## 3 Results and Discussion
### In Vitro Antifungal Activity of Propolis Extracts Against Stemplyium vesicular  
All propolis extracts assayed were able to inhibit in vitro _S. vesicularium_ mycelial growth in all the concentrations tested (Figure 1). For E2 the inhibition of mycelial growth increased significantly with the increase of propolis concentration in the culture medium (Figures 1 and 2). However, for the other propolis extracts (E1, E3, and E4) there were no significant differences in the percentage of inhibition of mycelial growth between the two highest concentrations assayed (0.5 and 1.0 mg/mL) (Figures 1 and 2). After 14 days of incubation at 25 \({}^{\circ}\)C, and for the highest concentration tested (1.0 mg/mL), extracts E3 and E4 presented a percentage of mycelial growth inhibition (\(\sim\)90%) significantly higher than extracts E1 (\(\sim\)78%) and E2 (\(\sim\)74%) (Figure 2). Thus, results showed that, among the extracts assayed, extracts containing northern propolisis (E3 and E4) and/or prepared by the ultrasound-assisted extraction were more efficient in inhibiting _S. vesicularium_ growth in vitro. As mentioned above, the biological activity of propolis derive from the plants available in its production area and consequently, variations in the antimicrobial activity may occur according to the propolis origin. Therefore, the results obtained suggest that propolis from the northern region of Portugal could be a good source of compounds with antifungal properties. Moreover, as biological activities are also related to the extraction method, the results obtained may also be associated to the ultrasound-assisted extraction method used to prepare E3 and E4. Ultrasound enlarge the pores of the cells and the fragmentation of the extracted material, increasing their contact with the fresh solvent and enhancing the extraction yields [36]. While some authors reported that ultrasound-assisted extraction was more efficient than maceration in extracting propolis's antimicrobial compounds [37], others have reported that the antibacterial activity of extracts obtained by ultrasound-assisted extraction was similar to that of extracts obtained by maceration [36]. The activity of propolis against _S. vesicularium_ is in line with the literature data. In fact, several authors reported that propolis has a wide antimicrobial range of activity, being effective against different bacteria, yeasts, protozoa, and filamentous fungi [31; 38; 39; 40; 41], including phytopathogenic fungi [17; 19; 20; 42; 43]. The mechanism of microbial growth inhibition exerted by propolis has not yet been fully elucidated. Several studies point out that the phenolic compounds from propolis may interfere with the integrity of the cell membrane, thus making cell survival unviable. This interference may occur by destabilization of cytoplasmic membrane with alteration of its potential and permeability [32; 43]. The inhibition of nucleic acid synthesis (DNA and RNA), proteins and lipids, as well as the inhibition of several enzymes, has been other mechanisms of antimicrobial activity attributed to phenolic compounds [43]. Particularly, in the case of fungi, bioactive compounds from propolis may change cell surface hydrophobicity and charge, eventually triggering the leak of cytoplasmic content [44]. Several of these compounds can also interfere with cytoplasmatic membrane and cell wall integrity, by interfering with ergosterol or 1,3-\(\beta\)-glucan synthesis or through chitin-binding ability [44]. [PERSON] et al. [19] reported that Chinese propolis extracts present a strong inhibitory activity against _Penicillium digitatum_ and _Penicillium italicum_. The same authors later identified pinocembrane, pinobanksin, chrysin, and galangin as the compounds probably responsible for these antifungal activities [42]. Pinochernin isolated from propolis, showed strong antifungal effects against _P. italicum_ by interfering with energy homeostasis, causing disruption of cell membranes and metabolic disorders [45].