## 3 Results
### Yeast and Fungi Community during the Early Stages of Winemaking without Sulfites
#### 3.2.2 Yeasts and Fungal Diversity  
HTS of the 185 rDNA gene was used to evaluate yeast and fungi microbial diversity during the prefermentary stages. The start of alcoholic fermentation was not considered for HTS due to the inoculation of the yeast with ADY _Saccharomyces cerevisiae_. A total of 18 samples were sequenced, resulting in 190,016 paired-end reads assembled with 5\({}^{\prime}\) primers, 3\({}^{\prime}\)primers, with expected length (between 300 and 400 bps), without ambiguous base calls (N characters) in their sequence or barcode. After filtering of chimeras, singletons, clustering SWARM and affiliation OTUs, 161,667 sequences were assigned to 493 OTUs. After a blast filter step (for identity and coverage), a preprocess step, and a _Vitis_ sp. removal step (\(-\)17.90%), 123 OTUs with 131,864 sequences were obtained. Finally, 7716 sequences were retained on average for each sample, except for one replicate at 48 h of maceration (without SO2 treatment) that was deleted because of its low number of sequences (729). Different phyla were detected among all OTUs, mostly within _Ascomycota_ (95.9%), followed by _Basidiumycta_ (2.3%), _Cryptomycota_ (1.6%), and other phyla, but with an abundance below 1% of all OTUs (data not shown). The _Ascomycota_ phylum contained 11 classes, including 57.2% _Dothidomyces_ (represented by three genera: _Aurebosidium_ (63%), _Cludosporium_ (28%); _Alternaria_ (3.4%)), 25.9% _Saccharomyces_ (represented by the _Metschikowia_ (47.3%); _Torulaspora_ (46.8%) and _Hansenispora_ (3.2%) genera) and 12.8% _Leotionycetes_ (represented predominantly by the _Botrytis_ genus (99.5%)) (Table 13). Among the eight major genera within the _Ascomycota_ phylum (Figure 3A and Table 13), five belonged to models previously reported on the grape berry (_Alternaria_, _Aurebosidium_, _Cladosporium_, _Botrytis_, and _Dipaldia_) (47,48). The _Aurebosidium_, _Cladosporium_, and _Botrytis_ genera were dominant whether the mustus were splitted or not. In accordance with population dynamic results, the _Torulaspora_ and _Metschikowia_ genera were most abundant for the bioprotection treatment. At the vatting stage, _Torulaspora_ represented 25% of the total relative abundance and this percentage remained stable during prefermerary maceration. By contrast, _Metschikowia_ represented approximately 40% of the total relative abundance and then decreased during maceration (30%), according to the Q-PCR results. Bioprotection led to a decrease in the relative abundance of _Aurebosidium_, _Botrytis_, and _Cladosporium_ in comparison with the other treatments. The use of SO2 at vatting did not lead to any significant changes in the relative abundances obtained from the eight major genera in the samples. _Hansenispora_ was poorly represented, ranging from 0.1 to 2.4% for all samples. Among the percentage of \"Others\" in Figure 3A, twelve additional genera were represented, with six fungi genera (_Aspergillus_, _Ramularia_, _Pleospora_, _Colletotrichum_, _Taphrina_, and _Zopfia_) and five yeast genera commonly associated with the grape berry microbial community (_Kluyveromyces_, _Candida_, _Saccharomyces_, _Lachancea_, and _Pichia_ are grape yeasts) (Figure 3B). Surprisingly, the _Starmerella bucilaris_ species was not identified among the sequences. Figure 2: Population dynamics of _Hansenispora_ spp., _Metschikowia pulcherrima_, and _Torulaspora delbruuckii_. During prefermentary stages (vatting, 24 h, and 48 h of maceration, start of alcoholic fermentation). Bioprotection (BP), SO2, and without SO2 treatments. Values indicated as the mean of four technical replicates \(\pm\) standard deviation. Biodiversity indices were calculated based on high-throughput sequencing data (Figure 4). The number of OTUs at the vatting stage was significantly lower for the bioprotection treatment compared to the other treatments (43 \(\pm\) 4 OTUs versus 60 \(\pm\) 3 OTUs).