Revista Mexicana de Fitopatología ISSN: 0185-3309 [email protected] Sociedad Mexicana de Fitopatología, A.C. México

Lira Saldívar, Ricardo Hugo; Balvantín García, Gloria Franciela; Hernández Castillo, Francisco Daniel; Gamboa Alvarado, Roberto; Jasso de Rodríguez, Diana; Jiménez Díaz, Florencio Evaluation of Resin Content and the Antifungal Effect of tridentata (Sesse and Moc. Ex D.C.) Coville Extracts from Two Mexican Deserts Against Pythium sp. Pringsh Revista Mexicana de Fitopatología, vol. 21, núm. 2, julio-diciembre, 2003, pp. 97-101 Sociedad Mexicana de Fitopatología, A.C. Texcoco, México

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Evaluation of Resin Content and the Antifungal Effect of (Sesse and Moc. Ex D.C.) Coville Extracts From two Mexican Deserts Against Pythium sp. Pringsh. Ricardo Hugo Lira-Saldívar, Centro de Investigación en Química Aplicada, Blvd. Enrique Reyna Hermosillo 140, Saltillo, Coahuila, México CP 25100; Gloria Franciela Balvantín- García, Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila, Blvd. Venustiano Carranza y Francisco Cárdenas Valdés, Saltillo, Coahuila CP 25100; Francisco Daniel Hernández-Castillo, Roberto Gamboa-Alvarado, Diana Jasso-de-Rodríguez, Universidad Autónoma Agraria Antonio Narro, Buenavista, Saltillo, Coahuila CP 25315; y Florencio Jiménez-Díaz, INIFAP, Campo Experimental La Laguna, km 17 Carr. Torreón- Matamoros, Apdo. Postal 247, Matamoros, Coahuila CP 25630. Correspondence to: [email protected] (Received: October 7, 2002 Accepted: November 26, 2002)

Lira-Saldívar, R.H., Balvantín-García, G.F., Hernández- Additional keywords: Gobernadora, creosote bush, organic Castillo, F.D., Gamboa-Alvarado, R., Jasso-de-Rodríguez, D., fungicide, fungicidal activity. y Jiménez-Díaz, F. 2003. Evaluation of resin content and the antifungal effect of Larrea tridentata (Sesse and Moc. Ex Resumen. El efecto antifúngico de extractos de Larrea D.C.) Coville extracts from two mexican deserts against tridentata, planta localmente conocida como “gobernadora Pythium sp. Pringsh. Revista Mexicana de Fitopatología o arbusto de creosota” del norte de México fue investigado 21:97-101. mediante bioensayos inhibitorios para hongos, a dosis de 0, Abstract. The antifungal effect of Larrea tridentata extracts, 500, 1000, 2000, 4000 y 8000 µl l-1. Muestras de L. tridentata a locally known as “gobernadora or creosote bush” from se colectaron en los estados de Coahuila y Zacatecas del northern Mexico, was investigated using fungal inhibition Desierto Chihuahuense (DCh) y en Baja California Sur del bioassays at 0, 500, 1000, 2000, 4000, 6000, and 8000 µl l-1. Desierto Sonorense (DS). La resina de hojas y ramas pequeñas Samples of L. tridentata were collected from Coahuila and fue extraída con metanol, etanol y cloroformo. Los resultados Zacatecas states in the Chihuahuan Desert (ChD), and from mostraron que el valor promedio de resina de las muestras South Baja California state in the Sonoran Desert (SD). Resin colectadas en el DCh fue de 22.60%, mientras que las del DS from leaves and small twigs was extracted with methanol, resultaron de 25.49%. El crecimiento micelial del hongo ethanol, and chloroform. Results showed that the overall resin fitopatógeno Pythium sp. fue completamente inhibido con mean value for samples collected in the ChD was 22.6% los extractos evaluados. El efecto fungicida de los extractos whereas, those from the SD was 25.49%. Mycelial growth of de gobernadora se mostró consistente, independientemente the phytopathogen Pythium sp. was completely inhibited with del solvente usado para la extracción o del sitio geográfico the extracts evaluated. The fungicidal effect of gobernadora de colecta. Los extractos metanólicos de ambos desiertos extracts was consistent in spite of the solvent used for tuvieron un notable efecto a dosis relativamente bajas (500 extraction or location of sampling sites. Methanolic extracts µl l-1), ya que no hubo crecimiento del hongo in vitro. Los from both deserts had a notable effect at relatively low doses resultados indican que los extractos de resina del DS fueron (500 µl l-1), where fungal growth in vitro did not occur. The superiores en su efecto inhibitorio que los del DCh. Los overall results indicate that resin extracts from the SD were extractos de L. tridentata pueden ser de uso práctico como superior in its inhibitory effect than those from the ChD. L. compuestos antifúngicos de bajo impacto ambiental y con tridentata extracts could be of practical use as antifungal importantes posibilidades industriales. Se requieren estudios compounds with low impact on the environment with adicionales bajo condiciones de invernadero y campo, antes important industrial possibilities. Additional studies under de desarrollar nuevos fungicidas y otros agroquímicos con greenhouse and field conditions are required, before the base en extractos de Larrea. development of new agricultural based organic fungicides and other Larrea-based agrochemicals. Palabras clave adicionales: Gobernadora, creosote bush, fungicida orgánico, actividad fungicida. 98 / Volumen 21, Número 2, 2003

Larrea tridentata (Seese and Moc. ex D.C.) Coville, necessary to evaluate other alternatives to control this commonly named “gobernadora or creosote bush” is a pathogen (Kim et al., 1999; Russell et al., 1995; Tanaka and xerophytic perennial evergreen shrub, whose individual age Omura, 1993). Therefore, the aim of this study was to evaluate probably exceeds 100 years, although some clumps may the resin concentration extracted by three solvents, and to survive hundreds or thousands of years (Brinker, 1993). L. investigate the fungicidal effect of L. tridentata extracts from tridentata is ecologically dominant and widely distributed in two desertic regions of northern Mexico, on the in vitro the semi-arid regions of North America extending from inhibition of Pythium sp. southern United States to northern Mexico (Downum et al., 1988). The leaves of this xerophytic shrub are covered with MATERIALS AND METHODS a resinous coating that contains a complex mixture of Area of sampling. L. tridentata extracts were obtained from phenolics, saponins, terpenoids and wax esters that account approximately 10 kg of gobernadora foliage collected from for 10-20% of the leaf dry weight (Mabry et al., 1977; Seigler the Chihuahuan desert (ChD) located in the states of Zacatecas et al., 1974). Greenfield et al. (1987) reported that between and Coahuila in 2000, during November and December, from neighboring shrubs there are significant variations in resin four different sites (west of Paseo de San Juan, Guadalupe content. Over 80% of the resin is composed of phenolic Victoria, Las Esperanzas, and Monclova). Other samples were aglycones with the major component being also collected from four sites of South Baja California nordihydroguaiaretic acid (NDGA); this catechol lignan is a Sonoran desert (SD) (north of San Pedro, Loreto, Ciudad potent antioxidant and mediates important biocidal effects Constitución and south of Mulegé). Sampling areas were (Gnabre et al., 1995; Vargas-Arispuro et al., 1997; Verástegui located at similar latitudes (24, 25, 26, and 27°). et al., 1996). There is a difference in mean NDGA Larrea leaf resin content. Foliage samples were dried in an concentration among Larrea ploidy races depending on the oven at 65°C for five days to constant weight. The resin geographical area where gobernadora evolved; shrubs from determination for each sampling site was done independently the Chihuahuan desert report 2.62%, while those from the by triplicate with methanol, ethanol, and chloroform analytical Sonoran desert contain 3.84% (Gisvold, 1948). On the other grade (ACS regent), with a Soxhlet extraction equipment hand, the phytopathogenic fungus Pythium spp. Pringsh. (ASTM, 1993), placing 10 g of dry leaves in a extraction attacks a great number of crops, both annual and perennial, thimble and 250 ml of each solvent. The solvent was brought infecting in all growth stages, but it is most conspicuous to the boiling point during 8-10 h, and the pure solvent vapors as seed or seedling pathogen, and it is among the most produced a drip into the thimble. Resin content of leaves and widespread root pathogens (Wilhelm, 1984). Pythium spp. small twigs was calculated according to the following cause a pre-emergence and post-emergence seedling blight equation: Rc = W2 / W1 *100; where: Rc = Resin content or damping-off; in some parts of the United States, it is the (%); W1 = weight of sample prior to extraction (g); and W2 = pathogen most frequently isolated from germinated, but weight of sample after extraction (g). nonemerged diseased seedlings (DeVay et al., 1977). In Extract preparation. In order to produce enough resin for pepper nurseries, this fungus is responsible for the most the assays, L. tridentata leaves from each sampling site were troublesome damage to seedlings (Nuez et al., 1996). Control submerged into a 20 L container separately with each one of of this disease is primarily dependent upon continued the three solvents already mentioned, during 24 h at room applications of conventional synthetic fungicides, although temperature; the resulting product was separated from the effective, their continued or repeated use for several decades foliage with a cheesecloth, vacuum filtered through Watham has brought undesirable effects on the environment and human paper No. 1, and the solvent evaporated. After this procedure, health concerns. To minimize the adverse effects of synthetic the resin was treated with a 3% alkaline solution based on products on agro-ecosystems and emergence of sodium hydroxide, and evaporated to dryness in an oven at phytopatogens resistant to the currently used fungicides, it is 65°C during 5 to 7 days to obtain the solid extracts, that later

Table 1. Mean resin concentration extracted with three solvents from Larrea tridentata leaves collected in the Chihuahuan and Sonoran deserts of Mexico. Resin concentration (%) Mean Mean Solventy ChDz SD ChD SD ChD SD ChD SD ChD SD MeOH 26.83 37.00 31.43 37.43 19.16 35.26 29.23 35.4 26.66 36.27 EtOH 16.35 27.36 26.07 25.13 24.63 22.4 20.51 21.4 21.89 24.07

CHCl3 18.43 16.30 20.47 15.20 19.87 16.76 18.22 16.3 19.24 16.14 Mean 20.54 26.89 25.99 25.92 21.22 24.81 22.65 24.36 22.60 25.49 y MeOH = Methanol; EtOH = ethanol; CHCl3 = chloroform. zChD = Chihuahuan Desert; SD = Sonoran Desert. Resin values are the mean of three extractions performed with Soxhlet equipment from leaves collected at each sampling site in both deserts.

Revista Mexicana de FITOPATOLOGIA/ 101 chloroformic extracts. To our knowledge, this is the first Hurtado, L.M., Hernández, R., Hernández, F., and Fernández, report showing a clear fungicide effect of L. tridentata S. 1979. Fungi-toxic compounds in the Larrea resin. In: extracts on Pythium sp. growth at 500 µl l-1 concentration. L.E. Campos, T.J. Mabry, T.S. Fernández (eds.). LARREA. Our results supports the effectiveness of antifungal CIQA, Saltillo, Coahuila, México. pp. 327-341. compounds from gobernadora or creosote bush for in vitro Kim, B.S., Moon, S.S., and Hwang, B.H. 1999. Isolation, studies. For the convenient use of hydrosoluble extracts as antifungal activity, and structure elucidation of the commercial fungicides, they have to be further evaluated on glutarimide antibiotic, streptimidone, produced by in situ studies; greenhouse and field conditions will be Micromonospora coerulea. Journal of Agriculture and required in future work. Food Chemistry 47:3372-3380. 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