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Yeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis and rouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15.5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1.3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15.5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditions.

Trichoderma harzianum is a widely distributed soil fungus that antagonizes numerous fungal phytopathogens. The antagonism of T. harzianum usually correlates with the production of antifungal activities including the secretion of fungal cell walls that degrade enzymes such as chitinases. Chitinases Chit42 and Chit33 from T. harzianum CECT 2413, which lack a chitin-binding domain, are considered to play an important role in the biocontrol activity of this strain against plant pathogens. By adding a cellulose-binding domain (CBD) from cellobiohydrolase II of Trichoderma reesei to these enzymes, hybrid chitinases Chit33-CBD and Chit42-CBD with stronger chitin-binding capacity than the native chitinases have been engineered. Transformants that overexpressed the native chitinases displayed higher levels of chitinase specific activity and were more effective at inhibiting the growth of Rhizoctonia solani, Botrytis cinerea and Phytophthora citrophthora than the wild type. Transformants that overexpressed the chimeric chitinases possessed the highest specific chitinase and antifungal activities. The results confirm the importance of these endochitinases in the antagonistic activity of T. harzianum strains, and demonstrate the effectiveness of adding a CBD to increase hydrolytic activity towards insoluble substrates such as chitin-rich fungal cell walls.

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To clarify the role that respiration, the mitochondrial genome, and interactions of mitochondria and nucleus play on sporulation and to improve the sporogenic ability of several baker's yeasts, an investigation of the effects of different media and culture conditions on baker's yeast sporulation was undertaken. When standard protocols were followed, the sporulation frequency varied between 20 and 60% and the frequency of four-spore asci varied between 1 and 6%. Different presporulation and sporulation media, the use of solid versus liquid media, and incubation at 22 versus 30؇C were checked, and the cells were collected from presporulation media in either exponential or stationary phase. Best results, yielding sporulation and four-spore ascus formation frequencies up to 97 and 60%, respectively, were obtained by collection of the cells in exponential phase from liquid presporulation medium with 10% glucose and transfer of them to sporulation medium with 0.5% potassium acetate at 22؇C. Under these conditions, the most important factor was the growth phase (exponential versus stationary) at which cells from presporulation medium were collected. Changes in sporulation frequencies were also measured after transfer of mitochondria from different sources to baker's yeasts. When mitochondria from laboratory, baker's, and wine yeasts were transferred to baker's and laboratory petite strains, sporulation and four-spore ascus formation frequencies dropped dramatically either to no sporulation at all or to less than 50% in both parameters. This transfer also resulted in an increase in the frequency of petite mutant formation but yielded similar growth and respiration rates in glycerol. The strains tested recovered their ability to yield maximal sporulation and tetrad formation after recovering their own mitochondria.