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Laura Leticia Barrera Necha, Silvia Bautista Baños, Manuel Jiménez Estrada, Ricardo Reyes Chilpa
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. 66-71,
Sociedad Mexicana de Fitopatología, A.C.
México
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Revista Mexicana de Fitopatología,
ISSN (Printed Version): 0185-3309
mrlegarreta@prodigy.net.mx
Sociedad Mexicana de Fitopatología, A.C.
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2. / 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 04510. Correspondence to:
lbarrera@ipn.mx
Abstract.
Barrera-Necha, L.L., Bautista-Baños, S., Jiménez-Estrada,
M., and Reyes-Chilpa, R. 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. Revista Mexicana de Fitopatología 20:66-
71.
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 dose-effect
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 hexane-dicloromethane,
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 hexano-diclorometano,
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,
66
(Received: October 17, 2001 Accepted: January 28, 2002)

3. Revista Mexicana de FITOPATOLOGIA/
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 hexane-dicloromethane
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
67
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/cm2, 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 20oC, 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 ± 1oC 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 1H
NMR and carbon 13C NMR. The melting point was
determined in Fisher-Johns apparatus Mod. 12-144 (Fisher,

6. / 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 az 4.50 a 4.33 ab
With dicloromethane 4.50 a 4.50 a 4.26 ab
Fraction 12-13 2.63 def 4.50 a 3.66 bc
Fraction 15-22 2.50 ef 4.50 a 1.76 f
Fraction 23-37 3.96 abc 3.83 ab 2.70 de
Fraction 39-41 3.40 bcd 4.16 ab 2.83 de
Fraction 45 2.53 ef 4.50 a 4.50 a
Fraction 50-56 2.30 f 4.50 a 4.40 a
Fraction 58-62 3.20 cde 4.50 a 4.50 a
Fraction 63-73 2.83 def 3.83 ab 4.26 ab
Fraction 74-80 4.43 a 4.36 a 2.50 e
Fraction 83-90 2.63 def 1.5 c 2.76 de
Fraction 91-99 4.06 ab 3.06 b 2.70 de
Fraction 129-137 2.40 ef 3.50 ab 3.83 abc
Fraction 141 2.70 def 1.20 c 3.40 cd
yOn potato-dextrose-agar plates.
zMeans 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 C18 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.
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