Saccharomyces cerevisiae vineyard strains have different nitrogen requirements that affect their fermentation performances.

In this work the fermentation performances of seven vineyard strains, together with the industrial strain EC1118, have been investigated at three differing yeast assimilable nitrogen (YAN) concentrations (300 mg N l-1 , 150 mg N l-1 and 70 mg N l-1 ) in synthetic musts. The results indicated that the response to different nitrogen levels is strain dependent. Most of the strains showed a dramatic decrease of the fermentation at 70 mg N l-1 but no significant differences in CO2 production were found when fermentations at 300 mg N l-1 and 150 mg N l-1 were compared. Only one among the vineyard strains showed a decrease of the fermentation when 150 mg N l-1 were present in the must. These results contribute to shed light on strain nitrogen requirements and offer new perspectives to manage the fermentation process during winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected vineyard Saccharomyces cerevisiae strains can improve the quality and the complexity of local wines. Wine quality is also influenced by nitrogen availability that modulates yeast fermentation activity. In this work, yeast nitrogen assimilation was evaluated to clarify the nitrogen requirements of vineyard strains. Most of the strains needed high nitrogen levels to express the best fermentation performances. The results obtained indicate the critical nitrogen levels. When the nitrogen concentration was above the critical level, the fermentation process increased, but if the level of nitrogen was further increased no effect on the fermentation was found.

Comparative strain typing of Rhizobium leguminosarum bv. viciae natural populations.

372 natural isolates of Rhizobium leguminosarum bv. viciae, rescued from nodules of pea plants grown in an agricultural field in northern Italy, were analyzed by different methods. Three DNA-based fingerprinting techniques were lined up to compare their relative degree of resolution and possible advantages of each approach. The methods included (i) Eckhardt gel plasmid profiles, (ii) pulsed-field gel electrophoresis (PFGE) of genomic large fragment digests, and (iii) random amplified polymorphic DNA (RAPD) profiles, generated with arbitrary primers. The scheme also involved the isolation of a number of different isolates per nodule to estimate the level of intra-nodular variability. It was therefore possible to evaluate the frequency of double and multiple occupancies, and the proportion of the alternative profiles sharing the same nodule, generally resulting in a numerically dominant, main representative accompanied by a secondary one with a slightly different fingerprint. This finding revealed that the different profiles within a nodule are normally due to bacteria derived from the same single invader following genetic alterations possibly occurred during infection, e.g., by plasmid loss. The analysis of 31 nodules revealed 16 different patterns, representing the most frequently occurring nodulation-proficient isolates of the natural soil examined, five of which were found with frequencies around 15%. The sensitivity of the methods in differentiating isolates was compared. The relatedness of the different natural rhizobial isolates was investigated by densitometrical gel analysis of the fingerprints, allowing a comparison of the results. One of the most interesting conclusions was that the degree of information yielded by the plasmid gel profiling alone, carried out by simple visual inspection without software-aided analyses, was surprisingly high, as it enabled a placement of the isolates, whose accuracy, in terms of relatedness, was subsequently confirmed by each of the two genomic methods.

Aspects of Marker/Reporter Stability and Selectivity in Soil Microbiology.

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and b-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacteria while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.