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Starmerella bacillaris is a non-Saccharomyces yeast recently proposed for grape fermentation in association with Saccharomyces cerevisiae. Due to its high glycerol and moderate volatile acidity production this yeast can contribute to improving wine quality. Some strains have been demonstrated to exhibit antifungal activity against grey mould on grape, which is caused by Botrytis cinerea. The simultaneous presence of these traits in S. bacillaris is of great interest. Indeed, this yeast can be potentially used as a biocontrol agent in vineyards. Research on the ability of S. bacillaris to survive or, even to grow on the surface of grapes is a starting point in the evaluatation of its potential use in vineyards. The preliminary results of our study showed that when applied to the grape surface under laboratory condictions, inoculum sized S. bacillaris with antifungal activity developed and lasted for at least 6 days in high concentrations. In addition, it positively influenced the fermentation process by producing high concentrations of glycerol (average value 4.89 ± 0.47 g/L). Interestingly, a positive effect on wine quality was also observed when the inoculum size was 10 times higher or lower than the reference concentration. When sprayed on the vines in the vineyard and present on the grape skin surface after harvest, S. bacillaris cells can start alcoholic fermentation.

In Saccharomyces cerevisiae, the fermentation rate and the ability to complete the sugar transformation process depend on the glucose and fructose transporter set-up. Hexose transport mainly occurs via facilitated diffusion carriers and these are encoded by the HXT gene family and GAL2. In addition, FSY1, coding a fructose/H+ symporter, was identified in some wine strains. This little-known transporter could be relevant in the last part of the fermentation process when fructose is the most abundant sugar. In this work, we investigated the gene expression of the hexose transporters during late fermentation phase, by means of qPCR. Four S. cerevisiae strains (P301.9, R31.3, R008, isolated from vineyard, and the commercial EC1118) were considered and the transporter gene expression levels were determined to evaluate how the strain gene expression pattern modulated the late fermentation process. The very low global gene expression and the poor fermentation performance of R008 suggested that the overall expression level is a determinant to obtain the total sugar consumption. Each strain showed a specific gene expression profile that was strongly variable. This led to rethinking the importance of the HXT3 gene that was previously considered to play a major role in sugar transport. In vineyard strains, other transporter genes, such as HXT6/7, HXT8, and FSY1, showed higher expression levels, and the resulting gene expression patterns properly supported the late fermentation process.

Glutathione (GSH) is an antioxidant molecule of great technological interest due to its wide range of applications in the food and beverage industry. In winemaking, although glutathione is produced during fermentation, its addition is possible for the control of oxidative spoilage of wine. Recently, to improve wine quality, mixed fermentation has been proposed by introducing a selection of non-Saccharomyces yeasts as complementary starters to the oenological species S. cerevisiae. Among them, Starmerella bacillaris, an osmophilic and high glycerol producer yeast, has been extensively studied. In the present study, the genomes of two S. bacillaris strains were compared with S. cerevisiae to identify the GSH metabolic pathway. The results showed that GSH biosynthesis includes the GSH1 and GSH2 genes in both species. The identification of a new transcription factor which binds sites in the promoter region of these genes underlined differences in the transcriptional regulation of both species. Additionally, between S. bacillaris strains, a high number of polymorphisms was found in genes involved in GSH redox balance. Preliminary laboratory scale fermentations revealed marked differences in the cell glutathione content of the two S. bacillaris strains. By comparing genomes, it was possible to gain a better understanding of the genes involved in the GSH metabolism pathway in S. bacillaris.

In wine production, sulfites are widely used as antimicrobials and antioxidants, whereas copper is associated with fungicides and wine fining treatments. Therefore, wine yeasts are constantly exposed to these agents. Copper tolerance is related to the copy number of the CUP1 gene, encoding for a metallothionein involved in copper detoxification. In wine yeasts, sulfite resistance mainly depends on the presence of the translocation t(XVI;VIII) in the promoter region of the SSU1 gene. This gene encodes for a plasma membrane sulfite pump involved in sulfite metabolism and detoxification. Recently, a new translocation, t(XVI;VIII), was identified. In this work, 253 Saccharomyces cerevisiae strains, representing three vineyard populations from two different continents, were analyzed, along with 20 industrial starters. Copper and sulfites tolerance as well as distribution of CUP1 gene copy-number, t(XVI;VIII)and t(XVI;XV) of SSU1 gene were studied to evaluate the impact of these genomic variations on population phenotypes. The CUP1 gene copy-number was found to be highly variable, ranging from zero to 79 per strain. Moreover it differently impacted the copper tolerance in the populations of the two continents. The diffusion of t(XVI;VIII) and, for the first time, t(XVI;XV) was determined in the three vineyard populations. The correlation between the presence of the translocation and strain sulfite tolerance levels was significant only for the t(XVI;VIII).

Aim: In this work, seven strains of Starmerella bacillaris were analysed for their ability to release polysaccharides during alcoholic fermentation (AF), both in single-strain and in sequential AF together with Saccharomyces cerevisiae.Methods and Results: A synthetic polysaccharide-free must was used to characterise the mannoproteins (MPs) released. The MPs were quantified, characterised in terms of carbohydrate composition, and tested to assess their ability to reduce protein and tartrate instabilities and their ability to affect the foaming properties of wine.Conclusions: All the tested strains in sequential AF increased the total MPs production. Moreover, the strains affected the MPs properties in different ways regarding tartaric and protein stabilities. The MPs released in sequential AF by some S. bacillaris strains showed a significant effect on protein stabilisation and tartaric stability. An effect on the foamability was found for MPs obtained in single-strain AFs of S. bacillaris.Significance and impact of the study: An improvement in wine stability can be achieved using the sequential AF.

During white-grape winemaking, grape marc is separated from juice immediately after crushing. Both mark and juice are obtained from the same grapes, but they differ strongly for their physical and chemical properties. Marc is mainly composed of solid residues. Its pH is usually higher than that of the juice and Saccharomyces cerevisiaestrains are largely present. Therefore, it can be considered as a potential alternative environment for the selection of industrial yeasts. In order to evaluate the effect of different pH and physical state of the two matrices on grapes yeast population composition, the isolation of S. cerevisiae, from both grape juice and marc during simultaneous fermentations, was performed. After yeast identification and genotyping, strains present at high frequencies were tested in fermentation at different pH values. Biofilm production was also tested to evaluate strain ability to develop on a solid matrix. Genotype analysis showed that high-frequency strains were always more abundant in one of the two environments, suggesting the existence of a selective effect. Generally, fermentations at different pH revealed that the best fermentation performance of each strain, in terms of CO2production, was in the pH range of its original environment. Only one strain, mostly present in grape marc, produced a high biofilm level. Therefore, biofilm production does not seem to favor strain adaptation to grape marc condition. These results demonstrate that grape juice and marc represent two different environments able to influence yeast strains distribution. The pH level can be included among the selection factors acting on yeast strains distribution. Grape marc can be considered a yeasts reservoir and its fermentation can be used for the development and isolation of new strains, genetically and physiologically different from those present in the grape juice.

In this study, two vineyards of different age were chosen. During three years, a sampling campaign was performed for isolating vineyard-associated Saccharomyces cerevisiae (S. cerevisiae) strains. Bark portions and, when present, grape bunches were regularly collected from the same vine plants during the overall sampling period. Each bark portion was added to a synthetic must, while each grape bunch was manually crushed, and fermentations were run to isolate S. cerevisiae strains. All collected yeasts were identified at different species and strain levels to evaluate the genetic variability of S. cerevisiae strains in the two vineyards and strains dynamics. Moreover, bark-associated strains were compared with those isolated from spontaneous fermentations of grapes collected during the two harvests. Regarding the youngest vineyard, no S. cerevisiae was identified on bark and grape surface, highlighting the importance of vine age on yeast colonization. Results reported the isolation of S. cerevisiae from vine bark of the old vineyard at all sampling times, regardless of the presence of the grape bunch. Therefore, this environment can be considered an alternative ecological niche that permanently hosts S. cerevisiae. Bark-associated strains were not found on grape bunches and during pilot-scale vinifications, indicating no significative strain transfer from vine bark to the grape must. Commercial starters were identified as well both in vineyards and during vinifications.

Among the viticultural techniques developed to obtain wine with reduced alcohol content, the use of unripe grapes with low sugar and high malic acid concentration, harvested at cluster thinning, was recently explored. So far, no studies have evaluated the fermentation performances of Saccharomyces in unripe grape musts, in terms of fermentation ability and reducing malic acid contents, to improve the quality of this low-alcohol beverage. In this work, we evaluated 24 S. cerevisiae strains isolated from Italian and Croatian vineyards with different fermentation aptitudes. Moreover, four S. paradoxus were considered, as previous works demonstrated that strains belonging to this species were able to degrade high malic acid amounts in standard musts. The industrial strain S. cerevisiae 71B was added as reference. Sugar and malic acid contents were modified in synthetic musts in order to understand the effect of their concentrations on alcoholic fermentation and malic acid degradation. S. cerevisiae fermentation performances improved when glucose concentration decreased and malic acid level increased. The conditions that simulate unripe grape must, i.e. low glucose and high malic acid content were found to enhance S. cerevisiae ability to degrade malic acid. On the contrary, S. paradoxus strains were able to degrade high amounts of malic acid only in conditions that resemble ripe grape must, i.e. high glucose and low malic acid concentration. In fermentation trials when low glucose concentrations were used, at high malic acid levels S. cerevisiae strains produced higher glycerol than at low malic acid condition. Malic acid degradation ability, tested on the best performing S. cerevisiae strains, was enhanced in fermentation trials when unripe grape must was used.

Starmerella bacillaris is an osmotolerant yeast with interesting winemaking traits such as low-ethanol and high-glycerol production, previously considered as wine spoilage and recently proposed to improve the sensory quality of wine. This is the first work performing a whole-genome analysis of the variants identified by comparing two S. bacillaris strains (PAS13 and FRI751). Additionally, an extensive search for orthologous genes against Saccharomyces and non-Saccharomyces yeasts produced a detailed reconstruction of the pan-genome for yeast species used in winemaking. Starmerella bacillaris PAS13 was able to produce 36% more glycerol than S. bacillaris FRI751 without increasing ethanol level over 5% (v/v). Orthologous genes revealed new insights in the response to osmotic stress determined by the mitogen-activated protein kinase (MAPK) from S. bacillaris strains. The comparison between the two S. bacillaris genomes revealed 33 771 high-quality variants that were ranked considering their predicted impact on gene functions. Furthermore, analysis of structural variations in the genome revealed five translocations. The absence of some transcriptional factors involved in the regulation of GPD (glycerol-3-phosphate dehydrogenase), like the protein kinases YpK1p and YpK2p, and the identification of a tandem duplication increasing the GPP1 (glycerol-3-phosphate phosphatase) gene copy number suggest a remarkably different regulation of the glycerol pathway for S. bacillaris in comparison to S. cerevisiae.

AIMS Selection projects aiming at the identification of new Saccharomyces strains are always on going as the use of the suitable yeast can strongly improve fermented food production, particularly winemaking. They are mainly targeted on Saccharomyces cerevisiae, but other species in the Saccharomyces genus are of interest. For this reason, more and more efficient molecular techniques for yeast identification able to accelerate yeast selection process are always needed. Among the Saccharomyces genus, four yeasts are widespread in natural environments: S. cerevisiae; S. uvarum; S. kudriavzevii and S. paradoxus. Therefore, among the Saccharomyces species, their discrimination is of great interest. METHODS AND RESULTS A two-step protocol is proposed. Firstly the Saccharomyces genus identification is achieved by multiplex PCR analysis. Then, the Saccharomyces species is determined by a new method based on high-resolution melting analysis (HRMA). CONCLUSIONS For HRMA two primer pairs have been proposed. The first was able to achieve the simultaneous identification of the four widespread Saccharomyces species, the second was used for the unambiguous discrimination of S. cerevisiae within its taxonomical genus. SIGNIFICANCE AND IMPACT OF THE STUDY This assay allowed an easy, rapid and simultaneous discrimination of S. cerevisiae, S. uvarum and S. paradoxus during yeast selection programs.

Vale dos Vinhedos appellation of origin has a very recent history as industrial wine making region. In this study we investigated the genetic and phenotypic variability of Saccharomyces cerevisiae strains isolated from South-Brazilian vineyards in order to evaluate strain fermentation aptitude and copper and sulphites tolerance. Merlot grape bunches were collected from three vineyards and yeast isolation was performed after single bunch fermentation. High genotypic variability was found and most of the genotypes revealed to be vine-specific. No industrial strain dissemination was present in the sampled vineyards, although it has been wildly reported in traditional winemaking countries. From the phenotypic traits analysis these Brazilian native strains showed good fermentation performances, good tolerance to sulphites and, in particular, a high copper tolerance level. Copper is the most important metal in the formulation of fungicides against downy mildew (Plasmopara viticola), one of the most harmful disease of the vines, and other fungal pests. The high tolerance to copper suggests an environmental adaptation to the strong use of copper-based fungicides, requested by the wet subtropical climate.

From a technological point of view, yeast resistance to sulfite is of great interest and represents an important technological character for winemaking. Several mechanisms are involved, and strain-dependent strategies to obtain SO2 resistance can deeply influence wine quality, although this choice is less relevant in determining the technological performance of the strain during fermentation. In this study, to better understand the strain-specific mechanisms of resistance, 11 Saccharomyces cerevisiae strains, whose genomes have been previously sequenced, were selected. Their attitude towards sulfites, in terms of resistance and production, was evaluated, and RNA-sequencing of four selected strains was performed during fermentation process in synthetic grape must in the presence of SO2. Results demonstrated that at molecular level, the physical effect of SO2 triggered multiple stress responses in the cell and high tolerance to general enological stressing condition increased SO2 resistance. Adaptation mechanism due to high basal gene expression level rather than specific gene induction in the presence of sulfite seemed to be responsible in modulating strain resistance. This mechanism involved higher basal gene expression level of specific cell wall proteins, enzymes for lipid biosynthesis, and enzymes directly involved in SO2 assimilation pathway and efflux.

Starmerella bacillaris is a fermentative yeast commonly found in vineyards. Here, we present the draft genome sequence of S. bacillaris PAS13, a nonconventional enological yeast with a potential role as a biocontrol agent. This gene sequence will provide insights into the genetic basis of yeast activity against gray mold disease ( Botrytis cinerea ).

In recent years the interest for natural fermentations has been re-evaluated in terms of increasing the wine terroir and managing more sustainable winemaking practices. Therefore the level of yeast genetic variability and the abundance of Saccharomyces cerevisiae native populations in vineyard are becoming more and more crucial at both ecological and technological level. Among the factors that can influence the strain diversity, the commercial starter release that accidentally occur in the environment around the winery, has to be considered. In this study we led a wide scale investigation of S. cerevisiae genetic diversity and population structure in the vineyards of three neighboring winemaking regions of Protected Appellation of Origin, in North-East of Italy. Combining mtDNA RFLP and microsatellite markers analyses we evaluated 634 grape samples collected over three years. We could detect major differences in the presence of S. cerevisiae yeasts, according to the winemaking region. The population structures revealed specificities of yeast microbiota at vineyard scale, with a relative Appellation of Origin area homogeneity, and transition zones suggesting a geographic differentiation. Surprisingly, we found a widespread industrial yeast dissemination that was very high in the areas where the native yeast abundance was low. Although geographical distance is a key element involved in strain distribution, the high presence of industrial strains in vineyard reduced the differences between populations. This finding indicates that industrial yeast diffusion it is a real emergency and their presence strongly interferes with the natural yeast microbiota.

Appl. Microbiol. Biotechnol.ISI Document Delivery No.: AF0VXTimes Cited: 0Cited Reference Count: 53Treu, Laura Campanaro, Stefano Nadai, Chiara Toniolo, Chiara Nardi, Tiziana Giacomini, Alessio Valle, Giorgio Blondin, Bruno Corich, VivianaUniversity of Padua [CPDA082991]; Provincia di TrevisoRNA-seq was performed at the CRIBI Biotechnology Centre (University of Padua) and we acknowledge the support of Michela D'Angelo in processing our samples. Fermentation curves and enological analysis were performed at the CIRVE laboratories of Conegliano (Treviso, Italy) and we acknowledge the support of Barbara Bovo and Milena Carlot. This work was supported by the University of Padua [CPDA082991] and by the Provincia di Treviso providing PhD fellowship. We express thanks to Georgine Faulkner of the Department of Biology (University of Padua) for her assistance in the manuscript revision.SpringerNew york; We used RNA-sequencing (RNA-seq) to analyze the expression profile of four vineyard strains of Saccharomyces cerevisiae having different fermentation performances. The expression profiles obtained in two steps of the fermentation process were compared with those obtained for the industrial wine strain EC1118 and for the laboratory strain S288c. The two strains with low fermentation efficiency, namely, S288c and the vineyard strain R103, exhibited markedly different expression profiles when compared to the other four strains. We also found that the vineyard strains P283 and P301 are characterized by a high expression of the transcription factor Met32p in the first step of the fermentation. Met32p, in coordination with the Hap4p transcription factor, determined the over-expression of the genes involved in the respiration processes, in the response to oxidative stress and in the sulfur amino acids biosynthesis. These combined actions are likely to increase the level of antioxidants whose protective effect could contribute to improve the fermentation process. Gene expression and phenotypic data revealed that the vineyard strain P301 has low nitrogen utilization in comparison to the other wine strains, combined with high fermentation efficiency. Analysis of the genes involved in fermentation stress response revealed a lower expression in strains characterized by low fermentation efficiency, particularly in the first fermentation phase. These findings evidenced the high variability of transcriptional profiles among different wine yeast strains and clarify their connection with complex phenotypic traits, such as the fermentation efficiency and the nitrogen sources utilization.

Summary Environmental Saccharomyces 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.

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.

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.

Sulfur dioxide is extensively used during industrial fermentations and contributes to determine the harsh conditions of winemaking together with low pH, high sugar content and increasing ethanol concentration. Therefore the presence of sulfite has to be considered in yeast gene expression studies to properly understand yeast behavior in technological environments such as winemaking. A reliable expression pattern can be obtained only using an appropriate reference gene set that is constitutively expressed regardless of perturbations linked to the experimental conditions. In this work we tested 15 candidate reference genes suitable for analysis of gene expression during must fermentation in the presence of sulfite. New reference genes were selected from a genome-wide expression experiment, obtained by RNA sequencing of four Saccharomyces cerevisiae wine strains grown in enological conditions. Their performance was compared to that of the most common genes used in previous studies. The most popular software based on different statistical approaches (geNorm, NormFinder and BestKeeper) were chosen to evaluate expression stability of the candidate reference genes. Validation was obtained using other wine strains by comparing normalized gene expression data with transcriptome quantification both in the presence and absence of sulfite. Among 15 reference genes tested ALG9, FBA1, UBC6 and PFK1 appeared to be the most reliable while ENO1, PMA1, DED1 and FAS2 were the worst. The most popular reference gene ACT1, widely used for S. cerevisiae gene expression studies, showed a stability level markedly lower than those of our selected reference genes. Finally, as the expression of the new reference gene set remained constant over the entire fermentation process, irrespective of the perturbation due to sulfite addition, our results can be considered also when no sulfite is added to the must.