Revista Mexicana de Fitopatología Sociedad Mexicana de Fitopatología, A.C.guillermofuentes_davila@hotmail.com ISSN (Versión impresa): 0185-3309MÉXICO

2004 Laura Leticia Barrera Necha / Silvia Bautista Baños / Leticia Bravo Luna / Francisco Javier Leobardo

García Suárez / Dagoberto Alavez Solano / Ricardo Reyes ChilpaANTIFUNGAL ACTIVITY OF SEED POWDERS, EXTRACTS, AND SECONDARY METABOLITES OF

PACHYRHIZUS EROSUS (L.) URBAN (FABACEAE) AGAINST THREE POSTHARVEST FUNGI Revista Mexicana de Fitopatología, diciembre, año/vol. 22, número 003

Sociedad Mexicana de Fitopatología, A.C. Ciudad Obregón, México

pp. 356-361

Red de Revistas Científicas de América Latina y el Caribe, España y Portugal

Universidad Autónoma del Estado de México

http://redalyc.uaemex.mx

Antifungal Activity of Seed Powders, Extracts, and SecondaryMetabolites of Pachyrhizus erosus (L.) Urban (Fabaceae) Against

Three Postharvest FungiLaura Leticia Barrera-Necha, Silvia Bautista-Baños, Leticia Bravo-Luna, FranciscoJavier Leobardo García-Suárez, Instituto Politécnico Nacional, Centro de Desarrollode Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San Isidro Yautepec, Morelos, MéxicoCP 62731; Dagoberto Alavez-Solano, y Ricardo Reyes-Chilpa, Universidad NacionalAutónoma de México, Instituto de Química, Circuito Exterior, Ciudad Universitaria,Coyoacán, México, D.F., CP 04510. Correspondencia: lbarrera@ipn.mx

Barrera-Necha, L.L., Bautista-Baños, S., Bravo-Luna, L.,García-Suárez, F.J., Alavez-Solano, D., y Reyes-Chilpa, R.2004. Antifungal activity of seed powders, extracts, andsecondary metabolites of Pachyrhizus erosus (L.) Urban(Fabaceae) against three postharvest fungi. Revista Mexicanade Fitopatología 22:356-361.Abstract. The antifungal effects of seed powders, extracts,and secondary metabolites of Pachyrhizus erosus fromMorelos state, Mexico, was investigated using mycelialinhibition bioassays on Colletotrichum gloeosporioides,Fusarium oxysporum, and Rhizopus stolonifer. In general, adose-effect curve for concentrations 0.5, 2.0, 5.0, and 10 mg/ml was observed on the fungi evaluated. Seed powders hadan 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 inhibitedthe three fungi at 2.0, 5.0, and 10 mg/ml. The greatestfungistatic effects were achieved with dicloromethane extracton R. stolonifer (-64.97%), on F. oxysporum (-37.8%), andC. gloeosporioides (-36.4%). These results suggest thatfungicidal compounds might be extracted with this solvent.The dicloromethane extract was subjected to chromatographyisolating rotenone (1), erosone (3), paquirrizone (5),dolineone (6), and paquirrizine (4). The isoflavonedehydroneotenone (2) was isolated from the acetone extract.The identity of the components was determined by infrared,ultraviolet, 1H, and 13C nuclear magnetic resonance. Thesesecondary metabolites significantly inhibited the three fungiat 250 µg/ml. The best fungicidal effect was achieved withrotenone 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 dePachyrhizus erosus cosechadas en el estado de Morelos,México, usando bioensayos de inhibición micelial deColletotrichum gloeosporioides, Fusarium oxysporum, yRhizopus stolonifer. En general, se observó una curva de dosisefecto a las concentraciones de 0.5, 2.0, 5.0, y 10 mg/ml enlos hongos evaluados. Los polvos de semilla tuvieron unefecto 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 acetonainhibieron significativamente a los tres hongos aconcentraciones de 2.0, 5.0, y 10 mg/ml. Se obtuvo el mayorefecto fungistático con el extracto de diclorometano sobre R.stolonifer (-64.97%), F. oxysporum (-37.8%) y C.gloeosporioides (-36.4%). Estos resultados sugieren que loscompuestos fungistáticos pueden ser extraídos con estesolvente. El extracto de diclorometano se sometió a unacromatografía en columna, aislándose rotenona (1), erosona(3), paquirrizona (5), dolineona (6), y paquirrizina (4). Laisoflavona dehidroneotenona (2) fue aislada del extracto deacetona. La identidad de los componentes se determinó porespectrometría de infrarrojo, ultravioleta, y resonanciamagnética nuclear de 1H y 13C. Estos metabolitos secundariosinhibieron significativamente a los tres hongos a unaconcentración de 250 µg/ml. Se obtuvo el mayor efectofungistático con rotenona sobre R. stolonifer, con paquirrizinasobre 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

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

356 / Volumen 22, Número 3, 2004

serious economic losses to worldwide horticulturalproduction. Fungi such as Colletotrichum gloeosporioides(Penz.) Penz. y Sacc., Fusarium oxysporum Schlechtend.:Fr.,and Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. cause diseaseson different fruits and vegetables, and all are considered majorplant pathogens (Farr et al., 1989). There is a world need todevelop new and acceptable postharvest disinfestationmethods. To minimize the adverse effects of syntheticproducts on agro-ecosystems and because of the emergenceof plant pathogens resistant to the currently used fungicides,it is necessary to evaluate other alternatives such as naturalproducts. Plants synthesize a vast array of organic compounds,commonly called secondary metabolites, that play animportant role in the complex interactions between plantsand other organisms. One of their many functions is a chemicaldefense against pathogens and herbivores (McLaren, 1986).The seed of yam bean, Pachyrhizus erosus (L.) Urban(Fabaceae), is of interest as a potential source of insecticidalmaterial, because it contains rotenone a compound which ishighly toxic to insect pests, mites, and fish, but not tomammals. In folk medicine, the pulverized seed of this plantis applied for treatment of skin eruptions and as fish poison(Perry and Metzger, 1980). More than a decade ago it wasdiscovered that rotenone has potential antitumor activity(Kardono et al., 1990). This plant has been well studiedphytochemically, and rotenone and its derivatives have beenfound to be the constituents responsible for its biologicalactivity (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 effectof seed powders, extracts, and secondary metabolites ofPachyrhizus erosus on the in vitro mycelial growth of threepostharvest fungi.

MATERIALS AND METHODSPlant material. Seeds of P. erosus variety Agua Dulce weredonated by farmers to the Centro de Desarrollo de ProductosBióticos in Yautepec, State of Morelos, Mexico. They weredipped in 1% sodium hypochlorite solution, rinsed withdistilled water, and air-dried. To obtain a better extraction ofthe active compound, seeds were finely ground and then storedat room temperature (26°C) in amber bottles until further use.Microorganisms. The following postharvest pathogens wereisolated from infected papaya (Carica papaya L.): C.gloeosporioides, F. oxysporum, and R. stolonifer. Each funguswas frequently inoculated and reisolated from its host in orderto maintain pathogenicity.In vitro bioassay. Seed powders were prepared at fourconcentrations (0.5, 2.0, 5.0, and 10.0 mg/ml), added to 24ml of potato-dextrose-agar (PDA), and autoclaved (15 lb/cm2, 15 min). After sterilization media were poured into Petriplates (60 x 15 mm). A five mm agar disc containing therespective pathogen was placed at the center of each platewhich 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) wasmeasured at the end of the incubation time. Six replicationswere run simultaneously for each concentration of seedpowder. Control Petri plates contained only PDA. Tests werefinished when mycelium of the control plates reached theedge 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 growthin treatment.Extraction. Seed powders (1.5 kg) were extracted withhexane, dicloromethane, and acetone for 48 h in each solventsystem at room temperature. After each extraction step, theseed extracts were concentrated in a rotary evaporator (BüchiR-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, andacetone extract had 1.21% yield. Three concentrations (2.0,5.0, and 10.0 mg/ml) of each extract were used to amend 24ml of PDA, which was then autoclaved and poured into Petriplates. Growth of each test fungus was recorded at the end ofeach incubation time. Each treatment was replicated six times.Isolation, separation and identification of secondarymetabolites. Organic extracts were subjected to columnchromatography (CC) on silica gel 60 (Merck 0.2-0.5), atthe ration (1:20). Elution was carried out with a mixture ofhexane-dicloromethane in order to increase polarity in a lineargradient. The identity of components was determined byinfrared, ultraviolet, nuclear proton magnetic resonance, andcarbon13 spectroscopic analysis. The melting point wasdetermined in a Fisher-Johns apparatus Mod. 12-144 (Fisher,USA). The yellow dark precipitate and supernatant obtainedwith dicloromethane extract, and the acetone extract, weresubjected to column chromatography to isolate differentisoflavonoids. The isolated compounds and certificaterotenone (1) were used as reference to determine theirpresence in dicloromethane and acetone extract by thin layerchromatography (TLC) using silica gel plates (Merck, 0.25mm), and eluted with dicloromethane. Developed TLC platessprayed afterwards with a reagent (Cerium IV dehydratesulphate-Baker 2% in 2N H2SO4) and warmed up on a hotplate (150°C, 1 min) were evaluated in UV light. The Rf-value = Solute front/ solvent front of six compounds wasdetermined in these conditions. The secondary metabolitesamounts were then dissolved in 1 ml of acetone to obtain afinal concentration of 250 µg/ml,, added to the PDA media,and tested for fungicidal activity as described previously. Twodifferent controls were used: the first containing only PDA,and the second containing PDA amended with 1.0 ml ofacetone. Petri dishes were incubated in darkness at 25 ± 1°C,and the antifungal properties of the six compounds elutedfrom CC were tested against C. gloeosporioides, F.oxysporum, and R. stolonifer measuring mycelial growth aspreviously described. Three plates were run per treatment.

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