Revista Mexicana de Fitopatología

ISSN: 0185-3309

mrlegarreta@prodigy.net.mx

Sociedad Mexicana de Fitopatología, A.C.

México

Bautista Baños, Silvia; Lucca, Anthony J. De; Wilson, Charles L.

Evaluation of the Antifungal Activity of Natural Compounds to Reduce Postharvest Blue Mould

(Penicillium expansum Link.) of Apples (Malus domestica Borkh.) During Storage

Revista Mexicana de Fitopatología, vol. 22, núm. 3, diciembre, 2004, pp. 362-369

Sociedad Mexicana de Fitopatología, A.C.

Texcoco, México

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Silvia Bautista-Baños, Instituto Politécnico Nacional, Centro de Desarrollo de ProductosBióticos, km. 8.5 Carr. Yautepec-Jojutla, San Isidro Yautepec Morelos, Mexico CP 62731;Anthony J. De Lucca, USDA-ARS, Southern Regional Research Center, 1100 Robert E.Lee Blvd., P.O. Box 19687, New Orleans, LA, USA 70124; and Charles L. Wilson,USDA-ARS, Appalachian Fruit Research Station, 45 Wiltshire Road, Kearneysville, WestVirginia, USA 25430. Correspondence to: sbautis@ipn.mx

(Received: December 1, 2003 Accepted: February 23, 2004)

Evaluation of the Antifungal Activity of Natural Compounds toReduce Postharvest Blue Mould (Penicillium expansum Link.) of

Apples (Malus domestica Borkh.) During Storage

Bautista-Baños, S., De Lucca, A.J., and Wilson, C.L. 2004.Evaluation of the antifungal activity of natural compounds toreduce postharvest blue mould (Penicillium expansum Link.)of apples (Malus domestica Borkh.) during storage. RevistaMexicana de Fitopatología 22:362-369.Abstract. Several natural compounds were evaluated for thecontrol of Penicillium expansum on apples during green-ripeor ripe maturity stages and stored at ambient or coldtemperature. Semipure or crude derivatives of Capsicumfrutescens completely inhibited conidial germination of P.expansum at all concentrations. The crude derivative at 5.0µg/L concentration reduced fruit infection. Overall, applestreated with chitosan at 1.0% had less infection at bothmaturity stages and storage temperatures. The extract of seedof yam bean stimulated disease incidence at both maturitystages and storage temperatures as compared to the remainingtreatments. Hinokitiol was not effective in reducing fruitinfection. During cold storage, the highest disease incidencewas recorded after 8 week storage on apples treated with theC. frutescens derivatives, and after 16 weeks when treatedwith chitosan, hinokitiol, or the fungicide Mertec.

Additional keywords: Capsicum frutescens, chitosan, yambean root, hinokitiol, Pachyrrizhus erosus, plant derivatives.

Resumen. Se evaluaron varios compuestos naturales paracontrolar Penicillium expansum durante el almacenamientoa temperatura ambiente o durante el almacenamiento en frío,en manzanas en dos estados de madurez (verde-madura omadura). La germinación de los conidios de P. expansum fuecompletamente inhibida con los derivados semipuros o crudosde Capsicum frutescens y en todas las concentracionesevaluadas. El porcentaje de infección se redujo cuando lafruta se trató con el derivado crudo en concentraciones de 5µg/L. En general, las manzanas tratadas con quitosano al 1.0%tuvieron menor infección en ambos estados de madurez ytemperaturas de almacenamiento. Comparado con el resto

de los tratamientos, el extracto de semilla de jícama estimulóla incidencia de la enfermedad en ambos estados de madurezy temperaturas de almacenamiento. El hinokitiol no fueefectivo en reducir la infección de la fruta. Durante elalmacenamiento en frío, la incidencia más alta de laenfermedad se registró después de 8 semanas dealmacenamiento en manzanas tratadas con los derivados deC. frutescens, y después de 16 semanas cuando se trató conquitosano, hinokitiol o el fungicida Mertec.

Palabras clave adicionales: Capsicum frutescens, quitosano,jícama, hinokitiol, Pachyrrizus erosus, derivados de plantas.

Penicillium expansum Link. is a major postharvest pathogenof apples (Malus domestica Borkh.) during fruit cool storage.P. expansum often infects through wounds, fruit calyx or core(Snowdon, 1990). At present, control of P. expansum reliesheavily on synthetic fungicides (Koffmann and Penrose,1987). Recently, there has been an increased interest in thedevelopment of fungicides derived from natural compoundsas an alternative to synthetic fungicides for the control ofpostharvest microorganisms (El-Ghaouth et al., 2002).Natural plant-animal-compounds have shown a great potentialto control postharvest pathogens of various horticulturalcommodities (Wilson et al., 1994). Among these, chitosanand plant derivatives have become an important source ofbiofungicides. Plants have long been recognized to contain anumber of secondary metabolites with antimicrobialproperties (Grange and Ahmed, 1988). The insecticidalactivity of seed derivatives and powders of the root yam bean[Pachyrrhyzus erosus (L.) Urban] against various pests hasbeen proven (Alavez-Solano et al., 1998; Hansberry, 1947;Hansberry and Lee, 1943). Studies carried out in ourlaboratory also confirmed the fungicidal potential of seedpowders of yam bean on mycelial development ofColletotrichum gloeosporioides (Penz.) Penz. and Sacc.,Fusarium oxysporum Schlechtend.:Fr., and Rhizopus

362 / Volumen 22, Número 3, 2004

stolonifer (Ehrenb.:Fr.) Vuill. (data not published). Hinokitiol(α-thujaplacin) a plant derivative obtained from the bark orroots of the Hinoki tree [Thujopsis dolabrata (L.f.) Sieb. andZucc.] (Osaka Organic Chemical Industry LTD, 2003) or redcedar (Thuja plicata Donn ex D. Don) (Trust and Coombs,1973) has been effectively proven to control in vitro growthof various fungi such as Penicillium citrinum Thom.,Aspergillus niger van Tieghem, Botrytis cinerea Pers.:Fr.,Monilinia fructicola (G. Wint.) Honey, and Rhizopus oryzaeWent. and Prinsen Geerligs, and to inhibit growth of a widerange of phytopathogenic bacteria (Sholberg and Shimizu,1991; Trust and Coombs, 1973). The fungicidal propertiesof the saponin CAY-1 a derivative of the pepper cayenne(Capsicum frutescens L.) have been reported by De Lucca etal. (2002). The in vitro fungicidal activity of this saponinwas confirmed on germinating conidia of various species ofAspergillus. Chitosan produced by the deacetylation of chitinhas shown to supress development of important postharvestfungi of numerous crops. Their benefitial effects have beenreported on fruits to control B. cinerea, R. stolonifer, M.fructicola, and C. gloeosporioides (Bautista-Baños et al.2003b; El-Ghaouth et al., 1991; 1992; Li and Yu, 2000; Lunaet al. 2001), and in vegetables against B. cinerea andAlternaria alternata (Fr.:Fr.) Keissler, among others(Bhashkara Reddy et al., 2000; El-Ghaouth et al., 1997). Theobjectives of this work were: a) to assay the fungicidal effectof two derivatives of the pepper cayenne and one saponin onP. expansum development, and b) to evaluate the diseaseprogress and incidence of treated apples with aqueous extractof seed of yam bean, two derivatives of pepper cayenne,hinokitiol, chitosan, and a commecial fungicide, stored at twotemperatures.

MATERIALS AND METHODSMicroorganism, reagents, fruit, and plant material. Theisolate of Penicillium expansum was obtained from infectedapple fruit and maintained on potato dextrose agar (PDA).To fulfill Koch´s postulates, subcultures of the fungus weremade with a sterile nailbrush applying mycelia to artificiallywounded ripened apple fruit, which was kept in a humiditychamber at 20°C to observe symptoms and re-isolate thefungus. Chitosan, high molecular weight was obtained fromSigma-Chemical Co. (St. Louis, MI, USA), the commercialfungicide Mertec (Thiabendazole), was purchased fromGreenChemicals (Winchester, VA, USA) and a wetting agent(surfactant) was obtained from Spray N-Grow, Inc (Cary Il.USA). Two maturity stages of apple fruit cv. GoldenDelicious, green-ripe (green color) and ripe (green-yellow)were obtained from a commercial orchard in Kearnesville,West Virginia, USA. Yam bean seeds of cv. Agua Dulce wereobtained from a commercial seed producer in the state ofMorelos, Mexico, the saponine CAY-1 and the semipure andcrude derivatives of C. frutescens were provided by Dr. A.De Lucca (Southern Regional Research Center, USDA, ARS),and the sodium salt of hinokitiol was provided by Osaka

Organic Chemical Industry Ltd. (Kashiwara Osaka, Japan).Fruit inoculation and treatment. Healthy fruit wasinoculated at one site, midway between the pole and theequator, using a metal bar which was pressed on the skinuntil it ruptured, making triangular wounds of about 7 to 9mm on each side. Each wound was flooded with 20 µl ofsterile water containing a spore suspension (1 x 105 spores/mL) of P. expansum, and let dry at 20°C ± 2 for 1h(McLaughlin et al., 1990).Screening for in vitro and in situ antifungal activity ofpepper cayenne derivatives. The fungicidal properties ofsaponin CAY-1 and the semipure and crude derivatives ofpepper cayenne were determined in vitro. For in situ studies,only the semipure and crude derivatives were tested. Twenty-five µl of P. expansum conidia (1 x 105/ml) were added tosterile 500 µl volume microfuge tubes (Eppendorf, Darmstadt,Germany) containing each different concentrations of 221,214, 203, 181, 137, 71, and 5 µl of nutrient yeast-dextrosebroth (NYDB, Difco). CAY-1 and the semipure and crudederivatives were added to the microfuge tubes proportionallyto the NYDB concentrations at 4.4, 11, 22, 44, 88, 157, and220 µl. After mixing, the samples were constantly shakenand incubated for 24 h at 20°C. Control consisted of NYDBmedium (225 µl) and conidial suspension of P. expansum(25 µl). Fungicidal activity was measured as percentage ofconidia germination. Each treatment was evaluated twice (n= 3). For the in situ experiments, only the semipure and crudederivatives were tested using the following concentrations0.5, 1.0, 2.5, 5.0, 7.5, 10, 20, and 40 µg/L. Derivatives weremixed in 7 L distilled water, with 5 mL of a surfactant. Theinoculated control fruit was treated with both distilled wateror with a commercial fungicide (Mertec). After inoculation,the fruit was dipped in the aqueous mixture for 15 min, driedat ambient temperature (18-20°C), and placed in perforatedplastic bags. Ripe fruit were stored at 18-20°C, and green-ripe fruit at 4°C under high relative humidity (95%). Diseaseincidence was evaluated after 2, 10, and 15 days of storage atambient temperature, and after 4, 8, and 12 weeks when storedat 4°C. Each treatment was applied to three replicates of 22fruits each within each experiment, which were repeated twice.Screening for fungicidal activity of chitosan, hinokitioland yam bean against P. expansum rots. Chitosan-propionate concentrations of 0.5% and 1.0% were preparedby adding equal amounts (w/v 1:1) of propionic acid tochitosan. The chitosan-propionic acid mixture was added tothe total volume of distilled water and stirred overnight. Thesodium salt of hinokitiol was dissolved in distilled water at1%. Seed powder of yam bean was mixed with distilled water(2:10 w/v) and left at 18-20°C for 24 h, and filtered. Theinoculation and the control fruit was the same as describedpreviously. After inoculation, treatments were pippeted (250µl) directly onto apple wounds. After one hour, the fruit waspackaged and stored at 18-20°C or 4°C. For both maturitystages (green-ripe or ripe), disease incidence was evaluatedafter 4, 8, and 12 days of storage at ambient temperature and

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4, 6, 12, and after 16 weeks when stored at 4°C. Eachtreatment was applied to three replicates of 20 fruits eachwithin each experiment. The experiment was repeated twice.Statistical analysis. Treatments were arranged in acompletely randomized design. Percentage disease wasanalysed using the Chi square procedure. Treatments meanswere separated by Duncan’s multiple range (P = 0.05).

RESULTSScreening for in vitro and in situ antifungal activity ofpepper cayenne derivatives. With the exception of thesaponin CAY-1 at 4.4 µg/L concentration, no germination ofP. expansum was recorded for the remaining treatments (Table1). Significantly higher (P = 0.001) germination (100%) wasobserved for the untreated P. expansum conidia comparedwith those treated with CAY-1, and the semipure or crude C.frutescens derivatives. For the in situ experiment, percentage

infection was significantly different among treatments (P =0.01 or P = 0.001) (Table 2). Overall, apple fruit stored atambient temperature had less infection than that stored at4°C. There were progressively lower levels of diseaseinfection, when treatment concentration increased for thesemipure or crude derivatives in fruit cool stored or left atambient temperature, respectively. The least fruit infection(32%) was in fruit dipped in the crude treatment atconcentrations between 5.0 to 40 µg/L. No significantdifferences were observed on fruit treated with the crudederivative and stored at 4°C. Compared to the remainingtreatments, the fungicide Mertec was not effective in reducingstorage rots of apples. The highest fruit infection was shownafter 10 day storage at ambient temperature for both thesemipure and crude derivatives (Fig. 1). Overall, the highestinfection levels on fruit stored at 4°C ocurred after eight weeksof storage (Fig. 2). During the 12 weeks of cold storage,disease incidence of untreated fruit and those treated with

Table 1. Percent conidia germination of Penicillium expansumtreated with saponin CAY-1, and the semipure and crudepepper cayenne (Capsicum frutescens) derivatives at variousconcentrations, after incubation at 20°C for 24 h.Pepper cayenne derivatives Conidia germination (%) Concentration (µl) P = 0.001CAY-1 4.4 37 bz

11 0 c22 0 c44 0 c88 0 c157 0 c220 0 cControl 100 a

P = 0.001Semipure

4.4 0 b11 0 b22 0 b44 0 b88 0 b157 0 b220 0 bControl 100 a

P = 0.001Crude

4.4 0 b11 0 b22 0 b44 0 b88 0 b157 0 b220 0 bControl 100 a

zMeans followed by the same letter are not significantlydifferent (p = 0.05) as determined by Duncan´s multiple rangetest.

Table 2. Percent infection of apple (Malus domestica) fruitcv. Golden Delicious by Penicillium expansum after treatmentwith semipure or crude pepper cayenne (Capsicum frutescens)derivatives, and left at ambient temperature and cool storagefor 15 days and 12 weeks, respectively . Infection (%) Treatment Semipure Crude

Ambient temperature P = 0.01 P = 0.001(18-20°C)Concentrations (µg/L):0.5 64 bcz 64 bc1.0 64 bc 64 bc2.5 64 bc 41 d5.0 68 b 32 e7.5 68 b 32 e10 82 a 32 e20 82 a 32 e40 82 a 32 eMertec 82 a 68 bControl 82 a 86 aCool storage (4°C) P=0.001 NSConcentrations (µg/L):0.5 89 bc 98 ab1.0 87 bc 98 ab2.5 87 bc 96 abc5.0 87 bc 96 abc7.5 79 bcd 96 abc10 73 d 96 abc20 67 e 96 abc40 65 e 96 abcMertec 95 ab 99 aControl 96 a 99 a

zMeans followed by the same letter are not significantlydifferent (P = 0.05) as determined by Duncan´s multiple rangetest.

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