Gray mold is one of the most important diseases of grapevine in temperate climates. This plant pathogen affects plant growth and reduces wine quality. The use of yeasts as biocontrol agents to apply in the vineyard have been investigated in recent years as an alternative to agrochemicals. In this work, fermenting musts obtained from overripe grape berries, therefore more susceptible to infection by fungal pathogens such as Botrytis cinerea, were considered for the selection of yeasts carrying antifungal activity. Thirty-six isolates were identified as Starmerella bacillaris, a species recently proven to be of enological interest. Among them 14 different strains were studied and antifungal activity against B. cinerea was demonstrated, for the first time, to be present in S. bacillaris species. The production of volatile organic compounds (VOCs), tested in vitro, was found to be the main responsible of S. bacillaris antifungal effects. All the strains were able to reduce B. cinerea decay on wounded grape berries artificially inoculated with gray mold. The colonization level of wound was very high reaching, after five days, a concentration of 106 cells per ml of grape juice obtained after berry crushing. At this cell concentration S. bacillaris strains were used to ferment synthetic and natural musts. The sequential yeast inoculation, performed by adding S. cerevisiae 48 h after S. bacillaris, was needed to complete sugar consumption and determined a significant increase in glycerol content and a reduction of ethanol and acetic acid concentrations. The high wound colonization ability, found in this work, together with the propensity to colonize grape berry and the interesting enological traits possessed by the selected S. bacillaris strains allow the use of this yeast as biocontrol agent on vine and grape berries with possible positive effects on must fermentation, although the presence of S. cerevisiae is needed to complete the fermentation process. This work introduces new possibilities in wine yeast selection programs in order to identify innovative wine yeasts that are simultaneously antifungal agents in vineyards and alternative wine starters for grape must fermentation and open new perspective to a more integrated strategy for increasing wine quality.

Summary: Environmental S accharomyces cerevisiae strains are crucially important, as they represent the large pool from which domesticated industrial yeasts have been selected, and vineyard strains can be considered the genetic reservoir from which industrial wine strains with strong fermentative behaviour are selected. Four vineyard strains with different fermentation performances were chosen from a large collection of strains isolated from Italian vineyards. Their genomes were sequenced to identify how genetic variations influence gene expression during fermentation and to clarify the evolutionary relationship between vineyard isolates and industrial wine strains. RNA sequencing was performed on the four vineyard strains, as well as on the industrial wine yeast strain EC1118 and on the laboratory strain S288c , at two stages of fermentation. We showed that there was a large gene cluster with variable promoter regions modifying gene expression in the strains. Our results indicate that it is the evolvability of the yeast promoter regions, rather than structural variations or strain‐specific genes, that is the main cause of the differences in gene expression. This promoter variability, determined by variable tandem repeats and a high number of single‐nucleotide polymorphisms together with 49 differentially expressed transcription factors, explained the strong phenotypic differences in the strains.

The reduction of chemical fungicides in agriculture has led to the use of microorganisms as biocontrol agents. Starmerella bacillaris is a non-Saccharomyces yeast associated with overripe and botrytized grape berries microbiota. Its use has been proposed for wine fermentation because of yeast fructophilic character and high glycerol production. Recently, S. bacillaris has been demonstrated to possess antifungal activity against Botrytis cinerea on the grape. Penicillium expansum is the pathogen responsible for the blue mold rot, the most important postharvest disease of apples. These fruits are the raw material of the cider, an alcoholic beverage commonly produced using S. cerevisiae starter cultures. In this study 14 S. bacillaris strains have been studied to evaluate their postharvest antifungal activity against P. expansum on apples. Moreover, the fermentation performances in apple juice of these non-Saccharomyces strains were tested, both in single-strain fermentation and in sequential fermentation, together with S. cerevisiae. Four S. bacillaris strains, able to significantly decrease blue mold rot symptoms and to increase glycerol content during fermentation have been selected to improve apple and cider quality.