Use of Killer Yeast in the Management of Postharvest Papaya Anthracnose

5/20/2018 Use of Killer Yeast in the Management of Postharvest Papaya Anthracnose

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Postharvest Biology and Technology83 (2013) 5864

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Postharvest Biology and Technology

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Use ofkiller yeast in the management ofpostharvest papaya anthracnose

J.R. Limaa,, D.M.F. Gondime,J.T.A. Oliveirab, F.S.A. Oliveirad, L.R.B. Goncalvesc, F.M.P. Vianad

a Programa de ps-graduaco em Biotecnologia, RENORBIO, Universidade Federal do Cear, Empresa Brasileira de Pesquisa Agropecuria, EMBRAPA Laboratrio de Patologia

Ps-colheita, RuaDra. Sara Mesquita, 2270, Planalto do Pici, CEP 60511-110, Fortaleza, CE, Brazilb Universidade Federal do Cear UFC, Dep. de Bioqumica e BiologiaMolecular, Campus do PICI, CEP 60451-970, Fortaleza, CE, Brazilc Departamento Engenharia Qumica, Universidade Federal do Cear UFC, Campus do PICI, CEP60455-760, Fortaleza, CE, Brazild Empresa Brasileirade PesquisaAgropecuria,EMBRAPAAgroindstria Tropical, Laboratriode PatologiaPs-colheita, RuaDra. SaraMesquita,2270, Planaltodo Pici,CEP 60511-110,

Fortaleza, CE, Brazile Universidadede Fortaleza UNIFOR, Fortaleza, CE, Brazil

a r t i c l e i n f o

Article history:

Received30 October 2012Accepted17March 2013

Keywords:

PostharvestMycoparasitismScanning electronmicroscopy SEMHydrolytic enzymes

a b s t r a c t

Theefficiencyoftwokilleryeast strains,Wickerhamomyces anomalus (strain422)andMeyerozymaguillier-mondii (strain 443), as biocontrol agents against Colletotrichumgloeosporioides, a postharvest anthracnoseagent ofpapaya and other tropical fruit, was assessed. These strains were previously selected throughin vitro assays, but in the present study, their in vivo action was assessed. In addition, the influence ofphytopathogen inoculation time on the fruit in combination with the use of the biocontrol agent wasalso assessed. We assessedmycoparasitism as an antagonistic mechanism ofaction by scanning electronmicroscopy (SEM). In addition, two hydrolytic enzymes, chitinase and -1,3 glucanase, were assayed.Our results indicated that W. anomalus (strain 422) andM. guilliermondii (strain 443) reduced diseaseincidence by 24.62%and 20.68%, respectively, for up to 6d after inoculation, when applied 3h before thephytopathogen and incubated in a wet chamber (95% RH) at 28 C. The time ofyeast inoculation had asignificant effect on its antagonistic action. Application ofthe yeasts 12 or 24h before the phytopathogeninoculation resulted in 13.75% and 30% ofdisease reductions for W. anomalus (strain 422) and 31.35%and 41.17% reductions for M. guilliermondii (strain 443), respectively. Electron micrographs confirmed

mycoparasitism by showing the interaction ofthe yeasts with C. gloeosporioideshyphae, causing in somecases, a loss of turgor and yeast penetration ofwalls with marked concavity formation on hypha cellwalls.

2013 Elsevier B.V. All rights reserved.

1. Introduction

The susceptibility of papaya to postharvest diseases is high,thus necessitating careful management. Among the postharvestpapaya diseases, anthracnose, caused by the fungal species Col-letotrichumgloeosporioides, isconsideredoneofthemostsignificantin northeastern Brazil (Serra and Silva, 2004; Vida et al., 2004). C.gloeosporioides strains are also associated with the occurrence ofthe so-called chocolate spot disease, which is characterized bysmallsurfacepatchesof reddish-browncolor(Andradeetal.,2007),in addition to anthracnose.

Postharvest losses are economicallymore damaging than thosein the field because they add to the costs of production, fruittransportation and storage. Such losses are one of the factors thatinfluence thedecisiontousechemicalagentsto control postharvestdiseases. However, there are growing concerns about the safety of

Corresponding author. Tel.: +55085 3391 7264; fax: +55085 33917222.E-mail addresses:[email protected],[email protected] (J.R. Lima).

chemical agents(Jamalizadehet al., 2008; Robiglio et al., 2011), andthe appearance of resistant phytopathogens (Pimenta et al., 2010;Lima et al., 2011) has boosted the demand for control alternativeswith low risks (Spadaro et al., 2004; Kefialew andAyalew, 2008) tohumansandother animals (Jamalizadeh et al., 2011; Li et al., 2011;Manso and Nunes, 2011), and that will not contaminate soil andgroundwater (Veiga et al., 2006; Neto and Sarcinelli, 2009).

Several alternative strategies to the use of fungicides has beenreported, among which physical control with the use of gammaand ultra violet radiation(Cia et al., 2007), use of edible coatingsbased on chitosan (Ali et al., 2010, 2012), useof essential oils alone(Bosquez-Molina et al., 2010) or in combination with other non-toxic compounds, such as gum arabic (Maqbool et al., 2011) andbiological control (Platania et al., 2012). Theuseofmicroorganismsas biocontrol agents representsone of themost viable alternatives(Janisiewicz and Korsten, 2002; Romeiro and Garcia, 2007; Rosaet al., 2010; Zhang et al., 2010).

Yeasts stand out among microorganisms as potential agents tocontrol plant diseases due to their ability to colonize the surface ofleaves and fruit (Rosa-Magri et al., 2011), high reproductive rate,

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J.R. Lima et al. / Postharvest Biology and Technology 83 (2013) 5864 5

lownutritional requirement(Droby andChalutz, 1994) andknowninnocuity, widely recognized by the use of yeast in industrial fer-mentation for hundreds of years (Coelho et al., 2003; Druvefors,2004). Furthermore, yeasts do not produce spores or mycotoxins,unlike other potential biocontrol microorganisms such as fila-mentous fungi (Fan and Tian, 2000). Yeast-based or yeast-likecommercial formulations forpostharvestrotbiocontrolarealreadyavailable in the market, such as Nexy, whose active ingredient is

CandidaoleophilayeaststrainO,andCandifruit

,registeredinSpainforuseasafungicideonfruitandwhoseactiveingredientis Candidasake yeast CPA-1 (Sundh and Melin, 2011).

In general, biological control occurs through a wide variety ofmechanisms in addition to competition for space and nutrients(Wang et al. , 2010; Rosa et al. , 2010). Several studies indicate theexistence of other biocontrol mechanisms, such as production ofenzymes that degrade the pathogen cell walls (Chanchaichaovivatet al., 2008), induction of resistance (Zhao et al., 2008; Nantawanitetal., 2010), generation of reactive oxygenspecies (Macarisinet al.,2010), mycoparasitism(Longet al., 2005) andproduction of toxins,which act on the cellwalls of phytopathogens (Coelho et al., 2003).

In the last two decades, several scientific studies have demon-strated the efficiency of yeasts as biocontrol agents against awide variety of phytopathogens (Wang et al., 2008; Hashem andAlamri, 2009; Rosa et al., 2010; Zhang et al., 2010; Kong et al.,2010; Li et al., 2011; Manso and Nunes, 2011; Robiglio et al.,2011; Rosa-Magri et al., 2011; Zhang et al., 2011). However, moststudies have focused on the search for biocontrol agents thatact on the pathogens of temperate climate fruit, for example,apples, pears, strawberries and grapes, but there remains onlylimited research on biocontrol agents toward tropical climate fruitpathogens.

The present study was aimed at assessing the in vivo effect oftwo killer yeast strains, Wickerhamomyces anomalus (strain 422)andMeyerozyma guilliermondii (strain 443), previously selected inan in vitro study, on the control of C. gloeosporioides, a papayaanthracnose-causing phytopathogen. In addition, the influence ofinoculation time on the biocontrol effectwasexamined alongwith

theoccurrenceof mycoparasitismbyscanningelectronmicroscopy(SEM). Lastly, the activities of the hydrolytic enzymes -1,3-glucanase and chitinasewere assayed.

2. Materials and methods

2.1. Fruit

Healthy Gold papayas (Carica papaya L.), with a uniform sizeand ripening stage, and without spots or bruises, were harvestedat the Cacimbo Farm, located in the municipality of Paraibapa,approximately 90km from Fortaleza, capital of the state of Cear,Northeastern Brazil. The fruit were taken to the Laboratory of

PostharvestPathologyatEmbrapaTropicalAgroindustry, Fortaleza,sanitizedwith 2.0% (v/v) sodium hypochlorite for 3min and left todry naturally.

2.2. Phytopathogen

A fungal strain was isolated from papaya with anthracnosesymptoms and identified as C. gloeosporioides Penz. The phy-topathogen wasmaintained on Potato Dextrose Agar (PDA; Difco)at 4 C. Conidial suspensions (1.0105 conidia/mL)were preparedfrom new cultures incubated for 10d at 28 C. The cells werecounted using a Neubauer chamber under optical microscope(Nikon E-200) and concentration adjusted to 1.0108 cell/mL

(Zhao et al., 2008).

2.3. Killer yeast

Thekilleryeastsisolatedandidentifiedinapreviousstudy(Limet al., 2012),W. anomalus (strain 422) andM.guilliermondii (strain443), were maintained at 4 C on PDA (Difco) until use. The rRNAD1/D2-region sequenceswere deposited into GenBankwith the IDnumbers JN627213 and JN627210, for strain 422 ofW. anomaluand strain 443 ofM. guilliermondii, respectively.

Yeast liquid cultures were grown in Falcon flasks containin30mLof yeast extract peptone dextrose (YEPD)media (10g/L yeasextract, 20g/L peptone, 20g/L glucose). The cultures were incubatedfor24hat28C.Medium-freesuspension cellswere obtainedby cell culture centrifugation at 1.5 g for 10min followed by successive washing (twice) with sterile distilled water. The pelletwere resuspended in steriledistilledwater up to a concentration o1.0108 cells/mL. Thecells were counted using a Neubauer chamber under optical microscope (Nikon E-200) and concentrationadjusted to 1.0108 cells/mL (Zhao et al., 2008).

2.4. Efficiency of W. anomalus (strain 422) and M.guilliermondii (strain 443) in papaya anthracnose control

In order to assess the efficiency of the yeasts as biocontroagents, themethoddescribedbyZhaoetal.(2008),withslightmodifications, was used. Afterwashing the fruit, two uniform wounds3mm deep and 5mm in diameter, were performed on theequatoof papaya with a sterile corkscrew. Twenty microliter suspension aliquots, containing 1.0108 cells/mL of each strain (strain422W. anomalus and strain 443M. guilliermondii) were inoculateinholes located8 cmapart fromeachother.After 3 h,20LoftheCgloeosporioidesconidial suspension(1.0105 cells/mL)wereaddedto each wound. The fruit were incubated under high temperatur(28 C) and95%RH to providefavorableconditions for thepostharvest onset of thedisease. Lesion progression was assessed daily, a3,4,5,and6dafterC. gloeosporioides inoculation,bymeasuring thdiameterof the damaged area relative to the initial wound. Subsequently, the percentage of disease inhibitionwas calculated usinthe following equation:

n =a b

a 100 (1

where n is the percentage of disease reduction, a the colony areofC. gloeosporioides in untreated fruit and b the colony area ofCgloeosporioides in killer yeast-treated fruit.Steriledistilledwater,inlieu of killeryeast,wasused as a negative control andthe fungicidTECTO (1000ppm), which has the active ingredient thiabendazole, was used as a positive control. Two replicates of 10 papayawere used foreach treatment (20 fruit/treatment). Theexperimenwas repeated twice.

2.5. Influence of the inoculation time of W. anomalus (strain 422)

andM. guilliermondii (strain 443) in the control of anthracnose

Papaya fruit were harvestedand processed as described in Section 2.1. Subsequently theywere wounded (Section 2.4) and 20suspension aliquots (1.0108 cells/mL) ofW. anomalus (strain422) and M. guilliermondii (strain 443) were inoculated at thinjured sites prior (3, 12, and 24h) and after (12 and 24h)respectively, inoculation with the C. gloeosporioides suspension(1.0105 conidia/mL). The fruit were incubated for 6 d after thonset of the treatments, at 95% RH and 28C, and necrotic lesionprogression was daily assessed by measuring the diameter of thnecrotic area relative to the initial wound, and the percentage odisease inhibition calculated using Eq. (1). Sterile distilled watewasusedasnegativecontrol andthe fungicideTECTO (1000ppm)

containing the active ingredient thiabendazole, as the positiv


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control. Two replicates of 20 papayas each were used for eachtreatment. The experiment was repeated twice.

2.6. W. anomalus (strain 422) andM.guilliermondii (strain 443)populations in the wounds

The establishment ofW. anomalus (strain 422) and M. guillier-mondii(strain443)killeryeastswereassessedonthesurfaceoffruit

wounds. A 20L aliquot containing 1.0108 cells/mL of yeast wasinoculated in thewound sites on thepapaya fruit. After0, 1, 2, 3, 4,5,6, 7 and 8d ofincubation at28 C, a tissue samplewas removed,andserialdilutionswere preparedusing a 0.9% salinesolution.Cellcounting was carried out by the spread plate method. Three repe-titions with three fruit were used for each strain. The experimentwas conducted twice (Zhang et al., 2007).

2.7. Examination of W. anomalus (strain 422) andM.

guilliermondii (strain 443) parasitism on C. gloeosporioides byscanning electron microscopy (SEM)

Yeasts were individually picked onto Petri dishes with PDAmediumintheformoftwoparallellines1cmapartfromeachother.

A C. gloeosporioidesmyceliumdiskof6mmindiameter, cultured inPDAfor10d,wasplacedatthecenteroftheplatepreciselybetweenthe two yeast strains streak spots. The plates were incubatedat 28 C until an interaction between the phytopathogen hyphaeand the test yeast (4d of incubation) was reached. Subsequently,samples of approximately 1 cm2 containing both organisms wereextractedwith a scalpel,washedtwicewithdistilledwaterandpre-pared for SEM imaging, according to Bozzola and Russell (1999).The positive control was a mycelium portion (1cm2) taken from aC. gloeosporioidesculture,whichwasgrownonPDAat28 Cfor10d.The samples were placed in a modifiedKarnovsky fixative for 48handpost-fixed in1%osmiumtetroxide in sodiumcacodylate buffer(0.1M, pH 7.2) for 1 h. Afterwards, the samples were dehydratedby immersion in crescent ethanol series (30%, 50%, 70% and 90%)

for 10min. Next, the samples were immersed in 3 pure ethanolbaths of 10min each and taken to a critical point dryer (EmitechK850), coatedwith gold sputter coating (Emitech K550) andexam-inedwith a SEM (ZeissDSM940 A)using anaccelerationvoltage of15kV.

2.8. Chitinase and -1,3-glucanase activities in the culturemediumof the W. anomalus (strain 422) andM.guilliermondii

(strain 443)

The yeast strainsW. anomalus (strain 422)andM. guilliermondii(strain 443) were cultured in assay tubes with 20mL of YEPDmedium (10g/L yeast extract, 20g/L peptone, 20g/L glucose) at28 C for 24h. Subsequently, the cultures were filtered through a

0.22mmembrane andthe cell-freefiltratedwasused tomeasurechitinase and -1,3-glucanase activities.-1,3-Glucanase (GLU) activity was determined by the rate

of glucose production from the degradation of laminarin(SigmaAldrich) used as substrate (Boller, 1992). Laminarin wasdissolved in ultrapure water (Milli-Q), heated to 60 C for 10min,and dialyzed exhaustively against ultrapure water (Milli-Q) forremoval of free glucose. In the assay, 0.1mLof the sample wasincubatedwith 0.9mLof laminarin (2.0mg/mL) at50Cfor30min.After adding the appropriate reagents to the reaction mixture,absorbance readingswere taken at520nm. Thequantity of glucosemonomers released was determined using a d-glucose standardcurve (330g/mL). The -1,3-glucanase activity was expressedin nanokatal per milligram of protein (nkatGlu/mgP). One nkat is

equivalent to 1.0nmol glucose released/mL/s.

Fig. 1. Effect of the pre- (3, 12, and 24h) and post- (12 and 24h) treatments ofpapaya fruits with W. anomalus (strain 422) andM. guilliermondii (strain 443) onthe progression of the lesion causedby C. gloeosporioides, 6d after the onset ofthetreatments. The vertical bars represent the standard error of the means (n=20).Different letters over the columns at each time point differ significantly (Tukeystest, P0.05).

Chitinase activity was assessed using a colorimetric method todetect N-acetyl-d-glucosamine (NAG) produced by the combinedhydrolyticactionofchitinaseand -glucuronidase(SigmaAldrich)on non-radioactive colloidal chitin (Molano et al., 1977) used assubstrate (Boller, 1992; Reissig et al., 1995). The concentrationof NAG was determined by spectrophotometry at 585nm against

a standard curve of NAG (SigmaAldrich), ranging from 100 to600mM. Thechitinaseactivitywasexpressed innanokatalpermil-ligramof protein (nkat/mgP). One nkat is equivalent to 1.0nmol ofNAG released/mL/s, under the assay conditions.

2.9. Statistical analyses

The data were subjected to analysis of variance (ANOVA) [SIS-VAR software, package (version 5.3; Federal University of Larvas[Universidade Federal de Lavras])] (Ferreira, 2000) and statisti-cal significance was set to the level P0.05. Replication averageswithsimilar resultswereanalyzed together.Averagesandstandarderrors were calculated and reported for each experiment. Whenaverageswere significantlydifferent, a comparisonwasperformed

using Tukeys test (P0.05).

3. Results

3.1. Influence of the inoculation time of W. anomalus (strain 422)andM. guilliermondii (strain 443) in the control anthracnose

The time of inoculation between the antagonist yeasts and thepathogen had a significant effect on control efficiency. Through-out the incubation period, the papaya fruit treated with the yeasts12 and 24h before C. gloeosporioides had significantly smaller(P0.05) infection lesion sizes than those inoculated with theyeasts 12 and 24h later (Fig. 1). Indeed, the fruit treated with W.anomalus (strain422) 24h beforeC. gloeosporioides inoculationhad

a necrotic lesion size 31.35% smaller than control samples after 6 dincubation, while those treatedwith the yeasts 24h after thephy-topathogen inoculationwere only 4% smaller. Similar results werefound for strain 443, inwhich the yeast application 24h before thephytopathogen reduced in 41.17% the lesion size after 6 d incuba-tion. However, when M. guilliermondii was applied 24h after thephytopathogen, the lesion size reduction was less than 10% com-pared to control fruit (Fig. 1).

3.2. Inhibitory effect of killer yeast W. anomalus (strain 422) andM. guilliermondii (strain 443) on C. gloeosporioides

The killer yeastsW. anomalus (strain 422) andM.guilliermondii(strain 443) demonstrated in vivo antagonistic activity against C.

gloeosporioideswhen inoculated 3h beforethephytopathogen. The


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Fig. 2. Effect of the 3-h pre-treatment of papaya fruits with W. anomalus (strain422) andM.guilliermondii (strain 443) on the evolution of the lesions causedby C.gloeosporioidesmeasured at 3, 4, 5, and 6d after conidum inoculation. The verticalbars represent the standard error of the means (n=20). Different letters over thecolumns at thesame time interval differsignificantly (Tukeys test, P0.05).

suspensions of both strains significantly reduced (P0.05) theinjury caused by C. gloeosporioides in papaya when inoculated 3hafter the yeast treatments up to 6d of the experimental period(Fig. 2).

Inoculation with antagonistic yeast delayed the onset of theanthracnose symptoms, and the diameter of infection remainedsignificantly lower (P0.05) than the control group up to the endof the experimental period (6d) and similarly to the fungicidethiabendazole. At this time, W. anomalus (strain 422) and M. guil-liermondii (strain 443) killer yeast reduced the wound diameter by24.62% and 20.68%, respectively, when inoculated 3h prior to theinoculationwith the phytopathogen (Fig. 2).

3.3. Progression of W. anomalus (strain 422) andM.guilliermondii (strain 443) populations on papaya surfacewounds

The growth dynamics ofW. anomalus (strain 422) and M.guil-

liermondii (strain 443) respectively on papaya surface woundsare displayed in Figs. 3 and 4. In both cases, the populationsincreased after an adjustment period, reaching the maximumgrowth between 3 and 4d after inoculation, at which time theytendedto stabilizeandthendecreasedto levelssimilar to the initiallevels at the end of the eighth day of incubation.

3.4. Parasitism of W. anomalus (strain 422) andM.guilliermondii (strain 443) on C. gloeosporioides evaluated by

scanning electron microscopy (SEM)

W. anomalus (strain 422) andM.guilliermondii (strain 443) col-onized C. gloeosporioideshyphae, leading to a loss of turgidity (seearrows in Fig. 5E and F). Moreover, mycoparasitism, characterized

Fig. 3. Population dynamics ofW. anomalus (strain 422) killeryeast onpapayafruit

woundsites. Vertical bars representthe standard deviations of themeans.

Fig. 4. Population dynamics ofM.guilliermondii (strain 443) killer yeast on papayfruit wound sites.Vertical bars representthe standard deviations of themeans.

by the strong colonization of the pathogen hypha by the yeascells (see arrows in Fig. 5C, D, G and H), was also observed. Mycoparasitism is one of the mechanisms involved in phytopathogenbiocontrolandthepresentstudyindicatedthattheyeastsparasitizeC. gloeosporioideshyphae and cause physical damage, possibly duto the production of hydrolyte enzymes that degrade the hyphacell walls, such as -1,3-glucanase. The yeast penetration of wallwithmarkedconcavityformationonhyphacellwalls ishighlighted

by arrows in Fig. 5G and H.

3.5. Hydrolytic enzyme production

Theactivity of-1,3-glucanase forW. anomalus (strain422)andM. guilliermondii (strain 443) was 1.18 and 0.78nkat/mgP, respectively.No chitinase activitywas found for the twoyeasts under thconditions used in the assay.

4. Discussion

In recent years, several studies have been conducted aiming tisolate microorganisms that protect postharvest fruit from fungadiseases (Dal Bello et al., 2008; Hashem and Alamri, 2009; Liu and

Tsao, 2009). However, these studies have been restrictedto a smalgroupof fruit,suchasapple,pear,strawberryandcitrus. To thebesof our knowledge, this study is the first report on the assessmenofW. anomalus and M. guilliermondii as biocontrol agents againsC. gloeosporioides in postharvest papayaand in the investigation othe mechanisms involved. W. anomalus (strain 422) and M. guilliermondii (strain 443) delayed the onset of disease symptoms andreducethe intensityofdisease lesions,after6dof incubation. Thestraits make these yeasts efficient biocontrol agents, when appliedsimultaneouslywithC. gloeosporioides fungus. Similar results werdescribed by Platania et al. (2012), who reported Penicilliumdigitatum biocontrol by using the killer yeastsW. anomalus, on Toroccoorange fruit. Wang et al. (2010) reported a reduction in infectionafter 5 d of incubation when assessing the efficiency ofRhodospo

rium paludigenum for the biocontrol of the green mold caused bBotrytis cinerea in tomatoes.Zhangetal.(2010) alsoreportedreductions by AP6- Pseudozyma fusiformata, AP47Metschnikowia sp. andPL5 Aureobasidium pullulans, respectively, in the control of brownrot caused byMonilinia spp. in peaches.

Reduction of the infection of papaya caused by C. gloeosporioides might be related to the high reproductive rate of the yeastcompared to the phytopathogen. Indeed the yeasts colonized thwound surface faster than the fungus when they were inoculatedsimultaneously, thus protecting the fruit.

Thus these yeasts should be employed as a pre-treatment tooas a way to reduce the anthracnose disease in papaya fruit. Zhaoet al. (2008) described similar results when assessing the in vivefficiency ofPichia guilliermondii against the phytopathogen Rhizo

pus nigricans. They reported higher efficiency for tomato infection


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Fig. 5. Electronmicrographs of healthy phytopathogen hyphae, still turgid andwithout wall rupture (A, B); interaction of the antagonistic yeastsW. anomalus (strain 422)(C, E,G) andM.guilliermondii (strain 443) (D, F, H)with the C. gloeosporioides phytopathogen hyphae after 4d of incubation at 28C; C. gloeosporioides hyphae colonizedbyyeast (C andD), loss of turgidity of yeast-colonizedhyphae(E andF);yeast penetrationof walls with markedconcavity formationonhypha cell walls (G andH).


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control when the yeast was inoculated 24h prior to the pathogen.They further reported that the inhibition of lesions by 75.5%with simultaneous inoculation of both organisms, against 14.7% ofreduction with yeast inoculation 24h after phytopathogen inocu-lation. Therefore, this supports thehypothesis that competition fornutrients and space plays a key role in the biocontrol effect.Whenyeasts are inoculated prior to the phytopathogen, they have moretime to colonize the fruit and, thus, to reduce the availability of

nutrients to the pathogen.Wang et al. (2008) and Gholamnejad et al. (2010) reportedhighercontrolefficiencywhen employingRhodotorulapaludigenumin the control ofAlternaria alternata on cherry tomatoes and Can-dida membranifaciens and Rhodotorula mucilaginosa in the controlofPenicillium expansum, respectively, when the application of thebiocontrol agents preceded phytopathogen inoculation.

Inaddition to theestablishment ofmethodologiesfor theimple-mentation of selected agents, studies on the influence of theinoculation time on the effectiveness of the biocontrol agents areessential for developing control strategies. In the present study,the highest efficiency of the yeasts used was observed when theywere applied prior to C. gloeosporioidesestablishment. This findingdemonstrates the importance of the applicationof theantagonisticyeasts immediately after the fruit harvest. The loss of biocontrolefficiency when the pathogen is inoculated previously may berelated to theantagonisticyeastmechanismofaction. Competitionfor space and nutrients has a key role indisease control, as demon-strated in several studies (Zhao et al., 2008; Chanchaichaovivatet al., 2008; Droby et al., 2009; Rosa et al., 2010; Wang et al.,2010; Jamalizadeh et al., 2011; Li et al., 2011). Such a mechanismis advantageous for farming due to the lack of human exposure topotentially toxic compounds.

The killer yeasts studied,W. anomalus (strain 422) andM. guil-liermondii (strain 443), were able to survive quite well in papayawounds, reaching scores above the initial inoculum by approxi-mately 24h after inoculation in the fruit. This finding confirms theimportance of carrying out a survey of biocontrol agents withinthe pathogen niche, which increases their beneficial effects due

to a greater adaptation to environmental conditions (Janisiewiczand Korsten, 2002; Droby et al., 2009; Gholamnejad et al., 2010;Robiglio et al., 2011).

Abundant colonization and strong yeast binding to C. gloeospo-rioides hyphae was revealed by electron microscopy. Thisobservation confirmed the occurrence of mycoparasitism in therelationship between C. gloeosporioides and the killer yeast W.anomalus (strain 422) and M.guilliermondii (strain 443). Further-more, SEM also revealed turgidity loss, the occurrence of concaveareas and, in some cases, complete hypha rupturewithyeast pene-tration into thepathogenhyphae. These results areconsistent withthose ofHashemandAlamri (2009), whodescribed theoccurrenceof strong P. anomala Mob 93 yeast binding on Botryodiplodia theo-bromae hyphae after 4d of incubation as well as concave areas on

the surface of hyphae and evidence of penetration. These authorsfurther reported the occurrence of extracellular matrix accumula-tion aroundthecolonizedhyphae. Chan andTian (2005) reported astrong binding capacityofPichiamembranefaciens toMonilinia fruc-ticola, P. expansum and Rhizopus stoloniferhyphae and suggestedthat this binding was related to the production of lytic enzymesdemonstrated in their study.

The evidence of-1,3-glucanase production by W. anomalus(strain 422) and M.guilliermondii (strain 443) reinforces the pos-sibility of using both organisms as a biocontrol agent because thisenzymehydrolyzes glucans, themost abundant componentof fun-gal cell walls (Peberdy, 1990; Iorio et al., 2008; Fleuri and Sato,2008). This enzyme degrades the cell walls of filamentous fungiand some yeasts, and its activity is likely related to the C. gloeospo-

rioides hyphae lysis andpenetration observed in thepresentstudy.

Bauermeister et al. (2010) further noted that the commerciaproduct Aspire, recommended for the biological control of thpostharvest rot of citrus and other fruits such as apple and pearis based onC. oleophila-glucanase production.

There are several putative mechanisms of biological controThese mechanisms represent activities involving a variety of ecological relationships not only between biocontrol agents andphytopathogens but also the entire resident biota. Therefore

according to Jamalizadeh et al. (2011), knowing the main mechanisms involved in the action of a specific agent is key for thdevelopment of formulations based on these agents and methodologies of its application. Therefore, in the present study,we optedto useyeastswithkiller activity already reported in previous studies. Although these studies have not shown any direct toxin effecon thepathogen, itsactionon thefruit-residentmicrobiotamayacsynergicallywith yeast action on the pathogen.

The present study demonstrated that W. anomalus (strain 422andM.guilliermondii (strain443) killeryeast, isolated frompapayawere able to reduce the wounds caused by C. gloeosporioideswheninoculated on a fruit wound. This action is influenced by threlationbetweenthe time of biocontrol agent application andphytopathogen inoculation. It was also possible to demonstrate thathe effect ofW. anomalus (strain 422) andM.guilliermondii (strain443) yeast was due to multiple factors, including competition fospace andnutrients,mycoparasitismandsecretionof enzymesthalyse the phytopathogen cell wall.

Acknowledgments

Theauthorswouldlike to thankthe FundacoCearensedeApoiao Desenvolvimento Cientfico e Tecnolgico (FUNCAP) (CearState Foundation for the Support of Scientific and Technological Development) and the Conselho Nacional de DesenvolvimentCientifico e Tecnolgico (CNPq) (NationalCouncil for Scientific anTechnological Development) forproviding research grants and thEMBRAPA Tropical Agroindustry, whose invaluable support madthis study possible.

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Use of Killer Yeast in the Management of Postharvest Papaya Anthracnose

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