Fungal isolate

Bell peppers (Capsicum annuum) showing gray rot were collected at the Centro de Investigación y Transferencia de Tecnología (CITT) “Rancho El Islote” of the Instituto de Investigación y Capacitación Agropecuaria, Acuícola y Forestal del Estado de México (ICAMEX), in Villa Guerrero, State of Mexico, at 18° 58’ 04’’ North latitude and 99° 39’ 21’’ West longitude. Then, at ICAMEX’s Laboratory of Phytopathology, tissue fragments, measuring approximately 5 × 5 mm, were taken from the diseased bell peppers and disinfected with 1 % (v/v) sodium hypochlorite for 3 min; they were rinsed three times with sterile distilled water, and transferred to Petri dishes containing acidified at pH 4.5, with 25 % (v/v) lactic acid, oat-agar culture medium (MCAvA), and, then, incubated at 26±2 °C for 8 days. Later, using the hyphal tip technique, a portion of mycelial tissue was transferred to another Petri dish containing MCAvA to obtain a pure isolate of the fungus. To confirm the isolates’ pathogenicity, we inoculated healthy (Fragaria × ananassa) cv. Festival strawberries that had been previously disinfected as described earlier. Three perforations measuring approximately 2 mm in diameter × 2 mm deep were made on the strawberries using previously sterilized dissection needles. The perforations were filled with 20 µL of a suspension of 1×10⁶ conidia per millimeter. Once the typical symptoms of gray rot appeared, the fungus was identified by comparing the morphological structures observed (mycelium type and color, septation, conidiophores, conidiophore branching, conidia length and width) using the ^{Barnett and Hunter keys (1998)}. The specialized descriptions by ^{Ellis (1971)} and ^{Crous et al. (2007)} were used to determine the species.

Samples of the fungus were also sent to the Laboratory of Phytosanitary Diagnosis of Colegio de Postgraduados, Montecillo, Mexico, where they were molecularly analyzed. The ITS sequence obtained from the fungus was compared to sequences of the most similar reference organisms through a search in the BLAST database of GenBank® (National Institutes of Health (NIH), Bethesda, MD, USA). Once the species was confirmed, the fungus was grown to perform the tests of this experiment.

Obtaining vegetable extracts

Healthy leaves of uniform size and color were obtained (^{Franco et al., 2012}) from three wild grapevine accessions: (1) E-200, from Tejupilco, State of Mexico, (2) TN-4, from Tenancingo, State of Mexico, and (3) P-178, from Hueytamalco, Puebla; all the accessions were grown at the wild grapevine germplasm bank in Zumpahuacán, Mexico. The vegetable tissue was moved to the Laboratory of Horticulture of the Universidad Autónoma del Estado de México, in Toluca, State of Mexico, where 200 g of leaves per accession were macerated in 250 mL of 99 % methanol; these mixtures were left standing in darkness for a week. Afterwards, the extract was filtered and concentrated in a rotary evaporator until 50 mL of stock solution free of the extraction solvent was obtained. The extracts were poured into amber glass jars and placed in cold storage (4 °C) for conservation.

Identification of polyphenols

Using gallic acid as a standard, the total amount of phenols contained in extracts of wild grapevine leaves (EHVS) was determined using the method of Folin-Ciocalteu (^{Mora et al., 2009}) at 760 nm on a spectrophotometer. The result was expressed in gallic acid equivalents (EAG). Some polyphenolic compounds of EHVS of each individual accession were identified using an HPLC Shimadzu isocratic pump (serial 42205) and a reversed-phase Spherisorb C18 1 μm × 250 mm × 4.6 mm column (Waters, USA). For the mobile phase we used water: acetonitrile: acetic acid (70:29.9:0.1). The flow level was 1 mL min^-1 and we allowed a five minute stabilization interval between each sample. The signal was monitored at 270 nm (^{Lorrain et al., 2013}). The samples were injected three times in the chromatographic system and the compounds detected using the standard internal method were quantified.

Antifungal activity

The respective amounts (Table 1) of the EHVS of each accession, resveratrol (RVS) (Sigma), citric extract (EC) (Tecnosafe), and cyprodinil+ fludioxonil (fungicide Switch^® 62.5 WG) (SW) were poured into flasks containing unsolidified MCAvA (pH 4.5); MCAvA with no ingredients added was used as the absolute tester (TA).

Table 1. Concentration of phenolic compounds in oat-agar adding extracts of leaves of wild grapevine (Vitis spp). 

EAG: Equivalents of gallic acid. Data are the average of three replications; values with the same letter show a statistical difference at P≤0.05.

From each EHVS, we took 6, 8 and 12 mL for dilution at 100 mL of MCAvA at pH 4.5. Each solution was poured into properly labeled Petri dishes and allowed to solidify. Treatments with RVS (60, 90 and 120 µg/mL), EC (3, 5 and 8 % (v/v)) and SW (500, 800 and 1000 µg/mL) were prepared in the same way, using RVS diluted in: methanol 1:1, SW in distilled water, while EC was used in its liquid commercial form. Each of the 19 treatments were repeated four times using three Petri dishes per replication; each of the Petri dishes was an experimental unit. The experiment was performed twice, and because of the similarity of both results, the statistical analysis included in this report corresponds to only one experiment.

Later, a disk 5 mm in diameter of MCAvA with 5-day old active B. cinerea mycelium was placed in the middle of each Petri dish containing MCAvA and its corresponding treatment. The dishes were incubated at 26±2 °C and the diameter of each B. cinerea colony was measured every 48 h using a digital Vernier caliper. Measurement of each replication ended when the surface of MCAvA in all the TA dishes was completely covered by mycelium. Average values of mycelium growth were converted to percentage inhibition of mycelial growth compared (ICM) to TA by applying the formula: ICM (%) = [(dTa − dt)/dTa (ICM)] × 100, where dTa and dt denote the diameter of the mycelium growth of TA and of each of the other treatments, respectively (^{Soylu et al., 2010}). The Petri dishes within the incubator were arranged in a completely randomized design.

At the end of the experiment, MCAvA spores were collected from each treatment using a glass rod and sterile distilled water. For each previously described experimental unit, an aliquot of the suspension containing conidia was taken and transferred to a Neubauer chamber to count the conidia following the methodology of ^{Moo-Koh et al. (2014)}. Data were converted to percentages, and the results were reported as inhibition of sporulation (IE) following the formula: IE (%) = [(ETa − Et)/ETa] × 100, where ETa and Et denote the number of conidia in TA and in each of the remaining treatments, respectively (^{Soylu et al., 2010}).

On the other hand, using a sporulated culture medium (8 days old), a suspension of 1 × 10⁶ conidia per milliliter was prepared in sterile distilled water. Two hundred microliters of the suspension were poured in Petri dishes with MCAvA supplemented with the different doses of each of the 19 treatments. After 48 h of incubation at 22±2 °C, germination of 100 randomly placed conidia was evaluated. The conidia were considered germinated when the length of the germ tube was equal to or greater than the diameter of the conidium. The percentage of spore germination inhibition (IGC) was determined with the formula used by ^{Soylu et al. (2010)}: IGC (%) = [(GTa − Gt)/GTa] × 100, where GTa and Gt denote the number of germinated conidia in TA and in each of the other treatments, respectively. The experimental design and number of replications per treatment were the same ones used for mycelium growth.

Statistical analysis

ICM, IE and IGC percentages were converted to y = arsin (sqrt (y/100)). The data were processed through analysis of variance of a complete randomized design with a single factor, and when significance was found, we compared the means using Tukey’s test (P≤0.05); all analysis were performed with the SAS^® statistical software (^{SAS Institute, 2002}).

Morphological-molecular identification.

Four days after inoculation with Botrytis sp., all strawberries showed symptoms of gray rot, as well as abundant fungal sporulation (Figure 1A). On the other hand, in MCAvA, colonies showed white concentric mycelium growth of velvety consistency; seven days later, the mycelium turned gray (Figure 1B). Under a light microscope, we observed long, septate, pigmented conidia with smooth walls, apically branched and with bunches of conidia (Figure 1C). The conidia were unicellular, ovoid, smooth, hyaline, and measured 8-15 × 6-9 µm (Figure 1D). All the features observed corresponded to B. cinerea, as described by ^{Ellis (1971)}. On the other hand, when we compared the sequence in BLAST, it was 100 % similar to the KR055051 sequence. Based on the morphological and molecular features, we confirmed that the causal agent of gray rot on the bell pepper at CITT “Rancho el Islote” of ICAMEX is Botrytis cinerea.

Figure 1. Isolate of Botrytis cinerea from Capsicum annuum fruits. A) Symptoms of gray rot in strawberry fruits. B). Four-day old colony cultivated in oat-agar. C) Branched tip of a conidiophore with conidia bunches; D) Botrytis cinerea conidia. 

Phenolic compounds

We found gallic and ferulic acids in the EHVS of the three accessions, and resveratrol in P-178. When the amount of EHVS per millimeter of MCAvA increased, the concentration of the different phenols increased (Table 1).

Antifungal activity

All the concentrations of EHVS of the three accessions showed ICM. Accession P-178 12 % showed higher ICM, 70 % higher than TA. SW at its two highest doses showed 90 % of ICM, while the ICM per RVS reactive grade was between SW and EHVS. EC inhibited no more than 25 % of mycelium growth (Table 2, Figure 2). SW had the highest IE, followed by RVS; the percentage of the EHVS was 53 to 75 % compared to TA.

Table 2. Inhibitory activity of different treatments on in vitro mycelial growth (ICM), sporulation (IE) and spore germination (IGC) of Botrytis cinérea. 

Data are the average ± SE of four replications, three Petri dishes per replication. Averages followed by the same letter in each column show no significant difference using Tukey’s test at P≤0.05.

Figure 2. Development of Botrytis cinerea in oat-agar 8 days after inoculation at 26±2 °C A: absolute tester; B: extract of leaves of the wild grapevine accession E-200 12 % (v/v) (Vitis spp.); C: extract of leaves of the wild grapevine accession TN-4 12 % (v/v); D: extract of leaves of the wild grapevine accession P-178 12 % (v/v); E: citrus extract 8 % (v/v); F: resveratrol 120 μg/mL; G: cyprodinil + fludioxonil 1000 μg/mL. 

On the other hand, all the treatments showed IGC. The three doses of SW inhibited more than 90 %, followed by RVS, whose average of the three doses was 64 %. The highest IGC of an EHVS was observed in P-178 12 %, whose percentage was higher than 60 % in relation to TA.