Revista Mexicana de Fitopatología ISSN: Sociedad Mexicana de Fitopatología, A.C. México

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1 Revista Mexicana de Fitopatología ISSN: 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 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México Available in: How to cite Complete issue More information about this article Journal's homepage in redalyc.org Scientific Information System Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Non-profit academic project, developed under the open access initiative

2 66 / Volumen 20, Número 1, 2002 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 Correspondence to: lbarrera@ipn.mx (Received: October 17, 2001 Accepted: January 28, 2002) Abstract. Barrera-Necha, L.L., Bautista-Baños, S., Jiménez-Estrada, M., and Reyes-Chilpa, R 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 20: Powders of Pithecellobium dulce leaves, fruit and seeds sequentially extracted with hexane-dicloromethane, acetone, and methanol-water were evaluated on mycelial growth of Alternaria sp., Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum, Pestalotiopsis sp. and Rhizopus stolonifer. In comparison to fruit and leaf powders, seeds had the highest fungistatic activity against the fungi tested. In general, a doseeffect curve was observed for the three concentrations (0.5, 2.0 and 5.0 mg/ml) evaluated. However, for P. digitatum and Alternaria sp., the lowest and highest concentrations respectively, increased mycelial growth. Depending on concentration, leaf and fruit powders inhibited or increased mycelial growth. For Pestalotiopsis sp., P. digitatum, F. oxysporum, Alternaria sp., and R. stolonifer mycelial growth increased on seed residues (10 mg/ml), after hexanedicloromethane, acetone, and methanol-water extractions of seed powder, suggesting that fungistatic compounds were removed by the dissolvent used. The hexane-dicloromethane extract was subjected to column chromatography, obtaining 13 fractions with similar pattern, which were evaluated using the mycelial growth responses of F. oxysporum, P. digitatum and R. stolonifer. Eleven and nine fractions inhibited F. oxysporum and R. stolonifer development, respectively. P. digitatum was the fungus least affected by all fractions. Preliminary analysis of the most active fraction by nuclear magnetic resonance indicated the presence of a tryacyl glycerol. Additional key words: Guamúchil, huamúchil, Madras thorn, manila tamarind, ojiuma, plant extracts, fractions, Alternaria sp., Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum, Pestalotiopsis sp., Rhizopus stolonifer. Resumen. Los polvos de hojas, frutos y semillas de Pithecellobium dulce y semillas extraídas secuencialmente con hexano-diclorometano, acetona y metanol-agua se evaluaron sobre el crecimiento micelial de Alternaria sp., Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum, Pestalotiopsis sp. y Rhizopus stolonifer. Los polvos de semillas tuvieron la más alta actividad fungistática contra los hongos probados en comparación con los polvos de fruto y hoja. En general, se observó una curva de dosis-efecto para las tres concentraciones evaluadas (0.5, 2.0 and 5.0 mg/ml). Sin embargo, para P. digitatum y Alternaria sp. la concentración más baja y la más alta, respectivamente, incrementaron el crecimiento micelial. Dependiendo de la concentración, los polvos de hoja y fruto inhibieron o incrementaron el crecimiento micelial. El crecimiento micelial de Pestalotiopsis sp, P. digitatum, F. oxysporum, Alternaria sp. y R. stolonifer se incrementó sobre los residuos de semillas (10mg/ml), después de la extracción de hexanodiclorometano, acetona y metanol-agua de polvos de semillas, sugiriendo que los compuestos fungistáticos fueron removidos por los disolventes usados. El extracto hexano-diclorometano fue sometido a una cromatografía en columna, obteniéndose 13 fracciones con patrones similares, las cuales fueron evaluadas usando la respuesta de crecimiento micelial de F. oxysporum, P. digitatum y R. stolonifer. Once y nueve de las fracciones inhibieron el crecimiento de F. oxysporum y R. stolonifer, respectivamente. P. digitatum fue el hongo menos afectado por todas las fracciones. El análisis preliminar de la fracción más activa por resonancia magnética nuclear indicó la presencia de un triacil glicerol. Palabras clave adicionales: Guamúchil, huamúchil, Madras thorn, manila tamarind, ojiuma, extractos vegetales,

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

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6 70 / Volumen 20, Número 1, 2002 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 a z 4.50 a 4.33 ab With dicloromethane 4.50 a 4.50 a 4.26 ab Fraction def 4.50 a 3.66 bc Fraction ef 4.50 a 1.76 f Fraction abc 3.83 ab 2.70 de Fraction bcd 4.16 ab 2.83 de Fraction ef 4.50 a 4.50 a Fraction f 4.50 a 4.40 a Fraction cde 4.50 a 4.50 a Fraction def 3.83 ab 4.26 ab Fraction a 4.36 a 2.50 e Fraction def 1.5 c 2.76 de Fraction ab 3.06 b 2.70 de Fraction ef 3.50 ab 3.83 abc Fraction def 1.20 c 3.40 cd y On potato-dextrose-agar plates. z Means 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 we used. The hexane-dicloromethane seed extract was subjected to column chromatography, obtaining 13 fractions (Table 1). The most sensitive fungus we tested was F. oxysporum, since eleven fractions retarded its growth. Nine fractions retarded growth of R. stolonifer. The least sensitive fungus was P. digitatum since only two fractions were fungistatic. Results suggest that various compounds in the fractions tested are effective against the three fungi. The most active fraction (50-56) against F. oxysporum had a white wax with a melting point of 31 C. Preliminary NMR studies indicated the presence of an uncommon tryacyl glycerol compound. Related studies have shown that lipophyllic substances can be fungitoxic. For instance, Ginkgo biloba leaves contain fungitoxic oil and wax that affect Monilinia fructicola development (Johnston and Sproston, 1965). Franich et al., (1983) reported that specific fatty and resin acids were highly fungistatic to Dothistroma pini (a needle pathogen of Pinus radiata) suggesting that these compounds could be pre-infectional barriers contributing to resistance. In rice leaves antifungal compounds against Pyricularia oryzae include twelve C 18 hydroxy and epoxy fatty acids based on linolenic acid (Kato et al., 1993). Our research was centerd on in vitro studies. However, having in mind that behaviour of the fungi can dramatically change when experiments are in vitro rather than in planta further investigation should be undertaken to determine the effects of these plant extracts with studies on intact fruit. Acknowledgements. This work was supported by the General Coordination of Posgraduate Study and Research, and the Commission of Operation and Development of Academic Activities from the National Polytechnic Institute (IPN), Mexico, F.D. LITERATURE CITED Aguilar, A., Camacho, J.R., Chino, S., Jacquez, P. y López, M.E Plantas Medicinales del Herbario del Instituto Mexicano del Seguro Social. Redacta S.A. ed. p. 63. Bautista-Baños, S., Hernández, L.M., and Barrera, N.L.L. 2000a. Antifungal screening of plants of the state of Morelos, México against four postharvest pathogens of fruits and vegetables. Revista Mexicana de Fitopatología 18: Bautista-Baños, S., Hernández-López, M., Díaz-Pérez, J.C., and Cano-Ochoa, C.F. 2000b. Evaluation of the fungicidal properties of plant extracts to reduce Rhizopus stolonifer of ciruela fruit (Spondias purpurea L.) during storage. Postharvest Biology and Technology 20: Bravo, L.L., Bermúdez, T.K. y Montes B.R Inhibición del crecimiento micelial y esporulación de Fusarium moniliforme Sheld. mediante aceites esenciales vegetales y algunos de sus componentes químicos. Revista Mexicana de Fitopatología 16: Farr, D.F., Bills, G.F., Chamuris, G.P., and Rossman, A.Y Fungi on plant and plant products in the United States. American Phytopathological Society Press. St. Paul, MN, USA p.

7 Revista Mexicana de FITOPATOLOGIA/ 71 Franich, RA, Gadget, P.D. and, Shain, L Fungistatic effects of Pinus radiata needle epicuticular fatty and resin acids on Dothistroma pini. Physiological Plant Patholology 23: Johnston, H.W., and Sproston, Jr. T The inhibition of fungal infection pegs in Ginkgo biloba. Phytopathology 55: Kato, T., Yamaguchi, Y., Nanai, T., and Hirukawa, T., Oxygenated fatty acids with anti-rice blast fungus activity in rice plants. Bioscience, Biotechnology and Biochemistry 57: Montes, B.R., Cruz, C.V. y Peralta, D.M Extractos vegetales para el control de la roya del frijol Uromyces appendiculatus. Agrociencia 1: Montes, B.R., Carvajal, M., Figueroa, R. y Méndez, I Extractos sólidos, acuosos y hexánicos de plantas para el combate de Aspergillus flavus Link. en maíz. Revista Mexicana de Fitopatología 15:26-30.