## 3 Results  
### Genotyping, Occurrence, H\({}_{2}\)S Production and Killer Activity of S. cerevisiae Strains  
Interdelta sequence analyses of the 206 _S. cerevisiae_ isolates (99 winery 1 and 107 winery 2) revealed a total of 19 different biotypes, as showed in Figure 2. Seven different biotypes had been found in winery 1, and 12 different biotypes in winery 2, highlighting more biodiversity in the lastly winery. Biotypes from the two wineries were different and both biotype groups were different from the more diffused commercial strains utilized in the Verdicchio wine area and used as reference.  
Regarding the occurrence of each biotype within the winery, in both there are two biotypes that represent the dominant population, biotype XIII and XIV in winery 1 and biotype I and IV in winery 2 (Table 1). In winery 1, the two biotypes represented more than 90% of the entire population of _S. cerevisiae_ strains while the occurrence of each of the other biotypes was \(\leq\)2%. Moreover, both dominant biotypes and the biotype XVI had been found in both two-year sampling campaigns, as \"resident yeasts\" (95%). Indeed, persistent biotypes XIII and XIV represented 68% and 19%, respectively, of the _S. cerevisiae_ population detected in 2016 while the same biotypes represented 42% and 43%, respectively, of the _S. cerevisiae_ population detected in 2019.  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline \multicolumn{1}{c}{**Winery**} & \multicolumn{1}{c}{**Biotype**} & \multicolumn{1}{c}{**Frequency (\%)**} & \multicolumn{1}{c}{**H\({}_{2}\)S Production**} & \multicolumn{1}{c}{**Killer Activity**} \\ \hline \multirow{8}{*}{\(1\)} & XIII * & 49.5 & 2.0 & \(\pm\) \\  & XIV * & 43.5 & 0.0 & + \\  & XV & 1.0 & 1.5 & + \\  & XVI * & 2.0 & 0.0 & + \\  & XVII & 1.0 & 0.5 & + \\  & XIX & 2.0 & 0.8 & + \\ \hline \multirow{8}{*}{\(2\)} & I & 23.4 & 0.5 & + \\  & II & 15.0 & 0.0 & \(-\) \\  & III & 16.8 & 0.0 & + \\  & IV * & 19.6 & 1.0 & + \\  & V & 2.8 & 0.5 & \(\pm\) \\  & VI & 7.5 & 0.5 & + \\  & VII & 1.9 & 1.5 & \(\pm\) \\  & VIII & 1.9 & 0.8 & \(\pm\) \\  & IX & 3.7 & 0.0 & \(\pm\) \\  & X & 0.9 & 1.0 & + \\  & XI & 5.6 & 1.0 & + \\  & XII & 0.9 & 1.0 & \(\pm\) \\ \hline \hline \end{tabular}
* Biotypes found in both sampling years within the same winery.  
\end{table}
Table 1: Biotypes occurring in wineries, their frequency and characterization in the H\({}_{2}\)S production and killer activity. H\({}_{2}\)S production was expressed as a score, using a hedonic scale from zero (no H\({}_{2}\)S production) to 5 (maximum level of H\({}_{2}\)S production). The presence of killer activity was expressed as “+”, the absence with “\(-\)”, and “\(\pm\)” indicates faint killer activity.  
Figure 2: Different interdelta profiles of biotypes of both wineries. Each biotype is represented as a Roman numeral, from XIII to XIX for winery 1 and from I to XII for winery 2. All biotype’s profiles were compared with those of commercial strains OKAY®, N96, EC1118 and VIN13.  
In winery 2, a wide biotype's variability was observed. The dominant biotypes represented c.a. 40% of the _S. cerevisiae_ population, followed by the biotype III (c.a. 17%) and biotype II (15%). The remaining 28% of the population was grouped in the other biotypes. Similarly to winery 1, in winery 2 a resident biotype (biotype IV) was also found that represented 18% and 6% of the _S. cerevisiae_ population detected in 2021 and 2022, respectively.  
The results (Table 1) show a low or absent production of H2S (rotten egg aroma) for all biotypes of both wineries. All of them showed H2S production with a score \(\leq\) 2.0. The evaluation of the presence of a killer phenomenon showed that there was a wide diffusion among the biotypes. Indeed, all biotypes of both wineries showed killer activity against the sensitive _S. cerevisiae_ yeast DiSVA 42, with the only exception of the biotype II belonging to winery 2 (Table 1).