Low fermentation temperatures are a crucial aspect of winemaking, influencing yeast metabolism, process efficiency, and aroma retention. However, it is a significant source of energy consumption. Here, we investigated the effect of high (20 °C ± 1 °C) and low (16 °C ± 1 °C) temperatures combined with multi-starter fermentations with Metschnikowia pulcherrima (Level² Flavia and Level² Initia) and Saccharomyces cerevisiae (EC 1118 and Zymaflore X5) strains on the aromatic profile of Lugana wine. Sequential inoculation at 20 °C shortened fermentation by five days compared to 16 °C, lowering energy consumption for cooling. Significant differences in key aromatic compound classes were observed between the two temperature trials. Wines fermented at 20 °C exhibited more intense tropical and fruity aromas, with 3-MH and 3-MHA concentrations up to ten times higher than those at 16 °C. Such an increase was particularly evident in the sequential fermentation, especially Level² Flavia/Zymaflore X5, compared to S. cerevisiae alone. In contrast, lower temperatures favored the production of ethyl esters and ethyl acetate, with increases of 14% for ethyl octanoate and 24% for ethyl butanoate. Sensory analysis confirmed these aromatic differences, with all trials at 20 °C enhancing white wine aroma complexity, particularly those involving multi-starter fermentations. These findings provide practical insights for the wine industry, validating sequential fermentation at moderately elevated temperatures as a strategy to improve wine quality while supporting energy-efficient and sustainable practices.

Metschnikowia pulcherrima is increasingly valued in sustainable vitiviniculture for its dual role as a biocontrol agent and as a contributor to wine quality. However, a coordinated dual-purpose selection strategy has not yet been systematically implemented for this species. This study aimed to identify native strains with combined wine-related traits and biocontrol potential by screening a collection of 179 isolates for key phenotypic traits—β-glucosidase and β-lyase activities, hydrogen sulfide (H₂S) production, and pulcherrimin biosynthesis—and assessing their genotypic diversity. Dereplication yielded 106 unique strains, from which five with the most favorable wine-related traits and distinct genotypic profiles were selected for subsequent evaluation of antagonistic potential. Safety-related traits, including growth at 37 °C, invasive growth, pseudohyphal formation, and proteolytic activity, were assessed to exclude virulence-associated behaviors. Antagonistic activity against Botrytis cinerea was evaluated through in vitro dual-culture assays and in vivo grape-berry inoculations, revealing strain- and pathogen-dependent inhibition, with volatile-mediated effects generally exceeding direct-contact interactions. Among the tested strains, NLSFS4 showed strong and consistent biocontrol potential. Microvinification trials further confirmed its oenological relevance, demonstrating the ability to modulate wine aroma composition while preserving fermentation performance. Overall, this study highlights the substantial functional diversity within M. pulcherrima and identifies a promising native strain for integrated use in wine fermentation and biological control in sustainable production systems.