## 3 Results  
Spontaneous Fermentation of Chambourcin Grape Must Provides Insights into Fungal Diversity of Red Hybrid Grapes  
One hundred and twenty isolates from Chambourcin grape must were obtained from three vineyards during the 2019 vintage. Phylogenetic analysis of the fungal ITS region clustered these isolates into four main clades (Figure 1). Forty isolates were identified from each location by colony morphology and Sanger sequencing (see Section 2). _Hanseniispora_ spp. and _Pichia_ spp. were the most abundant populations followed by _Sporididobus pararoseus_, _Sarmerella bacillaris_, and _Aureobasidium pullulans_ (Figure 2A). Observations from all three regional wineries suggests that _H. uvarum_, _H. meyeri_, and _H. oportuniae_ were the most abundant (41.5%) in grape must. _Pichia fermentatus_, _P. kluyveri_, _P. kudriavzevii_, and _P. terricola_ were the second most abundant species (10.8%), as well as _S. pararoseus_. _S. bacillaris_ (9%), _Aureobasidium pullulans_, and _Fibosidium floridrome_ were also identified with equal relative abundance (5%). In PAV1, fungal composition was most diverse (17 fungal species identified) highlighted by diversity of _Hanseniispora_ spp. and _Pichia_ spp. as well as the presence of filamentous fungi such as _Caldosporium angustisporum_, _Epicoccum sorghumum_, _Ncopestalotiopsis clavispora_, and _Pestalotiopsis visinae_. In PAV2, 11 fungal species were identified with _H. uvarum_, _S. pararoseus_, and _A. pullulans_ being the most dominant species. Finally, 12 fungal species were identified in PAV3 where _H. uvarum_ accounted for 50% of isolated strains and _Pichia_ spp., _S. bacillaris_ and _Candida_ spp. accounted for the remaining fungal community.  
Other yeast species isolated and identified from spontaneous fermentation include _Candida_ spp., _Meyerozyma caropphilis_, _Kregervanrija_ sp., and _Papiliotrema_ sp. _Candida_ species included _C. californica_ and _C. railenensis_. _Candida californica_ was identified only from PAV3. One strain of _Kregervanrija_ sp. and _Papiliotrema_ sp. was found in PAV1 and PAV2, respectively. Using this culture-based approach, filamentous fungi were less dominant and less consistent across vineyards compared to the abundance of yeast species present on grape berries. _Leptosphaerulina chartarum_ was the only species found across vineyards at PAV1 and PAV2. Other examples of filamentous fungi identified within individual vineyards include _Alternaria alternata_, _Caldosporium_ spp., _Epicoccum sorghum_, _Fusarium_ sp., _Mucor nidicola_, _Ncopestalotiopsis clavispora_, _Penicillium breciompactum_, _P. spinulosum_, and _Pestalotiopsis visinae_ (Table S1). After 24 h of spontaneous fermentation, decreased fungal diversity in Chambourcin must was observed and filamentous fungi were not identified in Chambourcin grape must. _Hanseniispora_ spp. and _Pichia_ spp. were the most dominant species in PAV1, and _Hanseniispora_ spp. and _S. bacillaris_ were dominant in PAV2 and PAV3 after 24 h of spontaneous fermentation (Figure 2B).  
Figure 1: Phylogenetic diversity of fungal strains associated with Chamboucrin hybrid grape must during spontaneous fermentation based on ribosomal internal transcribed spacer (ITS) regions and 5.8S rRNA gene sequences. Branches corresponding to partitions reproduced from less than 50% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. _Hanseniaspora_ clade was suppressed into a black empty triangle including all _Hanseniaspora_ species clustered with 96% bootstrap support. Subtrees including same species supported by 100% bootstrap value were suppressed and represented as solid black triangles, including _Starmerella bicillaris_, _Candida californica_, _Leptosphaerulina chartarum_, _Aurebosidium pullulans_, _Fibosidium floriforme_, and _Sporidiobolus pararoseus_ strains.