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R. H. Lira-Saldivar, M. Hernndez-Surez, F. D. Hernndez-CastilloActivity of Larrea tridentata (D.C.) Coville L. extracts and chitosan against fungi that affect horticultural crops

Revista Chapingo. Serie Horticultura, vol. 12, nm. 2, julio-diciembre, 2006, pp. 211-216,Universidad Autnoma Chapingo

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Revista Chapingo Serie Horticultura 12(2): 211-216, 2006.Recibido: 25 de mayo, 2006Aceptado: 26 de septiembre, 2006

ACTIVITY OF Larrea tridentata (D.C.) Coville L. EXTRACTS AND CHITOSAN AGAINST FUNGI

THAT AFFECT HORTICULTURAL CROPS

R. H. Lira-Saldivar1; M. Hernndez-Surez1; F. D. Hernndez-Castillo21Centro de Investigacin en Qumica Aplicada. Blvd. Enrique Reyna Nm. 140,

Saltillo, Coahuila. C. P. 25253, Mxico. Tel/Fax (844) 438-9830.E-mail: rhlira@ciqa.mx (Corresponding autor).

2Universidad Autnoma Agraria Antonio Narro, Departamento de Parasitologa Agrcola,Buenavista, Saltillo, Coahuila C. P. 23315, MXICO.

SUMMARY

The antifungal activity of gobernadora [Larrea tridentata (D.C.) Coville (L.)] hydrosoluble resin extract and chitosan solutions (Ch),alone and in combination, were investigated in vitro for their bioactivity against the fungi Botrytis cinerea, Colletotrichum coccodesand Fusarium oxysporum f. sp. lycopersici that were isolated from greenhouse roses, potatoes and tomatoes from commercial plots,respectively, and later purified. Both bioproducts demonstrated their fungicidal effect at 1000 and 2000 lliter-1; however, when theywere combined they exhibited synergistic fungicidal activity. These preliminary results indicate that L. tridentata hydrosoluble resinextract or the combination L-Ch could be considered as potential agrochemicals of low environmental impact for use as organicfungicides; however, further work is needed before this application could be used commercially. To the best of our knowledge, thisis the first time that a mixture of L. tridentata and chitosan has been reported acting as antifungal compound.

ADDITIONAL KEY WORDS: organic fungicides, biopolymers, creosote bush, plant extracts, synergistic effect.

ACTIVIDAD DE EXTRACTOS DE Larrea tridentata (D.C.) Coville (L.)Y QUITOSN CONTRA HONGOS QUE AFECTAN CULTIVOS HORTCOLAS

RESUMEN

La actividad antifngica de extracto de resina hidrosoluble de gobernadora [Larrea tridentata (D.C.) Coville (L)] y soluciones dequitosn (Ch), solos y combinados fueron investigados in vitro por su actividad antifngica contra Botrytis cinerea, Colletotrichumcoccodes y Fusarium oxysporum f. sp. lycopersici que fueron aislados de rosas de invernadero y de lotes comerciales de papa ytomate, respectivamente, mismos que posteriormente fueron purificados. Ambos bioproductos manifestaron su efecto funguicida a1,000 y 2,000 llitro-1, sin embargo, cuando fueron combinados mostraron una actividad fungicida sinrgica. Estos resultadospreliminares indican que el extracto hidrosoluble de L. tridentata o la combinacin L-Ch pudiesen ser considerados como agroqumicospotenciales de bajo impacto ambiental para ser usados como fungicidas orgnicos, pero se requiere ms trabajo de investigacinantes de que esto tenga una aplicacin comercial. Hasta lo mejor de nuestro conocimiento esta es la primera vez que se reporta a lamezcla L. tridentata y quitosn actuando como un compuesto antifngico.

PALABRAS CLAVE ADICIONALES: fungicidas orgnicos, biopolmeros, gobernadora, extractos vegetales efecto sinrgico.

INTRODUCTION

Botrytis cinerea Pers. is considered an omnipresentpathogen around the world that causes gray mold on a largenumber of economically important crops during the growingseason and throughout post harvest (Keller et al., 2003);also, it is the most common disease of glasshouse crops(Daugherty et al., 1995). Fungal diseases like black dot of

potatoes, caused by Colletotrichum coccodes (Wallr.) S. J.Hughes, was considered a minor disease worldwide untilthe early 1990s, when reports from several countries indi-cated increasing occurrences of the disease and greaterimpacts on potato production (Tsror et al., 1999). This dis-ease is now being found in certain potato growing areas ofMexico; it causes aerial infections, premature plant death,and, as a soil-borne pathogen, it can interact with other fungi

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to increase symptom expression and yield reduction onpotato (Lees and Hilton, 2003). One of the worldwidephytosanitary problems limiting tomato production is vascu-lar wilting caused by Fusarium oxysporum f. sp. lycopersiciSnyder and Hansen, which occurs more in warm weatherwhere it causes great economic losses (Carrillo-Fasio etal., 2003). This fungus causes severe crown and root rot oftomato, as well as, extensive necrotic lesions in the basalportion of the stem, wilting, and plant death. Fusariumoxysporum f. sp. radicis-lycopersici is another very damag-ing fungus that has reduced yield of tomato up to 50 % inthe state of Sinaloa (Apodaca-Sanchez et al., 2002), whichis the most important tomato producing region of Mexico.Sanitation, cultural practices and chemical fungicides areused as control measures against B. cinerea, C. coccodes,and F. oyxysporum in Mexico and elsewhere, but growersdepend chiefly on synthetic fungicides (Romanazzi et al.,2002; Lees and Hilton, 2003). Although effective, their con-tinued or repeated use for several decades has disruptednatural control and has lead to outbreaks of diseases, unde-sirable effects on no target organisms, and environmentaland human health concerns.

Botanical product-based pesticides offer advantagesin that they can sometimes be specific to the target speciesand typically have unique modes of action with little mam-malian toxicity. Furthermore, they generally do not persistin the environment (Duke et al., 2003). Creosote bush orgobernadora [Larrea tridentata (D.C.) Coville (L.)] is the mostabundant shrub on millions of hectares of the North Ameri-can warm deserts (Chihuahuan, Sonoran and Mohave). Theleaves of this shrub are covered with a resinous coating thatcontains a complex mixture of phenolics, saponins, terpe-noids and wax esters that account for 20 - 35 % of the leafdry weight (Table 1). Over 80 % of Larrea resin is composedof phenolic aglycones with the major component beingnordihydroguaiaretic acid (Brinker, 1993), this catechol lignanis a potent antioxidant and mediates important biocidal ef-fects in diverse microorganisms (Gnabre et al., 1995). Anotable phytochemical attribute of gobernadora shrubs isthat they produce a thick and water insoluble resin that ac-cumulates in leaves and small twigs. The resin has shownto have fungicidal and bactericidal properties in many patho-gens (Verstegui et al., 1996; Lira-Saldivar et al., 2002; Lira-Saldivar et al., 2003a). At present, an increasing interest isbeing devoted to the use of natural substances such aschitosan a biopolymer derived from chitin which is very simi-lar to cellulose (Figure 1). This high molecular weight cat-ionic polysaccharide that occurs in fungal cells walls andarthropods exoskeletons had been considered as a validalternative to synthetic fungicides (Romanazzi et al., 2003).Indeed, chitosan is an ideal preservative coating for freshfruit and vegetables because of its film-forming and biochemi-cal properties (Muzzarelli, 1986). Also, it is reported to pro-long storage life and control decay of several fruits(Romanazzi et al., 2002). Therefore, the objective of thisresearch was to find out the amount of leaf resin concentra-tion from different native populations of L. tridentata shrubs

located in northern Mexico, and whether Larrea extract andchitosan, alone and combined, could prove to be effective asantifungal agents, and lastly to determine if mixtures of bothbiocompounds can have a synergistic effect against threefungi that have a harmful effect on many horticultural crops.

MATERIALS AND METHODS

Sampling sites and climatic characteristic

L. tridentata foliage for the resin extracts was collected

TABLE 1. Main phytochemical constituents of Larrea tridentata(Brinker, 1993).

Dry weight Class/type Compound (%)

16 - 21 Phenolics/ lignans Dihydroguaiaretic acidHemi-norisoguaiacinNordihydroguaiaretic acidNorisoguaiacin

5 - 7.5 Flavonoids/aglycone ApigeninKaempferol

Flavonoids/glycosides ChrysoeriolQuercetin

10 - 15 Saponins/triterpenes Larreagenin ALarreic acid

70.1 (of stems) Lipids/wax esters Alkyl esters

FIGURE 1. Chemical structures of the biopolymers cellulose(a), chitin (b), and its derivative chitosan (c).

a)

b)

c)

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Revista Chapingo Serie Horticultura 12(2): 211-216, 2006.

during March 2004 from a zone of the Chihuahuan Desertlocated in the states of Zacatecas and Coahuila. Four places(San Tiburcio, La Pardita, Pabelln Hidalgo and Concepcindel Oro) were situated in Zacatecas, and another four (Encar-nacin de Guzmn, Gmez Faras, Tanque de Emergenciaand Agua Nueva) in Coahuila. All sites were located betweenthe geographic coordinates 24 and 25 N and 101 and102 W, at an altitude range from 2,200 to 2,600 m. Accord-ing to the classification of Kppen, modified by Garca (1988)the climate of this region is type BWw (x) h (e), which isvery arid with scarce and torrential rains during the long sum-mer and cold temperatures during the winter.

L. tridentata resin content and chitosan preparation

Foliage samples were dried in an oven at 65 C for fivedays to constant weight. Resin determination for each sam-pling site was done independently by triplicate with ethanolanalytical grade (ACS regent), with a Soxhlet extractionequipment (ASTM, 1993), placing 10 g of dry leaves in anextraction thimble and 250 ml of methanol as the solvent.The solvent was brought to a boiling point for 8-10 h and thepure solvent vapors produced a drip into the thimble. Resincontent of leaves and small twigs was calculated accordingto the following equation: Rc = W2 / W1 * 100; where: Rc =Resin content (%); W1 = weight of sample prior to extrac-tion (g); and W2 = weight of sample after extraction (g). Inorder to produce enough resin for the assays, L. tridentataleaves from each sampling site were submerged during 24 hat room temperature into a 20-liter container separately withthe organic solvent already mentioned; the resulting productwas separated from the foliage with cheesecloth, vacuumfiltered through Waltham No. 1 paper and the solvent evapo-rated in a rotary evaporator. Crab-shell chitosan, purchasedfrom Sigma Aldrich Co. (Sheboygan WI, USA), was arrangedas described by El Ghaouth et al. (1992). For experimentaluse the stock solution, (1.0 %, w/v) was prepared by dis-solving purified chitosan in 0.5 % (v/v) glacial acetic acid un-der continuous stirring during 16 h, and the pH was adjustedto 5.6 using 1 N NaOH (Romanazzi et al., 2003). The stocksolution was autoclaved and appropriate concentrations wereobtained by dilution with deionized distilled water.

Isolation and identification of plant pathogens

B. cinerea was isolated from diseased greenhouseroses (Rosa spp.); C. coccodes from diseased potatoes(Solanum tuberosum L.) and F. oxysporum from diseasedtomatoes (Lycopersicon esculentum) obtained both from com-mercial plots. To isolate the fungi from vegetative material,pieces of infected tissue were taken and disinfested withsodium hypochlorite at 1.5 % during 5 min and later wereplanted in growth medium potato dextrose agar (PDA) andincubated at 25 C. In order to carry out fungal identification,sporulation was induced under continuous artificial light; oncethe fungi growth fully covered Petri dishes and spores wereproduced, microscopic preparations were made with bluelactophenol, and with the aid of a microscope the fungi wereidentified using the key of Barnett and Hunter (1998).

Detection of antifungal activity in semi-solid medium

To study the inhibitory effects of the bioproducts, as-says were performed with Petri dishes in which dissolved L.tridentata (L) extract and chitosan (Ch) were added alone ormixed to potato dextrose agar (PDA) growth medium (Bioxon;Becton Dickinson of Mexico). Chitosan solutions and PDAwere autoclaved separately and combined after autoclaving.Equal volumes of acid were used for all concentrations ofchitosan, adjusted to pH 5.6 to keep the salt concentrationconstant. Eleven treatments were evaluated with each plantpathogen: three L doses (1,000, 2,000 and 4,000 lliter-1),two Ch concentrations (1,000 and 2,000 lliter-1), four L-Chmixtures (1,000-1,000; 2,000-1,000; 1,000-2,000 and 2,000-2,000 lliter-1) plus two controls: (i) distilled water (dw) and(ii) a synthetic fungicide (Chlorotalonil and Prozycar; 2000 lliter-1). After thorough mixing, the molten medium was dis-pensed into 9-cm diameter Petri dishes (approximately 20mlplate1). In vitro experiments were carried out using 7-day-old cultures of B. cinerea, 10-day-old for C. coccodes,and 13-day-old for F. oxysporum. Mycelia plugs of each ofthe fungi were placed at plate centers and incubated at 25C during 5, 20 and 14 days for each fungi, respectively; atthe end of the incubation period growth of each pathogenwas determined by measuring fungal radial growth with theaid of a vernier. Percent inhibition (PI) was determined byusing the following equation: PI = 100 [(Mrg * 100) / Pr]; inwhich Mrg = mycelia radial growth (mm) and Pr = plate ra-dius (mm). Spore counts per ml of each fungus were perfor-med with a Neubauer hematocytometer using a microscopewith the 40 X objective. A completely randomized experimen-tal design with six replicates was used for the resulting treat-ments. In order to determine whether a synergistic interac-tion was produced by the interaction between L. tridentataand chitosan treatments, Limpels method as described byRomanazzi et al. (2003) was used. Limpels formula is Ee =X+Y-(XY/100), in which Ee is the expected effect from addi-tive responses of two inhibitory agents, and X and Y are thepercentages of inhibition relative to each agent used alone.Thus, if the combination of the two agents produces anyvalue of inhibition greater than Ee, then synergism exists.

RESULTS AND DISCUSSION

Leaf resin content

L. tridentata resin extracted with methanol ranged from19.45 to 31.23 %, with a mean value of 21.75 and 26.30 %,respectively, for the Zacatecas and Coahuila populations(Table 2). Our L. tridentata leaf resin content values reportedhere are higher than 10 to 26 % extracted with diethyl etheras an extraction solvent reported by Rhoades (1977) fromArizona populations of L. tridentata. A former study reportedby Lira-Saldivar et al. (2003b) pointed out that mean leafresin content extracted with methanol and ethanol rangedfrom 36.27 to 34.43 %, respectively for Larrea populationsbelonging to the Chihuahuan and Sonoran deserts. This dif-ference among resin concentrations could be attributed to

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the higher polarity and extractive power of methanol as asolvent; and also to the fact that foliage was collected afterthe rainy season, when the production of secondary me-tabolites was at the highest point. Place-to-place variationsin foliage resin content is normally related to a variety ofabiotic and biotic factors such as levels of soil moisture, soilslope, air temperature; leaf development, plant age and leafmaturation (Lira-Saldivar et al., 2003a); therefore, the inter-and intrapopulation variation detected for resin content wasdependent upon L. tridentata populations and ecologicalcharacteristics of the sampling sites. In this work we ob-served that the proportion of resin content follows a generalpattern of decrease among populations and along a soil andair humidity gradient from northern Zacatecas to southeast-ern Coahuila. Gonzlez-Coloma et al. (1988) investigatedthe impact of ozone concentration on NDGA levels in Larrearesin; they also reported a substantial geographic and in-trapopulation variation, which in turn depended on abioticfactors such as soil moisture and soil temperature.

Mycelia radial growth inhibition

The effect of L. tridentata extract, chitosan and combi-nations of both bioproducts on Mrg varied according to thetreatment and fungi. The data shows that the level of inhibi-tion by L. tridentata and chitosan treatments by themselveswere not as great as that of mixtures of both products on C.coccodes and F. oxysporum (Table 3). Mean value for Mrginhibition was 98.86 % on B. cinerea, followed by 82.75 %and 65.61 % on C. coccodes and F. oxysporum respec-tively. The maximum mycelia inhibition (100 %) on B. ci-nerea was obtained with L-Ch combination at the concen-trations 2000-1000; 1000-2000 and 2000-2000 lliter-1; thesetreatments significantly (P

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served with the fungicide chlorothalonil. When L. tridentataextract was applied separately, sporulation was partially di-minished; therefore, compared to the distilled water controlno statistical differences were experienced due to Larreaextract. On F. oxysporum the combinations L-Ch 2,000-1,000and 2,000-2,000 lliter-1 showed a total inhibitory effect onsporulation. It is noteworthy that these treatments, includ-ing L. tridentata extract at 4,000 lliter-1, resulted in statis-tically superior fungitoxic activity (P

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fore, we detected a synergistic effect between L. tridentatahydrosoluble extract and chitosan solutions. To the best ofour knowledge, the synergy linking these biocompounds isreported here for the first time. Our results suggest that thecombination L-Ch could be of practical use as antifungalcompound for its utilization against diseases that affect manyhorticultural crops in Mexico and elsewhere. Therefore, thesuitable use of these organic products as commercial pes-ticides requires having them further evaluated through in vivostudies under greenhouse and field conditions.

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