).ForC. musea,only104and105conidia.ml-1weretestedbecausethatpathogendidnotproduceenoughsporesfor thepreparationofa concentration of 106conidia.ml-1. Furthermore, alowin vitrogrowthofC. museawasobservedontheELISAplate.

For the 168-196h period, conidia concentrations(104 and 105conidia.ml-1) had no significant effectonP. italicum andP. digitatum growth, but they hadahighlysignificanteffecton thegrowthofC. musea(Table 3). Given the strong decrease in growth fordilutionshigher than3x 10-2andgiventhesufficientgrowth of the studied fungi with a concentration of104conidia.ml-1,theseparameterswereselectedforthesubsequentexperiments.

The orange juice and banana juice allowed aquantifiable and sufficient growth of pathogens.Comparing natural and synthetic media, Kuhajeket al.(2003)showedthatgrowthfromzoosporeswashigherinanaturalmediumthanthegrowthobservedina syntheticmedium.However,whenvariability inbatch-to-batch composition is observed in a naturalmedium, a completely synthetic, chemically definedmediumshouldbeconsidered.


The effect of methanol concentration on fungalgrowth is shown infigure 4a (P. italicum),figure 4b(P. digitatum) andfigure 4c (C. musea).Avariabilityoffungalgrowthwasobserveddependingonthetypeof blocks used. However, this variability was lessimportant in Penicillium strains than in C. musea.Nevertheless, trends in average growth can beclearly defined. The sensitivity of each pathogen tomethanol differed depending on concentration. Highconcentrations ofmethanol either partially or totallyinhibited thegrowthofpathogens.Thiswas thecaseforthehighestconcentration(33.0%),whichpartially


Figure 2. MeanstandarderrorofreplicatesofPenicillium italicum(a),Penicillium digitatum(b)andColletotrichum musea(c)—Erreur standard moyenne des répétitions dePenicilliumitalicum(a),Penicilliumdigitatum(b)andColletotrichum musea(c).-+-:theoreticalstandarderror—erreur standard théorique;-o-:observedstandarderror—erreur standard observée.

Number of replications


Sta

ndar

d e

rror

of a

vera

ge o

ptic

al d

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ty


Sta

ndar

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vera

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tand

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inhibitedP. italicumat48.1%andP. digitatumat46.8%andwhich totally inhibitedC. musea after168-192hofincubation.ThemixedmodelshowedaveryhighlysignificanteffectofthemethanolconcentrationonthefungalODforbothperiods(72-96hand168-192h)andonthethreefungi(Fmin=F6,42=6.87,p-value<0.001)

(Table 4).Thelossofgrowthinthepathogensduetomethanolwashigherfortheperiod72-96hthanfortheperiod168-192h.Althoughthislossofgrowthinthepathogenswassignificantforallconcentrations(except0.5%)duringtheperiod72-96h,therewasnofurtherloss of growth during the period 168-196h (except

Figure 3. Growth (OD) of Penicillium italicum (a),Penicillium digitatum (b) and Colletotrichum musea (c)from three initial conidia concentrations (106, 105, 104conidia.ml-1) in three concentrations (3 x 10-2, 3 x 10-3,3x10-4v/v)oforange juice(Penicillium)orbanana juice(C. musea) — Croissance (DO) de Penicillium italicum (a), Penicilliumdigitatum (b) et Colletotrichummusea (c) pour trois concentrations initiales de conidies (106, 105, 104 conidies.ml-1) dans trois concentrations (3 x 10-2, 3 x 10-3, 3 x 10-4 v/v) de jus d’orange (Penicillium) ou jus de banane (C.musea).Growthwasmonitoredat24-hintervalsfor192hofincubationat23°CbymeasuringODat492nm—La croissance a été suivie à intervalle de 24 h pendant 192 h d’incubation à 23 ° C en mesurant la DO à 492 nm;OD:OpticalDensity—Densité Optique.

Hours Hours

Gro

wth

(OD

)

Gro

wth

(OD

)

a b0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.0

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.00 24 48 72 96 120 144 168 192 216

Hours

Gro

wth

(OD

)

c 0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.00 24 48 72 96 120 144 168 192 216

0 24 48 72 96 120 144 168 192 216

3 x 10-2

3 x 10-2

3 x 10-2

3 x 10-3

3 x 10-3

3 x 10-3

3 x 10-4

3 x 10-4

3 x 10-4

106

105

104

105

104

106

105

104

Table 3.Growth(OD)offungiat168-192hofincubationfor each conidia concentration—Croissance (DO) des champignons pendant à 168-192 h d’incubation pour chaque concentration de conidies.Conidia.ml-1 Growth (OD) of fungi

P. italicum P. digitatum C. musea104 0.325a 0.203a 0.061a

105 0.320a 0.219a 0.124b

106 0.346a 0.247b ntF 0.940 8.570 34.27p-value 0.396 0.001 <0.001Valuesinthesamecolumnmarkedwithdifferentlettersaresignificantlydifferentat5%—Les valeurs de la même colonne affectées de lettres différentes sont significativement différentes au seuil de 5 %;OD:OpticalDensity—Densité Optique.

Table 2. Growth (OD) of fungi at 168-192 h ofincubation for each juice dilution — Croissance (DO) des champignons à 168-192 h d’incubation pour chaque dilution de jus.Juices dilution(v/v)

Growth (OD) of fungiP. italicum P. digitatum C. musea

3x10-2 0.601a 0.577a 0.171a

3x10-3 0.299b 0.079b 0.065b

3x10-4 0.091c 0.016b 0.042b

F 316.23 1,584.46 55.19p-value <0.001 <0.001 <0.001Valuesinthesamecolumnmarkedwithdifferentlettersaresignificantlydifferentat5%—Les valeurs de la même colonne affectées de lettres différentes sont significativement différentes au seuil de 5 %;OD:OpticalDensity—Densité Optique.


in thecaseof thehighest concentrationand including5%,10%and17%inthecaseofC. musea).Thisresultshowsthatmethanolcausedasignificantslowdowninthe growth of pathogens but that it had no fungicidaleffect (except in the highest concentration) given thatthisgrowthretardationwasrecoveredattheendoftheobservationperiod,asshownbyanalysisoftheresultsfor the 168-192h period. C. musea seems from thisperspective to be more sensitive to alcohol. Indeed,growthretardationwasstilldetectableattheendoftheobservation period for the majority of concentrationstested.

For the six combinations of time period andfungi, only the 0.5% methanol concentration did notsignificantlyaffectgrowthcomparedtothecontrol(0%).


Theeffectofessentialoilconcentrationonfungalgrowthfor168-192hofincubationispresentedinfigure 5.Thethirtyessentialoilswerefirstlytestedat1,000ppmforeach pathogen. Only essential oils that inhibited thegrowthofpathogensformorethan70%ofthereferencegrowthwereretainedforthetestat500ppm.Thesamegrowthlimitwasusedtoselecttheessentialoilsforthe100ppmtestfromthe500ppmresults.Itemergedthatthe most effective oils against the three pathogens at1,000ppmwerethoseofOrmenis mixta,Cymbopogon martinii, Monarda fistulosa, Cymbopogon nardus,Thymus satureioides, Cinnamomum zeylanicum,Cinnamomum verum,Thymus vulgaris,Trachyspermum ammi,Eugenia caryophyllus,Corydothymus capitatus,Origanum compactum and Origanum heracleoticum.As can be seen in table 1, the main constituents of

theseessentialoilsarealdehydes,alcoholsandphenols(forming the largest constituent).Thehighpercentageinhibitionofthesemosteffectiveessentialoilscouldbeduetotheirhighcontentofmajorcompounds.ThisresultisinagreementwiththosereportedbyFaidetal.(1996)andDormanetal.(2000).Theseauthorsalsoobserveda relationship between the high activity of essentialoils and the presence of oxygenated compounds.Antimicrobialactivityofmajoressentialoilcompoundsis in theorder:phenols>alcohols>aldehydes. In thisstudy,despitethefactthatsomeessentialoilswererichinalcoholoraldehydecompounds, theydidnotshowinteresting antifungal activity. This was the case forMentha piperita, Melaleuca alternifolia, Origanum majoranaandCymbopogon citratus.

Thustherelativeantifungalactivityofessentialoilsmaynotbeeasilycorrelatedwiththemajorcompoundbutwithamixtureofcompoundsactinginsynergy.

Pennicilium digitatum and C. musea presentedsimilar sensitivity towards essential oils. HoweverP. italicumwasmore resistant tocertainessentialoils.This was the case, for example, with Cymbopogon nardus and Thymus satureioides, which inhibitedcompletely the growth of P. digitatum and C. museaat500ppm,whereas inhibitionwas less than60%forP. italicum.

Among the eight essential oils still effective at100ppm,onlyCinnamomum zeylanicum,Cinnamomum verumandEugenia caryophylluswereactiveagainstthethreepathogens(P. italicum,P. digitatumandC. musea).Atsuchaconcentration,essentialoilsaregenerallynotdangerousortoxicforhumans(Isman,2000;Couderc,2001).Furthermore,ahighquantityofessentialoilscanbeextractedfromplants:15to20%yieldforEugenia

Table 4. Growth (OD) of fungi at differentmethanol concentrations at 72-96 h and 168-192 h of incubation and testof significance of the difference with 0%methanol concentration—Croissance (DO) des champignons à différentes concentrations de méthanol à 72-96 h et 168-192 h d’incubation et le test de différence significative par rapport à la concentration de 0 % de méthanol.Methanol concentrations (%) Growth (OD) of fungi

P. italicum P. digitatum C. musea72-96 h 168-192 h 72-96 h 168-192 h 72-96 h 168-192 h

0 0.272 0.658 0.336 0.487 0.191 0.2930.5 0.246ns 0.647ns 0.298ns 0.484ns 0.159ns 0.271ns

1 0.197** 0.619ns 0.271* 0.463ns 0.132* 0.225ns

5 0.174*** 0.585ns 0.259* 0.458ns 0.089*** 0.145***10 0.160*** 0.603ns 0.238** 0.455ns 0.106** 0.166**17 0.167*** 0.607ns 0.222*** 0.442ns 0.076*** 0.137***33 0.019*** 0.341*** 0.054*** 0.259*** 0.022*** 0.035***ns:notsignificant—non significatif(p-value≥0.05);*:significant—significatif(p-value<0.05);**:highlysignificant—très significatif(p-value<0.01);***:veryhighlysignificant—hautement significatif(p-value<0.001);OD:OpticalDensity—Densité Optique.


Figure 4.Growth(OD)ofPenicillium italicum,Penicillium digitatumandColletotrichum museaatdifferentconcentrationsofmethanol(0,0.5,1,5,10,17and33%,v/v)—Croissance (DO) de Penicilliumitalicum, Penicilliumdigitatum et Colletotrichummusea à différentes concentrations de méthanol (0, 0,5, 1, 5, 10, 17 et 33 %, v/v).

Thegrowth(ODat490nm)wasassessedusingmicroplatesat24-hintervalsduring192hofincubationat23°Cinthedark.[Block1(+)Block2(o)block3(Δ)block4()]—La croissance (DO à 490 nm) a été évaluée en utilisant des microplaques, à intervalle de 24 h durant 192 h d’incubation à 23 °C dans l’obscurité. [Bloc 1 (+) Bloc 2 (o), bloc 3 (Δ) bloc 4 ()].

Time (h)

Gro

wth

(OD

)

Time (h)

Gro

wth

(OD

)G

row

th (O

D)

Time (h)

Penicillium italicum

Penicillium digitatum

Colletotrichum musea


Figu

re 5.Growthinhibition(%)ofP

enic

illiu

m it

alic

um,P

enic

illiu

m di

gita

tum,andC

olle

totr

ichu

m m

useaby

essentialoils(EOs)after168-192hofincubation—

Inhi

bitio

n de

la c

rois

sanc

e (%

) de Penicillium

italicum

, Penicilliumdigitatum

et C

olletotrichum

musea

par

les h

uile

s ess

entie

lles a

près

168

-192

h d

’incu

batio

n.

Thenumbersrepresenttheessentialoilsfrom

the

tabl

e 1—L

es n

umér

os re

prés

ente

nt le

s hui

les e

ssen

tielle

s men

tionn

ées d

ans l

e ta

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rowth

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a va

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nég

ativ

e in

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e un

e st

imul

atio

n de

la c

rois

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e.

Mean % inhibitor Mean % inhibitor

Mean % inhibitorMean % inhibitorMean % inhibitor

Mean % inhibitor Mean % inhibitor Mean % inhibitor

Mean % inhibitorP

enic

illiu

m it

alic

um -

EO

s at

1,0

00 p

pm

Pen

icill

ium

dig

itatu

m -

EO

s at

1,0

00 p

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Col

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otric

hum

mus

ea -

EO

s at

1,0

00 p

pm

Col

letr

otric

hum

mus

ea -

EO

s at

500

pp

m

Col

letr

otric

hum

mus

ea -

EO

s at

100

pp

m

Pen

icill

ium

dig

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EO

s at

500

pp

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Pen

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ium

dig

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EO

s at

100

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Pen

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ital

icum

- E

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at 5

00 p

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Pen

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- E

Os

at 1

00 p

pm


caryophyllus and 1 to 2% yield for the other two.Eugenia caryophyllus andCinnamomum essentialoilshavebeenreportedtoinhibitthegrowthofP. digitatum(Yahyazadeh et al., 2008) and C. musea (Ranasingheetal.,2002),respectively,inin vitroconditions.Usinga solid culture medium (PDA) on Petri dishes and atechnique based on the determination ofmycelial dryweights respectively, these authors obtained highervaluesofinhibitionconcentrationthanours.

5. CONCLUSION

In this investigation, a microbioassay procedure wasdeveloped for evaluating the efficacy of antifungalessential oils against C. musea, P. italicum, andP. digitatum.The procedure reported here incorporatesa standardized template for rapid in vitro screening.Due to the in vitro fungicidal effect of the essentialoils C. zeylanicum, C. verum and E. caryophyllus, apossibilityexiststodevelopaneffectivetreatmentsystemto control postharvest diseases of banana and citrus.However,thepotentialuseoftheseessentialoilsrequiresa detailed examination of their biological activity anddispersioninfruittissues.

Acknowledgements

WethankPranarômInternationalS.A.fortheircollaborationand all those who provided technical assistance. The PhDscholarshipofthefirstauthorwasprovidedbytheMinistryofScientificResearchofCôted’Ivoire.

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