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Author's personal copy Industrial Crops and Products 31 (2010) 134–138
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Antimicrobial activities of cascalote (Caesalpinia cacalaco) phenolics-containing extract against fungus Colletotrichum lindemuthianum Rafael Veloz-García b , Raúl Marín-Martínez b , Rafael Veloz-Rodríguez b , Raúl Rodríguez-Guerra c , Irineo Torres-Pacheco a , Mario M. González-Chavira d , José L. Anaya-López d , Lorenzo Guevara-Olvera b , ˜ e , Ramón G. Guevara-González a,∗ Ana A. Feregrino-Pérez a , Guadalupe Loarca-Pina a
Laboratorio de Biosistemas. Facultad de Ingeniería, Universidad Autónoma de Querétaro, Centro Universitario Cerro de las Campanas, s/n, Querétaro, Qro 76010, Mexico Departamento de Ingeniería Bioquímica, Instituto Tecnológico de Celaya, Av. Tecnológico s/n, Celaya, Gto, Mexico c INIFAP-Campo Experimental General Terán, Km. 31 Carretera Montemorelos-China, 67400 General Terán, NL, Mexico d Unidad de Biotecnología, Campo experimental Bajío, INIFAP, Km 6.5 Carretera Celaya-San Miguel de Allende, Celaya, Gto 38010, Mexico e Programa de Posgrado en Alimentos del Centro de la República (PROPAC), Universidad Autónoma de Querétaro, Centro Universitario Cerro de las Campanas, s/n, Querétaro, Qro 76010, Mexico b
a r t i c l e
i n f o
Article history: Received 5 May 2009 Received in revised form 11 September 2009 Accepted 21 September 2009
Keywords: Cascalote Phenolics Anthracnose Antimicrobials
a b s t r a c t Cascalote (Caesalpinia cacalaco) is a tree located in Pacific Mexican coast, and it is an excellent source of phenolics as gallic and tannic acids used in Mexican tannery industry, and with a potent antioxidant and antimutagenic activities. Based on the phenolics properties, these compounds present potential antimicrobial activity against a myriad of microorganisms. Thus, in this study it was evaluated the antimicrobial activity of cascalote phenolics against phytopathogenic fungus Colletotrichum lindemuthianum which causes anthracnose disease in common beans (Phaseolus vulgaris). The results indicate that cascalote phenolics have in vitro fungistatic activity against C. lindemuthianum races R-0 and R-1472. Moreover, these compounds provoked on the whole, in vitro inhibition of spores germination and cellulase and polygalacturonase activities in these fungi. In vivo assays with cascalote phenolics under greenhouse conditions using susceptible cv. PI 206272 of common bean resulted in a good protection against anthracnose severity especially as a preventive treatment. Several aspects of the results and implications on the potential use of cascalote phenolics as organic antifungal are discussed. © 2009 Elsevier B.V. All rights reserved.
1. Introduction In a previous study, phenolic extracts of cascalote (Caesalpinia cacalaco) pods displayed potent antimutagenic and antioxidant activities in in vitro assays, as well as to be a rich source of gallic and tannic acids (Veloz-García et al., 2004). Phenolics partake mainly in plant protection, structural, photosynthesis and nutrient uptake, among other functions in vascular plants and are classified into simple phenols and polyphenols (Marín-Martínez et al., 2009). Moreover, it has been demonstrated that induction of resistance in several plant species is followed by production of phenolics with antimicrobial activity (Luzzatto et al., 2007). Many plant phenols are known to possess antimicrobial properties, so they might change the composition of microflora in any environment in which these compounds are applied and/or induced in a proper kind and concentration (Puupponen-Pimiä et al., 2001; Heinäaho et al., 2006). In fact, the antimicrobial activity of phenolics is well known
∗ Corresponding author. E-mail address:
[email protected] (R.G. Guevara-González). 0926-6690/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2009.09.013
and it is related to their ability to denature proteins, being generally classified as surface-active agents (Sousa, 2006). On the other hand, there is an increasing worldwide consumer demand for organically produced food, and hence, there is also an increasing interest in exploiting natural products (plant extracts) that have antimicrobial activities against plant pathogens, which then eventually might be used in organic crop production (Wallace, 2004; Heinäaho et al., 2006). As aforementioned, the pods of cascalote tree are a rich source of gallic and tannic acids, which in Mexico are currently only used in tannery industry (Veloz-García et al., 2004). In addition, it has been estimated that around 20 000 tons of cascalote pods are annually produced in México (Veloz-Rodríguez, 1993). Common bean (Phaseolus vulgaris) is considered the third main crop in México due to its importance in the diet, and it is widely consumed (Cardador-Martínez et al., 2006). Anthracnose caused by C. lindemuthianum is one of the most prominent diseases in common beans worldwide reducing pod and seed yield in susceptible cultivars (Rodríguez-Guerra et al., 2003; Hernández-Silva et al., 2007; Awale et al., 2008). In México it has been reported losses by this disease up to 47%, and the most commonly used control strategy is to employ resistant common bean cultivars (Rodríguez-Guerra
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et al., 2003). Resistance to conventional fungicides causes a poor disease control in agriculture, and natural products from plants have great potential as novel antimicrobial sources for controlling pathogenic fungi (Chang et al., 2008). Thus, this work aimed to evaluate the antimicrobial activity of cascalote phenolics against two races of the phytopathogen fungus Colletotrichum lindemuthianum in in vitro and in vivo assays under greenhouse conditions. 2. Materials and methods 2.1. Biological material Cascalote pods (300 kg) were collected in the Michoacán state of Mexico during September and October 2006. The pods were dried at 80 ± 3 ◦ C, reducing their moisture from 15 to 3–5 ± 0.5%. Dried pods were ground to obtain a 40-mesh size powder (Scientific Apparatus, Philadelphia, PA, USA). Powdered samples were liophylized and stored in the dark at 5 ◦ C until phenolics extraction, chemical analysis and antimicrobial and enzymatic assays were carried out (Cardador-Martínez et al., 2006). Races R-0 (non-pathogenic) and R-1472 (highly pathogenic) of C. lindemuthianum were kindly provided by Dr. June Simpson of CINVESTAV-Irapuato. Susceptible cultivar PI207262 of common bean to anthracnose by C. lindemuthianum race R-1472, but not by race R-0 was provided by Campo Experimental Bajío INIFAP. 2.2. Cascalote extracts Samples of ground cascalote pods were extracted essentially as described in Veloz-García et al. (2004). Briefly, cascalote pods were crushed and dried at 80 ± 3 ◦ C (in a stove), reducing their moisture from 15 to 3–5 ± 0.5%. Dried pods were ground to obtain a 40-mesh size powder. Powdered samples were liophylized and stored in the dark at −70 ◦ C until phenolics extraction was carried out. Powdered cascalote pods were extracted for 2.5 h with a water–methanol solution (8:2, v/v) with rapid shaking. The proportion of extraction solution and powdered cascalote pods was 4 L/kg. After extraction, the samples were filtered in Whatman 41 paper. The filtrate was spray-dried in a Niro atomizer (Mazal, Germany) under the following conditions: outlet temperature 90 ◦ C, 4.5 kg/cm2 (air pressure) and 15–18 mL/min (yield on a dry powdered base: 896 ± 2.3 g/kg). Spray-dried extract samples were stored at −70 ◦ C until the different assays of this work were carried out. Extractions were carried out in three different samples which were used as replicas for the HPLC analysis and for the enzymatic and antimicrobial assays. Total phenolics of cascalote extracts were determined using the method described by Deschamps et al. (1983) and reported as gallic acid equivalents (GAE)/kg of sample (GAE/kg). In the HPLC analysis of extracts, 50 L of the pure phenolic acids: caffeic, chlorogenic, gallic, p-cumaric and tannic acid were injected and eluted isocratically with a mobile phase of water and acetonitrile (70:30, v/v) at a flow rate of 1 mL/min at wavelength 280 nm. Phenolics were identified and quantified by comparison of retention times with those of standard compounds aforementioned prepared in the same way as the samples. Quantification was related to peak areas of corresponding compounds. The linear standard calibration curves (0.9909R2 ) were generated by injecting 1.5–20 g/mL of a specific phenolic acid in 50 L of 70% H2 O–30% acetonitrile. 2.3. In vitro antimicrobial assays The antifungal properties of cascalote extracts were tested in vitro according to the poisoned-food technique by evaluating concentrations of 70, 210 and 350 g GAE/mL disolved in potato dextrose agar (Invitrogen Corporation, Carlsbad, CA, USA) (MüllerRiebau et al., 1995). Three independent experiments with four
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replicates of each concentration were evaluated inoculating with 7 mm discs of fungus mycelium of either races R-0 or R-1472 of C. lindemuthianum and incubated in the dark at 28 ◦ C, and mycelial growth was measured after 7 days. The fungistatic–fungicidal nature of cascalote extracts were tested by observing revival of growth of the inhibited mycelial disc following its transfer to nontreated potato dextrose agar. In this sense, extract was classified as fungistatic if any mycelial growth was observed after 10 days. Benlate (ampule of 100 mg, active ingredient benomyl: methyl1-butylcarbamoyl-benzimidazolecarbamato; Sigma–Aldrich, St. Louis, MO, USA) as a commercial fungicide used against anthracnose of common bean was evaluated in this assays for comparison. 2.4. Cellulase and polygalacturonase activity assays The effect of cascalote phenolics extract on the in vitro activity of extracellular cellulase and polygalacturonase enzymes of C. lindemuthianum Races 0 and 1472, was carried out using a viscosimetric assay according to Pandita and Jindal (1991). Three mycelial discs of 7 mm diameter were inoculated into Erlen Meyer flasks (125 mL) containing 25 mL of minimal medium M9 with 1% of citric pectin or carboxymethylcellulose (Sigma–Aldrich, St. Louis, MO, USA) either if polygalacturonase or cellulase activity was going to be measured. Flasks were incubated during 6 days at 28 ◦ C, then centrifuged and collected the supernatant. Loss of viscosity of 130 mL of a 1.2% solution of citric pectine or 0.8% solution of carboxymethylcellulose occasioned by 20 mL of corresponding supernatant was measured during 1 h of incubation at 30 ◦ C in a viscosimeter model viscolan (LABOLAN, Spain). One unit of enzyme was considered as the enzyme quantity causing a decrease in 1% of viscosity of either citric pectin or carboxymethylcellulose solution/h/mL of supernatant aforementioned. For controls and calibration of viscosimeter for the assays, 100% viscosity was considered as the viscosity of pectic or carboxymethyl cellulosic solution after 1 h at the mentioned conditions additioning 20 mL of boiled extracts (100 ◦ C during 5 min), and 0% viscosity considered only measurements of deionized water. Inhibition of enzymatic activities assays included the aforementioned system, and addition of 70, 210 or 350 g GAE/mL of cascalote phenolic extract into the pectic or carboxymethylcellulose solutions. These experiments were carried out by triplicate, and using four replicates of each phenolics dose evaluated. 2.5. Assays of inhibition of in vitro germination of C. lindemuthianum spores Preparation of C. lindemuthianum spores (R-0 and R-1472 races) was carried out according to Rodríguez-Guerra et al. (2003). The in vitro germination assays were carried out by triplicate in petri dishes containing corn flour medium (French and Hebert, 1980; Rodríguez-Guerra et al., 2003) and 70, 210 or 350 g GAE/mL of cascalote phenolic extract and 20 000 spores/plate. Control was also carried out by triplicate in petri dishes without phenolics. The percentage of germination was carried out by microscopic (Carl Zeiss, Germany) observations at 40× and counting germinated spores in five fields after 72 h of incubation at 28 ◦ C. 2.6. Antimicrobial assays in bean plants Collection of spores of both C. lindemuthianum races were carried out as described by Rodríguez-Guerra et al. (2003). The spores suspensión for the assays contained 1 500 000 spores/mL. Common bean plants of cv. PI207262 in the 4–6 true leaves-stage were used in the antimicrobial assays. In preventive treatments 2 mL of cascalote phenolic solutions either of 70, 210 or 350 g GAE/mL were applied on each plant and 24 h post-application 2 mL
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of the aforementioned spore suspensión was sprayed on them. In corrective treatments, spore suspensión was first applied as mentioned above, and 72 h post-application 2 mL of each phenolic solution was sprayed. Plants were incubated under greenhouse conditions at 28 ◦ C during 10 days and then evaluated for symptoms severity using a five-level scale reported by Garrido-Ramírez and Romero-Cova (1989), where 0–2 = resistant and 3–4 = susceptible. Twenty plants per treatment were used with three replicates in a randomized array of the experiment. Benlate (ampule of 100 mg, active ingredient benomyl: methyl-1-butylcarbamoylbenzimidazolecarbamato; Sigma–Aldrich, St. Louis, MO, USA) as commercial fungicide was used for comparison in both preventive and corrective treatments. Control common bean plants of cv. PI207262, were inoculated either with non-pathogenic race (R0) or highly pathogenic race (R-1472), for negative and positive controls of the experiments without using any antimicrobial (only water sprayed). 3. Experimental design and statistical analysis In all experiments, the size of samples was chosen according to previously established methodologies. For the in vitro antimicrobial and enzymatic activities, the size of the sample and number of replicates was according to Hayashi (2005). On the other hand, experiments with plants were designed according to Snedecor and Cochran (1976). Data were subjected to analysis of variance by the general linear models (GLM) procedure, means comparison by Tukey’s test according to SAS methods (1990). 4. Results and discussion 4.1. Characterization of cascalote extracts Total phenolic content of cascalote pods extracts expressed as GAE, was 703.5 ± 1.36 g/kg of dry matter. This content was similar to that reported for cascalote by Veloz-García et al. (2004), when used cascalote pods collected in the same region during the year 2000, 6 years before the collection used in this study. According to HPLC analysis, the chromatogram obtained showed two peaks: the major one represented 90% of the total area, while the minor represented the rest 10% (Fig. 1A). Comparisons of retention times and peak areas with those of standard phenolics used (Fig. 1B), it can be concluded that gallic acid is the major component in cascalote extract (410.5 g/L). Meanwhile, tannic acid is the minor component of this extract (45.6 g/L). All these studies also agree with the previous reports by Veloz-García et al. (2004), for HPLC characterization of cascalote phenolic extracts. Taken together these results indicate that cascalote phenolic extract presents similar characteristics at least when pods are collected in the same region and stage of the year, because the results were highly similar with a difference of 6 years in time of collection when comparing with those obtained by Veloz-García et al. (2004) and this study. 4.2. In vitro inhibition of mycelial growth of C. lindemuthianum races R-0 and R-1472 Three concentrations of phenolic extract (70, 210 and 350 g GAE/mL) were used in this work based on the concentrations of active ingredients of several commercial fungicides as benomyl used in Mexico against antrachnose (Gutiérrez-Alonso and Gutiérrez-Alonso, 2003). Thus, according to the results the inhibition of mycelial growth of both races of C. lindemuthianum studied was dose dependent, the higher was the concentration of phenolics, the higher was the inhibition of mycelial growth in vitro (Table 1). In addition, all doses evaluated of cascalote pheno-
Fig. 1. HPLC chromatogram of cascalote phenolics extract. Panel A: HPLC separation of cascalote phenolics. Panel B: HPLC profile of mixture of gallic and tannic acid standards.
lics showed only fungistatic activity, in contrast with benomyl 100 ppm, which acted as fungicide (Table 1). As in other plant phenolic extracts in which only fungistatic activity against several phytopathogenic fungi was detected (Müller-Riebau et al., 1995; Rauha et al., 2000; Amzad-Hossain et al., 2008), cascalote phenolics in the concentrations evaluated presented fungistatic activity against both evaluated races of C. lindemuthianum (Table 1). Meanwhile, it is well known the fungicide activity of benomyl on a great spectrum of phytopathogenic fungi likely due to its ability to be absorbed by the phytopathogen cells (Gutiérrez-Alonso and Gutiérrez-Alonso, 2003). Thus, the fact that cascalote phenolics showed only fungistatic activity could suggest that this extract was either unable or poorly absorbed by the pathogen cells and the in vitro antimicrobial activity observed corresponded to surfacecontact actions likely on several proteins, as reported for other phenolics (Sousa, 2006). It has been suggested that fungistatic compounds are a less selective pressure than fungicide ones regarding Table 1 Percentage of inhibition of mycelial growth of C. lindemuthianum races R-0 and R1472 in in vitro assays. Phenolics concentrationa (g/mL) 0 70 210 350 Benomyl (100 ppm)
R-0 0d 71.3 ± 2.3cb 89.4 ± 1.8bb 100ab 100ac
R-1472 0d 68.4 ± 3.1cb 89.1 ± 2.2bb 94.9 ± 0.7ab 100ac
a Concentration of phenolics is expressed as g equivalents of gallic acid/mL of culture media (PDA). Results are the average of four replicates and three independent experiments. Different letters in each column mean significant difference (P < 0.05). b Only fungistatic activity. c Fungicide activity.
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Table 2 Effect of cascalote phenolics on in vitro cellulase and polygalacturonase activities of C. lindemuthianum races R-0 and R-1472. Enzymatic activity (AU/mL)a
R-0
R-1472
Cellulase Control (C)b C + 70 C + 210 C + 350
1.3 ± 0.5a 0b 0b 0b
1.4 ± 0.3a 0b 0b 0b
Polygalacturonase Control (C)b C + 70 C + 210 C + 350
1.4 ± 0.4a 0.1 ± 0.02b 0c 0c
1.5 ± 0.3a 0.1 ± 0.03b 0c 0c
a One enzyme activity unit (AU) was considered as the enzyme quantity causing 1% of viscosity loss in 1 h assay/mL of supernatant of enzymatic extract. Different letters in the same column in each enzymatic activity mean significant difference (P < 0.05). b Control indicates supernatant extract boiled 100 ◦ C during 5 min. Data shown in this table are from three independent experiments, and four replicates for each phenolics dose evaluated.
fungus resistance (Gamboa-Alvarado et al., 2003). In this sense, cascalote phenolics displayed a similar protection to anthracnose than those provided with a commercial fungicide (benlate). Thus, it could be expected similar or improved protection of common beans to anthracnose, but with a lesser resistance selection in the fungus. 4.3. Cellulase and polygalacturonase activity As aforementioned, the fungistatic in vitro activity of cascalote phenolics on R-0 and R-1472 races of C. lindemuthianum suggests a surface-contact action probably on proteins and/or enzymatic functions. Thus, the effect of this extract on the cellulase and polygalacturonase activities, which are considered degrading extracellular enzymes produced during infection and are thought to play a major role in anthracnose pathogenesis was evaluated (Acosta-Rodríguez et al., 2005; Hernández-Silva et al., 2007). As displayed in Table 2, both R-0 and R-1472 races produced similar levels of cellulases and polygalacturonases, which agree with previous reports (Acosta-Rodríguez et al., 2005; Hernández-Silva et al., 2007). Besides, the three concentrations of cascalote phenolics evaluated occasioned a significant inhibition of activity of both enzymes (Table 2). These results agree with those reported by Gamble et al. (2000), in which flax bast phenolics inhibited both cellulases and pectinases activities of this plant. It is well indicated that phenolics as tannic and gallic acids can form complex via covalent or non-covalent interactions with several proteins (including enzymes as cellulases and polygalacturonases), and moreover both phenolics are assumed to be one of the major barriers against pathogen infections (Krause et al., 2005; Chung et al., 2008). 4.4. Effect of cascalote phenolics on spores’ germination Several chemical compounds as benomyl among others used against phytopathogenic fungi, have the capacity to inhibit spore germination (Chiocchio et al., 2000). Thus, this possibility in the case of cascalote phenolics against C. lindemuthianum R-0 and R-1472 races was evaluated. As shown in Fig. 2A, the minimal concentration evaluated of cascalote phenolics with a significant inhibition of spore’s germination for both races was 210 g GAE/mL. Moreover, in control treatments, both fungi races displayed a typical germination (Fig. 2B, left picture) which was 100% at 72 h of incubation, except when used benomyl 100 ppm, which provoked a total inhibition of spores germination in both races at the same incubation time (Fig. 2B, right picture). This result is inter-
Fig. 2. Effect of cascalote phenolics on germination in vitro of C. lindemuthianum R-0 and R-1472 spores. Panel A: Effect on % germination after 72 h post-incubation in in vitro assays. Panel B: Typical germination of spores of C. lindemuthianum (R-1472 as example) (left) and no germination (right) at 72 h post-incubation. As comparison, benomyl 100 ppm caused a 0% of germination at 72 h in all cases (not shown). Data on panel A are from three independent experiments.
esting because although at 70 g GAE/mL, there were no observed inhibition of spore germination of R-0 and R-1472 races of C. lindemuthianum, however, this concentration provoked a significant inhibition in mycelial growth (Table 1) and abolished both extracellular cellulase and polygalacturonase activities in vitro (Table 2). Taken together these results agree with reports of successful inhibition of spores germination using phenolics from different plant sources against phytopathogen Botrytis cinerea in in vitro assays (Yildirim and Yapici, 2007). 4.5. Antimicrobial assays in common bean plants In order to evaluate the effect of cascalote phenolics in vivo on the anthracnose severity, common bean plants (cv. PI 206272) were studied in corrective and preventive treatments with these extracts under greenhouse conditions. In preventive treatments, all concentrations of cascalote phenolics and benomyl 100 ppm displayed a significant decrease in symptoms severity on common beans cv. PI 206272 in comparison to controls (Table 3). On the other hand, in corrective assays, only treatments with 350 g GAE/mL and benomyl 100 ppm significantly diminished anthracnose severity (Table 3). It is noteworthy that no phytotoxic effect was observed in cv. PI 206272 plants with any of the treatments Table 3 Symptoms severityb in common bean plants at greenhouse level for anthracnose protection using cascalote phenolics. Concentrationa
Preventive
Corrective
Negative controlc Positive controlc 70 210 350 Benomyl (100 ppm)
0c 3.6 ± 0.3a 1.0 ± 0.2b 0.6 ± 0.2b 0.6 ± 0.3b 0.5 ± 0.3b
0c 3.4 ± 0.5a 3.4 ± 0.3a 2.9 ± 0.4a 1.6 ± 0.4b 0.8 ± 0.5b
Concentration expressed as g equivalents of gallic acid/mL. Severity of anthracnose symptoms according to Garrido-Ramírez and RomeroCova (1989). c Negative control, common bean plants cv. PI 207262 were only inoculated with non-pathogenic R-0 race of C. lindemuthianum; positive control, common bean plants cv. PI 207262 were only inoculated with pathogenic R-1472 race of C. lindemuthianum. Both controls were sprayed with distilled water. Results shown are the average of three replicates of the experiment. Different letters in the same column mean significance difference (P < 0.05). a
b
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evaluated in this study (data not shown). Thereby, cascalote phenolics showed to be a good anthracnose control strategy under greenhouse conditions evaluated in this study in common bean cv. PI 206272, especially as a preventive treatment, although at the highest concentration evaluated they also confered significant decrease in anthracnose levels as corrective treatment. It could be hypothesized that cascalote phenolics presented a better effect as preventive rather than corrective treatment because of they might poorly be absorbed by fungi and plant cells, such as the main possible antifungal effect is by complexing important proteins used by the pathogens for plant cell wall degradation (i.e. cellulases and pectinases, among others) and penetration (i.e. appresoria formation). In fact, it has been demonstrated for fungicides as benomyl, that its potential is similar in preventive as well as corrective treatments due to its effective absortion by both plant and fungi cells (Gutiérrez-Alonso and Gutiérrez-Alonso, 2003). Besides, it has been demonstrated that phenolics as gallic acid, methyl gallate and likely tannic acid, can inhibit appressoria formation in rice pathogen Magnaporthe grisea, acting on a cAMP-related signaling pathway regulating appressorium formation in this phytopathogen (Ahn et al., 2005). 5. Conclusions The results of this research suggest that cascalote phenolics might be a potential organic antimicrobial agent (fungistatic) against anthracnose in common bean caused by C. lindemuthianum under greenhouse conditions. Acknowledgements Authors acknowledge Fondo de Investigación de la Facultad de Ingeniería de la UAQ (FIFI)-2009, Proyecto de Equipamiento del Laboratorio de Biosistemas 2008 and PROMEP/103.5/08/3320 for support this research. Special thanks to Adriana Medellín-Gómez and Silvia C. Stroet of the Translation Edition Office of UAQ for technical review of English writing. References ˜ Acosta-Rodríguez, I., Pinon-Escobedo, C., Zavala-Páramo, M.G., López-Romero, E., Cano-Camacho, H., 2005. Degradation of cellulose by the bean-pathogenic fungus Colletotrichum lindemuthianum: production of extracellular cellulolytic enzymes by cellulose induction. Antoine van Leeuwenhoek 87, 301–310. Ahn, Y.-J., Lee, H.-S., Oh, H.-S., kim, H.-T., Lee, Y.-H., 2005. Antifungal activity and mode of action of Galla rhois-derived phenolics against phytopathogenic fungi. Pest. Biochem. Physiol. 81, 105–112. Amzad-Hossain, M., Ismail, Z., Rahtman, A., Kang, S.C., 2008. Chemical composition and antifungal properties of the essential oils and crude extracts of Orthosiphon stamineus Benth. Ind. Crops Prod. 27, 328–334. Awale, H., Falconi-Castillo, E., Villatoro-Mérida, J.C., Kelly, J., 2008. Caracterización de aislamientos de Colletotrichum lindemuthianum de Ecuador y Guatemala para identificar genes de resistencia. Agron. Mesoamer. 19, 1–6. ˜ Cardador-Martínez, A., Albores, A., Bah, M., Calderón-Salinas, V., Castano-Tostado, ˜ G., 2006. RelationE., Guevara-González, R., Shimada-Miyasaka, A., Loarca-Pina, ship among antimutagenic, antioxidant and enzymatic activities of methanolic extract from common beans (Phaseolus vulgaris L.). Plant Foods Hum. Nutr. 61, 161–168. Chang, H.-T., Cheng, Y.-H., Wu, C.-L., Chang, S.-T., Chang, T.-T., Su, Y.-C., 2008. Antifungal activity of essential oil and its constituents from Calocedrus macrolepis var. Formosana Florin leaf against plant pathogenic fungi. Bioresour. Technol. 99, 6266–6270. Chiocchio, V., Venedikian, N., Martínez, A.E., Menendez, A., Ocampo, J.A., Godeas, A., 2000. Effect of the fungicide benomyl on spore germination and hyphal length of the arbuscular mycorrhizal fungus Glomus mosseae. Int. Microbiol. 3, 173–175. Chung, H.J., Kwon, B.R., Kim, J.M., Park, S.M., Park, J.K., Cha, B.J., Yang, M.S., Kim, D.H., 2008. A tannic acid-inducible and hypoviral-regulated Laccase3 contributes to
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