This document summarizes a study that investigated the antifungal effects of seed powders, extracts, and secondary metabolites from Pachyrhizus erosus against three postharvest fungi. Seed powders and extracts showed inhibitory effects on the fungi in a dose-dependent manner. The dicloromethane extract showed the greatest fungistatic effects. Six secondary metabolites were isolated including rotenone, erosone, paquirrizone, dolineone, paquirrizine, and dehydroneotenone. These metabolites significantly inhibited the fungi. Rotenone showed the best effect against Rhizopus stolonifer while paquirrizine and dehydroneotenone were most effective against

1. Revista Mexicana de Fitopatología
Sociedad Mexicana de Fitopatología, A.C.
guillermofuentes_davila@hotmail.com
ISSN (Versión impresa): 0185-3309
MÉXICO
2004
Laura Leticia Barrera Necha / Silvia Bautista Baños / Leticia Bravo Luna / Francisco Javier Leobardo
García Suárez / Dagoberto Alavez Solano / Ricardo Reyes Chilpa
ANTIFUNGAL 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

2. 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 IsidroYautepec, 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: 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, and
secondary metabolites of Pachyrhizus erosus (L.) Urban
(Fabaceae) against three postharvest fungi. Revista Mexicana
de Fitopatología 22:356-361.
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
(Received: July 19, 2004 Accepted: October 12, 2004)
356 / Volumen 22, Número 3, 2004

3. 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 varietyAgua 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/
cm2
, 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 0.2-0.5), 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
carbon13
spectroscopic analysis. The melting point was
determined in a Fisher-Johns apparatus Mod. 12-144 (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 H2
SO4
) and warmed up on a hot
plate (150°C, 1 min) were evaluated in UV light. The Rf-
value = 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.
Revista Mexicana de FITOPATOLOGIA/ 357

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