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Contamination of vineyards from black Aspergilli is a well-known condition that cause the accumulation of ochratoxin A (OTA) in grapes and derived products. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, although, great variations may occur from one year to another. Among the black Aspergilli commonly found in infected grapes, Aspergillus carbonarius is considered the main responsible of OTA contamination, with A. niger at secondary extent. To minimise the black Aspergilli infection and limit OTA concentrations in grapes, several strategies are commonly adopted, including the implementations of good agricultural practices and the use of pesticides and fungicides. These strategies are essential to manage the problem, but since they are insufficient when extremely favourable condition occurs in the vineyard, new strategies, aimed to reduce OTA risk in vineyards, are necessary. In this respect, implementation of electrolysed oxidising water (EOW) in agriculture has arising during the last decade as an interesting alternative to replace or limit the use of chemicals. The efficacy of EOW was also demonstrated in post-harvest for reduction of gray mold and brown rot on surfaces of peaches and grapes. In this study, we screened for the first time the efficacy of EOW generated by EVA System® 100 apparatus (Industrie De Nora S.p.A., Milan, Italy) at different concentrations of free chlorine (ranging from 0.0125 to 0.4 g/L) on conidial germination and growth of A. carbonarius and A. niger. A good fungicidal activity was achieved after 2-10 minutes treatment with EOW containing 0.4-0.2 g/L of free chlorine, although A. carbonarius conidia were more resistant to EOW than A. niger conidia. Then EOW at 0.4 g/L free chlorine was tested on detached berries of Primitivo and Aglianico wine grape varieties that were singularly infected with A. niger and A. carbonarius. Treatments with Switch (cyprodinil and fludioxonil) at 0.8 g/L, a fungicide regularly used in vineyard against black Aspergilli, were used as positive controls. Percentage of infected berries, McKinney index, and OTA concentrations were used to evaluate the efficacy of the EOW treatment. On Aglianico grape berries EOW and Switch produced an almost complete reduction on percentage of infection, McKinney index and OTA concentration compared to the control. On Primitivo grape berries EOW treatment reduced more than half the percentage of infections and McKinney index for both A. carbonarius and A. niger, although Switch showed a better performance. A significant reduction of OTA concentration was observed for EOW and Switch treatments. These results evidence for the first time that EOW is effective to reduce black Aspergilli inoculum and OTA contamination on grape berries.

In winemaking, the utilization of mixed starter cultures of Saccharomyces and non-Saccharomyces yeasts is a methodology that allows the enhancement of sensory quality and complexity of the final product, without compromising the general quality and safety of the oenological products. The aim of this study was to assess the oenological potential of Candida zemplinina in mixed cultures (co-inoculation) and sequential inoculation with S. cerevisiae for the industrial wine production. Three previously characterized S. cerevisiae strains [1] were separately used, as multistarter, together with an autochthonous C. zemplinina strain [2] in lab-scale microvinification trials of Negroamaro must. The fermentation course and the quality of final product indicated that C. zemplinina strain 35NC1 positively contributed to increasing the wine organoleptic quality. At the best of our knowledge, the present report is the first study regarding the utilization of a selected C. zemplinina strain in multistarter inoculation with S. cerevisiae for the production of Negroamaro wine. The results of this investigation corroborate the concept that non-Saccharomyces yeasts play a essential role in wine-bouquet formation and their action could reduce the risks for wine quality and safety often associated with uncontrolled vinification. This research was partially supported by the Apulian Region Projects "Sviluppo di approcci microbiologici innovativi per il miglioramento della qualità di vini tipici regionali (NEWine)". References 1.Chiriatti M., et al. (2012). Identification, characterization and application of autochthonous fermentative starter cultures for the industrial production of Negroamaro and Primitivo wine Proceedings of the III SIMTREA Congress, June 26-28, Bari, Italy, pp. 93-94. 2.Tristezza M., et al. (2016). Caratterizzazione molecolare e tecnologica di lieviti vinari autoctoni appartenenti alla specie Candida zemplinina. Proceedings of the "11° Convegno Nazionale sulla Biodiversità", 9-10 giugno, Matera, Italy, p. 211.

In conventional winemaking, grape must fermentations are carried out by a succession of different yeast species. The Saccharomyces cerevisiae completes the process, whereas its initial stage is dominated by non-Saccharomyces strains, whose by-products contribute to the composition of the wine bouquet. In this study, we evaluated the performance of two selected strains of Hanseniaspora uvarum and S. cerevisiae as multistarters for inoculation of Negroamaro must vinification.

Indigenous yeasts are present on the surfaces of grapes and their success in surviving and driving fermentation depends on the sum of various physical, chemical and biotic factors. The analysis of berry-resident strain diversity and the relationship between genotype and phenotype can be used in the development and identification of specific non-Saccharomyces strains provided with interesting technological properties. In this contribution, during a large-scale study on vineyard-associated yeast strains from Apulia (Southern Italy), we isolated a new yeast species from "Negroamaro" grape berries.

Spontaneous grapes must fermentations are promoted by the indigenous yeasts, that are able to confer a distinctive style and quality to the produced wine. The spontaneous fermentations of grape must are at first dominated by non-Saccharomyces yeasts and, in a final stage the alcoholic fermentation process is completed by dominant S. cerevisiae strains (Bauer and Pretorius, 2000).The autochthonous yeast strains are associated to a specific vineyard niche habitat and their role in natural fermentation allows the production of wines with particular features in each microclimatic area (Pérez-Coello et al., 1999). However, in order to avoid the unpredictability of must spontaneous fermentation, the winemakers employ commercial dry active yeast culture for wine industrial productions. Increasing interest in the application of locally selected yeasts for fermentation management has been reported (Tristezza et al., 2012). The employment of autochthonous strains of S. cerevisiae as starters seems to be preferable since they are adapted to all the constraints related to a specific wine-production area (Lopes et al., 2007) and are thus capable to dominate more efficiently the indigenous microflora during the fermentation process. Moreover, autochthonous yeast strains can assure the preservation and/or the enhancement of the typical oenological and sensory features which could be considered representative of an oenological region (Rodríguez et al., 2010). In the present study, we developed and applied a strategy to select S. cerevisiae strains from a larger number of yeast isolates. This was achieved adopting a number of key parameters indicative of the strains technological and enological properties. S. cerevisiae population has been isolated from natural fermentations of grape musts, which derive from grapes sampled from the six most representative Negroamaro and Primitivo producing-areas in Apulia (Southern Italy). The yeast populations were identified by molecular assays (AFLP and sequencing) and some selected representative strains were subjected to physiological, oenological and technological characterization. At the end of the selection procedure, that lasted three years, three indigenous S. cerevisiae strains (one for Primitivo and two for Negroamaro), characterized by interesting technological and oenological properties, were selected. The three selected strains were evaluated by both laboratory tests and semi-industrial scale fermentations to confirm their ability to act as autochthonous fermentation starters. An optimized procedure was worked out for the production of starter biomasses, in order to test them in Negroamaro and Primitivo wine production, at an industrial scale, in six different wineries. The employment of autochthonous starter cultures for the industrial production of typical wines in Apulia will be discussed.

The conversion of grape must to wine is a complex biochemical process mediated by highly specialised micro organisms, yeasts and bacteria. The primary role of wine yeast is to catalyse the rapid, complete and efficient conversion of grape sugar into ethanol, carbon dioxide and other minor, but important, metabolites without the development of off-flavours. Winemakers have started to use strains isolated from their own geographical regions to produce wines with peculiar sensorial properties and reproducible quality. Indigenous yeasts with oenological potential are considered autochthonous and are present on the surfaces of undamaged grapes and their success depends on the sum of physical, chemical and biotic factors. In the spontaneous fermentation of grape must, there is a progressive growth pattern of indigenous yeast, with the final stage invariably being dominated by alcohol-tolerant strains of Saccharomyces cerevisiae. Traditionally, the identification and characterisation of yeast species and strains has been based on morphology and physiological properties. In recent years, techniques developed in molecular biology have provided an alternative to these traditional methods and are an important tool in solving industrial problems. An investigation was carried out in order to characterize the yeast population present in the spontaneous fermentation of "Negroamaro" must, an important grape cultivar for the industrialized production of The Salento (south-eastern Italy) wine. The objective of this study was the identification and characterization of natural yeast flora with peculiar technological and oenological properties from "Negroamaro" grapes collected from different vineyards of the The Salento and grown in organic and non-organic conditions. Identification and characterisation of yeast species and strains was carried out by morphological, physiological, biochemical and molecular methods by sampling the micro flora present on grape skin and in the must during spontaneous fermentation, at different stages. The colonies from each sample were selected on the bases of morphological and physiological characteristics and the isolated population was identified at genus level by PCR analysis of the internal transcribed ITS1- 5,8S - ITS2 region. Further identification was carried out by RFLP analysis of the above amplified fragments and of the amplified d interspersed element. On non-organic cultivated grape skin Hanseniaspora uvarum, Metschnikowia pulcherrima and Issatchenkia orientalis were observed, whereas on the skin of organic cultivated grapes Kluyveromyces thermotolerans, Issatchenkia terricola, Candida incomunis and Candida stellata were also found. In the first stage of fermentation (d=1.125 g/l) a population similar to that present on the grape skin was identified. In the following fermentation stages (d= 1070-1.000 g/l) only Saccharomyces cerevisiae was found and a higher number of strains was identified in must from non-organic grapes than in that from organic grapes. The technological properties (ability of the strains to utilise different carbon sources, production of ethanol, glycerol and hydrogen sulphite, surviving in dry conditions, etc.) of the selected S. cerevisiae strains were studied. The dominant S. cerevisiae strains identified in fermented musts showed potentially important enological characteristics.