Directory UMM :HenikSukorini:

and education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling or

licensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of the

article (e.g. in Word or Tex form) to their personal website or

institutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies are

encouraged to visit:

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

Control

of

postharvest

green

mold

of

citrus

fruit

with

yeasts,

medicinal

plants,

and

their

combination

Henik

Sukorini

a,b,∗

,

Somsiri

Sangchote

c

,

Netnapis

Khewkhom

c

a_{DepartmentofGraduateSchool,TropicalAgriculture,KasetsartUniversity,Bangkhen,Bangkok10900,Thailand} b_{DepartmentofAgrotechnology,FacultyofAgriculture,UniversityofMuhammadiyahMalang,65141,Indonesia} c_{DepartmentofPlantPathology,FacultyofAgriculture,KasetsartUniversity,Bangkhen,Bangkok10900,Thailand}

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received30July2012 Accepted5January2013

Keywords: Penicilliumdigitatum Greenmold Citrus Medicinalplant Yeast

a

b

s

t

r

a

c

t

Theuseofbio-fungicidesandaplantextractstocontrolpostharvestdiseasewasinvestigatedasan alter-nativetochemicalcontrol.Thecombinationofapromisingplantextractandyeastwereselectedthrough

invitroandinvivotechniques.AcombinationofCandidautilisTISTR5001andEugeniacaryophyllatacrude extractwasthebestcombinationtoattainareductionindiseaseincidenceanddiseaseseverityof Peni-cilliumdigitatumoncitrusfruit.ColonizationwasthelowestonfruittreatedwiththecombinationofE. caryophylatacrudeextractandC.utilisTISTR5001,andsurvivalofC.utilisTISTR5001wasthehighest. ThecombinationofE.caryophylatacrudeextractandC.utilisTISTR5001significantlyreducedthenatural developmentofgreenmoldofcitrusfruit,andhadnoeffecttofruitquality.TheactivecompoundofE. caryophylatawasfoundtobeeugenol,basedonHPLCandNMR(1_Hand13_{C).Hence,theresultsindicate}

thatacombinationofplantextractsandyeastspossesantifungalactivitythatcanbeexploitedasanideal treatmentforfutureplantdiseasemanagement.

©2013ElsevierB.V.Allrightsreserved.

1. Introduction

GreenmoldrotdiseasecausedbyPenicilliumdigitatumisthe mostimportantpostharvest diseaseof citrus fruit(Kinayetal., 2007).Thispathogenhasarelativelyshortdiseasecycle:3–5days at25◦_{C.Eachconidialheadisabletoproduceupto2billionconidia} andcanbespreadbywind(HolmesandEckert,1999).Harvested fruitareverysusceptibletothepathogen(Sommeretal.,2002), andthusthegreatestlossofcitrusfruitbyP.digitatumcanoccur afterharvest,duringtransport,storageandmarketing(Eckertand

Eaks,1989).P.digitatumisresponsiblefor90%ofcitrusfruitloss

(Macarisinetal.,2007),andminimizingfruitinjury,precise

tem-peraturemanagement,andpostharvestfungicidetreatments,are theprimarymethodstoreducetheselosses(EckertandEaks,1989). In the many citrus packing-houses, fungicides are used to treatthefruit.Continuoususeofafungicidesuchasimazalil,o -phenylphenol,orthiabendazoleformorethanthreedecadeshas resultedinpathogenresistance(HolmesandEckert,1999).Kinay

etal.(2007)reportedthatisolatesofP.digitatumcollectedfrom

Californiapacking-housesandorchardswereresistanttotwoor

∗Correspondingauthorat:DepartmentofPlantPathology,FacultyofAgriculture, KasetsartUniversity,Bangkhen,Bangkok10900,Thailand.Tel.:+6625799550; fax:+6629428044.

E-mailaddresses:hsukorini@yahoo.com(H.Sukorini),agrsrs@ku.ac.th (S.Sangchote),agrnpk@ku.ac.th(N.Khewkhom).

morefungicides.Effortstofindaneffectivecontroltechniquewhich issafeforworkers,theenvironmentandconsumers,areneeded

(JanisiewiczandKorsten,2002).Biologicalcontrol(Pimentaetal.,

2010),plantextracts(Mekbib etal., 2007),low toxicity

chemi-cals(Smilanicketal.,1999),andphysicaltreatments(Poratetal.,

2000) are possible methods to control P. digitatum.The use of antagonisticbacteriaandfungiareoftenusedinbiological con-trol methods. Wilsonand Wisniewski (1989) summarizedthat antagonistmicrobescouldbepotentialantagonists;theantagonist shouldhavetheabilitytocolonizeandsurvivein the commod-ity,andbecompatiblewithotherpostharvestpractices,processes, andothercontrolmethodsincludingchemicals.Inaddition, orga-nismsmustbesuitableforlarge-scaleproductionusinglowcost substrates.Amongmodesofactions,competitionfornutrientsand spacebetweenpathogenandantagonistisconsideredadesirable modeofactioninpostharvestcontrol(Ippolitoetal.,2000).

Manystudiesusingplantextractssuchasessentialoilsandpure compoundsagainstplantpathogenicfungihavebeenconducted. A number ofantifungal compounds havebeen foundinplants, derivedfromsecondarymetabolitessuchastannins,terpenoids, alkaloids,andflavonoids(Arifetal.,2009).

Thecombinationofdifferentmethodscouldprovidemore effec-tivecontrolandconsistentresultscomparedwithsingletreatments

(El-Ghaouth et al., 2004).Treatments withmixed aqueous leaf

extracts of Solonum virginionum and Trichoderma viridae were foundtoeffectivelycontrolanthracnosediseaseoftomatoby70%

(Mogle,2011).AtreatmentcomprisingBacillusamyloliquefaciens

HF-01combinedwith50␮gmL−1_{teasaponinwasaseffectiveas} afungicidetreatmentandsubsequentlyresultedinexcessof90% controlofgreenandbluemoldsandsourrot(Haoetal.,2011). Theapplicationofcarnaubawaxformulacontainingeitheryeast

SaccharomycescerevisiaeorthecommercialbackingyeastofS.

cere-visiae mixture(CBY) combinedwithpeppermint oil(1%) had a

superioreffectinreducinggraymold,softrotandblackrot inci-denceintomatoes(Abd-Allaetal.,2009).

Theobjectiveofthepresentresearchwastoselectplantextracts andyeastsbothinvitroandinvivotocontrolP.digitatumincitrus fruit.

2. Materialsandmethods

2.1. Pathogeninoculum

AhighlyvirulentstrainofP.digitatumwasobtainedfrom decay-ing“SaiNumPung”tangerinefruit.Thepathogenwasgrownon potatodextroseagar(PDA)at25◦_{Cforsevendaysandaconidial} suspensionwasprepared.Conidiaconcentrationwasdetermined bya haemacytometerandadjustedto1×105conidiamL−1 with steriledistilledwater.

2.2. Antagonists

Sevenantagonistyeastswereusedinthisresearch,including

Candida tropicalis TISTR5010/ATCC13803 (http://www.biotec.

or.th/tncc/dbstore/detail.asp?DB=TISTR) used for production of

xylitol (Choi et al., 2000), Pichia membranefaciens TISTR 5093 isolatedfromcoconuttoddyandusedforesterproduction(http://

www.tistr.or.th/tistrculture/listen.php?type=y&key=P), Candida

utilis TSITR 5001, used for production of single cell protein

from cassava (Trien et al., 2000), Aureobasidium pullulans

TISTR 3384 (http://www.straininfo.net/strains/505092) used forfructo-oligosaccharide(FOS)productionforprebioticpurposes

(Fungsin et al., 2012), Candida guillermondii BCC 5389 (http://

www.biotec.or.th/tncc/dbstore/detail.asp?pg=113&wr=&DB=BCC),

CandidasakeTISTR5143fromdecayedfruit(http://www.biotec. or.th/tncc/dbstore/StrainDetails.asp?Genus=Candida&Species=

sake&id=376&DB=TISTR),andDebaryomyceshanseniiTISTR5155

(http://www.tistr.or.th/tistrculture/listen.php?type=y&key=D)

usedforeconomicalkefiranproduction(CheirsilpandRadchabut, 2011).Alltheaboveinformationwasaccessedon18December 2012.

TheyeastswereacquiredfromtheNationalScienceand Tech-nologyDevelopmentAgency(NSTDA)andtheThailandInstituteof ScientistandTechnologicalResearch(TISTR).Theyeastcellswere culturedonyeastmaltextractagar(YMA)andincubatedat28◦_C for48h.Thecellsuspensionwasthenpreparedandaddedto10mL ofsteriledistilledwaterandcountedwithahaemocytometer.The cellsuspensionwasadjustedto1×108cells/mL(Nunesetal.,2001;

Yuetal.,2007).

2.3. Fruitpreparation

“SaiNumPung”tangerinefruit(CitrusreticulataBlanco)were obtainedfromacommercialorchardinFang,ChiangMaiprovince, Thailand, and fruit free of defects were chosen. The fruit sur-facesweredisinfectedbyimmersionin1%sodiumhypochlorite for3min,rinsedwithsterilewater,anddryinginasterile cham-ber.Thesemi-commercialtest didnotimmersion in1%sodium hypochlorite.

2.4. Plantextractmaterials

Thefollowingplantextracts:Cymbopogoncitratusstem,Zingiber officinalerhizome,Momordicacharantiafruit,Curcuma longa rhi-zome,driedEugeniacaryophyllataflowerbud,Cinnamomumcassia

bark,andTinosporacrispabarkwereusedinthisresearch.Thefresh plantsweredriedundershadeandblendedtopowder.Powdered materialwithamassof300gwassoakedin400mL96%ethanol forthree dayswithfrequentagitation.Themixturewasfiltered throughWhatmanno.1filterpaperandthecrudeextractwas col-lected.Thecrudeextractwasdistilledat40◦_{Cwitharotavaporat} 200mbar.Anextractwascollectedandmixedinaratioof1:3of CH2Cl2andleftfor30minpriortofiltration.Thefiltratewasdried

inarotaryevaporatorandsubsequentlyaddedto20%ethanoland keptat−20◦Cuntilused.

2.5. Plantcrudeextractscreening

The screening of the plant crude extracts for their efficacy againstthefungalpathogenP.digitatumwasconductedwiththe poisonousfoodtechnique.Forthispurpose,potatodextroseagar wasamendedwithplantcrudeextractataconcentrationof0(no plantextractadded),5000,10,000,15,000,and20,000mg/Lplus 2%citrusjuice.Asmallblock(2cm×2cm)wasasepticallycutand inoculatedwiththeP.digitatumonthesidesofanagarblockand thenincubatedonamoistplatefor48h.P.digitatumgrowthwas investigatedbyaddinglactophenolcottonblue(Harris,1986;Woo etal.,2010).Fivereplicateswereusedforeachplantextract. Inhi-bition(%)offungalradialgrowthwascalculatedusingtheformula: ((radiusofcontrol−radiusoftreatment)/radiusofcontrol)×100%

(SkidmoreandDickinson,1976).

2.6. Plantcrudeextractscreeningoncitrusfruit

Ten␮Lofconidialsuspension(1×105conidia/mL)wasadded, usingasterilepipette,tothecitruswoundtoadepthof3mmand allowedtodryunderasepticconditions.Then10␮Lofplantcrude extractor10␮Lofsterilewater(controltreatment)wasappliedto thewound.Theinoculatedfruitwereincubatedina100%RH cham-berat25◦_{Cfor7days.Eachtreatmenthadthreereplicationsand} theexperimentwasarrangedinarandomizedblockdesign.Disease incidenceanddiseaseseveritywereobservedafter7daysof incu-bation.Tocalculatediseaseincidence,thenumberofinfestedcitrus fruitwasobserved.Thediseaseincidencewascalculatedusingthe formula:

Diseaseincidence

=(numberofinfectedfruit/totalnumberoffruitassessed)×100.

Thediseaseseveritywasdeterminedaccordingtotheportionof theinfectedareaofthefruit.Thediseaseseveritywascalculated usingtheformula(Masoodetal.,2010):

Diseaseseverity=(infectedtissuearea/totaltissuearea)×100.

2.7. Antagonistscreening

Interaction between yeasts and P. digitatum hyphae was assessedfordirectparasitismin90mmdiameterPetridisheswhich containedPDAmediumwithdualculturessegregatedby3cm.The radialgrowthofthemyceliumtowardtheyeaststripwas mea-suredafterstorageatroomtemperaturefor7days.TenPetridishes per treatmentwereused.Thepercentageofinhibition ofradial growth(PIRG)wasrecorded.Thescreeningtestswereperformed toselecttwopromisingyeasts.TheformulaofPIRGwasdefinedas: ((radiusofcontrol– radiusoftreatment)/radiusofcontrol)×100%

2.8. AntagonistandplantcrudeextractstocontrolP.digitatum oncitrusfruit

Twopromisingplantcrudeextractsandtwoyeastsweretested oncitrusfruitbothindividuallyandincombination.Ten␮Lof coni-dialsuspension (1×105conidia/mL)of P.digitatumwereadded usingasterilepipette tothecitrus woundtoa depthof3mm, madewithasterileneedle,andallowedtodryunderaseptic con-ditions.Ten␮Lofcellsuspensionofyeastat1×108cells/mLwere alsoaddedonthewound.Afterdrying,10␮Loftheplantcrude extractwereaddedtothewoundsite,andthenincubatedunder a100%RH,at25◦_{C.Diseaseincidenceanddiseaseseveritywere} observedafter7daysincubation.Eachtreatmenthadthree repli-cationsandtheexperimentwasarrangedinarandomizedblock design.

2.9. EffectsofantagonistandplantextractsonP.digitatumand yeastcolonizationoncitrusfruit

The colonization ability of P. digitatum and yeast on citrus woundswasobservedinthetreatedfruit(addedwith10␮Lof coni-dialsuspensionofP.digitatumat1×105conidia/mL,then10␮L ofcellsuspensionofyeastat1×108cells/mL,andfinally,10␮L ofplantcrudeextract).In addition,thecolonizationabilitywas observedin thecontrol (addwith10␮L of sterilewater).Both thetreatedandthecontrolfruitwereincubatedat100%RHand 25◦_{C.Thecolonizationabilitywasobserved2,4,6,and8hafter} treatment.Themethodusedwasbasedonthemethoddescribed

byPimentaetal.(2010);1cm2_{ofcitruspeelaroundprickedarea}

wascutintosmallpieces(0.5mm×5mm)andthensubsequently culturedinbothPDAandYMAmedia.ThenumberofP.digitatum

andyeastcolonizedpieceswereobservedduring7days incuba-tion.Eachtreatmenthad5replicationsandtheexperimentwas arrangedinarandomizedblockdesign.ColonizationofP. digita-tumandyeastwasdeterminedwithaformuladefinedas:number ofcolonizedofcitruspeel/totalpiecesofcitruspeel×100%.

2.10. Efficacyofyeastandplantextractsinreducingnatural decaydevelopmentoncitrusfruit

Theeffectivenessofyeastandplantextractstocontrolnaturally infectedcitrusfruitwasdeterminedinsemi-commercial experi-ment.Thebestcombinationofplantextractandyeastwastested andcomparedtoImazalil(150mg/L),waterand20%ethanol.Allthe treatments200fruitweredippedinthesuspensionofyeastand plantextract,imazalil,wateror20%ethanolfor2min,thendried andpackedintoplasticboxesandcoverwithplasticbags.After storageat25◦_{C,95%RHfor2weeks,diseaseincidenceanddisease} severitywererecorded.Eachtreatmenthadfourreplicationsand theexperimentwasarrangedinarandomizedblockdesign.

2.11. Effectsofyeastandplantextractsonpostharvestqualityof citrusfruit

Theeffectofyeastandplantextractonpostharvestqualityof citrusfruitwasexamined.Weightloss(%),fruitfirmness(kg),total juicepercentage(%),totalsolublesolids(%),titratableacidity(%), andascorbicacidcontent(mg/100g)offruitweredeterminedfrom 20 symptomlessfruit, randomly selectedfrom each replication afterstorageat25◦_{Cforthreeweeks.Forweightloss,fruitwere} weighedbeforetreatment(A)andafterstorage(B),andtheweight losscalculated as (A/B)/A. Fruit firmnesswasmeasured atfour pointsoftheequatorialregionusingafirmnesstester(N.O.W., FHR-5)witha5mmprobe.Totaljuicepercentagewascalculatedusing theformula: (juiceweight/fruitweight)×100. Thetotalsoluble solidsinjuiceweredeterminedwithadigitalrefractometer(N1-E,

AtagoCo.,Tokyo,Japan) (Laceyetal.,2009).Thetitratable acid-itywasmeasuredbytitrationwith0.1MNaOHpH8.3(Hernández

etal.,2006).

2.12. Determinationofactiveingredientsofplantextracts

Athinlayerchromatography(TLC)methodwasusedfor frac-tionation of plant crude extracts. The solvent combinations of toluene, dichloromethane, and acetone were tested to obtain the bestseparationof active ingredients. Purityof the isolated compoundswasconfirmedbyhighperformanceliquid chromatog-raphy(HPLC).TheHPLCmeasurement wascarried outusingan Agilent1100serieswithUV-DADdetectionat230nmwitha ref-erencewavelengthat600nm.Theseparationwasaccomplished by using a Hypersil BDS C-18 column with the dimensions of 250mm×4.6mmand5␮mparticlesize.Themobilephasewas anaqueousbuffercontaining15mMortho-phosphoricacidand 1.5mMtetrabutylammoniumhydroxide(A)andmethanol(B)with a linear gradient 0–15min: 20–90% B followed by 100% Bfor 5min.Thiswaskeptforafurther8min.Theflowratewassetto 0.8mL/minandtheinjectionvolumewas10␮L.Identificationof thechemicalformulaofthecompoundwasobtainedusingnuclear magneticresonancespectroscopy(NMRs),usingaBrukerDRX-400 AVANCEspectrometer(Bruker,Rheinstetten,Germany)equipped with400.13MHz(1_{H)or100.61MHz(}13_{C),andTopspin1.2}

soft-warewasused.

2.13. Statisticalanalysis

All data were analyzed by statistical analysis of variance (ANOVA)andregressionanalysisusingStatisticalAnalysisSystem (SAS)software.Statisticalsignificancewasassessedatp<0.05and Tukey’SHSDmultiplerangetestwasusedtoseparatemeans.

3. Resultsanddiscussion

3.1. Effectsofplantcrudeextractsonpathogengrowth(invitro)

The plant crude extracts at concentrations of 10,000–20,000mg/L reduced P. digitatum hyphal growth. The resultsshowedE.caryophyllataandC.longacrudeextractswere effectiveto reduceP.digitatumhyphalgrowthby100% and up to 70%, respectively, more than the other plantcrude extracts

(Table 1).As such, E. caryophyllata and C. longa crude extracts

wereestablishedasthemostpromisingcrudeextractstocontrol

P.digitatuminvitro.Theantifungal activityofclovemaybedue toitsactivecompound,eugenol,whichisthemainconstituentof cloveoil.Themechanismofeugenolasaneffectiveantagonistis inincreasingthepermeabilityof thecells andcausing irregular branchingof hyphae in theapical partsand a loss of linearity, withcollapsingandsquashingduetothelackofcytoplasm(Xing

etal.,2012).LaksanaphisutandSangchot(2010)reportedthata

crudeextractofturmericpowderin20%ofethanolat30,000ppm reduceddiseaseincidenceofgreenmolddiseaseontreatedfruit by25%.

3.2. EffectsofyeastonP.digitatumgrowth(invitro)

C.tropicalisTISTR5010andotherCandidaspp.werefoundto inhibitmycelialgrowthofP.digitatumby78.0%andP. membrane-faciensTISTR5093by69.3%(Fig.2).Furthertestsrevealedthatboth

Table1

Invitroscreeningofsevenplantcrudeextractsdissolvedin20%ethanolatdifferentconcentrationsmixedwithPDAplus2%citrusjuiceontheirinhibitionofhyphalgrowth ofP.digitatumat24hat25◦_C.

Treatment PercentageofinhibitionofP.digitatumhyphalgrowth(%)

5000mg/L 10,000mg/L 15,000mg/L 20,000mg/L

Cymbopogoncitratus 31.35c 40.08e 49.21fg 53.97h

Zingiberofficinale 53.97h 55.95hi 55.30hi 69.05m

Momordicacharantia 23.89b 33.73d 58.89j 64.29kl

Curcomalonga 46.67f 64.68k 73.41m 78.97o

Eugeniacaryophyllata 100.00p 100.00p 100.00p 100.00p

Cinnamomum 0.00a 0.00a 50.86g 75.00n

Tinosporacrispa 42.86e 55.95hi 56.75ij 65.87l

20%ethanol 0.00a 0.00a 0.00a 0.00a

ValueswiththesameletterswerenotsignificantdifferentfromeachotherbasedontheTukey’SHSDmultiplerangetest(p=0.05).Eachvaluewasmeanoffivereplicates.

0

Fig.1.Effectofplantcrudeextractsondiseaseseverity(%)anddiseaseincidence(%)ofgreenmoldoncitrusfruitswhichweretreatedwithP.digitatum(1×105conidia/mL, 10␮L),incubatedat25◦_{Cand95%RHforsevendays.(Control)ethanol20%(A)crudeextractofEugeniacaryophyllata(15,000mg/L,10␮L);(B)Curcumalonga(30,000mg/L,}

10␮L).Eachvalueismeanofthreereplicates.Barsrepresentthestandarddeviationsofthemean.Statisticalsignificancedeterminedatp<0.05accordingtoTukey’sHSD test.

3.3. Effectsofplantcrudeextractsandyeastsongreenmoldrot control

E. caryophylata at 15,000mg/L and C. longa at 30,000mg/L reduceddiseaseseverityanddiseaseincidence(Fig.1).Allofthe combinationsofplantcrudeextractsandyeastsreduceddisease incidenceinexcessof70%.ThecombinationofE.caryophylataat 15,000mg/L,10␮L,andC.utilisTITR5001at1×108,10␮L,was

Fig.2.EffectofyeastonhyphalradialgrowthinhibitionofP.digitatumwithdual culturessegregatedbythreecentimeters,incubatedat25◦_{Cand95%RHforseven}

days.(A)CandidasakeTISTR5143;(B)CandidatropicalisTISTR5010;(C)P. mem-branefaciensTISTR5093;(D)DebaryomyceshanseniiTISTR5155;(E)Aureobasidium pullulansTISTR3389;(F)CandidaguillermondiiBCC5389;(G)CandidautilisTISTR 5001.Eachvalueismeanoffivereplicates.Barsrepresentthestandarddeviations ofthemean.Statisticalsignificancedeterminedatp<0.05accordingtoTukey’sHSD test.

established tobethebestcombination toattain a reduction in disease incidenceby 90.3%, while thecombinations of C. longa

at30,000mg/L,10␮L,andC.utilisTITR5001at1×108,10␮L,E.

caryophylataat15,000mg/L,10␮L,andC.tropicalisTISTR5010at 1×108,10␮L,C.longaat30,000mg/L,10␮L,andC.tropicalisTISTR 5010at1×108,10␮L,resultedinareductioninthedisease inci-denceby80.7%,77.4%,and71%,respectively(Fig.4a).Likewise,for

0

Fig.3. Effectofyeast(s)onhyphalradialgrowthinhibitionofP.digitatumwithdual culturessegregatedbythreecentimeters,incubatedat25◦_{Cand95%RHforseven}

0

Green mold rot incidence (%)

Treatment

Green mold rot severity (%)

Treatment

Fig.4.Effectofplantextract(Eugeniacaryophylatacrudeextractat15,000mg/L, andCurcumalongacrudeextractat30,000mg/L)andyeast(CandidautilisTISTR 5001at1×108_{andCandidatropicalisTISTR5010at1×10}8_{)oncontrolofgreen} moldrot(Penicilliumdigitatum)incidence(a)anddiseaseseverity(b)inwounded citrusfruit,incubatedat25◦_{Cand95%RHforsevendays.(A)E.caryophylataand} C.utilisTISTR5001;(B)C.longaandC.utilisTISTR5001;(C)E.caryophylataand C.tropicalisTISTR5010;(D)C.utilisTISTR5001;(E)C.longaandC.tropicalisTISTR 5010;(F)E.caryophylata;(G)C.longa.Eachvalueismeanofthreereplicates.Bars representthestandarddeviationsofthemean.Statisticalsignificancedetermined atp<0.05accordingtoTukey’sHSDtest.

diseaseseverity,thecombinationofE.caryophylataat15,000mg/L, 10␮L,andC.utilisTITR5001at1×108,10␮L,wasthebest combi-nationtoattainareductionindiseaseseverityby96.26%,whilethe combinationsofC.longaat30,000mg/L,10␮L,andC.utilisTITR 5001at1×108,10␮L,E.caryophylataat15,000mg/L,10␮L,and

C.tropicalisTISTR5010at1×108,10␮L,C.longaat30,000mg/L, 10␮L, and C.tropicalisTISTR 5010at1×108,10␮Lresulted in reductionsindiseaseseverityby93.2%,90.3%,and89.4%, respec-tively(Fig.4b).

Citrus peels werecultured both onPDA and YMAmediato observeP.digitatum and yeastscolonizationin the wound site after2,4,6,and8hofapplication.P.digitatumcolonizationwas thelowestoncitrustreatedwiththecombinationofE. caryophy-latacrudeextractat15,000mg/L,10␮L,andC.utilisTITR5001at 1×108,10␮L(Fig.5).SurvivalofC.utilisTITR5001wasthehighest onfruittreatedwithE.caryophylatacrudeextractat15,000mg/L, 10␮L,andC.utilisTITR5001at1×108,10␮L(Fig.6).Inthe infec-tionsite,colonizationoftheantagonistwashigherthanpathogen colonization,showingastrongrelationship(R2_{=0.8287)(Fig.7).}

AcombinationofE.caryophylatacrudeextractandC.utilisTITR 5001reducednaturaldevelopmentofgreenmoldrotincidenceby 90%andseverityby86%,and150mg/LImazalil,and20%ethanol,by 10%and5%ofdiseaseincidenceand25.4%,8.76%ofdisease sever-ity,respectively.Theweightlossandjuicecontentofcitrusfruit beforeandafterstorageat25◦_{C,95%RHfor3weekswerenot} dif-ferent(Fig.8).Ontheotherhand,fruitfirmness,totalsolublesolids,

0

Fig.5.P.digitatumcolonizationoncitruspeel,treatedwithEugeniacaryophyllaat 15,000mg/LandCandidautilisTISTR5001at1×108attwo,four,six,andeighthours

afterinoculationat25◦_{C.Eachvalueismeanoffivereplicates.C=E.caryophylla;}

Y=C.utilisTISTR5001;P=P.digitatum.

Fig.6. CandidautilisTISTR5001colonizationoncitruspeel,treatedwithEugenia caryophyllaat15,000mg/LandCandidautilisTISTR5001at1×108attwo,four,

six,andeighthoursafterinoculationat25◦_{C.Eachvalueismeanoffivereplicates.}

C=Eugeniacaryophylla;Y=CandidautilisTISTR5001;P=P.digitatum.

andascorbicacidcontentweredifferentbetweenbeforeandafter storage(Table2).Bardinetal.(2003),reportedthatabiocontrol agentandsyntheticchemicalorplantmaterialcombined applica-tions,providedbetterplantprotectionthanindividualtreatments. Thecombinationof Paeoniasuffruticosa(medicinalplant)andT. harzianumwasmoreeffectivethaneithertreatmentwitha sin-gleagenttocontrolRhizoctoniadamping-off(Leeetal.,2008).The resultsagreewithLeeetal.(2011)whoinvestigated55species ofmedicinalplantsfortheirantifungalactivityagainstRhizoctonia solaniAG2-1toimprovethebiocontrolefficacyofT.harzianum invitro.Sixspecieswerefoundtobeeffectiveandamongthese,E. caryophyllataflowerbud,andCinnamomumloureiriistembarkwere

y = -0.014x2_{+ 1.228x + 11.35}

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00

P

. digitatum

Coloniz

ation (%

)

C. utilis colonization (%)

0 2 4 6 8 10 12

Disease Incidence (%)

Treatment

0 1 2 3 4 5 6 7 8 9

Dsease Severity (%)

Treatment a

A B C D

a

A B D C

b

b _b

b

b b

(a) _(b)

Fig.8. EffectofEugeniacaryophylatacrudeextractat15,000mg/L,andyeast(CandidautilisTISTR5001at1×108)forreducingnaturalrotdevelopmentofthedisease

incidence(a)anddiseaseseverity(b).(A)EugeniacaryophylataandCandidautilisTISTR5001;(B)Imazalil150mg/L;(C)water;(D)ethanol20%.Eachvalueismeanoffour replicates.Barsrepresentthestandarddeviationsofthemean.Statisticalsignificancedeterminedatp<0.05accordingtoTukey’sHSDtest.

Fig.9.HPLCchromatogramofEugeniacaryophyllaextract,recordedat254nm.

Table2

EffectofE.caryophylataandC.utilisTISTR5001onpostharvestqualitiesofcitrusfruits.

Treatment Weightloss

(%)

Fruitfirmness (kg)

Juicecontent (%)

Totalsolublesolid (%)

Titratableacidity (%)

Ascorbicacidcontent (mg/100g)

E.caryophylataandC.utilis 1.96a 1.59b 36.53a 9.15b 0.33a 16.61b

Imazalil150mg/L 1.91a 1.60b 36.73a 9.80b 0.19a 15.01b

Water 2.17a 1.51b 36.20a 9.70b 0.18a 15.09b

Ethanol20% 3.84a 1.57b 37.74a 9.65b 0.19a 16.15b

Beforestorage 1.34a 40.61a 5.90a 1.73b 12.15a

ValueswiththesamelettersincolumnwerenotsignificantdifferentfromeachotherbasedontheTukey’SHSDmultiplerangetest(p=0.05).Eachvaluewasmeanoffour replicates.

themosteffectiveagainstR.solaniAG2-1mycelialgrowth,withan inhibitoryefficacyof73.7%and71.1%,respectively(Table3).

3.4. Determinationofactiveingredientsofplantextracts

The chromatograms showed the presence four peaks with retentiontimesbetween15and20min.Thepeakoftheisolated compound at 16.085min was detected by HPLC and identified bycomparisonofretentiontimeandUVspectraofthepeaksin thesamplesolutionwiththestandardeugenol.Thestandardand isolatedcompoundshowedasinglepeakthatconfirmedthe com-poundwaseugenol(Figs.9and10).The1_HNMRandthe13_CNMR

providedfurtherevidencefortheconfirmationofthestructureof

Table3

13_Cand1_{HchemicalshiftofNMRspectraofEugeniacaryophyllataextract.}

Atomnumber 13_C 1_H

1 143.85 –

2 146.39 –

3 111.06 6.73a

4 131.86 –

5 121.12 6.72a

6 114.21 6.89

1′ _{39.83 3.37}

2′ _{137.78 6.00}

3′ _{115.46 5.11}

Fig.10.UVspectraofthepeaksshownintheHPLCchromatogramofEugenia caryophyllaextract(a);reference(b).

theisolatedcompound. Basedonthemassspectraldata,itwas concludedthattheisolatedcompoundwaseugenol.

4. Conclusions

ThecombinationofE.caryophylatacrudeextractandC.utilis TITR5001hadsignificanteffectsasanalternativetreatmentin con-trollinggreenmold.E.caryophylatacrudeextractandC.utilisTITR 5001couldbeanidealtreatmentfor futureplantdisease man-agement.Furthermore,furtherstudy shouldbefocused onthis combinationwithothercontrolstrategies,suchaswaxing, atmo-sphereconditions,carrierandadhesivematerials,todevelopan effectiveapproachforpostharvestdiseasecontrolofcitrusfruit.

References

Abd-Alla,M.A.,Nehal,S.E.M.,Nadia,G.E.G.,2009.Formulationofessentialoilsand yeastforcontrollingpostharvestdecayoftomatofruits.PlantPathol.Bull.18, 23–33.

Arif,T.,Mandal,T.K.,Dabur,R.,2009.Naturalproducts—antifungalagentsderived fromplants.J.AsianNat.Prod.Res.11,621–638.

Bardin,S.D.,Huang,H.C.,Koyer,J.R.,2003.ControlofPythiumdampingoffofsugar beetbyseedtreatmentwithcropstrawpowdersandabiocontrolagent.Biol. Contr.4,1–8.

Cheirsilp,B.,Radchabut,S.,2011.Wheylactosefromdairyindustryforeconomical kefiranproductionbyLactobacilluskefiranofaciensinmixedcultureswithyeasts. NewBiotechnol.28,574–580.

Choi,J.H.,Moon,K.H.,Ryu,Y.W.,Seo,J.H.,2000.Productionofxylitolincellrecycle fermentationsofCandidatropicalis.Biotechnol.Lett.22,1625–1628.

Eckert,J.L.,Eaks,I.L.,1989.Postharvestdisordersanddiseasesofcitrusfruits.In:W. Ruther,W.,Calaran,E.C.,Carman,G.E.(Eds.),TheCitrusIndustry:Crop Protec-tion,PostharvestTechnologyandEarlyHistoryofCitrusResearchinCalifornia. UniversityofCalifornia,Berkeley,pp.179–260.

El-Ghaouth,A.,Wilson,C.L.,Wisniewski,M.E.,2004.Biologicallybasedalternatives tosyntheticfungicidesforthepostharvestdiseasesoffruitandvegetables.In: Naqvi,S.A.M.H.(Ed.),DiseaseofFruitandVegetables:Diagnosisand Manage-ment.KluwerAcademicPublisher,TheNetherlands,pp.511–535.

Fungsin, B., Saman, P., Meeploy, M., Chatanon, L., Srichuai, A., Sukcharurn, J., Artjariyasripong, S., 2012. Study on fungi in their ability of fructo-oligosaccharides (FOS) production for prebiotic purpose. In: The 8th International Symposium on Biocontrol and Biotechnology, Thailand, pp.190–196.

Hao,W.,Li,H.,Hu,M.,Yang,L.,Rizwan,M.,2011.Integratedcontrolofcitrusgreen andbluemoldandsourrotbyBacillusamyloliquefaciensincombinationwith teasaponin.PostharvestBiol.Technol.59,316–323.

Harris,J.L.,1986.Modifiedmethodforfungalslideculture.J.Clin.Microbiol.24, 460–461.

Hernández,Y.,Lobo,M.G.,González,M.,2006.DeterminationofvitaminCintropical fruits:acomparativeevaluationofmethods.FoodChem.98,654–664. Holmes, G.J., Eckert, J.W., 1999. Sensitivity of Penicillium digitatum and P.

italicum to postharvestcitrus fungicides inCalifornia. Phytopathology 89, 716–721.

Ippolito,A.,El-Ghaouth,A.,Wilson,C.L.,Wisniewski,M.A.,2000.Controlof posthar-vestdecayofapplefruitbyAureobasidiumpullulansandinductionofdefense responses.PostharvestBiol.Technol.19,265–272.

Janisiewicz,W.J.,Korsten,L.,2002.Biologicalcontrolofpostharvestdiseasesoffruit. Annu.Rev.Phytopathol.40,411–441.

Kinay,P.,Mansourc,M.F.,Gablerb,F.M.,Margosanc,D.A.,Smilanick,J.L.,2007. Char-acterizationoffungicide-resistantisolatesofPenicilliumdigitatumcollectedin California.CropProt.26,647–656.

Laksanaphisut,S.,Sangchot,S.,2010.Controlofgreenmoldrotofcitruscaused byPenicilliumdigitatum,withpartialpurifiedextractofturmericandchitosan. Agric.Sci.J.41,287–290.

Lee,T.O.,Khan,Z.,Kim,S.G.,Kim,Y.H.,2008.Amendmentwithpeonyrootbark improvesthebiocontrolefficacyofTrichodermaharzianumagainstRhizoctonia solani.J.Microbiol.Biotechnol.18,1537–1543.

Lee,H.M.,Khan,Z.,Kim,S.G.,BaekNam-InKim,Y.H.,2011.Evaluationofthe bio-controlpotentialofsomemedicinalplantmaterialsaloneandincombination withTrichodermaharzianumagainstRhizoctoniasolaniAG2-1.PlantPathol.J. 27,68–77.

Lacey,K.,Hancock,N.,Ramsey,H.,2009.Measuringinternalmaturityofcitrus.Farm Note354,1–4.

Macarisin, D., Cohen, L., Eick, A., Rafael, G., Belausov, E., Wisniewski, M., Droby, S., 2007. Penicillium digitatum suppresses production of hydrogen peroxideinhosttissueduringinfectionofcitrusfruit. Phytopathology97, 1491–1500.

Masood,A.,Saeed,S.,Iqbal,N.,2010.Methodologyfortheevaluationof symp-tomsseverityofmangosuddendeathsyndromeinPakistan.Pak.J.Bot.42, 1289–1299.

Mekbib,S.B.,Thierry,J.C.,Regnier,I.,Korsten,L.,2007.ControlofPenicillium digita-tumoncitrusfruitusingtwoplantextractsandstudyoftheirmodeofaction. Phytoparasitica35,264–276.

Mogle,U.P.,2011.Efficacyofbiofertilizerandplantextractagainstantracnose dis-easeoftomato.Biosci.Discov.2,104–108.

Nunes,C.,Teixido,N.,Usall,J.,Vinas,I.,2001.Biologicalcontrolofmajorpostharvest diseasesonpearfruitwithantagonisticbacteriumPantoeaagglomerans(CPA-2). ActaHortic.553,403–404.

Pimenta,Silva,R.S.,Coelho,J.F.M.,Morais,C.M.,Rosa,P.B.,CorrêaJr.,C.A.,Braz,A., 2010.IntegratedcontrolofPenicilliumdigitatumbythepredaciousyeast Sac-charomycopsiscrataegensisandsodiumbicarbonateonoranges.J.Microbiol.41, 404–410.

Porat,R.,Lers,A.,Dori,S.,Cohen,L.,Ben-Yehoshua,S.,Fallik,E.,Droby,S.,Lurie,S., 2000.InductionofresistanceagainstPenicilliumdigitatumandchillinginjury instarrubygrapefruitbyashorthotwater-brushingtreatment.J.Hort.Sci. Biotechnol.75,428–432.

Skidmore,A.M.,Dickinson,C.H.,1976.Colonyinteractionsandhyphalinterference betweenSeptenanodurumandphylloplanefungi.Trans.Br.Mycol.Soc.66, 57–64.

Smilanick,J.L.,Margosan,D.A.,Milkota,F.,Usall,J.,Michael,I.F.,1999. Control of citrus green mold by carbonate and bicarbonate salts and the influ-ence ofcommercial postharvestpractices ontheir efficacy.Plant Dis. 83, 139–145.

Trien,L.H.,Tasakorn,P.,Chavadej,S.,2000.Productionofsinglecellproteinfrom cassavabymixedcultureofEndomycopsisfibuligeraTISTR5097andCandida utilisTISTR5001.SCIRESCU25,137–144.

Wilson,C.L.,Wisniewski,M.E.,1989.Biologicalcontrolofpostharvestdiseases offruitandvegetables:anemergingtechnology.Annu.Rev.Phytopathol.27, 425–441.

Woo,P.C.Y.,Ngan,A.H.Y.,Chui,H.,Lau,S.K.P.,Yuen,K.,2010.Agarblocksmear prepa-ration:anovelmethodofslidepreparationforpreservationofnativefungal

structuresformicroscopicexaminationandlong-termstorage.J.Clin.Microbiol. 48,3053–3061.

Xing,Y.,Xu,Q.,Li,X.,Che,Z.,Yun,J.,2012.Antifungalactivitiesofcloveoilagainst Rhi-zopusnigricans,AspergillusflavusandPenicilliumcitrinuminvitroandinwounded fruittest.J.FoodSafety32,84–93.