## 3 Results and Discussion
### In vivo Antifungal Activity of Propolis Extracts Against Stemphylium vesicularium  
The ability of propolisols to control _S. vesicularium_ was also assayed in vivo in \"Rocha\" pears, by assessing the decrease of the number of infected wounds (disease incidence) and the reduction of lesions diameter over time (treatment efficacy), in relation to control, in curative and prophylactic assays. Propolis extract E4 was selected for the in vivo assay because of its high total phenolic content and in vitro inhibitory activity against _S. vesicularium_. Aiming to evaluate a potential cytotoxic effect of propolis on pear pulp, a control in which the wounds were treated only with undiluted propolisols extract (without pathogen inoculation) was performed. No symptoms of apparent toxicity were observed in the pear tissues, the wounds were healed, and no browning was observed. The curative assay was performed with two different concentrations of E4, namely 30.0 mg/mL (undiluted extract) and 6.0 mg/mL (1:5 dilution of initial extract). After 14 days of incubation the disease incidence decreased significantly in propolis-treated fruits in a dose-dependent manner (Figure 3). Comparing to the control, the disease incidence decreased 25.0% in fruits treated with the highest extract concentration (30.0 mg/mL) and 15.0% in fruits treated with the lowest extract concentration (6.0 mg/mL). The extract concentration with the higher inhibitory effect in the curative assay (30.0 mg/mL) was selected for the prophylactic assay. In this case, the disease incidence also decreased in propolis-treated fruits, but this reduction was less pronounced (15.0%) (Figure 3). Figure 2: Percentage of _Stemphylium vesicularium_ mycelial growth inhibition in the presence of different concentrations (0.2, 0.5, and 1.0 mg/mL) of the four propolisols extracts (E1, E2, E3, and E4) after 14 days of incubation at 25 \({}^{\circ}\)C. Results are presented as means \(\pm\) standard deviation of three replicates. Different lower-case letters denote significant differences among the percentage of mycelial growth inhibition obtained with different concentration of each propolisols extract (\(p<0.05\)). Different upper-case letters denote significant differences among the percentage of mycelial growth inhibition obtained with the same concentration of the different propolisols extracts (\(p<0.05\)). In agreement with the results obtained in the in vitro assay, propolis was also able to reduce the in vivo growth of _S. vesicularium_ since, compared to the control, lesion diameter decreased significantly in propolis-treated fruits in a dose-dependent manner (Figure 4). In the curative assay, the lesion diameter decreased 56.6% in fruits treated with the highest extract concentration (30.0 mg/mL) and 34.2% in fruits treated with the lowest extract concentration (6.0 mg/mL). In the prophylactic assay, the lesion diameter decreased 55.9% in propolis-treated fruits. Accordingly, there were no significant differences in the efficacy of prophylactic and curative treatments (Figure 4). Thus, under the conditions of the present study, it seems that in vivo propolis is effective in reducing both the incidence of infection, especially if applied curatively, and the disease severity. As mentioned above, fungicides vary in efficacy and with disease pressure [2]. Moreover, repeated use of some types of fungicides has been restricted by the emergence of fungicide-resistant strains [46]. Thus, there is a need for the search of new strategies to control _S. vesicularium_. To the best of authors' knowledge, so far, no studies focused on the use of propolis extracts to control _S. vesicularium_ growth in \"Rocha\" pear. However, several in vivo studies describe the activity of  
Figure 4: Reduction of lesions diameter (treatment efficacy) relative to control after 14 days of incubation at 20 \({}^{\circ}\)C in the curative (left) and prophylactic (right) assays Results are presented as means \(\pm\) standard deviation of two replicates.