Non-conventional yeasts present a huge, yet barely exploited, resource of yeast biodiversity for industrial applications. This presents a great opportunity to explore alternative ethanol-fermenting yeasts that are more adapted to some of the stress factors present in the harsh environmental conditions in second-generation (2G) bioethanol fermentation. Extremely tolerant yeast species are interesting candidates to investigate the underlying tolerance mechanisms and to identify genes that when transferred to existing industrial strains could help to design more stress-tolerant cell factories. For this purpose, we performed a high-throughput phenotypic evaluation of a large collection of non-conventional yeast species to identify the tolerance limits of the different yeast species for desirable stress tolerance traits in 2G bioethanol production. Next, 12 multi-tolerant strains were selected and used in fermentations under different stressful conditions. Five strains out of which, showing desirable fermentation characteristics, were then evaluated in small-scale, semi-anaerobic fermentations with lignocellulose hydrolysates. Our results revealed the phenotypic landscape of many non-conventional yeast species which have not been previously characterized for tolerance to stress conditions relevant for bioethanol production. This has identified for each stress condition evaluated several extremely tolerant non-Saccharomyces yeasts. It also revealed multi-tolerance in several yeast species, which makes those species good candidates to investigate the molecular basis of a robust general stress tolerance. The results showed that some non-conventional yeast species have similar or even better fermentation efficiency compared to S. cerevisiae in the presence of certain stressful conditions. Prior to this study, our knowledge on extreme stress-tolerant phenotypes in non-conventional yeasts was limited to only few species. Our work has now revealed in a systematic way the potential of non-Saccharomyces species to emerge either as alternative host species or as a source of valuable genetic information for construction of more robust industrial S. serevisiae bioethanol production yeasts. Striking examples include yeast species like Pichia kudriavzevii and Wickerhamomyces anomalus that show very high tolerance to diverse stress factors. This large-scale phenotypic analysis has yielded a detailed database useful as a resource for future studies to understand and benefit from the molecular mechanisms underlying the extreme phenotypes of non-conventional yeast species.

Flowers offer a wide variety of substrates suitable for fungal growth. However, the mycological study of flowers has only recently begun to be systematically addressed from an ecological point of view. Most research on the topic carried out during the last decade has focused on studying the prevalence and diversity of flower-inhabiting yeasts, describing new species retrieved from floral parts and animal pollinators, and the use of select nectar yeasts as model systems to test ecological hypotheses. In this primer article, we summarize the current state of the art in floral nectar mycology and provide an overview of some research areas that, in our view, still require further attention, such as the influence of fungal volatile organic compounds on the foraging behavior of pollinators and other floral visitors, the analysis of the direct and indirect effects of nectar-inhabiting fungi on the fitness of plants and animals, and the nature and consequences of fungal-bacterial interactions taking place within flowers.

Floral nectar is commonly inhabited by microorganisms, mostly yeasts and bacteria, which can have a strong impact on nectar chemistry and scent. Yet, little is known about the effects of nectar microbes on the behavior and survival of insects belonging to the third trophic level such as parasitoids. Here, we used five nectar-inhabiting yeast species to test the hypothesis that yeast species that almost solely occur in nectar, and therefore substantially rely on floral visitors for dispersal, produce volatile compounds that enhance insect attraction without compromising insect life history parameters, such as survival. Experiments were performed using two nectar specialist yeasts (Metschnikowia gruessii and M. reukaufii) and three generalist species (Aureobasidium pullulans, Hanseniaspora uvarum, and Sporobolomyces roseus). Saccharomyces cerevisiae was included as a reference yeast. We compared olfactory responses of the generalist aphid parasitoid Aphidius ervi (Haliday) (Hymenoptera: Braconidae) when exposed to these microorganisms inoculated in synthetic nectar. Nectar-inhabiting yeasts had a significant impact on nectar chemistry and produced distinct volatile blends, some of which were attractive, while others were neutral or repellent. Among the different yeast species tested, the nectar specialists M. gruessii and M. reukaufii were the only species that produced a highly attractive nectar to parasitoid females, which simultaneously had no adverse effects on longevity and survival of adults. By contrast, parasitoids that fed on nectars fermented with the reference strain, A. pullulans, H. uvarum or S. roseus showed shortest longevity and lowest survival. Additionally, nectars fermented by A. pullulans or S. roseus were consumed significantly less, suggesting a lack of important nutrients or undesirable changes in the nectar chemical profiles. Altogether our results indicate that nectar-inhabiting yeasts play an important, but so far largely overlooked, role in plant-insect interactions by modulating the chemical composition of nectar, and may have important ecological consequences for plant pollination and biological control of herbivorous insects.