Recent advances in biocontrol and other alternative strategies for the management of postharvest decay in table grapes.

During postharvest, table grapes are often spoiled by molds. Aspergillus sp., Alternaria sp., Botrytis sp., Cladosporium sp. and Penicillium sp. are different mold genera frequently related to table grape rot. Fungal spoilage affects nutritional value and organoleptic properties while also producing health hazards, such as mycotoxins. Traditionally, synthetic fungicides have been employed to control fungal diseases. However, possible negative effects on health and the environment are a serious concern for consumers and government entities. This review summarized data on innovative strategies proposed to diminish postharvest losses and extend table grape shelf life. Among physical, chemical, and biological strategies, either alone or in combination, the integrated management of fungal diseases is a sustainable alternative to synthetic fungicides. However, to date, only a few alternative technologies have succeeded on a commercial scale. Recent research aimed at increasing the competitiveness of alternative technologies has led to the development of integrated management strategies to prevent postharvest decay and increase the safety and quality of table grapes.

Biocontrol of Alternaria alternata in cold-stored table grapes using psychrotrophic yeasts and bioactive compounds of natural sources.

Alternaria alternata is a common fungal pathogen causing postharvest decay in table grapes. This study addressed the potential of autochthonous yeasts and bioactive compounds of natural sources to act as biocontrol agents (BCAs) against A. alternata in cold-stored table grapes. With this purpose, 19 yeast capable of growing at 0-1 °C were isolated from the surface of Red Globe table grapes. These isolates, along with the pre-isolated strain Metschnikowia pulcherrima RCM2, were evaluated as BCAs in wounded berries. From these results, six yeast isolates were pre-selected to be combined with bioactive compounds of natural sources, like phenolic compounds (PCs) of side streams of wine industry, including bunch stem extract (BSE) (5-25 %), and cane extract (CE) (5-25 %), and functional polysaccharides from shrimp waste such as chitosan (CH) (0.5 %). Then, the biocontrol efficacy of combined treatments beyond individual ones was compared. The results revealed that 4 yeast isolates, namely M. pulcherrima RCM2 and ULA146, and Aureobasidium pullulans FUL14 and FUL18, were the most effective. However, when combined with the natural bioactive compounds, their efficacy against A. alternata did not increase significantly. Notably, ULA146 and FUL18 demonstrated a biocontrol efficacy of 36-37 %, comparable to that of the treatment with commercial doses of SO2, which only showed a 27 % reduction in the lesion diameter. These findings highlight the potential of using psychrotrophic yeasts as BCAs against A. alternata in cold-stored table grapes. Combining these yeast strains with BSE, CE and CH did not increase BCAs efficacy against this pathogen at the concentrations tested. The development of effective biocontrol strategies for A. alternata could contribute to reducing reliance on chemically synthesized fungicides, promoting sustainable practices, aiming to improve the quality and safety of cold-stored table grapes.

Evaluation of different nitrogen sources on growth and fermentation performance for enhancing ethanol production by wine yeasts.

The utilization of grape juice from low oenological value grape varieties for bioethanol production represent an alternative for diversification and value addition in viticulture. Optimizing Very High Gravity (VHG) fermentation can significantly increase ethanol productivity while reducing water and energy consumption. In this study, the impact of different nitrogen sources on growth and fermentative performance of locally selected yeast strains was investigated. Five yeast strains of species Saccharomyces cerevisiae and Zygosaccharomyces rouxii were cultured in both synthetic culture media and natural grape juice supplemented with ammonium sulfate (NH), yeast extract (YE), Fermaid K (FERM), and urea (U) at varying concentrations. Due to the very low fermentation rate, the Z. rouxii strain was excluded from the selection. The results obtained in synthetic medium showed that nitrogen sources that promoted growth (NH and YE) had minimal effects on fermentative performance and were highly dependent on the specific yeast strain. However, the combination of urea and ammonium favored the rate of sugar consumption. When validated in natural grape juice, urea combined with ammonium (U + NH 300 + 75 mg/L) improved both growth parameters and ethanol yield. Doubling the concentration (U + NH 600 + 150 mg/L) further enhanced sugar consumption and ethanol production while reducing unwanted by-products. The combined use of urea and ammonium exhibited a synergistic effect, making it a cost-effective nitrogen supplement for VHG fermentations.

Mutagenesis, screening and isolation of Brettanomyces bruxellensis mutants with reduced 4-ethylphenol production.

The use of non-conventional yeast species to obtain interesting flavors and aromas has become a new trend in the fermented beverages industry. Among such species, Brettanomyces bruxellensis (B. bruxellensis) has been reported as capable of producing desirable or at least singular aromas in fermented beverages like beer and wine. However, this yeast can also produce an aromatic defect by producing high concentrations of phenolic compounds like, 4-ethylguaiacol and particularly 4-ethylphenol (4-EP). In the present study, we designed a mutant screening method to isolate B. bruxellensis mutants with reduced 4-EP production. More than 1000 mutants were screened with our olfactory screening method, and after further sensory and chemical analysis we were able to select a B. bruxellensis mutant strain with a significant reduction of 4-EP production (more than threefold) and less phenolic perception. Notably, the selected strain also showed higher diversity and concentration of ethyl esters, the most important group of odor active compounds produced by yeasts. Based on these results, we consider that our selected mutant strain is a good candidate to be tested as a non-conventional yeast starter (pure or in co-inoculation) to obtain wines and beers with novel aromatic properties.

Generation of a Non-Transgenic Genetically Improved Yeast Strain for Wine Production from Nitrogen-Deficient Musts.

The yeast Saccharomyces cerevisiae is the main species responsible for the process that involves the transformation of grape must into wine, with the initial nitrogen in the grape must being vital for it. One of the main problems in the wine industry is the deficiency of nitrogen sources in the grape must, leading to stuck or sluggish fermentations, and generating economic losses. In this scenario, an alternative is the isolation or generation of yeast strains with low nitrogen requirements for fermentation. In the present study, we carry out a genetic improvement program using as a base population a group of 70 strains isolated from winemaking environments mainly in Chile and Argentina (F0), making from it a first and second filial generation (F1 and F2, respectively) based in different families and hybrids. It was found that the trait under study has a high heritability, obtaining in the F2 population strains that consume a minor proportion of the nitrogen sources present in the must. Among these improved strains, strain "686" specially showed a marked drop in the nitrogen consumption, without losing fermentative performance, in synthetic grape must at laboratory level. When using this improved strain to produce wine from a natural grape must (supplemented and non-supplemented with ammonium) at pilot scale under wine cellar conditions, a similar fermentative capacity was obtained between this strain and a widely used commercial strain (EC1118). However, when fermented in a non-supplemented must, improved strain "686" showed the presence of a marked floral aroma absent for EC1118 strain, this difference being probably a direct consequence of its different pattern in amino acid consumption. The combination of the capacity of improved strain "686" to ferment without nitrogen addition and produce floral aromas may be of commercial interest for the wine industry.

Optimization of fermentation-relevant factors: A strategy to reduce ethanol in red wine by sequential culture of native yeasts.

Current consumer preferences are determined by well-structured, full-bodied wines with a rich flavor and with reduced alcohol levels. One of the strategies for obtaining wines with reduced ethanol content is sequential inoculation of non-Saccharomyces and Saccharomyces cerevisiae yeasts. However, different factors affect the production of metabolites like ethanol, glycerol and acetic acid by inoculated yeasts. In order to obtain low alcohol wines without quality loss, the aims of our study were: i) to determine optimum conditions (fermentation temperature and time of permanence and initial inoculum size of the non-Saccharomyces population at the beginning of the process, prior to inoculation with S. cerevisiae); ii) to validate the optimized factors; and iii) to assess sensory quality of the wines obtained after validation. Two combinations of yeasts were used in this study: Hanseniaspora uvarum BHu9/S. cerevisiae BSc114 and Candida membranaefaciens BCm71/S. cerevisiae BSc114. Optimization of three fermentation factors that affect to non-Saccharomyces yeasts prior to S. cerevisiae inoculation was carried out using a Box-Behnken experimental design. Applying the models constructed by Response Surface Methodology, the lowest ethanol production by H. uvarum BHu9/S. cerevisiae BSc114 co-culture was obtained when H. uvarum BHu9 was inoculated 48 h 37 min prior to S. cerevisiae inoculation, at a fermentation temperature of 25 °C and at an initial inoculum size of 5 × 106 cells/mL. Lowest alcohol production with C. membranaefaciens BCm71/S. cerevisiae BSc114 was observed when C. membranaefaciens BCm71 was inoculated 24 h 15 min prior to S. cerevisiae at a fermentation temperature of 24.94 °C and at an initial inoculum size of 2.72 × 106 cells/mL. The optimized conditions of the two co-cultures were subsequently submitted to lab-scale validation. Both proposed strategies yielded ethanol levels that were significantly lower than control cultures (S. cerevisiae). Wines fermented with non-Saccharomyces/Saccharomyces co-cultures under optimized conditions were also associated with higher aromatic complexity characterized by the presence of red fruit aromas, whereas wines obtained with S. cerevisiae BSc114 were described by parameters linked with high ethanol levels.

Construction of low-ethanol-wine yeasts through partial deletion of the Saccharomyces cerevisiae PDC2 gene.

We propose an alternative GMO based strategy to obtain Saccharomyces cerevisiae mutant strains with a slight reduction in their ability to produce ethanol, but with a moderate impact on the yeast metabolism. Through homologous recombination, two truncated Pdc2p proteins Pdc2pΔ344 and Pdc2pΔ519 were obtained and transformed into haploid and diploid lab yeast strains. In the pdc2Δ344 mutants the DNA-binding and transactivation site of the protein remain intact, whereas in pdc2Δ519 only the DNA-binding site is conserved. Compared to the control, the diploid BY4743pdc2Δ519 mutant strain reduced up to 7.4% the total ethanol content in lab scale-vinifications. The residual sugar and volatile acidity was not significantly affected by this ethanol reduction. Remarkably, we got a much higher ethanol reduction of 10 and 15% when the pdc2Δ519 mutation was tested in a native and a commercial wine yeast strain against their respective controls. Our results demonstrate that the insertion of the pdc2Δ519 mutation in wine yeast strains can reduce the ethanol concentration up to 1.89% (v/v) without affecting the fermentation performance. In contrast to non-GMO based strategies, our approach permits the insertion of the pdc2Δ519 mutation in any locally selected wine strain, making possible to produce quality wines with regional characteristics and lower alcohol content. Thus, we consider our work a valuable contribution to the problem of high ethanol concentration in wine.

Selection of non-Saccharomyces yeasts to be used in grape musts with high alcoholic potential: a strategy to obtain wines with reduced ethanol content.

Ethanol content of wine has increased over the last decades as consequence of searching phenolic maturity, requiring increased grape maturity. This may result in the production of wines with excessive alcohol levels (sometimes more than 15% (v/v)), sluggish and stuck fermentations and excessive volatile acidity. Many strategies to reduce ethanol in wines are being studied, and microbial methods have some additional advantages. However, because of the broad intra- and interspecies variability, new selection criteria should be included. Therefore, the goal of the present work was to design and evaluate a simple and integral procedure for non-Saccharomyces yeast selection. This strategy allowed selection of yeasts that presented successful implantation in grape must with high alcohol potential and their use in co-cultures could reduce the ethanol in wines. A total of 114 native non-Saccharomyces yeasts were assayed to determine their respiratory, fermentative and physiological characteristics of enological interest. Hanseniaspora uvarum BHu9 and BHu11, H. osmophila BHo51, Starmerella bacillaris BSb55 and Candida membranaefaciens BCm71 were selected as candidates to design co-culture starters.

Culture-dependent and independent techniques to monitor yeast species during cold soak carried out at different temperatures in winemaking.

Transformation of grape must into wine is a process that may vary according to the consumers' requirements. Application of cold soak prior to alcoholic fermentation is a common practice in cellars in order to enhance flavor complexity and extraction of phenolic compounds. However, the effect of this step on wine yeast microbiota is not well-known. The current study simultaneously analyzed the effect of different cold soak temperatures on the microbiological population throughout the process and the use of culture-dependent and independent techniques to study this yeast ecology. The temperatures assayed were those normally applied in wineries: 2.5, 8 and 12°C. PCR-DGGE allowed detection of the most representative species such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae. As could be expected, highest diversity indices were obtained at the beginning of each process, and survival of H. uvarum or S. bacillaris depended on the temperature. Our results are in agreement with those obtained with culture independent methods, but qPCR showed higher precision and a different behavior was observed for each yeast species and at each temperature assayed. Comparison of both culture-independent techniques can provide a general overview of the whole process, although DGGE does not reveal the diversity expected due to the reported problems with the sensitivity of this technique.

Molecular and enological characterization of a natural Saccharomyces uvarum and Saccharomyces cerevisiae hybrid.

Saccharomyces cerevisiae plays a main role in the winemaking process, although other species, like Saccharomyces uvarum or Saccharomyces paradoxus, have been associated with must fermentations. It has been reported in recent years, that yeast hybrids of different Saccharomyces species might be responsible for wine productions. Although S. cerevisiae×Saccharomyces kudriavzevii hybrids have been well studied, very little attention has been paid to S. cerevisiae×S. uvarum hybrids. In this work we characterized the genomic composition of S6U, a widely used commercial S. cerevisiae×S. uvarum yeast hybrid isolated in wine fermentations containing one copy of the genome of each parental species, which suggests a relatively recent hybridization event. We also studied its performance under diverse enological conditions. The results show enhanced performance under low temperature enological conditions, increased glycerol production, lower acetic acid production and increased production of interesting aroma compounds. We also examined the transcriptomic response of the S6U hybrid strain compared with the reference species under enological conditions. The results show that although the hybrid strain transcriptome is more similar to S. uvarum than to S. cerevisiae, it presents specifically regulated genes involved in stress response, lipids and amino acid metabolism. The enological performance and aroma profile of this S. cerevisiae×S. uvarum hybrid makes it a good candidate for participating in winemaking, especially at low temperatures.

Yeast population dynamics during prefermentative cold soak of Cabernet Sauvignon and Malbec wines.

Prefermentative cold soak is a widely used technique in red wine production, but the impact on the development of native yeast species is hardly described. The aim of this work was to analyse the dynamics and diversity of yeast populations during prefermentative cold soak in red wines. Three different temperatures (14 ± 1 °C; 8 ± 1 °C and 2.5 ± 1 °C) were used for prefermentative cold soak in Cabernet Sauvignon and Malbec grape musts. Saccharomyces and non-Saccharomyces populations during cold soak and alcoholic fermentation were analysed. In addition, the impact on chemical and sensory properties of the wines was examined. Yeast dynamics during prefermentative cold soak were temperature dependent. At 14 ± 1 °C, the total yeast population progressively increased throughout the cold soak period. Conversely, at 2.5 ± 1 °C, the yeast populations maintained stable during the same period. Prefermentative cold soak conducted at 14±1°C favoured development of Hanseniospora uvarum and Candida zemplinina, whereas cold soak conducted at 8 ± 1 °C favoured growth of Saccharomyces cerevisiae. At 2.5 ± 1 °C, no changes in yeast species were recorded. Acidity and bitterness, two sensory descriptors, appear to be related to wines produced with prefermentative cold soak carried out at 14 ± 1 °C. This fact could be associated with the increase in non-Saccharomyces during the prefermentation stage. Our results emphasise the importance of the temperature as a determinant factor to allow an increase in non-Saccharomyces population during prefermentative cold soak and consequently to modify sensorial attributes of wines as well as their sensorial impact.

Biodiversity of Aspergillus section Nigri populations in Argentinian vineyards and ochratoxin A contamination.

Aspergillus section Nigri are described as the main source of ochratoxin A (OTA) contamination in grapes and wine worldwide. The knowledge of the factors affecting grape contamination by species included in this section and OTA production is essential to be able to reduce their presence, not only to improve wine quality, but also to maintain their safety. Therefore, the aims of this study were to determine the incidence of Aspergillus section Nigri species harvested in different grape-growing regions from Argentina, their ability to produce OTA, to correlate with meteorological conditions and geographical coordinates with their prevalence and to evaluate the OTA natural occurrence in grapes and wines. The morphological identification showed that Aspergillus niger aggregate species were the most prevalent ones, followed by Aspergillus carbonarius and Aspergillus uniseriate. These populations were confirmed through using AFLP markers and sequencing and, Aspergillus tubingensis was separated from A. niger aggregate. Climatic factors, altitude, longitude and latitude have influenced on the distribution of species included in the section. A. carbonarius and A. niger were OTA producers but differed in their OTA producing ability. Temperature was the factor which influenced the most over the highest incidence of A. carbonarius in La Rioja and San Juan regions. The trellis system in vineyards and drip irrigation also influenced the species isolation. The OTA levels detected in grapes and wines were low, but grape variety was more important in susceptibility to fungal infection and OTA levels.

Evaluation of Saccharomyces cerevisiae strains as probiotic agent with aflatoxin B1 adsorption ability for use in poultry feedstuffs.

In this study the aflatoxin B1 (AFB1) removal capacity, the tolerance to salivary and gastrointestinal conditions, autoaggregation and coaggregation with pathogenic bacteria of Saccharomyces cerevisiae strains isolated from broiler feces, were evaluated. Only four of twelve isolated strains were identified as Saccharomyces cerevisiae using molecular techniques. The results obtained in AFB1 binding studies indicated that the amount of AFB1 removed was both strain and mycotoxin-concentration dependent. Therefore, a theoretical model was applied in order to select the most efficient strain to remove AFB1 in a wide range of mycotoxin concentration. The results indicated that S. cerevisiae 08 and S. cerevisiae 01 strains were the most efficient microorganisms in the mycotoxin removal. Viability on simulated salivary and gastrointestinal conditions was investigated and S. cerevisiae 08 strain showed the best results, achieving 98% of total survival whereas S. cerevisiae 01 reached only 75%. Autoaggregation and coaggregation assays showed S. cerevisiae 08 as the most appropriate strain, mainly because it was the unique strain able to coaggregate with the four bacterial pathogens assayed. Consequently, S. cerevisiae 08 is the best candidate for future in vivo studies useful to prevent aflatoxicosis. Further quantitative in vitro and in vivo studies are required to evaluate the real impact of yeast-binding activity on the bioavailability of AFB1 in poultry. However, this study could be useful in selecting efficient strains in terms of AFB1 binding and provide an important contribution to research into microorganisms with potential probiotic effects on the host.

Genome-wide gene expression of a natural hybrid between Saccharomyces cerevisiae and S. kudriavzevii under enological conditions.

The species Saccharomyces cerevisiae plays a predominant role in the wine making process. However, other species have been associated with must fermentation, such as Saccharomyces uvarum (Saccharomyces bayanus var. uvarum) or Saccharomyces paradoxus. Recently, yeast hybrids of different Saccharomyces species have also been reported as responsible for wine production. Yeast hybrids between the species S. cerevisiae×S. kudriavzevii isolated in wine fermentations show enhanced performance in low temperature enological conditions and increased production of interesting aroma compounds. In this work, we have studied the transcriptomic response in enological conditions of a S. cerevisiae×S. kudriavzevii hybrid strain and compared it with the reference species of S. cerevisiae and S. kudriavzevii. The results show that the hybrid strain presents an up-regulation of genes belonging to functional group translation and amino-acid metabolism. Moreover, key genes related to cold stress and production of glycerol and aroma compounds were also up-regulated. While some genes inherited regulation patterns from one of the parents, most of the up-regulated genes presented a new gene expression pattern, probably generated during the hybridization and adaptation process.

Monitoring of killer yeast populations in mixed cultures: influence of incubation temperature of microvinifications samples.

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae. Samples were periodically extracted during vinification, seeded on Petri dishes and incubated at 25 and 37 °C; the latter temperature was assayed for possible inactivation of killer toxin production. Colonies developed under the described conditions were randomly transferred to killer phenotype detection medium. Significant differences in the killer/sensitive ratio were observed between both incubation temperatures in all microvinifications. These results suggest that 37 °C seems a better option to determine the biomass of sensitive yeasts, in order to avoid underestimation of sensitive cells in the presence of killer yeasts during fermentations. Incubation at a toxin-inhibiting temperature clearly showed the real ratio of killer to sensitive cells in fermentation systems.

Control of ochratoxin A production in grapes.

Ochratoxin A (OTA) is a mycotoxin commonly present in cereals, grapes, coffee, spices, and cocoa. Even though the main objective of the food and feed chain processors and distributors is to avoid the extended contamination of plant-derived foods and animal feeds with mycotoxins, until now, complete OTA removal from foods and feedstuffs is not feasible. Prevention through pre-harvest management is the best method for controlling mycotoxin contamination. However, in the case that the contamination occurs after this stage, the hazards associated with OTA must be managed through post-harvest strategies. Due to the increasing number of fungal strains resistant to chemical fungicides and the impact of these pesticides on the environment and human health, maximum levels of chemical residues have been regulated in many products. Alternative methods are necessary to substitute or complement treatments with fungicides to control fungi under field or storage conditions. Yeasts are considered one of the most potent biocontrol agents due to their biology and non-toxic properties. Epiphytic yeasts are the major component of the microbial community on the surface of grape berries and they are evolutionarily adapted to this ecological niche. Nowadays, several yeast species included in different genera are considered as potential biocontrol agents to control both, growth of ochratoxigenic Aspergillus species and OTA accumulation.

Multi-enzyme production by pure and mixed cultures of Saccharomyces and non-Saccharomyces yeasts during wine fermentation.

Saccharomyces and non-Saccharomyces yeasts release enzymes that are able to transform neutral compounds of grape berries into active aromatic compounds, a process that enhances the sensory attributes of wines. So far, there exists only little information about enzymatic activity in mixed cultures of Saccharomyces and non-Saccharomyces during grape must fermentations. The aim of the present work was to determine the ability of yeasts to produce extracellular enzymes of enological relevance (ß-glucosidases, pectinases, proteases, amylases or xylanases) in pure and mixed Saccharomyces/non-Saccharomyces cultures during fermentation. Pure and mixed cultures of Saccharomyces cerevisiae BSc562, Hanseniaspora vinae BHv438 and Torulaspora delbrueckii BTd259 were assayed: 1% S. cerevisiae/99% H. vinae, 10% S. cerevisiae/90% H. vinae, 1% S. cerevisiae/99% T. delbrueckii and 10% S. cerevisiae/90% T. delbrueckii. Microvinifications were carried out with fresh must without pressing from Vitis vinifera L. c.v. Pedro Jiménez, an autochthonous variety from Argentina. Non-Saccharomyces species survived during 15-18days (BTd259) or until the end of the fermentation (BHv438) and influenced enzymatic profiles of mixed cultures. The results suggest that high concentrations of sugars did not affect enzymatic activity. ß-Glucosidase and pectinase activities seemed to be adversely affected by an increase in ethanol: activity diminished with increasing fermentation time. Throughout the fermentation, Saccharomyces and non-Saccharomyces isolates assayed produced a broad range of enzymes of enological interest that catalyze hydrolysis of polymers present in grape juice. Vinifications carried out by a pure or mixed culture of BTd259 (99% of T. delbrueckii) showed the highest production of all enzymes assayed except for ß-glucosidase. In mixed cultures, S. cerevisiae outgrew H. vinae, and T. delbrueckii was only detected until halfway the fermentation process. Nevertheless, their secreted enzymes could be detected throughout the fermentation process. Our results may contribute to a better understanding of the microbial interactions and the influence of some enzymes on vinification environments.

Biodiversity of Saccharomyces cerevisiae populations in Malbec vineyards from the "Zona Alta del Río Mendoza" region in Argentina.

The "Zona Alta del Río Mendoza" (ZARM) is the major Malbec grape viticulture region of Argentina. The aim of the present study was to explore Saccharomyces cerevisiae biodiversity in ZARM vineyards. Interdelta PCR and RFLP mtDNA molecular markers were applied to differentiate S. cerevisiae strains. The presence of commercial strains on ZARM vineyards was also assessed. Our results reveal a highly diverse, but genetically closely related, S. cerevisiae population (containing more than 190 molecular patterns among 590 S. cerevisiae isolates). According to the S. cerevisiae strain diversity found in vineyards, they were classified as vineyards with high and low polymorphic S. cerevisiae populations. Six vineyards showed a high polymorphic population, with more than 20 different S. cerevisiae molecular patterns. S. cerevisiae populations in these vineyards were diverse and irregularly distributed, with different strains in each vineyard site. Low polymorphic S. cerevisiae population vineyards displayed very low yeast diversity, with only 9 to 10 different S. cerevisiae strains and presence of two commercial strains widely distributed. Population diversity estimators were calculated to determine the population structure of S. cerevisiae in the ZARM vineyards. The obtained values support the hypothesis that the eight sampled subpopulations come indeed from a larger population.

Biocontrol as a strategy to reduce the impact of ochratoxin A and Aspergillus section Nigri in grapes.

The efficacy of two strains of Kluyveromyces thermotolerans in preventing the growth and ochratoxin A (OTA) accumulation of ochratoxigenic fungi both "in vitro" and "in situ" was evaluated. The data from this study showed that both yeast strains were able to control Aspergillus carbonarius and A. niger aggregate species growth and ochratoxin A accumulation. The inhibitory effects were dependent on the ochratoxigenic species, yeast strains, a(w) and temperature evaluated and their interactions. Over all conditions assayed, ochratoxin A accumulation was reduced from 3% to 100% and the growth rate from 11% to 82.5%, depending on conditions. These results are promising for future development of a bio-pesticide.