Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México

Barrera Necha, Laura Leticia; Bautista Baños, Silvia; Jiménez Estrada, Manuel; Reyes Chilpa, Ricardo Influence of Leaf, Fruit and Seed Powders and Extracts of Pithecellobium dulce (Roxb.) Benth. (Fabaceae) on the in vitro Vegetative Growth of Seven Postharvest Fungi Revista Mexicana de Fitopatología, vol. 20, núm. 1, enero-junio, 2002, pp. 66-71 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México

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Influence of Leaf, Fruit and Seed Powders and Extracts of Pithecellobium dulce (Roxb.) Benth. (Fabaceae) on the in vitro Vegetative Growth of Seven Postharvest Fungi Laura Leticia Barrera-Necha, Silvia Bautista-Baños, Instituto Politécnico Nacional, Centro de Desarrollo de Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San Isidro Yautepec, Morelos, México CP 62731; Manuel Jiménez-Estrada and Ricardo Reyes- Chilpa, Universidad Nacional Autónoma de México, Instituto de Química, Circuito Exterior, Ciudad Universitaria, Coyoacán, México, D.F., CP 04510. Correspondence to: [email protected] (Received: October 17, 2001 Accepted: January 28, 2002)

Abstract. sp., Botrytis cinerea, Colletotrichum gloeosporioides, Barrera-Necha, L.L., Bautista-Baños, S., Jiménez-Estrada, Fusarium oxysporum, Penicillium digitatum, Pestalotiopsis M., and Reyes-Chilpa, R. 2002. Influence of leaf, fruit and sp., Rhizopus stolonifer. seed powders and extracts of Pithecellobium dulce (Roxb.) Benth. (Fabaceae) on the in vitro vegetative growth of seven Resumen. Los polvos de hojas, frutos y semillas de postharvest fungi. Revista Mexicana de Fitopatología 20:66- Pithecellobium dulce y semillas extraídas secuencialmente 71. con hexano-diclorometano, acetona y metanol-agua se Powders of Pithecellobium dulce leaves, fruit and seeds evaluaron sobre el crecimiento micelial de Alternaria sp., sequentially extracted with hexane-dicloromethane, acetone, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium and methanol-water were evaluated on mycelial growth of oxysporum, Penicillium digitatum, Pestalotiopsis sp. y Alternaria sp., Botrytis cinerea, Colletotrichum Rhizopus stolonifer. Los polvos de semillas tuvieron la más gloeosporioides, Fusarium oxysporum, Penicillium alta actividad fungistática contra los hongos probados en digitatum, Pestalotiopsis sp. and Rhizopus stolonifer. In comparación con los polvos de fruto y hoja. En general, se comparison to fruit and leaf powders, seeds had the highest observó una curva de dosis-efecto para las tres fungistatic activity against the fungi tested. In general, a dose- concentraciones evaluadas (0.5, 2.0 and 5.0 mg/ml). Sin effect curve was observed for the three concentrations (0.5, embargo, para P. digitatum y Alternaria sp. la concentración 2.0 and 5.0 mg/ml) evaluated. However, for P. digitatum and más baja y la más alta, respectivamente, incrementaron el Alternaria sp., the lowest and highest concentrations crecimiento micelial. Dependiendo de la concentración, los respectively, increased mycelial growth. Depending on polvos de hoja y fruto inhibieron o incrementaron el concentration, leaf and fruit powders inhibited or increased crecimiento micelial. El crecimiento micelial de mycelial growth. For Pestalotiopsis sp., P. digitatum, F. Pestalotiopsis sp, P. digitatum, F. oxysporum, Alternaria sp. oxysporum, Alternaria sp., and R. stolonifer mycelial growth y R. stolonifer se incrementó sobre los residuos de semillas increased on seed residues (10 mg/ml), after hexane- (10mg/ml), después de la extracción de hexano- dicloromethane, acetone, and methanol-water extractions of diclorometano, acetona y metanol-agua de polvos de semillas, seed powder, suggesting that fungistatic compounds were sugiriendo que los compuestos fungistáticos fueron removidos removed by the dissolvent used. The hexane-dicloromethane por los disolventes usados. El extracto hexano-diclorometano extract was subjected to column chromatography, obtaining fue sometido a una cromatografía en columna, obteniéndose 13 fractions with similar pattern, which were evaluated using 13 fracciones con patrones similares, las cuales fueron the mycelial growth responses of F. oxysporum, P. digitatum evaluadas usando la respuesta de crecimiento micelial de F. and R. stolonifer. Eleven and nine fractions inhibited F. oxysporum, P. digitatum y R. stolonifer. Once y nueve de las oxysporum and R. stolonifer development, respectively. P. fracciones inhibieron el crecimiento de F. oxysporum y R. digitatum was the fungus least affected by all fractions. stolonifer, respectivamente. P. digitatum fue el hongo menos Preliminary analysis of the most active fraction by nuclear afectado por todas las fracciones. El análisis preliminar de la magnetic resonance indicated the presence of a tryacyl fracción más activa por resonancia magnética nuclear indicó glycerol. la presencia de un triacil glicerol.

Additional key words: Guamúchil, huamúchil, Madras thorn, Palabras clave adicionales: Guamúchil, huamúchil, Madras manila tamarind, ojiuma, plant extracts, fractions, Alternaria thorn, manila tamarind, ojiuma, extractos vegetales, Revista Mexicana de FITOPATOLOGIA/ 67 fracciones, Alternaria sp., Botrytis cinerea, Colletotrichum sapote mamey (Pouteria sapota), R. stolonifer, P. digitatum, gloeosporioides Fusarium oxysporum, Penicillium digitatum, F. oxysporum and C. gloeosporioides from infected papaya Pestalotiopsis sp., Rhizopus stolonifer. (Carica papaya), and Botrytis cinerea from infected strawberry (Fragaria X ananassa). To maintain Failure to control postharvest pathogenic fungi can result in pathogenicity, each fungus was frequently inoculated and serious economic losses to worldwide horticultural reisolated from its indicated host. production. Fungi such as Alternaria sp., Botrytis cinerea, In vitro bioassay. Powders of leaves, fruit and seeds were Colletotrichum gloeosporioides, Fusarium oxysporum, prepared at three concentrations (0.5, 2.0 and 5.0 mg/ml in Penicillium digitatum, Pestalotiopsis sp. and Rhizopus the growing media) and amended with 16 ml of Potato stolonifer, cause diseases to different fruits and vegetables dextrose agar (PDA) and autoclaved (15 lb/cm2, 15 min.). and all are considered major plant pathogens (Farr et al., After sterilization media were poured into Petri plates (60 x 1989). Natural products may offer a new approach for control 15 mm). A five mm disc agar containing the respective of postharvest diseases. Pithecellobium dulce (Roxb.) Benth. pathogen was placed at the centre of each plate which was (Fabaceae) (common names: guamúchil, huamúchil, manila then incubated as follows: One day for R. stolonifer, two days tamarind, Madras thorn, ojiuma) is an evergreen tree for Alternaria sp., Pestalotiopsis sp., P. digitatum and B. indigenous to the Americas and widely distributed throughout cinerea and four days for F. oxysporium and C. Mexico. It has also been introduced into Asia, Africa and gloeosporioides. Except for B. cinerea, whose incubation Australia. An ethnobotanical survey of medicinal plants temperature was at 20oC, the other fungi were incubated at conducted by the Mexican Social Security Institute (IMSS) 25ºC. Mycelial growth (colony diameter) was measured at in the State of Morelos, Mexico revealed that the boiled fruit the end of the incubation time. Three replications were run peel of P. dulce was able to cure cough among people of simultaneously for each treatment (leaves, fruit and seeds at Tamaulipas, Chiapas and Guerrero (Aguilar et.al., 1996). We different concentrations). Petri plates of controls only have previously reported that the powder and aqueous extract contained PDA media. Tests were finished when mycelium from the leaves of P. dulce inhibited at some stage the growth of the control plates reached the edge of the dishes. Growth of four important postharvest fungal pathogens of fruits and inhibitory effects were calculated as fallow: % Inhibition = vegetables (Bautista-Baños et al., 2000a). It is noteworthy Mycelial growth in control – Mycelial growth in treatment/ that P. dulce was the most effective among twenty plants Mycelial growth in control X 100 tested. For example, sporulation of R. stolonifer previously Extraction. Seed powders (200 g) were successively isolated from ‘ciruela’ (Spondias purpurea) was completely extracted at room temperature with hexane-dicloromethane inhibited, while percentage infection after storage was (2:8), acetone and methanol-water (8:2) for 48 h in each significantly reduced in two varieties ciruelas: red and yellow solvent system. After each extraction step, a sample of 10 previously dipped in leaf extracts of P. dulce as compared mg/ml was amended with 16 ml of PDA, autoclaved and with control fruit (Bautista-Baños et al., 2000b). Montes et. poured onto Petri plates (60 x 15 mm). Mycelial growth of al., (1990), reported significant antifungal activity of leaf each test fungus was recorded at the end of each incubation extracts of P. dulce against Uromyces appendiculatus on bean time. Three replicates (three plates) were carried out for each crops as well. The objectives of this work were: a) To treatment. determine the effect of powders of P. dulce leaves, fruit and Separation. Seed extracts were concentrated in a rotary seeds and the removal of antifungal compounds from powders evaporator. The hexane-dicloromethane extract (25 g) was seeds, on the mycelia growth of seven fungal postharvest subjected to column chromatography (CC) on 300 g silica pathogens and b) To identify the active fractions of the hexane- gel with mixture of hexane-dicloromethane. Each fraction dicloromethane seed extract and testing their antifungal effect. was then dissolved in 1 ml of dicloromethane, amended with PDA media and tested as described previously. The resulting MATERIALS AND METHODS fractions were calculated to obtain a final concentration of Plant Material. Leaves, fruits and seeds of P. dulce were 6.4mg/ml. Two different controls were run: the first containing collected in February and March at the Centro de Desarrollo only PDA, and the second containing PDA amended with de Productos Bióticos in Yautepec, State of Morelos, Mexico. 1.0 ml of dicloromethane. Dishes were incubated in darkness Leaves, fruits and seeds were dipped in 1% sodium at 25 ± 1oC and the mycelial growth was measured after 96 h. hypochlorite solution, rinsed with distilled water and air-dried. The antifungal properties of thirteen fractions eluted from To obtain a better extraction of the active compound leaves, CC, were tested against F. oxysporum, P. digitatum and R. fruits or seeds were finely grounded with the aid of a grinder stolonifer measuring mycelial growth as previously described. and then stored at ambient temperature in amber bottles until Three plates were run per treatment. 2 Fractions eluted with further use. hexane-dicloromethane (2:8) contained a white wax which Microorganisms. Postharvest pathogens were isolated from was analyzed by nuclear magnetic resonance for proton 1H fruits as follow: Alternaria sp. from infected tomato NMR and carbon 13C NMR. The melting point was (Lycopersicum esculentum), Pestalotiopsis sp. from diseased determined in Fisher-Johns apparatus Mod. 12-144 (Fisher,

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Table 1. Effect of column chromatographic fractions of Pithecellobium dulce seed extract eluted with hexane-dicloromethane on mycelial growth (cm) of three postharvest pathogens. Mean colony diameter (cm)y Treatment Fusarium Penicillium Rhizopus oxysporum digitatum stolonifer No solvent 4.50 az 4.50 a 4.33 ab With dicloromethane 4.50 a 4.50 a 4.26 ab Fraction 12-13 2.63 def 4.50 a 3.66 bc Fraction 15-22 2.50 ef 4.50 a 1.76 f Fraction 23-37 3.96 abc 3.83 ab 2.70 de Fraction 39-41 3.40 bcd 4.16 ab 2.83 de Fraction 45 2.53 ef 4.50 a 4.50 a Fraction 50-56 2.30 f 4.50 a 4.40 a Fraction 58-62 3.20 cde 4.50 a 4.50 a Fraction 63-73 2.83 def 3.83 ab 4.26 ab Fraction 74-80 4.43 a 4.36 a 2.50 e Fraction 83-90 2.63 def 1.5 c 2.76 de Fraction 91-99 4.06 ab 3.06 b 2.70 de Fraction 129-137 2.40 ef 3.50 ab 3.83 abc Fraction 141 2.70 def 1.20 c 3.40 cd yOn potato-dextrose-agar plates. zMeans followed by the same letter are not significantly different (p < 0.05) as determined by Tukey’s multiple range test. other compounds not removed with the three solvent systems Acknowledgements. This work was supported by the General we used. The hexane-dicloromethane seed extract was Coordination of Posgraduate Study and Research, and the subjected to column chromatography, obtaining 13 fractions Commission of Operation and Development of Academic (Table 1). The most sensitive fungus we tested was F. Activities from the National Polytechnic Institute (IPN), oxysporum, since eleven fractions retarded its growth. Nine Mexico, F.D. fractions retarded growth of R. stolonifer. The least sensitive fungus was P. digitatum since only two fractions were LITERATURE CITED fungistatic. Results suggest that various compounds in the Aguilar, A., Camacho, J.R., Chino, S., Jacquez, P. y López, fractions tested are effective against the three fungi. The most M.E. 1996. Plantas Medicinales del Herbario del Instituto active fraction (50-56) against F. oxysporum had a white wax Mexicano del Seguro Social. Redacta S.A. ed. p. 63. with a melting point of 31°C. Preliminary NMR studies Bautista-Baños, S., Hernández, L.M., and Barrera, N.L.L. indicated the presence of an uncommon tryacyl glycerol 2000a. Antifungal screening of plants of the state of compound. Related studies have shown that lipophyllic Morelos, México against four postharvest pathogens of substances can be fungitoxic. For instance, Ginkgo biloba fruits and vegetables. Revista Mexicana de Fitopatología leaves contain fungitoxic oil and wax that affect Monilinia 18:36-42. fructicola development (Johnston and Sproston, 1965). Bautista-Baños, S., Hernández-López, M., Díaz-Pérez, J.C., Franich et al., (1983) reported that specific fatty and resin and Cano-Ochoa, C.F. 2000b. Evaluation of the fungicidal acids were highly fungistatic to Dothistroma pini (a needle properties of plant extracts to reduce Rhizopus stolonifer pathogen of Pinus radiata) suggesting that these compounds of ‘ciruela’ fruit (Spondias purpurea L.) during storage. could be pre-infectional barriers contributing to resistance. Postharvest Biology and Technology 20:99-106. In rice leaves antifungal compounds against Pyricularia Bravo, L.L., Bermúdez, T.K. y Montes B.R. 1998. Inhibición oryzae include twelve C18 hydroxy and epoxy fatty acids based del crecimiento micelial y esporulación de Fusarium on linolenic acid (Kato et al., 1993). Our research was centerd moniliforme Sheld. mediante aceites esenciales vegetales on in vitro studies. However, having in mind that behaviour y algunos de sus componentes químicos. Revista Mexicana of the fungi can dramatically change when experiments are de Fitopatología 16:18-23. in vitro rather than in planta further investigation should be Farr, D.F., Bills, G.F., Chamuris, G.P., and Rossman, A.Y. undertaken to determine the effects of these plant extracts 1989. Fungi on plant and plant products in the United with studies on intact fruit. States. American Phytopathological Society Press. St. Paul, MN, USA. 1252 p. Revista Mexicana de FITOPATOLOGIA/ 71

Franich, RA, Gadget, P.D. and, Shain, L. 1983. Fungistatic in rice plants. Bioscience, Biotechnology and Biochemistry effects of Pinus radiata needle epicuticular fatty and resin 57:283-287. acids on Dothistroma pini. Physiological Plant Patholology Montes, B.R., Cruz, C.V. y Peralta, D.M. 1990. Extractos 23:183-195. vegetales para el control de la roya del frijol Uromyces Johnston, H.W., and Sproston, Jr. T. 1965. The inhibition of appendiculatus. Agrociencia 1:99-106. fungal infection pegs in Ginkgo biloba. Phytopathology Montes, B.R., Carvajal, M., Figueroa, R. y Méndez, I. 1997. 55:225-227. Extractos sólidos, acuosos y hexánicos de plantas para el Kato, T., Yamaguchi, Y., Nanai, T., and Hirukawa, T., 1993. combate de Aspergillus flavus Link. en maíz. Revista Oxygenated fatty acids with anti-rice blast fungus activity Mexicana de Fitopatología 15:26-30.