Botrytis cinerea Pears

Botrytis cinerea was isolated from infected grapes of the Flame Seedless variety in a vineyard located in the wine region of Pesqueira, Sonora. The strain was identified by its morphological and molecular characteristics such as conidia size, colony appearance and ITS1-5.8S RNA-ITS2 sequence (access AY568636). For growth, it was sown in agar-potato-dextrose (PDA) medium culture and kept in darkness at 25 °C from 7 to 9 days (^{Tzortzakis et al., 2007}). The fungus was sown again by taking a piece of mycelium and inoculating it into PDA at 25 °C from 7 to 9 days, or until the fungus produced enough spores to prepare a 1x10⁶ spore/mL suspension. To prepare the suspension, 5 mL of distilled water with 0.01% of 0.02% Tween 80 were poured on the surface of the plate containing the fungus; then, it was scratched using a glass rod, the liquid containing fungal spores was collected and filtered through muslin cloth to remove the mycelium, and the mixture was centrifuged at 5000 x g. The collected spores were re-suspended in sterile water until a 1x10⁶ spores/mL concentration was obtained, and quantified in a Neubauer chamber, according to the methodology of ^{Moo-Koh et al. (2014)}.

Hydroalcoholic extract of garlic (EHA) and its derived sulphur compounds (DADS, DATS and allicin)

The extract was prepared using garlic of the Regional variety purchased at the local market. Ten gram of garlic cloves were blended with 14 mL of distilled water for 1 min at room temperature. The mixture was centrifuged at 15 000 x g at 4 °C for 20 min, then 6 mL of ethanol were added to the supernatant and the mixture was centrifuged again at 8000 x g at 4 °C for 20 min (^{Jansen et al., 1987}). The supernatant represented the hydroalcoholic extract of garlic. Standards with purity above 98% allicin, dialyldisulfide (DADS) and dialyltrisulfide (DTS) were purchased from Neem Biotech (Cardiff, UK).

In vitro evaluation of the effect of EHA, DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on grape clusters

Grape clusters of the Flame Seedless variety were collected at harvest in vineyards in the wine region of Pesqueira, Sonora. The clusters were reduced to 10 berries per cluster, ensuring that they did not show visible symptoms of gray mold caused by B. cinerea. The clusters were disinfected by immersion in a 2% (v/v) sodium hypochlorite solution for 5 min, rinsed with sterile distilled water and dried on paper towels at room temperature. A puncture wound near the peduncle of each berry was made with a sterile scalpel. Each cluster was sprayed with 1 mL of the 1x10⁶ spores/mL suspension of B. cinerea prepared in dextrose sabouraud agar (CDS). The inoculated clusters were placed in 50 cm³ plastic chambers (one cluster per chamber) and exposed to volatile compounds from EHA, DADS, DATS and allicin at the doses per treatment shown in Table 1. The compounds were impregnated in cellulose pads (6x2 cm) that had been previously stuck on the coverlid of the chambers. The control treatment was impregnated with a water-glycerol solution because glycerol was used as a retention agent to control the release of the emitter’s volatiles. This compound was incorporated into the treatments before impregnating the emitter. The disease incidence was quantified every other day during 14 days at 4 and 25 °C. Berries of each cluster with gray mold symptoms were counted. The results are presented as the disease incidence (percentage of infected berries) calculated using the formula proposed by ^{Nally et al. (2012)}. Three replications per treatment were prepared and the evaluation was performed three times.

Table 1 Treatments used to evaluate in vitro incidence of gray mold on table grape clusters at 4 and 25 °C. 

Evaluation of the synergistic effect among DADS, DATS and allicin on gray mold caused by Botrytis cinerea on grape clusters

To determine a possible synergistic effect among DADS, DATS and allicin compounds, a mixture containing 20 μL of each compound and 90 μL of glycerol was prepared. The mixture was homogenized with gentle agitation and kept in a cold-water bath until it was applied to the emitter. The treatments used are shown in Table 2. The same procedure described for independent compounds was used to determine the effect of the compound mixture on the incidence of gray mold on grape clusters.

Table 2 Treatments used to evaluate the synergy among DADS, DATS and allicin in the 

Experiment design and statistical analysis

Independent compounds were arranged in a completely randomized design with 5 treatments and 3 replications; the mixture of compounds was arranged in a completely randomized design with 3 replications and 3 treatments. The experiment unit in both treatments was a 10-berry cluster, and the response variable the number of berries with gray mold symptoms. The percentage of disease incidence was calculated using the equation of ^{Nally et al., 2012}. Data of the percentage of gray mold incidence were converted to y = arsin (sqrt (y/100)) and then subjected to an analysis of variance with a factor and, in case of significance, a media comparison was made using Tukey-Kramer test (P≤0.05) and NCSS statistical program (^{Number Cruncher Statistical System, 2007}).

Effect of EHA, DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on grape clusters

The effect of volatiles emitted by the hydroalcoholic extract of garlic and DADS, DATS and allicin on gray mold development on table grape clusters evaluated during 14 days after treatment application is shown in Figure 1. The figure shows that volatiles emitted by T2 (EHA) controlled gray mold development on grape table clusters both at 4 °C and 25 °C. From T3, T4 and T5 treatments evaluated at 25 °C (Figure 1B), only T4 showed a similar effect (p≤0.05) to EHA on gray mold development. Clusters treated with T3 showed 70% of gray mold incidence, and T5 a similar incidence (p≤0.05) to that of the control, reaching 100% on day 10 of the 14-day evaluation.

The effect of the treatments evaluated at 4 °C showed that T2, T3 and T4 controlled gray mold development (Figure 1A), and although T5 allowed 10% of disease incidence, the statistical analysis (p≤0.05) was the same as that of T2, T4 and T5. Special importance should be given to the effect of the treatments evaluated at 4 °C, because during the first 8 days under treatment, T5 showed only 5% of gray mold incidence, whereas the control presented 30%.

Effectiveness of the treatments evaluated to inhibit gray mold development on table grape clusters is shown in Table 3. Results show that T2 was more effective in controlling the disease during 14 days at 25 and 4 °C, followed by T4 treatment that controlled the disease 8 and 12 more days than the control at 25 and 4 °C, respectively.

Figure 1 Effect of volatiles emitted by EHA, DADS, DATS and allicin during gray mold development on table grape clusters evaluated at 25 and 4 °C during 14 days. Values represent the media of three replications of 10 berries each. Vertical bars represent the standard deviation (n=3). Values with the same letter are not statistically different from each other for P≤0.05, according to Tukey-Kramer. The experiment was conducted three times. 

Table 3 Evaluation in vitro of the effectiveness of treatments to protect clusters of table grape from gray mold. 

z Disease development in EHA started 14 days after.

Synergistic effect of a mixture of DADS, DATS and allicin on gray mold development caused by Botrytis cinerea on table grape clusters

The mixture of DADS, DATS and allicin had a similar effect (P≤0.05) that of EHA on gray mold development on grape clusters (Figure 2). When evaluated at 4 °C, both EHA and the mixture of compounds inhibited disease development during 14 days (Figure 2A), while when evaluated at 25 °C only EHA controlled the disease during 14 days. The mixture of DADS, DATS and allicin resulted in 9% of gray mold incidence at day 10; this value remained until day 14, when the evaluation ended.

Figure 2 Synergistic effect of a mixture of DADS, DATS and allicin on gray mold development evaluated at 25 and 4 °C during 14 days, compared with EHA and its control. Vertical bars represent the standard deviation (n=3). Values with the same letter are not statistically different from each other for P≤0.05, according to Tukey-Kramer.