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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,

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mrlegarreta@prodigy.net.mx

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/ Volumen 20, Número 1, 2002

Influence of Leaf, Fruit and Seed Powders and Extracts ofPithecellobium dulce (Roxb.) Benth. (Fabaceae) on the in vitro

Vegetative Growth of Seven Postharvest FungiLaura Leticia Barrera-Necha, Silvia Bautista-Baños, Instituto Politécnico Nacional,Centro de Desarrollo de Productos Bióticos, km 8.5 Carr. Yautepec-Jojutla, San IsidroYautepec, Morelos, México CP 62731; Manuel Jiménez-Estrada and Ricardo Reyes-Chilpa, Universidad Nacional Autónoma de México, Instituto de Química, CircuitoExterior, 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 andseed powders and extracts of Pithecellobium dulce (Roxb.)Benth. (Fabaceae) on the in vitro vegetative growth of sevenpostharvest fungi. Revista Mexicana de Fitopatología 20:66-71.Powders of Pithecellobium dulce leaves, fruit and seedssequentially extracted with hexane-dicloromethane, acetone,and methanol-water were evaluated on mycelial growth ofAlternaria sp., Botrytis cinerea, Colletotrichumgloeosporioides, Fusarium oxysporum, Penicilliumdigitatum, Pestalotiopsis sp. and Rhizopus stolonifer. Incomparison to fruit and leaf powders, seeds had the highestfungistatic 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 andAlternaria sp., the lowest and highest concentrationsrespectively, increased mycelial growth. Depending onconcentration, leaf and fruit powders inhibited or increasedmycelial growth. For Pestalotiopsis sp., P. digitatum, F.oxysporum, Alternaria sp., and R. stolonifer mycelial growthincreased on seed residues (10 mg/ml), after hexane-dicloromethane, acetone, and methanol-water extractions ofseed powder, suggesting that fungistatic compounds wereremoved by the dissolvent used. The hexane-dicloromethaneextract was subjected to column chromatography, obtaining13 fractions with similar pattern, which were evaluated usingthe mycelial growth responses of F. oxysporum, P. digitatumand 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 nuclearmagnetic resonance indicated the presence of a tryacylglycerol.

Additional key words: Guamúchil, huamúchil, Madras thorn,manila tamarind, ojiuma, plant extracts, fractions, Alternaria

sp., Botrytis cinerea, Colletotrichum gloeosporioides,Fusarium oxysporum, Penicillium digitatum, Pestalotiopsissp., Rhizopus stolonifer.

Resumen. Los polvos de hojas, frutos y semillas dePithecellobium dulce y semillas extraídas secuencialmentecon hexano-diclorometano, acetona y metanol-agua seevaluaron sobre el crecimiento micelial de Alternaria sp.,Botrytis cinerea, Colletotrichum gloeosporioides, Fusariumoxysporum, Penicillium digitatum, Pestalotiopsis sp. yRhizopus stolonifer. Los polvos de semillas tuvieron la másalta actividad fungistática contra los hongos probados encomparación con los polvos de fruto y hoja. En general, seobservó una curva de dosis-efecto para las tresconcentraciones evaluadas (0.5, 2.0 and 5.0 mg/ml). Sinembargo, para P. digitatum y Alternaria sp. la concentraciónmás baja y la más alta, respectivamente, incrementaron elcrecimiento micelial. Dependiendo de la concentración, lospolvos de hoja y fruto inhibieron o incrementaron elcrecimiento micelial. El crecimiento micelial dePestalotiopsis 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 removidospor los disolventes usados. El extracto hexano-diclorometanofue sometido a una cromatografía en columna, obteniéndose13 fracciones con patrones similares, las cuales fueronevaluadas usando la respuesta de crecimiento micelial de F.oxysporum, P. digitatum y R. stolonifer. Once y nueve de lasfracciones inhibieron el crecimiento de F. oxysporum y R.stolonifer, respectivamente. P. digitatum fue el hongo menosafectado por todas las fracciones. El análisis preliminar de lafracción más activa por resonancia magnética nuclear indicóla presencia de un triacil glicerol.

Palabras clave adicionales: Guamúchil, huamúchil, Madrasthorn, manila tamarind, ojiuma, extractos vegetales,

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(Received: October 17, 2001 Accepted: January 28, 2002)

Revista Mexicana de FITOPATOLOGIA/

fracciones, Alternaria sp., Botrytis cinerea, Colletotrichumgloeosporioides Fusarium oxysporum, Penicillium digitatum,Pestalotiopsis sp., Rhizopus stolonifer.

Failure to control postharvest pathogenic fungi can result inserious economic losses to worldwide horticulturalproduction. Fungi such as Alternaria sp., Botrytis cinerea,Colletotrichum gloeosporioides, Fusarium oxysporum,Penicillium digitatum, Pestalotiopsis sp. and Rhizopusstolonifer, cause diseases to different fruits and vegetablesand all are considered major plant pathogens (Farr et al.,1989). Natural products may offer a new approach for controlof postharvest diseases. Pithecellobium dulce (Roxb.) Benth.(Fabaceae) (common names: guamúchil, huamúchil, manilatamarind, Madras thorn, ojiuma) is an evergreen treeindigenous to the Americas and widely distributed throughoutMexico. It has also been introduced into Asia, Africa andAustralia. An ethnobotanical survey of medicinal plantsconducted by the Mexican Social Security Institute (IMSS)in the State of Morelos, Mexico revealed that the boiled fruitpeel of P. dulce was able to cure cough among people ofTamaulipas, Chiapas and Guerrero (Aguilar et.al., 1996). Wehave previously reported that the powder and aqueous extractfrom the leaves of P. dulce inhibited at some stage the growthof four important postharvest fungal pathogens of fruits andvegetables (Bautista-Baños et al., 2000a). It is noteworthythat P. dulce was the most effective among twenty plantstested. For example, sporulation of R. stolonifer previouslyisolated from ‘ciruela’ (Spondias purpurea) was completelyinhibited, while percentage infection after storage wassignificantly reduced in two varieties ciruelas: red and yellowpreviously dipped in leaf extracts of P. dulce as comparedwith control fruit (Bautista-Baños et al., 2000b). Montes et.al., (1990), reported significant antifungal activity of leafextracts of P. dulce against Uromyces appendiculatus on beancrops as well. The objectives of this work were: a) Todetermine the effect of powders of P. dulce leaves, fruit andseeds and the removal of antifungal compounds from powdersseeds, on the mycelia growth of seven fungal postharvestpathogens and b) To identify the active fractions of the hexane-dicloromethane seed extract and testing their antifungal effect.

MATERIALS AND METHODSPlant Material. Leaves, fruits and seeds of P. dulce werecollected in February and March at the Centro de Desarrollode Productos Bióticos in Yautepec, State of Morelos, Mexico.Leaves, fruits and seeds were dipped in 1% sodiumhypochlorite 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 grinderand then stored at ambient temperature in amber bottles untilfurther use.Microorganisms. Postharvest pathogens were isolated fromfruits as follow: Alternaria sp. from infected tomato(Lycopersicum esculentum), Pestalotiopsis sp. from diseased

sapote mamey (Pouteria sapota), R. stolonifer, P. digitatum,F. oxysporum and C. gloeosporioides from infected papaya(Carica papaya), and Botrytis cinerea from infectedstrawberry (Fragaria X ananassa). To maintainpathogenicity, each fungus was frequently inoculated andreisolated from its indicated host.In vitro bioassay. Powders of leaves, fruit and seeds wereprepared at three concentrations (0.5, 2.0 and 5.0 mg/ml inthe growing media) and amended with 16 ml of Potatodextrose agar (PDA) and autoclaved (15 lb/cm2, 15 min.).After sterilization media were poured into Petri plates (60 x15 mm). A five mm disc agar containing the respectivepathogen was placed at the centre of each plate which wasthen incubated as follows: One day for R. stolonifer, two daysfor Alternaria sp., Pestalotiopsis sp., P. digitatum and B.cinerea and four days for F. oxysporium and C.gloeosporioides. Except for B. cinerea, whose incubationtemperature was at 20oC, the other fungi were incubated at25ºC. Mycelial growth (colony diameter) was measured atthe end of the incubation time. Three replications were runsimultaneously for each treatment (leaves, fruit and seeds atdifferent concentrations). Petri plates of controls onlycontained PDA media. Tests were finished when myceliumof the control plates reached the edge of the dishes. Growthinhibitory effects were calculated as fallow: % Inhibition =Mycelial growth in control – Mycelial growth in treatment/Mycelial growth in control X 100Extraction. Seed powders (200 g) were successivelyextracted at room temperature with hexane-dicloromethane(2:8), acetone and methanol-water (8:2) for 48 h in eachsolvent system. After each extraction step, a sample of 10mg/ml was amended with 16 ml of PDA, autoclaved andpoured onto Petri plates (60 x 15 mm). Mycelial growth ofeach test fungus was recorded at the end of each incubationtime. Three replicates (three plates) were carried out for eachtreatment.Separation. Seed extracts were concentrated in a rotaryevaporator. The hexane-dicloromethane extract (25 g) wassubjected to column chromatography (CC) on 300 g silicagel with mixture of hexane-dicloromethane. Each fractionwas then dissolved in 1 ml of dicloromethane, amended withPDA media and tested as described previously. The resultingfractions were calculated to obtain a final concentration of6.4mg/ml. Two different controls were run: the first containingonly PDA, and the second containing PDA amended with1.0 ml of dicloromethane. Dishes were incubated in darknessat 25 ± 1oC and the mycelial growth was measured after 96 h.The antifungal properties of thirteen fractions eluted fromCC, 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 withhexane-dicloromethane (2:8) contained a white wax whichwas analyzed by nuclear magnetic resonance for proton 1HNMR and carbon 13C NMR. The melting point wasdetermined in Fisher-Johns apparatus Mod. 12-144 (Fisher,

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/ Volumen 20, Número 1, 2002

other compounds not removed with the three solvent systemswe used. The hexane-dicloromethane seed extract wassubjected to column chromatography, obtaining 13 fractions(Table 1). The most sensitive fungus we tested was F.oxysporum, since eleven fractions retarded its growth. Ninefractions retarded growth of R. stolonifer. The least sensitivefungus was P. digitatum since only two fractions werefungistatic. Results suggest that various compounds in thefractions tested are effective against the three fungi. The mostactive fraction (50-56) against F. oxysporum had a white waxwith a melting point of 31°C. Preliminary NMR studiesindicated the presence of an uncommon tryacyl glycerolcompound. Related studies have shown that lipophyllicsubstances can be fungitoxic. For instance, Ginkgo bilobaleaves contain fungitoxic oil and wax that affect Moniliniafructicola development (Johnston and Sproston, 1965).Franich et al., (1983) reported that specific fatty and resinacids were highly fungistatic to Dothistroma pini (a needlepathogen of Pinus radiata) suggesting that these compoundscould be pre-infectional barriers contributing to resistance.In rice leaves antifungal compounds against Pyriculariaoryzae include twelve C18 hydroxy and epoxy fatty acids basedon linolenic acid (Kato et al., 1993). Our research was centerdon in vitro studies. However, having in mind that behaviourof the fungi can dramatically change when experiments arein vitro rather than in planta further investigation should beundertaken to determine the effects of these plant extractswith studies on intact fruit.

Acknowledgements. This work was supported by the GeneralCoordination of Posgraduate Study and Research, and theCommission of Operation and Development of AcademicActivities from the National Polytechnic Institute (IPN),Mexico, F.D.

LITERATURE CITEDAguilar, A., Camacho, J.R., Chino, S., Jacquez, P. y López,

M.E. 1996. Plantas Medicinales del Herbario del InstitutoMexicano del Seguro Social. Redacta S.A. ed. p. 63.

Bautista-Baños, S., Hernández, L.M., and Barrera, N.L.L.2000a. Antifungal screening of plants of the state ofMorelos, México against four postharvest pathogens offruits and vegetables. Revista Mexicana de Fitopatología18:36-42.

Bautista-Baños, S., Hernández-López, M., Díaz-Pérez, J.C.,and Cano-Ochoa, C.F. 2000b. Evaluation of the fungicidalproperties of plant extracts to reduce Rhizopus stoloniferof ‘ciruela’ fruit (Spondias purpurea L.) during storage.Postharvest Biology and Technology 20:99-106.

Bravo, L.L., Bermúdez, T.K. y Montes B.R. 1998. Inhibicióndel crecimiento micelial y esporulación de Fusariummoniliforme Sheld. mediante aceites esenciales vegetalesy algunos de sus componentes químicos. Revista Mexicanade Fitopatología 16:18-23.

Farr, D.F., Bills, G.F., Chamuris, G.P., and Rossman, A.Y.1989. Fungi on plant and plant products in the UnitedStates. American Phytopathological Society Press. St. Paul,MN, USA. 1252 p.

Table 1. Effect of column chromatographic fractions of Pithecellobium dulce seedextract eluted with hexane-dicloromethane on mycelial growth (cm) of threepostharvest pathogens. Mean colony diameter (cm)y

Treatment Fusarium Penicillium Rhizopus oxysporum digitatum stolonifer

No solvent 4.50 az 4.50 a 4.33 abWith dicloromethane 4.50 a 4.50 a 4.26 abFraction 12-13 2.63 def 4.50 a 3.66 bcFraction 15-22 2.50 ef 4.50 a 1.76 fFraction 23-37 3.96 abc 3.83 ab 2.70 deFraction 39-41 3.40 bcd 4.16 ab 2.83 deFraction 45 2.53 ef 4.50 a 4.50 aFraction 50-56 2.30 f 4.50 a 4.40 aFraction 58-62 3.20 cde 4.50 a 4.50 aFraction 63-73 2.83 def 3.83 ab 4.26 abFraction 74-80 4.43 a 4.36 a 2.50 eFraction 83-90 2.63 def 1.5 c 2.76 deFraction 91-99 4.06 ab 3.06 b 2.70 deFraction 129-137 2.40 ef 3.50 ab 3.83 abcFraction 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) asdetermined by Tukey’s multiple range test.

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Franich, RA, Gadget, P.D. and, Shain, L. 1983. Fungistaticeffects of Pinus radiata needle epicuticular fatty and resinacids on Dothistroma pini. Physiological Plant Patholology23:183-195.

Johnston, H.W., and Sproston, Jr. T. 1965. The inhibition offungal infection pegs in Ginkgo biloba. Phytopathology55:225-227.

Kato, T., Yamaguchi, Y., Nanai, T., and Hirukawa, T., 1993.Oxygenated fatty acids with anti-rice blast fungus activity

in rice plants. Bioscience, Biotechnology and Biochemistry57:283-287.

Montes, B.R., Cruz, C.V. y Peralta, D.M. 1990. Extractosvegetales para el control de la roya del frijol Uromycesappendiculatus. Agrociencia 1:99-106.

Montes, B.R., Carvajal, M., Figueroa, R. y Méndez, I. 1997.Extractos sólidos, acuosos y hexánicos de plantas para elcombate de Aspergillus flavus Link. en maíz. RevistaMexicana de Fitopatología 15:26-30.

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