ITS1 Vs. D2 for Assessing Yeast Diversity in Grape Berries and Fermentations in Washington State

Production and consumption of natural or native wines, made without introduced yeasts or amendments, have increased in the Pacific Northwest, USA in recent years. Aroma and flavor characteristics of natural wines arise from wild yeasts associated with berries during fermentation. To address a knowledge gap about the kinds and populations of wild yeasts on grape berries in the state of Washington (WA), we examined diversity on Cabernet Sauvignon berries in two WA American Viticultural Areas in the 2015 harvest season, and tracked their populations in small-scale fermentations. Half of the fermentations were amended with 50 ppm sulfur dioxide. 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 1,469 fungal OTUs, 102 of which were annotated as yeast genera or species. The D2 sequences yielded 286 fungal OTUs, 96 of which were yeast genera or species. The D2 analysis also revealed the extent to which grape berries harbored pollen, algae, mushroom spores and other airborne microbiota. Of 102 ITS-based OTUs, Aureobasidium pullalans was the most abundant in vineyard samples and persisted in early and mid-fermentation. Hanseniaspora spp., Metchnikowia spp., Meyerozyma guilliermondii, Wickerhamomyces anomalus and Saccharomyces cerevisiae also persisted. In contrast, Cryptococcus spp. did not persist during fermentation. Sulfur dioxide did not consistently affect yeast populations during fermentation. Additional comparisons of ITS1- and D2-based diversity will be reported.