Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] 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 erosus (L.) Urban () Against Three Postharvest Fungi Revista Mexicana de Fitopatología, vol. 22, núm. 3, diciembre, 2004, pp. 356-361 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México

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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 04510. Correspondencia: [email protected]

(Received: July 19, 2004 Accepted: October 12, 2004)

Barrera-Necha, L.L., Bautista-Baños, S., Bravo-Luna, L., Resumen. Se investigó el efecto antifúngico de polvos, García-Suárez, F.J., Alavez-Solano, D., y Reyes-Chilpa, R. extractos, y metabolitos secundarios de semillas de 2004. Antifungal activity of seed powders, extracts, and Pachyrhizus erosus cosechadas en el estado de Morelos, secondary metabolites of Pachyrhizus erosus (L.) Urban México, usando bioensayos de inhibición micelial de (Fabaceae) against three postharvest fungi. Revista Mexicana Colletotrichum gloeosporioides, Fusarium oxysporum, y de Fitopatología 22:356-361. Rhizopus stolonifer. En general, se observó una curva de dosis Abstract. The antifungal effects of seed powders, extracts, efecto a las concentraciones de 0.5, 2.0, 5.0, y 10 mg/ml en and secondary metabolites of Pachyrhizus erosus from los hongos evaluados. Los polvos de semilla tuvieron un Morelos state, Mexico, was investigated using mycelial efecto inhibitorio sobre C. gloeosporioides. Sin embargo, inhibition bioassays on Colletotrichum gloeosporioides, también se observaron efectos estimulatorios sobre F. Fusarium oxysporum, and Rhizopus stolonifer. In general, a oxysporum y R. stolonifer a 0.5 y 5.0 mg/ml, respectivamente. dose-effect curve for concentrations 0.5, 2.0, 5.0, and 10 mg/ Todos los extractos, hexano, diclorometano, y acetona ml was observed on the fungi evaluated. Seed powders had inhibieron significativamente a los tres hongos a an inhibitory effect on C. gloeosporioides; nevertheless, concentraciones de 2.0, 5.0, y 10 mg/ml. Se obtuvo el mayor stimulation effects were observed on F. oxysporum, and R. efecto fungistático con el extracto de diclorometano sobre R. stolonifer at 0.5, and 5.0 mg/ml, respectively. All extracts, stolonifer (-64.97%), F. oxysporum (-37.8%) y C. hexane, dicloromethane, and acetone, significantly inhibited gloeosporioides (-36.4%). Estos resultados sugieren que los the three fungi at 2.0, 5.0, and 10 mg/ml. The greatest compuestos fungistáticos pueden ser extraídos con este fungistatic effects were achieved with dicloromethane extract solvente. El extracto de diclorometano se sometió a una on R. stolonifer (-64.97%), on F. oxysporum (-37.8%), and cromatografía en columna, aislándose rotenona (1), erosona C. gloeosporioides (-36.4%). These results suggest that (3), paquirrizona (5), dolineona (6), y paquirrizina (4). La fungicidal compounds might be extracted with this solvent. isoflavona dehidroneotenona (2) fue aislada del extracto de The dicloromethane extract was subjected to chromatography acetona. La identidad de los componentes se determinó por isolating rotenone (1), erosone (3), paquirrizone (5), espectrometría de infrarrojo, ultravioleta, y resonancia dolineone (6), and paquirrizine (4). The isoflavone magnética nuclear de 1H y 13C. Estos metabolitos secundarios dehydroneotenone (2) was isolated from the acetone extract. inhibieron significativamente a los tres hongos a una The identity of the components was determined by infrared, concentración de 250 µg/ml. Se obtuvo el mayor efecto ultraviolet, 1H, and 13C nuclear magnetic resonance. These fungistático con rotenona sobre R. stolonifer, con paquirrizina secondary metabolites significantly inhibited the three fungi sobre F. oxysporum, y dehidroneotenona sobre C. at 250 µg/ml. The best fungicidal effect was achieved with gloeosporioides. rotenone on R. stolonifer, with pachyrrizine on F. oxysporum, and dehydroneotenone on C. gloeosporioides. Palabras clave adicionales: Jícama, extractos de semilla, Colletotrichum gloeosporioides, Fusarium oxysporum, Additional keywords: Yam bean, seed extracts, Rhizopus stolonifer. Colletotrichum gloeosporioides, Fusarium oxysporum, Rhizopus stolonifer. Failure to control postharvest pathogenic fungi can result in Revista Mexicana de FITOPATOLOGIA/ 357 serious economic losses to worldwide horticultural stolonifer, four days for F. oxysporum, and C. production. Fungi such as Colletotrichum gloeosporioides gloeosporioides. Mycelial growth (colony diameter) was (Penz.) Penz. y Sacc., Fusarium oxysporum Schlechtend.:Fr., measured at the end of the incubation time. Six replications and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. cause diseases were run simultaneously for each concentration of seed on different fruits and vegetables, and all are considered major powder. Control Petri plates contained only PDA. Tests were pathogens (Farr et al., 1989). There is a world need to finished when mycelium of the control plates reached the develop new and acceptable postharvest disinfestation edge of the dishes. The experiment was repeated twice. methods. To minimize the adverse effects of synthetic Growth inhibitory effects were calculated as follows: % I = products on agro-ecosystems and because of the emergence (MGC - MGT)/MGC x 100 where: % I = % Inhibition; MGC of plant pathogens resistant to the currently used fungicides, = Mycelial growth in control, and MGT = Mycelial growth it is necessary to evaluate other alternatives such as natural in treatment. products. synthesize a vast array of organic compounds, Extraction. Seed powders (1.5 kg) were extracted with commonly called secondary metabolites, that play an hexane, dicloromethane, and acetone for 48 h in each solvent important role in the complex interactions between plants system at room temperature. After each extraction step, the and other organisms. One of their many functions is a chemical seed extracts were concentrated in a rotary evaporator (Büchi defense against pathogens and herbivores (McLaren, 1986). R-114). The hexane extract was rich in yellow oil (14.3% The seed of yam bean, Pachyrhizus erosus (L.) Urban average yield of dry weight). From the dicloromethane extract, (Fabaceae), is of interest as a potential source of insecticidal a dark yellow precipitate was obtained with 4.88% yield, and material, because it contains rotenone a compound which is acetone extract had 1.21% yield. Three concentrations (2.0, highly toxic to pests, mites, and fish, but not to 5.0, and 10.0 mg/ml) of each extract were used to amend 24 mammals. In folk medicine, the pulverized seed of this plant ml of PDA, which was then autoclaved and poured into Petri is applied for treatment of skin eruptions and as fish poison plates. Growth of each test fungus was recorded at the end of (Perry and Metzger, 1980). More than a decade ago it was each incubation time. Each treatment was replicated six times. discovered that rotenone has potential antitumor activity Isolation, separation and identification of secondary (Kardono et al., 1990). This plant has been well studied metabolites. Organic extracts were subjected to column phytochemically, and rotenone and its derivatives have been chromatography (CC) on silica gel 60 (Merck 0.2-0.5), at found to be the constituents responsible for its biological the ration (1:20). Elution was carried out with a mixture of activity (Alavez-Solano et al., 1998; Gresshoff, 1890; hexane-dicloromethane in order to increase polarity in a linear Hansberry and Lee, 1943; Hansberry et al., 1947; Hwang, gradient. The identity of components was determined by 1941; Nag et al., 1936; Norton, 1943; Norton and Hansberry, infrared, ultraviolet, nuclear proton magnetic resonance, and 1945). The objective of this study was to investigate the effect carbon13 spectroscopic analysis. The melting point was of seed powders, extracts, and secondary metabolites of determined in a Fisher-Johns apparatus Mod. 12-144 (Fisher, Pachyrhizus erosus on the in vitro mycelial growth of three USA). The yellow dark precipitate and supernatant obtained postharvest fungi. with dicloromethane extract, and the acetone extract, were subjected to column chromatography to isolate different MATERIALS AND METHODS isoflavonoids. The isolated compounds and certificate Plant material. Seeds of P. erosus variety Agua Dulce were rotenone (1) were used as reference to determine their donated by farmers to the Centro de Desarrollo de Productos presence in dicloromethane and acetone extract by thin layer Bióticos in Yautepec, State of Morelos, Mexico. They were chromatography (TLC) using silica gel plates (Merck, 0.25 dipped in 1% sodium hypochlorite solution, rinsed with mm), and eluted with dicloromethane. Developed TLC plates distilled water, and air-dried. To obtain a better extraction of sprayed afterwards with a reagent (Cerium IV dehydrate the active compound, seeds were finely ground and then stored sulphate-Baker 2% in 2N H2SO4) and warmed up on a hot at room temperature (26°C) in amber bottles until further use. plate (150°C, 1 min) were evaluated in UV light. The Rf- Microorganisms. The following postharvest pathogens were value = Solute front/ solvent front of six compounds was isolated from infected papaya (Carica papaya L.): C. determined in these conditions. The secondary metabolites gloeosporioides, F. oxysporum, and R. stolonifer. Each fungus amounts were then dissolved in 1 ml of acetone to obtain a was frequently inoculated and reisolated from its host in order final concentration of 250 µg/ml,, added to the PDA media, to maintain pathogenicity. and tested for fungicidal activity as described previously. Two In vitro bioassay. Seed powders were prepared at four different controls were used: the first containing only PDA, concentrations (0.5, 2.0, 5.0, and 10.0 mg/ml), added to 24 and the second containing PDA amended with 1.0 ml of ml of potato-dextrose-agar (PDA), and autoclaved (15 lb/ acetone. Petri dishes were incubated in darkness at 25 ± 1°C, cm2, 15 min). After sterilization media were poured into Petri and the antifungal properties of the six compounds eluted plates (60 x 15 mm). A five mm agar disc containing the from CC were tested against C. gloeosporioides, F. respective pathogen was placed at the center of each plate oxysporum, and R. stolonifer measuring mycelial growth as which was then incubated at 25°C as follows: One day for R. previously described. Three plates were run per treatment.

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