Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains.

International audienceNatural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers the possibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was only effective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO(2) production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms

Yeast and filamentous fungi microbial communities in organic red grape juice : effect of vintage, maturity stage, SO2, and bioprotection

Changes are currently being made to winemaking processes to reduce chemical inputs [particularly sulfur dioxide (SO2)] and adapt to consumer demand. In this study, yeast growth and fungal diversity were investigated in merlot during the prefermentary stages of a winemaking process without addition of SO2. Different factors were considered, in a two-year study: vintage, maturity level and bioprotection by the adding yeast as an alternative to SO2. The population of the target species was monitored by quantitative-PCR, and yeast and filamentous fungi diversity was determined by 18S rDNA metabarcoding. A gradual decrease of the α-diversity during the maceration process was highlighted. Maturity level played a significant role in yeast and fungal abundance, which was lower at advanced maturity, while vintage had a strong impact on Hanseniaspora spp. population level and abundance. The presence of SO2 altered the abundance of yeast and filamentous fungi, but not their nature. The absence of sulfiting led to an unexpected reduction in diversity compared to the presence of SO2, which might result from the occupation of the niche by certain dominant species, namely Hanseniaspora spp. Inoculation of the grape juice with non-Saccharomyces yeast resulted in a decrease in the abundance of filamentous fungi generally associated with a decline in grape must quality. Lower abundance and niche occupation by bioprotection agents were observed at the overripened stage, thus suggesting that doses applied should be reconsidered at advanced maturity. Our study confirmed the bioprotective role of Metschnikowia pulcherrima and Torulaspora delbrueckii in a context of vinification without sulfites

Population dynamics and yeast diversity in early winemaking stages without sulfites revealed by three complementary approaches

Nowadays, the use of sulfur dioxide (SO2 ) during the winemaking process is a controversial societal issue. In order to reduce its use, various alternatives are emerging, in particular bioprotection by adding yeasts, with different impacts on yeast microbiota in early winemaking stages. In this study, quantitative-PCR and metabarcoding high-throughput sequencing (HTS) were combined with MALDI-TOF-MS to monitor yeast population dynamic and diversity in the early stages of red winemaking process without sulfites and with bioprotection by Torulaspora delbrueckii and Metschnikowia pulcherrima addition. By using standard procedures for yeast protein extraction and a laboratory-specific database of wine yeasts, identification at species level of 95% of the isolates was successfully achieved by MALDI-TOF-MS, thus confirming that it is a promising method for wine yeast identification. The different approaches confirmed the implantation and the niche occupation of bioprotection leading to the decrease of fungal communities (HTS) and Hanseniaspora uvarum cultivable population (MALDI-TOF MS). Yeast and fungi diversity was impacted by stage of maceration and, to a lesser extent, by bioprotection and SO2, resulting in a modification of the nature and abundance of the operational taxonomic units (OTUs) diversity

SSU1 Checkup, a Rapid Tool for Detecting Chromosomal Rearrangements Related to the SSU1 Promoter in Saccharomyces cerevisiae: An Ecological and Technological Study on Wine Yeast

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological Saccharomyces cerevisiae strains, CR involving the promoter region of the gene SSU1 lead to a higher sulfite tolerance by enhancing the SO2 efflux. To date, three different SSU1 associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method (SSU1 checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 S. cerevisiae strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the SSU1 promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged SSU1 promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of SSU1 promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO2 concentrations. The SSU1 checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers.Fil: Marullo, Philippe. Universite de Bordeaux; FranciaFil: Claisse, Olivier. Universite de Bordeaux; FranciaFil: Raymond Eder, María Laura. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Börlin, Marine. Universite de Bordeaux; FranciaFil: Feghali, Nadine. Lebanese University; LíbanoFil: Bernard, Margaux. Universite de Bordeaux; FranciaFil: Legras, Jean Luc. Université Montpellier II; FranciaFil: Albertin, Warren. Universite de Bordeaux; FranciaFil: Rosa, Alberto Luis. Universidad Católica de Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Recursos Naturales y Sustentabilidad José Sanchez Labrador S. J.; ArgentinaFil: Masneuf Pomarede, Isabelle. Universite de Bordeaux; Franci

L’identification des levures et bactéries œnologiques par spectrométrie de masse de type MALDI-TOF

La spectrométrie de masse de type MALDI-TOF a été adaptée afin d’être utilisée comme outil innovant d’identification au niveau de l’espèce des levures et bactéries isolées d’échantillons variés (moûts, vins, boissons). L’analyse d’un grand nombre de clones permet d’apprécier la diversité des espèces de levures, bactéries acétiques et lactiques présentes dès les phases pré-fermentaires, au cours des fermentations, pendant l’élevage ou après conditionnement. Dans le cas d’altération de produits, cet outil innovant participera à une meilleure maitrise des risques microbiologiques

Horticulture Research

Soil microbiota has increasingly been shown to play an integral role in viticulture resilience. The emergence of new metagenomic and culturomic technologies has led to significant advances in the study of microbial biodiversity. In the agricultural sector, soil and plant microbiomes have been found to significantly improve resistance to environmental stressors and diseases, as well as influencing crop yields and fruit quality thus improving sustainability under shifting environments. Grapevines are usually cultivated as a scion grafted on rootstocks, which are selected according to pedoclimatic conditions and cultural practices, known as terroir. The rootstock connects the surrounding soil to the vine’s aerial part and impacts scion growth and berry quality. Understanding rootstock and soil microbiome dynamics is a relevant and important field of study, which may be critical to improve viticulture sustainability and resilience. This review aims to highlight the relationship between grapevine roots and telluric microbiota diversity and activity. In addition, this review explores the concept of core microbiome regarding potential applications of soil microbiome engineering with the goal of enhancing grapevine adaptation to biotic and abiotic stress

Non-Saccharomyces yeasts as bioprotection in the composition of red wine and in the reduction of sulfur dioxide

Non-Saccharomyces yeasts have been used for many years due to their technological potential, particularly as a « booster » of wine fruity aroma in mixed fermentations with Saccharomyces cerevisiae. Recently, a new application has emerged, bioprotection, which consists in colonizing the environment in the context of sulfite reduction in wines. The chemical and sensory impact of non-Saccharomyces yeast according to different modes of application in a context of fermentation without addition of SO2 was evaluated through trial with Merlot N. (Vitis vinifera L.). An effective niche occupation by non-Saccharomyces yeasts was highlighted during the prefermentary stages by Quantitative-PCR and MALDI-TOF MS identification. Chemical analysis (GC-MS and GC MS/MS) of finish wine showed the significant impact of the dose applications, with bioprotection characterized by linear esters and sequential application by acetates of higher alcohol contents. Moreover, a separation according to the species used in bioprotection was revealed. Finally, using a panel trained, the sensory analysis confirmed that the use of non-Saccharomyces yeast was a fruity booster in sequential inoculation and, to a less extent, when used as bioprotection. This study shows for the first time that the use of non-Saccharomyces yeast as a bioprotection has a significant impact on the aromatic profile of wines

Grapevine decline is associated with difference in soil microbial composition and activity

Grapevine decline is a top concern in viticulture worldwide and is often associated with many biotic and abiotic factors. Grape trunk diseases and viruses are some of the most frequently identified causes of vine dieback. However, a decline is sometimes observed when no mineral deficiency or excess, or pathogenic causes can be identified. Soil enzymatic and microbial activities are relevant bio-indicators since they are known to influence vine health. Grapevine associated microbiota, linked to vine fitness, is known to be influenced by soil microbiota coming from the microbial pool inhabiting the vineyard. This work describes the microbial diversity and activity of four different vineyard plots of the Bordeaux region, selected due to the presence of localised declining areas unexplained yet by disease symptoms. Soils were sampled in declining areas and areas within the same plot showing no decline symptoms, during autumn and spring periods. Significant differences in enzymatic activities, microbial biomass and activity were found among soils even if those soils presented quite similar physicochemical characteristics that could not explain these observed declines. The results of enzymatic assays distinguished patterns in autumn and spring periods with an overall greater enzymatic activity in soils from non-declining areas. This work suggests that soils displaying decline symptoms present a dysbiosis in functionality and diversity which is linked to vine health

Front Microbiol

Chromosomal rearrangements (CR) such as translocations, duplications and inversions play a decisive role in the adaptation of microorganisms to specific environments. In enological strains, CR involving the promoter region of the gene lead to a higher sulfite tolerance by enhancing the SO efflux. To date, three different associated CR events have been described, including translocations XV-t-XVI and VIII-t-XVI and inversion inv-XVI. In the present study, we developed a multiplex PCR method ( checkup) that allows a rapid characterization of these three chromosomal configurations in a single experiment. Nearly 600 strains collected from fermented grape juice were genotyped by microsatellite markers. We demonstrated that alleles of the promoter are differently distributed according to the wine environment (cellar versus vineyard) and the nature of the grape juice. Moreover, rearranged promoters are significantly enriched among commercial starters. In addition, the analysis of nearly isogenic strains collected in wine related environments demonstrated that the inheritance of these CR shapes the genetic diversity of clonal populations. Finally, the link between the nature of promoter and the tolerance to sulfite was statistically validated in natural grape juice containing various SO concentrations. The checkup is therefore a convenient new tool for addressing population genetics questions and for selecting yeast strains by using molecular markers

Towards microbiota-based disease management: analysis of grapevine microbiota in plots with contrasted levels of downy mildew infection

Vineyards harbor a myriad of microorganisms that interact with each other and with the grapevines. Some microorganisms are plant pathogens, such as the oomycete Plasmopara viticola that causes grapevine downy mildew. Others, such as plant growth promoting bacteria and disease biocontrol agents, have a positive influence on vine health. The present study aims to (1) investigate whether vine-based culture media increase the cultivability of the grapevine microbiota, in comparison to standard culture media and (2) identify and isolate bacterial taxa naturally present in grapevine leaves and significantly more abundant in plots showing low susceptibility to downy mildew