Yeast Biodiversity in Vineyard during Grape Ripening: Comparison between Culture Dependent and NGS Analysis

In this study, the evolution of the yeast microflora present on the berry surface, during the ripening of Barbera grapes, was monitored. Sampling was performed in three vineyards located in the "Nizza" Barbera d'Asti DOC zone and different methodologies have been employed. A culture-dependent method based on the identification of strains grown on solid media by ARDRA (Amplified Ribosomal DNA Restriction Analysis) and the D1-D2 domain of ribosomal 26S DNA capillary sequencing was coupled to NGS (Next Generation Sequencing) targeting ITS (Internal Transcribed Sequence) amplicons with the Illumina MiSeq platform. By using culture-dependent techniques, the most frequently detected species was the yeast-like fungus Aureobasidium pullulans, which was dominant in the culturable fraction. Among yeasts, the presence of oligotrophic basidiomycetes such as Cryptococcus spp., Rhodotorula graminis and Sporidiobolus pararoseus was observed at the beginning of ripening. Afterward, upon approaching the harvest, a succession of oxidative or weakly fermentative copiotrophic species occurs, such as Saturnispora diversa, Issatchenkia terricola, Hanseniaspora opuntiae, Starmerella bacillaris and Hanseniaspora uvarum. The massive sequencing revealed a larger number of species, respect to the culture-dependent data. Comparing the two different approaches used in this work, it is possible to highlight some similarities since Aureobasidium, Rhodotorula and Sporobolomyces were detected by both methods. On the contrary, genera Hanseniaspora, Issatchenkia and Saturnispora were revealed by culture-dependent methods, but not by NGS, while Saccharomyces spp. were identified, with low frequency, only by NGS. The integrated application of NGS sequencing and culture-dependent techniques provides a comprehensive view of mycodiversity in the wine-growing environment, especially for yeasts with low abundance.