The control of postharvest blue and green molds of citrus in relation with essential oil-wax formulations, adherence and viscosity.

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ContentslistsavailableatSciVerseScienceDirect

Postharvest

Biology

and

Technology

j o u r n al hom ep age :w w w . e l s e v i e r . c o m / l o c a t e / p o s t h a r v b i o

The

control

of

postharvest

blue

and

green

molds

of

citrus

in

relation

with

essential

oil–wax

formulations,

adherence

and

viscosity

Kouadio

Hugues

Sosthène

Kouassi

a,b,∗,1

_,

_Mohammed

_Bajji

a,1,2

_,

_Haïssam

_Jijakli

a

a_{Phytopathology}_Unit,_Gembloux_Agro-Bio_Tech,_Liege_University,_Passage_des_Déportés,_2,_B-5030_Gembloux,_Belgium

b_Laboratory_of_Chemistry_and_Biochemistry_of_Natural_Products,_Faculty_of_Science_and_Technology,_Cheikh_Anta_Diop_University,_BP₅₀₀₅_Dakar-Fann,_Senegal

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received27March2012 Accepted20June2012 Keywords: Essentialoil Citrusfruit Biologicalactivity Penicilliumitalicum P.digitatum Wax

a

b

s

t

r

a

c

t

Theuseofwaxcoatingsenrichedwithantifungalshassignificantlycontributedtoqualitymaintaining ofharvestedcitrusfruit.Ontheotherhand,interestinessentialoils(EOs)asanalternativetosynthetic fungicideshasrecentlygainedmomentum.Inthisstudy,CinnamomumzeylanicumEOwasincorporated intoavarietyofcommercialcitruswaxes(shellac,carnauba,paraffinandpolyethylene).Thebiological activityoftheseformulationsagainstgreenandbluerotsaswellastheirviscosityandadherenceto theorangefruitsurfacewereevaluated.ExcellentdiseasecontrolwasachievedwithC.zeylanicumEO incorporatedinshellacand/orcarnaubawaxcomparedtootherEO–waxformulations.Diseasecontrolby EO–waxesseemstodependnotonlyonthevolumethatremainsonthefruitskin,butalso,probablyon theretentionofEOcomponentsonthefruit.Otherfactorssuchasformulationsolubility,permeability togases,andcompatibilitybetweenEOcompoundsandthoseofwaxesmayalsobeinvolvedinthe improvementofEOefficacy.Thepresentstudymaythereforeallowacarefulselectionofappropriate waxesfortheelaborationofeffectiveEO–waxformulations.

1. Introduction

The antifungal properties of essential oils (EOs) have been knownfor a long time, and studies on theireffectson several postharvestphytopathogens have beenreported (Bakkaliet al.,

2008;Sanchez-Gonzalezetal.,2011;Mareietal.,2012).

Antifun-galtestsininvitroconditionsofseveralEOsandtheircompounds

(Dafereraetal.,2003;Lindeetal.,2010;Soyluetal.,2010;Marei

etal.,2012;Kouassietal.,inpress)showedthattheycan(i)be

activeagainstsometypesofphytopathogenicfungiandnotactive againstothers,(ii)beactiveagainstthesamepathogen,butnotat thesameconcentration,and(iii)exhibitantifungalactivityagainst a certainpathogen in one fruit type, but not against thesame pathogeninanotherfruittype,oratleastnotatthesame concen-tration.Furthermore,complexityofEOsandtheircompoundsmake thestudyof theirmechanisms of action difﬁcult.Nevertheless, thepotentialuseofEOstocontrolpostharvestdiseasesrequires

∗ Correspondingauthorat:PhytopathologyUnit,GemblouxAgro-BioTech,Liege University,PassagedesDéportés,2,B-5030Gembloux,Belgium.

Tel.:+320477063353;fax:+32081610126.

E-mailaddress:sosthenehugues@yahoo.fr(K.H.S.Kouassi).

1 _These_authors_have_equally_contributed_to_this_work.

2 _Present_address:_{Bioengineering}_Unit,_Life_Sciences_Department,_Walloon

Agri-culturalResearchCentre(CRA-W),ChausséedeCharleroi234,B-5030Gembloux, Belgium.

adetailedexaminationoftheirbiologicalactivityandthe devel-opmentof formulationswhich inhibitthe growthofpathogens atnon-phytotoxicconcentrations. Inordertodevelop EO-based fungicideformulationsaspostharvesttreatments,astudywas con-ducted ontheantifungal activityininvitroconditions ofthirty EOsagainstPenicilliumitalicumWehmer,PenicilliumdigitatumSacc andColletotrichummusea(Berk.&M.A.Curtis)Arx,three posthar-vestfungalpathogensoffruit(Kouassietal.,inpress).Itshowed that Cinnamomumzeylanicum, Cinnamomumverum and Eugenia caryophyllataEOswerestilleffectiveat100ppmagainstthethree testedpathogens.Inanotherstudy(Kouassietal.,2010),theinvivo activityofthesethreeEOsagainstP.digitatumandP.italicumon orange fruit (preventive treatment) showedthat C.zeylanicum, C. verum and E.caryophyllata EOs dilutedin ethanol were able toeither partially(ata concentration<5%)or totally(ata con-centration≥5%)protectfruitfrominfectionsofbothpathogens. Furthermore,novisibledamagewasobservedontheorangecuticle orskinupto5%ofeachEO.AmongthethreeEOs,C.zeylanicumwas themosteffective.Theseresultsareinterestingfromascientiﬁc pointofview.However,thepracticalapplicationoftheseEOsasan alternativetochemicalfungicidesisnotyetfeasiblebecausetobe effective,excessiveconcentrationsarestillneeded.Inaddition,the methodusedforfruittreatment(localtreatmentofwoundsites) isnotapplicableunderconditionsoffruitexportandmarketing. Therefore, for EO-based fungicides to become realistic alterna-tivestochemicalfungicidesincitruspostharvestmanagement,itis

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necessarytoﬁndappropriatemethodsofEOapplicationfor protec-tionofcitrusfruitundercommercialconditions.

Chemical treatments are traditionally considered to be the cheapestandthemosteffectivemethodsforpostharvestdisease control.The fruit industry (CERAFRUITS.L.,TECNIDEX S.A., and FECOPA SARL) uses chemical fungicidesat effective concentra-tionsoflessthan1%fortheprotectionofcitrusfruit.Todevelop acompetitivealternative,itisalsonecessarythatsuchmethodsof applicationallowareductionineffectiveconcentrationofEOs.One disadvantageofEOsistheirvolatility.Theyvolatilizeanddegrade quicklyeven atroom temperature.Severalstudieshave shown thatininvitroconditions,EOsareexcellentbiofungicidesagainst plantpathogensandsomeareevenbetterthanchemicalfungicides

(Ranasingheetal.,2002;DeSousaetal.,2011;S¸erbanetal.,2011).

Underinvitroconditions,EOsaregenerallyeffectivefrom0.01%

orless(Bakkalietal.,2008;Combrincketal.,2011).Accordingto

otherstudies(Plottoet al.,2003;Kouassietal.,2010,in press), however,resultsobtainedininvivoconditionsshoweda signifi-cantdecreaseinEOefficiencycomparedtoinvitroconditions.This decreaseinefficiencyisduetogreatervolatilityofEOsininvivo thanininvitroconditions.Moreover,invitroconditionsprovidea greatercontactareabetweenEOsandpathogensandthusallowfor bettereffectiveness.Therefore,aproduct(i)allowingacloser con-tactbetweenEOsandfruitsurfaceand(ii)preventingthemfrom volatilizing,willprovidebetterprotectionoffruitbyEOsatlow concentrations.

Naturalorsyntheticfoodgradewaxesareusedforcoatingin thefoodindustry.Theyhavevisualcharacteristicsandgas perme-abilitieswhichareparticularlyadvantageous,especiallyforcontrol ofweightloss,maturationandqualityofcoatedfruitand

vegeta-bles(Sanchez-Gonzalezetal.,2011).TheincorporationofEOsin

waxesforthecoatingofpostharvestcitrusfruitcanbeasolution.

DuPlooyetal.(2009)showedthatLippiascaberrimaEO(at0.25%)in

combinationwithcarnaubatropicalwaxhasprovidedeither100% (preventivetreatment)or95%(curativetreatment)disease con-trolagainstP.digitatuminfectionofcitrusfruit.Theseresultswere encouragingandstrengthenedourobjectivetostudythe antifun-gal(preventiveandcurative)effectsofC.zeylanicumincombination withdifferenttypesofcommercialwax.Antifungaleffectsofa mix-tureofC.zeylanicumandalcoholorSimulgelwerealsoevaluated againstP.digitatumandP.italicuminfections.Simulgelisa thick-eningandemulsifyingpolymerwhichisoftenusedincosmeticsto solubilizeEOsintowater-basedproducts.Inaddition,viscosityand thevolumeofEOformulationsthatadheretothefruitsurfacewere assessed.

2. Materialsandmethods

2.1. C.zeylanicumEO-basedformulations

C.zeylanicumEO–ethanolformulation:C.zeylanicumwas pro-ducedbyPranarôminternationalSA(Ghislenghien,Belgium).Final EO–ethanolformulationscontained0.1–0.5%C.zeylanicumEOand 1.5%ethanolindistilledwater.

C.zeylanicumEO–Simulgelformulation:Simulgeliseasytouse ina liquid form.Finalconcentrations contained0.1%or 0.5%C. zeylanicumEOand1.5%Simulgelinwater.

C.zeylanicumEO–waxformulations:Sevencommercialwaxes were used, including XEDASOL M7 (Xeda International S.A., Andiol,France)made of shellac, carnauba wax, and fattyacids (shellac–carnauba–fatty acids); XEDASOL M15 (Xeda Interna-tional S.A., Andiol, France) made of shellac and carnauba wax (shellac–carnauba);XEDAFOS L(Xeda InternationalS.A., Andiol, France)madeofshellac(shellac);XEDASOLEC2(XedaInternational S.A.,Andiol,France)madeofcarnaubawax(carnauba);95369-1L

(Sigma–AldrichNV/SA,Belgium)madeofparafﬁnwax(parafﬁn) and Michem® _emulsion ₁₈₃₂₅ _(Michelman, _Aubange, _Belgium)

madeofpolyethylenewax(polyethylene).EO–waxformulations werecomposedofwaxsolutionsupplementedwithC.zeylanicum at0.1%or0.5%(v/v).

2.2. Pathogens

P.italicum(strainPIRBM1)andP.digitatum(strainPDRBM1) wereobtainedfromthePlantPathologyUnitcollection(Gembloux Agro-BioTech,Belgium).Penicilliumstrainswereisolatedfrom cit-rusinMorocco(ENA,Meknes).A14-d-oldcultureofthesestrains at25◦Cgrownonpotatodextroseagar(PDA)wasusedas coni-diasource.Conidiawerescrappedfromtheagarandsuspended indistilledwaterwith0.05%Tween20(v/v),thenthesuspension concentration(inoculum)wasadjustedto104_conidia/mL_using_a

haemocytometer(Bürkercell).

2.3. Citrusfruittreatmentandartiﬁcialinfections

Maturehealthyuntreatedorganicorangefruitwereusedinthe presentwork.Fruitweresurfacedisinfectedbydippingfor2minin a10%sodiumhypochloritesolutionandwererinsedtwicewith dis-tilledwaterbeforebeingdriedovernightatambienttemperature. Eachfruitwaswoundedontheoutersurfaceonthreelocationsat thesamesidetoadepthof3mmandadiameterof5mmusinga corkborer.Tenorangespertreatmentwereused.

Preventivetreatments werecarried outbydippingwounded fruitfor2minintothespeciﬁcformulationspreviouslydescribed (C.zeylanicumEO–ethanol,C.zeylanicumEO–SimulgelorC. zeylan-icumEO–wax)andallowedtoairdryfor4or24h.Thereafter,each woundsitewasinfectedbyinoculating25_␮Loftheinoculumof P.digitatumorP.italicum.Finally,eachfruitwasplacedinaplastic traycontainingaﬁlterpaperand3mLofsterilewatertoensure highrelativehumidity,andthenstoredat23◦C.Controlsconsisted offruittreatedwithdistilledwater,ethanol, Simulgelorwaxat thesameconcentrationsastheonesusedinformulations.A treat-mentwithachemicalfungicidethiabendazole(TBZ)usuallyused byFECOPASARL(Casablanca,Morocco)at0.4%inwax(citrashine) forcitruspostharvesttreatmentwasalsoincluded.

For curative treatments,fruit were woundedas above, then inoculatedwithP.italicum (25␮Lof inoculumperwound site) and incubatedfor24hat 23◦C. Fruitwereafterthat treatedas previouslydescribedandagainincubatedinthesameconditions asforpreventivetreatments.Twoindependentexperimentswere performedforbothpreventiveandcurativetreatments.

The effectoftreatments (preventiveand curative)was eval-uated after 7d of fruit incubation, and data were recorded as the percentage of disease control according to the formula (WIc−WIt)/WIc×100,whereWIcandWItrepresentwound

inci-dence for the control (distilled water) and for the treatment, respectively.Woundincidencewasdeterminedasthenumberof rottenwoundsforastudiedtreatmentpertotalnumberofwounds forthesametreatment.

2.4. Volumeoftreatmentperorange

Thevolumeoftreatmentusedperorange,whichrepresentthe volumeoftreatmentthatremainsonthefruitskin,wasdetermined byanempiricalmethodaccordingtotheformula(Vi−Vf)/Nt,where

Viistheinitialvolumebeforesoakingfruit(500mL/treatment),Vfis

theﬁnalvolumeaftersoakingandNtisthenumberoffruitsoaked

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Table1

IncidenceofblueandgreenmoldsofcitrusonorangesbyC.zeylanicumessentialoil(EO)amendedwithethanolapplied4hbeforeinoculationwithP.italicumorP.digitatum.

Treatment(4hbeforeinoculation) Bluemold(P.italicum) Greenmold(P.digitatum)

Woundincidencea _Disease_control_(%) _Wound_incidencea _Disease_control_(%)

Distilledwater(control) 1.00a 0 1.00a 0

Ethanol 1.00a 0 1.00a 0

C.zeylanicumEO–ethanol(0.1%) 0.80b 20 1.00a 0

C.zeylanicumEO–ethanol(0.4%) 0.80b 20 0.90b 10

C.zeylanicumEO–ethanol(0.5%) 0.43c 57 0.60c 40

Thiabendazole(0.4%)–water 0.00d 100 0.00d 100

a_Wound_incidence_was_determined_from_two_independent_experiments.

ValuesinthesamecolumnmarkedwithdifferentlettersaresigniﬁcantlydifferentatP≤0.05.

2.5. Rotationalrheologicalanalysis

Rheological measurementsof C. zeylanicum EO formulations (EO–ethanol, EO–Simulgel, and EO–wax) were performed in duplicatebya rotationalviscometer(BohlinInstrumentsCVO50 Rheometer)ﬁttedwithathermostaticbathfortemperature con-trol.Anintegratedcomputercontrollerwasusedtoprogramthe testsandthesensorSystemCVOutilizingacone/cylinder conﬁgu-rationwasusedformeasurement.Eachformulationsample(3mL) wasplacedinthesensorsystemformeasurementat25◦C.Curves ofinstantaneousviscosity(mPa)asafunctionoftheshearrate(D) wereobtainedwiththefollowingprogram:5minfrom0to300s−1 (themaximumshearrate).Viscosityofeachformulationat10s−1 and300s−1wasdeterminedfromthecurve.

2.6. Statisticalanalysis

Statisticalanalysisofvariance(ANOVA)wasperformedusing theSASsoftwareandmeanswereseparatedusingtheLeast Signif-icantDifference(LSD)testatP≤0.05.

3. Resultsanddiscussion

3.1. Citrusfruittreatmentandartiﬁcialinfection

Wefirststudiedthepreventiveactionofethanol-C.zeylanicum EO(0.1–0.5%)treatmentsagainstP.italicumandP.digitatum infec-tions(Table1).Distilled waterand TBZ (0.4%) treatmentswere alsoconsidered.TBZwasdilutedindistilled waterbeforebeing used.Pathogeninoculationsweredone4hafterfruittreatment. ThepathogenicityofP.italicum andP.digitatumwasconfirmed bythepresence(allwoundswererotten)respectivelyofblueand greenmoldsonorangefruittreatedwithdistilledwater(control). Likedistilledwater,ethanoltreatmentat1.5%hasnoeffectagainst thetwofungi(0%diseasecontrol).PreventivediptreatmentwithC. zeylanicumEO–ethanolformulationssignificantlyreducedwound incidenceagainstbothpathogenscomparedtothecontrol,except inthecaseofP.digitatumwherewoundincidencebyC.zeylanicum EOat0.1–0.3%wasasforthecontrol.C.zeylanicumEOat0.5%in ethanolwasthemosteffectivewithadiseasecontrolof57%(blue mold)and40.0%(greenmold).Thisresultshowedthatininvivo conditions,C.zeylanicumEOat0.5%mixedwithethanolwas par-tiallyeffective,whileininvitroconditions,thisEOevenat0.01% offeredtotalpathogeninhibition(Kouassietal.,inpress).Thefact that,forhigh-volumeformulation,ahomogenousaqueous disper-sionwasdifficulttoobtainbymixing1.5%ethanolwithEOcould haveaffectedtheeffectivenessofEOininvivoconditions.Forthe otherconcentrations(0.1–0.4%)ofC.zeylanicumEO,diseasecontrol wasequalorlowerthan20%forbothdiseases.

Accordingtotheliterature(Teuscheretal.,2005),themain com-poundsofthisEOare:cinnamaldehyde(42–82%),eugenol(1–11%),

cinnamicalcohol(8%),cinnamicacid(10%),cinnamylacetate, o-methoxycinnamaldehyde,benzylbenzoate,linaloolandsafrole(up to2%).Cinnamaldehydeandeugenolareknowntohavestrong anti-fungalactivity(Sheng-Yangetal.,2005;Amirietal.,2008;Sen-Sung

etal.,2008;Campanielloetal.,2010;Matanetal.,2011;Moonetal.,

2011).ThebiologicalactivityofC.zeylanicumEOmaybeduetothe synergisticactionofthesetwocompounds(Tsair-Borand

Shang-Tzen,2008).However,itwasreportedthatEOactivitywasmainly

duetoasynergisticortoantagonisticeffectsofitsdifferent com-pounds (HummelbrunnerandIsman,2001;Bakkalietal.,2008;

Sanchez-Gonzalezetal.,2011).Itisthuspossiblethattheactivity

ofthemaincomponentsismodulatedbyotherminormolecules. ControlofbothdiseaseswasmuchmoreenhancedbyC. zeylan-icumEO(0.5%)–ethanolinthepresentworkinwhichwoundedfruit weretreatedbydippingwholefruit(40%and56.7%,Table1) com-paredtoourpreviouswork(Kouassietal.,2010)inwhichtreatment wasappliedatthewoundsiteonly(only10%diseasecontrol).C. zeylanicumEO–ethanolformulationremainshoweverstillless efﬁ-cientthanthechemicalfungicidetreatment(100%diseasecontrol)

(Table1).Moreover,ethanolitselfisvolatileand,therefore,would

notallowmaintainingtheEOlongenoughontheskinoftheorange tobetterprotectit.

Asecondexperimentwasundertakentoevaluatepreventive and curativetreatments ofSimulgeland thecommercialwaxes (shellac,carnauba,shellac–carnauba,shellac–carnauba–fattyacids, parafﬁnandpolyethylene)supplementedwithC.zeylanicum EO (0.1%or0.5%)againstboth pathogeninfections. TBZ(0.4%) sup-plemented with shellac was included as a standard fungicide treatment.

Forthepreventiveeffect,fruittreatmentwascarriedout4h and24hbeforepathogeninoculationandtheresultsarepresented

in Table 2. Whateverthe time of inoculation, pathogenicity of

P.italicumand P.digitatumwasconﬁrmed bythedevelopment respectivelyofblueandgreenmoldsonallinfectedwoundsfor distilledwater,ethanol(1.5%),Simulgel(1.5%)andthecommercial waxes(100%incidence).Incontrast,treatmentwithTBZ–shellac inducedthehighestdiseasecontrolirrespectiveofthepathogen typeandtheinoculationtime.

For formulations based on ethanol, Simulgel, parafﬁn or polyethylenesupplementedwithC.zeylanicumEOat0.1%,wound incidencewastotal(allwoundswererotten)exceptinthecaseof inoculationbyP.italicum4haftertreatment.Inthiscase,wound incidencewasnotstatisticallydifferentforallC.zeylanicumEO for-mulationsat0.1%presentingadiseasecontrollessthan27%.For formulationsbasedonwax(shellac,carnauba,shellac–carnaubaor shellac–carnauba–fattyacids)supplementedwithC.zeylanicumat 0.1%,diseasecontrolwasnotbetter(equalorlessthan10%)inthe caseofinoculationbybothpathogens24haftertreatmentandby P.digitatum4haftertreatment.

With regard to treatment with C. zeylanicum EO at 0.5%, woundincidencewasgreatlyreducedincomparisonwiththatof

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Table2

IncidenceofblueandgreenmoldsofcitrusonorangesbyC.zeylanicumessentialoil(EO)formulationsapplied4hor24hbeforeinoculationwithP.italicumorP.digitatum (preventivetreatment).

Treatment Bluemold(P.italicum) Greenmold(P.digitatum)

4hbeforeinoculation 24hbeforeinoculation 4hbeforeinoculation 24hbeforeinoculation Wound incidencea Disease control(%) Wound incidencea Disease control(%) Wound incidencea Disease control(%) Wound incidencea Disease control(%)

Distilledwater(control) 1.00a 0 1.00a 0 1.00a 0 1.00a 0

Ethanol 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–ethanol 0.87b 13 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.5%)–ethanol 0.53c 47 0.87b 13 0.6c 40 0.87b 13

Simulgel 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–Simulgel 0.80b 20 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.5%)–Simulgel 0.33d 67 0.63c 37 0.43d 57 0.77b 23

Shellac–carnauba–fattyacids 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–shellac–carnauba–fattyacids 0.77b 23 0.90b 10 0.90b 10 1.00a 0

C.zeylanicumEO(0.5%)–shellac–carnauba–fattyacids 0.10e 90 0.37d 63 0.17ef 83 0.40c 60

Shellac–carnauba 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–shellac–carnauba 0.77b 23 0.97b 3 0.93b 7 1.00a 0

C.zeylanicumEO(0.5%)–shellac–carnauba 0.17e 83 0.37d 63 0.25e 75 0.40c 60

Shellac 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–shellac 0.73b 27 0.90b 10 0.90b 10 1.00a 0

C.zeylanicumEO(0.5%)–Shellac 0.00f 100 0.13e 87 0.00g 100 0.19d 81

Carnauba 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–carnauba 0.77b 23 0.90b 10 0.90b 10 1.00a 0

C.zeylanicumEO(0.5%)–carnauba 0.10e 90 0.37d 63 0.17ef 83 0.40c 60

Parafﬁn 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–parafﬁn 0.80b 20 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.5%)–parafﬁn 0.30d 70 0.58c 42 0.38d 62 0.73b 27

Polyethylene 1.00a 0 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.1%)–polyethylene 0.80b 20 1.00a 0 1.00a 0 1.00a 0

C.zeylanicumEO(0.5%)–polyethylene 0.30d 70 0.58c 42 0.38d 62 0.73b 27

Thiabendazole(0.4%)–shellac 0.00f 100 0.03f 97 0.00g 100 0.03e 97

ValuesinthesamecolumnmarkedwithdifferentlettersaresigniﬁcantlydifferentatP≤0.05afterLSDtest.

C. zeylanicum EO at 0.1%, regardless of the formulation, the pathogenspecies and theinoculation time.In addition, disease control wasfound tobe reduced when treatmentwas applied 24hinsteadof4hbeforeinoculation,whatevertheformulation andthepathogenspecies.Ingeneral,woundincidencewasmuch morereducedforC.zeylanicumEOat0.5%amendedwith Simul-gelorwaxthan thatamendedwithethanol, irrespectiveof the pathogenspeciesandtheinoculationtime.Therewere,however, someexceptions.InthecaseofinoculationbyP.digitatum24hafter treatment,woundincidencevaluesweresubstantiallyidenticalfor C.zeylanicumEO(0.5%)formulations(ethanol,Simulgel,parafﬁn andpolyethylene).Woundincidencewasalsomuchmorereduced forC.zeylanicumEOat0.5%amendedwithshellacand/orcarnauba thanwiththeotherwaxes(parafﬁnandpolyethylene)and Simul-gel,regardlessofthepathogenspeciesandtheinoculationtime. However,themostinterestingtreatmentwaswithC.zeylanicum EO (0.5%)–shellac,which allowed, as thefungicide (TZB),100% ofdiseasecontrolwheninoculationoccurred4haftertreatment whateverthepathogen.Inthiscaseofpreventivetreatment,the otherC.zeylanicumEO(0.5%)formulations(shellac–carnauba–fatty acids,shellac–carnaubaandcarnauba)offeredmorethan75%of diseasecontrol.

Regardingthecurativeeffect,fruittreatmentwasperformed 24h afterinoculation with P.italicum. Only a concentrationof 0.5%ofC.zeylanicumwastested.ResultsarepresentedinTable3. TheapplicationofC.zeylanicum EOsupplementedwithethanol andSimulgelresultedrespectivelyin67%and83%ofdisease con-trol.Theapplicationofshellac,carnauba,orshellac–carnauba–fatty acids(XEDASOLM7)amendedwithC.zeylanicumEO(0.5%)offered

atotaldiseasecontrollikethatofshellacamendedwithTBZat0.4%. Agooddiseasecontrol(97%)wasalsoobtainedwiththeapplication ofC.zeylanicumEO(0.5%)intoshellac–carnauba.Regarding paraf-ﬁn(95369-1L)orpolyethylene(Michem®_emulsion₁₈₃₂₅₎_mixed

withC.zeylanicumEO(0.5%);diseasecontrol(81%and85%, respec-tively)waslessthanthatoftheotherwaxesandwoundincidence wassigniﬁcantlydifferentfromthatoftheotherwaxes supple-mentedwithC.zeylanicum EObutstatisticallysimilartothatof SimulgelsupplementedwiththesameEO.Asinthecaseofthe preventivetreatment,themixtureofC.zeylanicumEOandshellac and/orcarnaubaresultedinagreaterreductionofwoundincidence comparedtothatofC.zeylanicumEOandethanol,Simulgel,parafﬁn orpolyethylene.

Finally,theseresults(Tables2and3)showedthatdisease con-trolagainstthetwomostimportantpostharvestpathogenswas improvedwithC.zeylanicum EOmixedwithSimulgel orwaxes comparedtothesameEOatthesameconcentrationmixedwith ethanol.Sinceethanol,Simulgelandwaxesalonehaveno antifun-galeffectonthepathogen,thisimpliesthatefficacyofformulations (EOintoethanol,Simulgelorwaxes)isonlyduetoEOandnotto asynergisticantifungaleffectbetweenEOandethanol,Simulgel orwaxes.Theimprovementofdiseasecontrolisthereforerelated totheimprovementoftheEOefficacy,itselfrelatedtothe prod-uctwithwhichEOisassociated(Simulgelandwaxesinourstudy). UnderstandingtheactionofsuchproductsinimprovingEOefficacy andthereforediseasecontrolisthusmorethannecessaryforthe developmentofeffectiveEO-basedbiofungicideformulations.This involvestherefore,thesearchforthecharacteristicsofproducts associatedwithEOs.

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Table3

IncidenceofbluemoldofcitrusonorangesbyC.zeylanicumessentialoil(EO) for-mulationsapplied24hafterinoculationwithP.italicum(curativetreatment).

Treatment(24hafter inoculation)

Woundincidencea _Disease_control_(%)

Distilledwater(control) 1.00a 0

Ethanol 1.00a 0 C.zeylanicumEO (0.5%)–ethanol 0.33b 67 Simulgel 1.00a 0 C.zeylanicumEO (0.5%)–Simulgel 0.17c 83 Shellac–carnauba wax–fattyacids 1.00a 0 C.zeylanicumEO (0.5%)–shellac–carnauba wax–fattyacids 0.00e 100 Shellac–carnauba 1.00a 0 C.zeylanicumEO (0.5%)–shellac–carnauba 0.03d 97 Shellac 1.00a 0 C.zeylanicumEO (0.5%)–shellac 0.00e 100 Carnauba 1.00a 0 C.zeylanicumEO (0.5%)–carnauba 0.00e 100 Parafﬁn 1.00a 0 C.zeylanicumEO (0.5%)–parafﬁn 0.19c 81 Polyethylene 1.00a 0 C.zeylanicumEO (0.5%)–polyethylene 0.15c 85 Thiabendazole (0.4%)–shellac 0.00e 100

Valuesinthesamecolumnmarkedwithdifferentlettersaresigniﬁcantlydifferent atP≤0.05afterLSDtest.

Unlike alcohol,Simulgelandwaxesincrease thesolubilityof EOsintowater-basedproductswithavisiblehomogeneous aque-ousdispersion,whichleadstoaconsistentdistributionofEOon thecitrusfruitsurface.Thischaracteristicmaycontributetothe improvementofdiseasecontrol.Inaddition,waxesform,after dry-ing,athinﬁlmaroundthefruitallowingaclosercontactofEOwith thefruitsurface,whichcertainlycontributestotheimprovement ofEOefﬁcacy.Waxeshavelowpermeabilityforwaterandgases

(Baldwin,1994;Baldwinetal.,1995;Hagenmaier,2002;Srinivasa

etal.,2004;Bourtoom,2008;Zhouetal.,2008;Farag,2010;Regnier

etal.,2010),particularlyadvantageousforcontrolofweightloss, maturationandqualityofcoatedfruitandvegetables.Ithasbeen reportedthattheincorporationofEOintofruitorvegetablecoating inducesasigniﬁcantreductioninO2andwatervapor

permeabil-itydependingonEOconcentration(Bosquez-Molinaetal.,2003;

DuPlooyetal.,2009;Regnieretal.,2010;Sanchez-Gonzalezetal.,

2010a,b,2011;Bonillaetal.,2012).Thischaracteristic(low

perme-abilitytogases)ofwaxesmayberesponsibleofthemaintainingof EOonthefruitsurface,therebykeepinghighconcentrationsofEO forextendedperiodsoftime.ItmayexplainwhyEOinwaxeswas moreeffectiveinreducingthelevelsofblueandgreenrotsthanEO atthesameconcentrationinalcoholorinSimulgel.

AmongEO–wax formulations,differentlevelsofwound inci-dence(diseasecontrol)wereobservedevenwhentheyallhavethe sameEOconcentration.DuPlooyetal.(2009)studiedtheeffect ofdifferentcommercialwaxes(MS1andcarnaubatropical formu-lations)supplementedwithdifferentEOs(e.g.L.scaberrimaand Menthaspicata)asanalternativetosyntheticfungicidesincitrus postharvestmanagementagainst P.digitatum.Asinthepresent work,excellent diseasecontrolwasachieveddepending onthe EO–waxcombination,whilewaxesaloneshowednodisease con-trol.Usingthesamecommercialcoatingssupplementedwiththe

Fig.1. Volume(mL)usedperorange(adherence)ofC.zeylanicumEOformulations. DifferentlettersindicatesigniﬁcantdifferencesatP≤0.05afterLSDtest.

sameEOs(2500␮L/L)asDuPlooyetal.(2009)andRegnieretal.

(2010)alsoobservedintheirstudythattheeffectivenessofanEOon

avocadofruitagainstColletotrichumgloeosporioidesand Lasiodiplo-diatheobromaedependedonthewaxused.L.scaberrimaEO-MS865 ismoreeffectivethanL.scaberrimaEO–carnaubawhereasM. spi-cataEO–carnaubaismoreeffectivethanM.spicataEO-MS865.In ourstudy,C.zeylanicumEO(0.5%)efﬁcacywasimproved(inthe preventivetreatment)whensupplementedwithshellacthanwith theotherwaxes(carnauba,parafﬁnandpolyethylene).Thismaybe duetothelowerpermeabilityforgasesofshellaccomparedtothe otherwaxes(MannheimandSoffer,1996;AlleyneandHagenmaier,

2000; Baiet al., 2003),thus keeping more EO onfruit surface.

Moreover,forthesameEO–waxformulation,diseasecontrolwas reduced whentreatmentwasdone24hversus4hbeforecitrus fruitinfection.ThisisprobablyduetothereleaseofEOovertime becauseofhighvolatility.Wearecurrentlystudyingtheeffectof thesewaxesonEOvolatilization.Preliminaryresultsshowedthat fruittreated 24hbeforeinoculationseemedtokeep lessEOon theirsurfaces,whichmakethemmorevulnerabletoinfectionthan fruittreated4hbeforeinoculation.Althoughthereisasigniﬁcant decreaseintheeffectivenessoftheEOafter24h,thedisease con-trolduetoEO–waxformulationsisrelativelyacceptablebecause morethanhalfofthewoundswereprotected.

Our resultsalsoshowedthatthecurativeeffectofC. zeylan-icumEOseemsmoremarkedthanitspreventiveeffect.Thismaybe explainedbytheimmediateeffectofEOformulationsonpathogen developmentin thecurativetreatment(no volatilizationeffect) and/orbythepossiblevulnerabilityofthepathogentothe treat-ment24hafteritsinoculation.

3.2. Volumeoftreatmentperorange(adherence)

Onlyaconcentrationof0.5%of C.zeylanicumEOwastested. ThevolumeoftreatmentusedperorangeispresentedinFig.1. The lowest volume of treatment (0.60mL) was recorded for EO–ethanolwhilethehighestone(2.80–2.84mL)wasforEO–wax (parafﬁn andpolyethylene). Thevolume oftreatmentofC. zey-lanicum EO–Simulgel (1.60mL) was more than double that of EO–ethanol.FortheotherC.zeylanicumEO–wax(shellac,carnauba, shellac–carnauba or shellac–carnauba–fatty acids)formulations, volumesrangingfrom2.31to2.35mLwererecorded.Theseresults, associated with those previously presented (Tables 1 and 2),

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Fig.2. Viscosity(mPas)(rotationalrheology)ofC.zeylanicumEOformulations. RheologicalmeasurementsofC.zeylanicumEOformulationswereperformedin duplicatebyarotationalviscometer(BohlinInstrumentsCVO50Rheometer)at 25◦_C._Curves_of_{instantaneous}_viscosity_(mPas)_as_a_function_of_the_shear_rate_(D)

wereobtainedwiththefollowingprogram:5minfrom0to300s−1_(the_maximum

shearrate).Viscosityofeachformulationat10s−1_and₃₀₀_s−1_was_determined

fromthecurve.Foreachshearrate,differentlettersindicatesigniﬁcantdifferences atP≤0.05afterLSDtest.

indicatethattreatmentefficacyiscorrelatedwiththevolumeof treatmentwhich remains on theorange fruit skin. Thegreater thevolumeofformulation treatmentadheringonthefruit sur-face, the better thedisease control. Indeed, once dried, waxes formedathinfilmaroundthefruit,keepingthereforemore for-mulation(i.e. more EO)onthefruit surface,resulting in better diseasecontrolthanethanolandSimulgel.WithregardtoC. zeylan-icumEOamendedwithwax(shellac,carnauba,shellac–carnaubaor shellac–carnauba–fattyacids),similarvolumesoftreatmentwere usedper orange (Fig. 1)while significantdifferencesin wound incidencewereobserved(Table2).Inaddition,thevolumeofthe treatmentofC.zeylanicumEOamendedwithparaffinor polyethy-lenewax wassignificantly superiortothat ofC. zeylanicumEO amendedwiththeotherwaxes.However,diseasecontrolafforded byformulationsbasedonC.zeylanicumEOandparaffinor polyethy-lenewaxeswasfoundtobelessimportantthanthosebasedonthe otherwaxes.ThisshowsthatdiseasecontrolbyEO–waxesisnot dependentonlyonthevolumethatremainsonthefruitskin,but also,probablyontheretentionofEOcomponentsonthefruit. Main-tainingEOonthefruitforextendedperiodsoftimemayberelated tothecompatibility(affinity)betweenEOcompoundsandthoseof waxes.Suchcompatibilitymayplayanimportantroleinimproving EOefficacy.

3.3. Rotationalrheological

Onlyaconcentrationof0.5%ofC.zeylanicumwastested. Viscos-ityofC.zeylanicumEOformulationsispresentedinFig.2.Whatever theEOformulation,therewasadecreaseofviscositywhengoing from10s−1 to300s−1;thisimpliesthatthebehaviorofEO for-mulationswasshear-thinning(orpseudoplastic)inallcasesand thuscouldbeusedduringsomeprocessingoperations(whenit ispumpedinamachine,forinstance).Theformulationbasedon C. zeylanicum EO–Simulgel wassigniﬁcantly more viscous than thatofC.zeylanicumEO–wax(shellac,carnauba,shellac–carnauba

or shellac–carnauba–fattyacids)and C.zeylanicum–ethanol, but less than that of C. zeylanicum EO–wax (paraffin or polyethy-lene).Intermsofviscosity,theformulationbasedonC.zeylanicum EO–Simulgelwassignificantlydifferentand∼5timessuperiorto that ofSimulgelalonewhiletherewasnosignificantdifference betweentheformulationbasedonC.zeylanicumEO–wax(shellac, carnauba,shellac–carnauba,shellac–carnauba–fattyacids,paraffin orpolyethylene)andwaxesalone(datanotshown).

If we compare the formulation based on C. zeylanicum EO–ethanolwiththeotherformulations,itseemsthatahigher vis-cosityoftheformulationleadstoitsgreateradherencetothefruit surface(i.e.moreEOs)andthereforebetterdiseasecontrol.ForC. zeylanicumEO–waxandC.zeylanicumEO–Simulgelformulations, however,thebestdiseasecontrolwasnotalwaysassociatedwith thehighestlevelsofadherenceand viscosity.Inaddition, what-evertheformulationofC.zeylanicumEO–wax(shellac,carnauba, shellac–carnaubaorshellac–carnauba–fattyacids),thevolumesof treatmentweresignificantlythesame,whiletheirviscosityand preventiveaction weresignificantlydifferent.Therefore, viscos-ity offormulationscannotalone explaintheimprovement ofC. zeylanicumEOefficacy.Itmaybeacombinedactionofthe solu-bility,viscosity,adherence,permeabilityofC.zeylanicumEO–wax formulations,andcertainlythecompatibility(affinity)betweenEO compoundsandthoseofwaxesthatallowretainingEOonthefruit surface,whichimproveEOefficacy.

3.4. Practicalaspects

EOsareconsideredasGRAS(GenerallyRecognizedAsSafe)and thus theirapplicationfor theprotectionoffruit andvegetables shouldnotposeregulatoryproblems.Butpracticalapplicationof EOsstillremainsexpensive,whichlimitstheiruseinthe preserva-tionoffruitandvegetables.TheincorporationofEOsintocoating formulationsappearstobeagoodstrategytoreduceapplication costssinceEOquantitiescanbereduced.Inthisstudy,theeffective concentrationofC.zeylanicumEOallowing100%diseasecontrol wasimproved(reduced)byupto0.5%byincorporatingC. zeylan-icumEOintoshellacwax(preventivetreatment),comparedtoour previousstudy(Kouassietal.,2010)whereaneffective concentra-tionofC.zeylanicumEOintoethanolwas5%.Atsuchaconcentration of0.5%,∼60mLofC.zeylanicumEOmixedwithabout11,840mLof shellacarenecessarytotreatonetonofcitrusfruit.Accordingto pricescurrentlyavailableonthemarket(Pranarôminternational SA,Sigma–Aldrich),theaveragecostofsucha quantityofEOis aboutsixtimesthatofthetestedchemicalfungicidenecessaryto treatthesamequantityoforangefruit.Itisthusclearthatforsuch biofungicides,otherexpensesrelatedtotheirdevelopment, reg-istration, distribution,etc.willbeadded tothepurchasecost.A possiblewaytoreducetreatmentcostsistocombineC.zeylanicum andotherrelativelycheaperEOs.Thishasprovedpossibleininvitro conditionswherethebiologicalactivityofC.zeylanicumalonewas similartothatofC.zeylanicum–C.verum–E.caryophyllatamixture (atequalvolumes)providedthatfinalconcentrationsaresuperior to0.02%(unpublishedresults).Iftheseresultsareconfirmedinvivo (ongoingstudy),theuseofthemixtureatequalvolumesofthese threeEOsatafinalconcentrationof0.5%wouldallowareductionof halfthecostofEOsnecessarytotreatatonoffruitcomparedwith theuseofC.zeylanicum alone.It isthusclearthattreatmentby EOswouldcostalittlemorethantreatmentbythetestedchemical fungicide.However,thebenefitsofEOs(eco-compatible,non-toxic atlow doses,biodegradable,andnoriskforresistance develop-ment)anddisadvantagesofchemicalfungicidesonhealthandon theenvironmentmakeEOsmoreinterestingforcitruspostharvest treatment.The preventiveand curativeeffectsofC. zeylanicum EOcouldmakeitanexcellentbiofungicidecandidate,becausea

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postharvesttreatmentbythisoilwouldpreventthedevelopment ofbothlatentpreharvestandpostharvestinfections.

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