Pat Okubara,* Xuefei Wang, Dan Schlatter, Charles Edwards, Timothy Paulitz, and Dean Glawe  
*USDA-ARS & Washington State University, 367A Johnson Hall, Washington State University, Pullman, WA 99164-6430 (patricia.okubara@ars.usda.gov)

Production and consumption of native wines, made without introduced microbes or amendments, has increased recently in the United States, including the Pacific Northwest. Aroma, flavor, and textural characteristics of native fermentations are driven by native yeasts associated with berries and winery environments. Native yeasts contribute to these characteristics throughout vinification, even in the presence of commercial Saccharomyces cerevisiae. To address a knowledge gap about populations of native yeasts in Washington State vineyards, we examined diversity on Cabernet Sauvignon berries from two vineyards located in different American Viticultural Areas in the 2015 harvest season, and tracked their populations in small-scale fermentations. We hypothesized that the berries from each vineyard would harbor yeast species reported in studies from other laboratories, but the species would vary between vineyards and some would be unique. Diversity 
was assessed using the ITS1 of the fungal internal transcribed spacer DNA and the D2 region of eukaryotic 26S rDNA. The ITS1 dataset yielded 1,467 fungal sequence similarity groups, called operational taxonomic groups (OTUs), 111 of which were annotated as yeast or yeast-like genera or species. The D2 sequence set yielded 286 fungal OTUs, 110 of which were yeast/yeast-like genera or species. The D2 analysis also revealed the extent to which grape berries harbored pollen, algae, mushroom spores, and other airborne microbiota. Of the ITS-based OTUs, S. cerevisiae, Metschnikowia chrysoperlae, Hanseniaspora uvarum, and Aureobasidium pullulans were most abundant in both vineyards, supporting our hypothesis of shared species. Five minor yeast species appeared to be unique to our sampled vineyards. Metschnikowia spp., Hanseniaspora spp., Meyerozyma guilliermondii, Wickerhamomyces anomalus, and A. pullulans persisted through early- or mid-stage fermentation. Amendment with 80 ppm SO2 affected populations of specific yeasts and non-yeast fungi, but had no global effects. Our findings provide a basis for subsequent yeast diversity and wine quality studies.

Funding Support: Washington State Grape and Wine Research Program, China Scholarship Council, and USDA-ARS CRIS Project 2090 22000 016 00D