Yeast community structures and dynamics in healthy and Botrytis-affected grape must fermentations

Indigenous yeast population dynamics during the fermentation of healthy and Botrytis-affected grape juice samples from two regions in Greece, Attica and Arcadia, were surveyed. Species diversity was evaluated by using restriction fragment length polymorphism and sequence analyses of the 5.8S internal transcribed spacer and the D1/D2 ribosomal DNA (rDNA) regions of cultivable yeasts. Community-level profiles were also obtained by direct analysis of fermenting samples through denaturing gradient gel electrophoresis of 26S rDNA amplicons. Both approaches revealed structural divergences in yeast communities between samples of different sanitary states or geographical origins. In all cases, Botrytis infection severely perturbed the bioprocess of fermentation by dramatically altering species heterogeneity and succession during the time course. At the beginning and middle of fermentations, Botrytis-affected samples possessed higher levels of biodiversity than their healthy counterparts, being enriched with fermentative and/or spoilage species, such as Zygosaccharomyces bailii and Issatchenkia spp. or Kluyveromyces dobzhanskii and Kazachstania sp. populations that have not been reported before for wine fermentations. Importantly, Botrytis-affected samples exposed discrete final species dominance. Selection was not species specific, and two different populations, i.e., Saccharomyces cerevisiae in samples from Arcadia and Z. baild in samples from Attica, could be recovered at the end of Botrytis-affected fermentations. The governing of wine fermentations by Z. baild is reported for the first time and could elucidate the origins and role of this particular spoilage microbe for the wine industry. This is the first survey to compare healthy and Botrytis-affected spontaneous fermentations by using both culture-based and -independent molecular methods in an attempt to further illuminate the complex yeast ecology of grape must fermentations.