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; Bravo Luna, Leticia; García Suárez, Francisco Javier Leobardo; Alavez Solano, Dagoberto; Reyes Chilpa, Ricardo Antifungal Activity of Seed Powders, Extracts, and Secondary Metabolites of Pachyrhizus erosus (L.) Urban (Fabaceae) Against Three Postharvest Fungi Revista Mexicana de Fitopatología, vol. 22, núm. 3, diciembre, 2004, 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

2 356 / Volumen 22, Número 3, 2004 Antifungal Activity of Seed Powders, Extracts, and Secondary Metabolites of Pachyrhizus erosus (L.) Urban (Fabaceae) Against Three Postharvest Fungi Laura Leticia Barrera-Necha, Silvia Bautista-Baños, Leticia Bravo-Luna, Francisco Javier Leobardo García-Suárez, Instituto Politécnico Nacional, Centro de Desarrollo de Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San Isidro Yautepec, Morelos, México CP 62731; Dagoberto Alavez-Solano, y Ricardo Reyes-Chilpa, Universidad Nacional Autónoma de México, Instituto de Química, Circuito Exterior, Ciudad Universitaria, Coyoacán, México, D.F., CP Correspondencia: lbarrera@ipn.mx (Received: July 19, 2004 Accepted: October 12, 2004) Barrera-Necha, L.L., Bautista-Baños, S., Bravo-Luna, L., García-Suárez, F.J., Alavez-Solano, D., y Reyes-Chilpa, R Antifungal activity of seed powders, extracts, and secondary metabolites of Pachyrhizus erosus (L.) Urban (Fabaceae) against three postharvest fungi. Revista Mexicana de Fitopatología 22: Abstract. The antifungal effects of seed powders, extracts, and secondary metabolites of Pachyrhizus erosus from Morelos state, Mexico, was investigated using mycelial inhibition bioassays on Colletotrichum gloeosporioides, Fusarium oxysporum, and Rhizopus stolonifer. In general, a dose-effect curve for concentrations 0.5, 2.0, 5.0, and 10 mg/ ml was observed on the fungi evaluated. Seed powders had an inhibitory effect on C. gloeosporioides; nevertheless, stimulation effects were observed on F. oxysporum, and R. stolonifer at 0.5, and 5.0 mg/ml, respectively. All extracts, hexane, dicloromethane, and acetone, significantly inhibited the three fungi at 2.0, 5.0, and 10 mg/ml. The greatest fungistatic effects were achieved with dicloromethane extract on R. stolonifer (-64.97%), on F. oxysporum (-37.8%), and C. gloeosporioides (-36.4%). These results suggest that fungicidal compounds might be extracted with this solvent. The dicloromethane extract was subjected to chromatography isolating rotenone (1), erosone (3), paquirrizone (5), dolineone (6), and paquirrizine (4). The isoflavone dehydroneotenone (2) was isolated from the acetone extract. The identity of the components was determined by infrared, ultraviolet, 1 H, and 13 C nuclear magnetic resonance. These secondary metabolites significantly inhibited the three fungi at 250 µg/ml. The best fungicidal effect was achieved with rotenone on R. stolonifer, with pachyrrizine on F. oxysporum, and dehydroneotenone on C. gloeosporioides. Additional keywords: Yam bean, seed extracts, Colletotrichum gloeosporioides, Fusarium oxysporum, Rhizopus stolonifer. Resumen. Se investigó el efecto antifúngico de polvos, extractos, y metabolitos secundarios de semillas de Pachyrhizus erosus cosechadas en el estado de Morelos, México, usando bioensayos de inhibición micelial de Colletotrichum gloeosporioides, Fusarium oxysporum, y Rhizopus stolonifer. En general, se observó una curva de dosis efecto a las concentraciones de 0.5, 2.0, 5.0, y 10 mg/ml en los hongos evaluados. Los polvos de semilla tuvieron un efecto inhibitorio sobre C. gloeosporioides. Sin embargo, también se observaron efectos estimulatorios sobre F. oxysporum y R. stolonifer a 0.5 y 5.0 mg/ml, respectivamente. Todos los extractos, hexano, diclorometano, y acetona inhibieron significativamente a los tres hongos a concentraciones de 2.0, 5.0, y 10 mg/ml. Se obtuvo el mayor efecto fungistático con el extracto de diclorometano sobre R. stolonifer (-64.97%), F. oxysporum (-37.8%) y C. gloeosporioides (-36.4%). Estos resultados sugieren que los compuestos fungistáticos pueden ser extraídos con este solvente. El extracto de diclorometano se sometió a una cromatografía en columna, aislándose rotenona (1), erosona (3), paquirrizona (5), dolineona (6), y paquirrizina (4). La isoflavona dehidroneotenona (2) fue aislada del extracto de acetona. La identidad de los componentes se determinó por espectrometría de infrarrojo, ultravioleta, y resonancia magnética nuclear de 1 H y 13 C. Estos metabolitos secundarios inhibieron significativamente a los tres hongos a una concentración de 250 µg/ml. Se obtuvo el mayor efecto fungistático con rotenona sobre R. stolonifer, con paquirrizina sobre F. oxysporum, y dehidroneotenona sobre C. gloeosporioides. Palabras clave adicionales: Jícama, extractos de semilla, Colletotrichum gloeosporioides, Fusarium oxysporum, Rhizopus stolonifer. Failure to control postharvest pathogenic fungi can result in

3 Revista Mexicana de FITOPATOLOGIA/ 357 serious economic losses to worldwide horticultural production. Fungi such as Colletotrichum gloeosporioides (Penz.) Penz. y Sacc., Fusarium oxysporum Schlechtend.:Fr., and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. cause diseases on different fruits and vegetables, and all are considered major plant pathogens (Farr et al., 1989). There is a world need to develop new and acceptable postharvest disinfestation methods. To minimize the adverse effects of synthetic products on agro-ecosystems and because of the emergence of plant pathogens resistant to the currently used fungicides, it is necessary to evaluate other alternatives such as natural products. Plants synthesize a vast array of organic compounds, commonly called secondary metabolites, that play an important role in the complex interactions between plants and other organisms. One of their many functions is a chemical defense against pathogens and herbivores (McLaren, 1986). The seed of yam bean, Pachyrhizus erosus (L.) Urban (Fabaceae), is of interest as a potential source of insecticidal material, because it contains rotenone a compound which is highly toxic to insect pests, mites, and fish, but not to mammals. In folk medicine, the pulverized seed of this plant is applied for treatment of skin eruptions and as fish poison (Perry and Metzger, 1980). More than a decade ago it was discovered that rotenone has potential antitumor activity (Kardono et al., 1990). This plant has been well studied phytochemically, and rotenone and its derivatives have been found to be the constituents responsible for its biological activity (Alavez-Solano et al., 1998; Gresshoff, 1890; Hansberry and Lee, 1943; Hansberry et al., 1947; Hwang, 1941; Nag et al., 1936; Norton, 1943; Norton and Hansberry, 1945). The objective of this study was to investigate the effect of seed powders, extracts, and secondary metabolites of Pachyrhizus erosus on the in vitro mycelial growth of three postharvest fungi. MATERIALS AND METHODS Plant material. Seeds of P. erosus variety Agua Dulce were donated by farmers to the Centro de Desarrollo de Productos Bióticos in Yautepec, State of Morelos, Mexico. They were dipped in 1% sodium hypochlorite solution, rinsed with distilled water, and air-dried. To obtain a better extraction of the active compound, seeds were finely ground and then stored at room temperature (26 C) in amber bottles until further use. Microorganisms. The following postharvest pathogens were isolated from infected papaya (Carica papaya L.): C. gloeosporioides, F. oxysporum, and R. stolonifer. Each fungus was frequently inoculated and reisolated from its host in order to maintain pathogenicity. In vitro bioassay. Seed powders were prepared at four concentrations (0.5, 2.0, 5.0, and 10.0 mg/ml), added to 24 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 agar disc containing the respective pathogen was placed at the center of each plate which was then incubated at 25 C as follows: One day for R. stolonifer, four days for F. oxysporum, and C. gloeosporioides. Mycelial growth (colony diameter) was measured at the end of the incubation time. Six replications were run simultaneously for each concentration of seed powder. Control Petri plates contained only PDA. Tests were finished when mycelium of the control plates reached the edge of the dishes. The experiment was repeated twice. Growth inhibitory effects were calculated as follows: % I = (MGC - MGT)/MGC x 100 where: % I = % Inhibition; MGC = Mycelial growth in control, and MGT = Mycelial growth in treatment. Extraction. Seed powders (1.5 kg) were extracted with hexane, dicloromethane, and acetone for 48 h in each solvent system at room temperature. After each extraction step, the seed extracts were concentrated in a rotary evaporator (Büchi R-114). The hexane extract was rich in yellow oil (14.3% average yield of dry weight). From the dicloromethane extract, a dark yellow precipitate was obtained with 4.88% yield, and acetone extract had 1.21% yield. Three concentrations (2.0, 5.0, and 10.0 mg/ml) of each extract were used to amend 24 ml of PDA, which was then autoclaved and poured into Petri plates. Growth of each test fungus was recorded at the end of each incubation time. Each treatment was replicated six times. Isolation, separation and identification of secondary metabolites. Organic extracts were subjected to column chromatography (CC) on silica gel 60 (Merck ), at the ration (1:20). Elution was carried out with a mixture of hexane-dicloromethane in order to increase polarity in a linear gradient. The identity of components was determined by infrared, ultraviolet, nuclear proton magnetic resonance, and carbon 13 spectroscopic analysis. The melting point was determined in a Fisher-Johns apparatus Mod (Fisher, USA). The yellow dark precipitate and supernatant obtained with dicloromethane extract, and the acetone extract, were subjected to column chromatography to isolate different isoflavonoids. The isolated compounds and certificate rotenone (1) were used as reference to determine their presence in dicloromethane and acetone extract by thin layer chromatography (TLC) using silica gel plates (Merck, 0.25 mm), and eluted with dicloromethane. Developed TLC plates sprayed afterwards with a reagent (Cerium IV dehydrate sulphate-baker 2% in 2N H 2 SO 4 ) and warmed up on a hot plate (150 C, 1 min) were evaluated in UV light. The Rfvalue = Solute front/ solvent front of six compounds was determined in these conditions. The secondary metabolites amounts were then dissolved in 1 ml of acetone to obtain a final concentration of 250 µg/ml,, added to the PDA media, and tested for fungicidal activity as described previously. Two different controls were used: the first containing only PDA, and the second containing PDA amended with 1.0 ml of acetone. Petri dishes were incubated in darkness at 25 ± 1 C, and the antifungal properties of the six compounds eluted from CC were tested against C. gloeosporioides, F. oxysporum, and R. stolonifer measuring mycelial growth as previously described. Three plates were run per treatment.

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7 Revista Mexicana de FITOPATOLOGIA/ 361 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. 450 p. Fukami, J.T Effects of some insecticides on the respiration of insect organs, with Special reference to the effects of rotenone. Botyu-Kagaku 21:122. Fukami, J.T., Shishido, T.S., Fukunaga, K., and Cosida J.E Oxidative metabolism of rotenone in mammals, fish and insects and its relation to selective toxicity. Journal of Agricultural and Food Chemistry 17: Fukami, J.T., and Nakajima, M Naturally Ocurring Insecticides. pp In: M. Jacobson, and D.G. Crosby, J. (eds.). Decker. New York, USA. 250 p. Gresshoff, M Eerste Verlagvan het Onderzoeek naar de Plantensoffen van Nederlandsch-Indië. Mededeelingen uit s Lands. Plantentuin (Batavia, Landsdrukkerij) 7: Haley, T.J A Review of the literature of Rotenone. Journal of Enviromental Pathology and Toxicology 1: Hansberry, R., and Lee, C The yam bean Pachyrhizus erosus (L.) Urban as a possible insecticide. Journal of Economic Entomology 36: Hansberry, R., Clausen, R.T., and Norton, J.B Variation in the chemical composition and insecticidal properties of the yam bean (Pachyrhizus erosus). Journal of Agricultural Research 74: Hwang, S.L A preliminary report on the chemical composition of yam bean, (Pachyrhizus erosus Urban). A new rotenone bearing plant. Kwangsi Agriculture 2: Kardono, L.B., Tsauri, S., Padmawinata, K., Pezzuto, J.M., and Kinghorn, A.D Cytotoxic constituents of the seed of Pachyrhizus erosus. Planta Médica 56: Leslie, A.R., ed Handbook of Integrated Pest Management. Lewis Publishers Inc. Washington, D.C., USA p. Matsumura, F Toxicity of insecticides. Plenum Press (eds.). New York, USA. 125 p. McLaren, J.S Biologically active substances from higher plants: status and future potential. Pesticide Science 17: Montes, B.R., Carvajal, M., Figueroa, B.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: Nag, N.C., Banerjee, H.N., and Pain, A.K Chemical examination of seeds of Pachyhrizus angulatus. Transactions of the Bose Research Institute 11: Norton, L.B Rotenone in the yam bean (Pachyrhizus erosus). Journal of the Association of Official Analytical Chemists 61: Norton, L.B., and Hansberry, R Constituents of the insecticidal resins of the yam bean, Pachyrhizus erosus. Journal of the American Chemical Society 67: Perry, L.M., and Metzger, J Medicinal Plants of East and South East Asia: Attributed Properties and Uses. MIT Press. Cambridge, Massachusetts, USA. 221 p.