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Differentiation of industrial wine yeast strains using microsatellite markers
Abstract
To differentiate nine industrial wine strains of Saccharomyces cerevisiae using microsatellite (simple sequence repeats, SSR) markers.
Six of the strains were indigenous yeasts currently used as high-density starter monocultures by the Uruguayan wine industry. Unequivocal differentiation of these six native strains and three commercial S. cerevisiae wine strains was achieved by PCR amplification and polymorphism analysis of loci containing microsatellite markers.
We recommend the use of this reproducible and simple molecular method to routinely discriminate wine yeast strains.
Microsatellites are superior to other methods for typing yeasts because the results can be exchanged as quantitative data. Knowledge of the frequencies of the alleles for different SSR markers will eventually lead to an accurate typing method to identify industrial wine yeast strains.
... Microsatellite markers, based on Simple Sequence Repeats (SSRs) scattered throughout the genome [69][70][71][72][73], represent the "gold standard" for this discrimination. Microsatellites are short DNA motifs, 2-6 bases (e.g GATA, GACA, etc.), tandemly repeated five to fifty times ( Table 2). ...
... Microsatellites are short DNA motifs, 2-6 bases (e.g GATA, GACA, etc.), tandemly repeated five to fifty times ( Table 2). Their sequence lengths are intraand interspecific polymorphic across species [56,[69][70][71][72][73]. Moreover, SSRs are characterised by higher mutation rate than the rest of the genome, representing a formidable tool for the genetic differentiation of S. cerevisiae strains, as reported by several papers in last 20 years [46,49,56,[69][70][71][72][73][74][75]. ...
... Their sequence lengths are intraand interspecific polymorphic across species [56,[69][70][71][72][73]. Moreover, SSRs are characterised by higher mutation rate than the rest of the genome, representing a formidable tool for the genetic differentiation of S. cerevisiae strains, as reported by several papers in last 20 years [46,49,56,[69][70][71][72][73][74][75]. Hence, they are optimal molecular markers for the strains typing due to their size polymorphism. ...
From the fundamental studies of Louis Pasteur in the XIX century to the current genomic analysis, the essential role of microorganisms in winemaking industry is well recognised. In the last decades, selected Saccharomyces cerevisiae strains with excellent fermentative behaviour have been widely commercialised in form of active dry yeasts. Currently, the production of organic and "natural" wines represents a new economically relevant trend in the wine sector. Based on this market demand, the use of industrial yeast starter could be perceived as non-organic practice and then, rejected. However, in order to preserve wines sensory quality, healthiness, and to avoid organo-leptic defects given by undesirable microorganisms, the "yeast factor" (S. cerevisiae or non-Saccharomyces) cannot be ignored. The purpose of this chapter is to describe the methods of selection of wine yeasts focusing the attention on indigenous S. cerevisiae strains. In fact, the use of ecotypic yeasts may represent a good compromise between the needs of microbiologically controlled fermentation and a modern vision of wine as natural expression of its "terroir", also from the microbiological point of view.
... Based on the length polymorphism of simple sequence repeat loci dispersed throughout the entire genome, microsatellite analysis has successfully been used to discriminate S. cerevisiae strains (Field and Wills, 1998;Gonzalez Techera et al., 2001;Pe´rez et al., 2001;Hennequin et al., 2001;Malgoire et al., 2005). Recently, multiplex PCR of microsatellite loci with high degrees of polymorphism has allowed the separation of a large number of commercial yeast strains (Schuller et al., 2004;Bradbury et al., 2005;Legras et al., 2005). ...
... To construct a rapid and sufficiently discriminatory routine method applicable even in low-budget, highthroughput laboratories, we have implemented multiplex amplification of three highly polymorphic microsatellite loci (Gonzalez Techera et al., 2001), followed by analysis of the band patterns generated by agarose gel electrophoresis. ...
... Three microsatellite loci, SC8132X, YOR267C and SCPTSY7, were used because of their high degree of polymorphism (Field and Wills, 1998;Gonzalez Techera et al., 2001). Amplification was performed with pairs of specific forward and reverse primers ( Table 2). ...
... Genetic characterization was performed using 11 highly polymorphic S. cerevisiae-specific microsatellite loci: ScAAT1, ScAAT2, ScAAT3, ScAAT4, ScAAT5, ScAAT6, ScYPL009c, ScYOR267c, C4, C5 and C11 (Field and Wills, 1998;Legras et al., 2005;Perez et al., 2001;Schuller et al., , 2012Techera et al., 2001). Multiplex PCR mixtures and cycling conditions were optimized and performed in 96-well PCR plates, as previously described (Franco-Duarte et al., 2009). ...
... All 172 strains were genetically characterized regarding allelic combinations for the previously described microsatellites ScAAT1, ScAAT2, ScAAT3, ScAAT4, ScAAT5, ScAAT6, ScYPL009c, ScYOR267c, C4, C5 and C11 (Field and Wills, 1998;Legras et al., 2005;Perez et al., 2001;Schuller and Casal, 2007;Schuller et al., , 2012Techera et al., 2001). As shown in Table 1, a total of 280 alleles was obtained; microsatellites ScAAT1 and ScAAT5 were the most and the least polymorphic, with 39 and 5 alleles, respectively. ...
... Summary of the distribution of alleles (indicated in numbers of repetitions) among 172 Saccharomyces cerevisiae strains from 11 microsatellite loci *A,Techera et al., 2001; B, Perez et al., 2001;C, Field and Wills, 1998;D, Legras et al., 2005. ...
Genome sequencing is essential to understand individual variation and to study the mechanisms that explain relations between genotype and phenotype. The accumulated knowledge from large-scale genome sequencing projects of Saccharomyces cerevisiae isolates is being used to study the mechanisms that explain such relations. Our objective was to undertake genetic characterization of 172 S. cerevisiae strains from different geographical origins and technological groups, using 11 polymorphic microsatellites, and computationally relate these data with results of 30 phenotypic tests. Genetic characterization revealed 280 alleles, with microsatellite ScAAT1 contributing the most to intra-strain variability, together with the alleles 20, 9 and 16, from the microsatellites ScAAT4, ScAAT5 and ScAAT6. These microsatellite allelic profiles are characteristic both for the phenotype and the origin of yeast strains. We confirm the strength of these associations by construction and cross-validation of computational models that can predict the technological application and origin of a strain from the microsatellite allelic profile. Associations between microsatellites and specific phenotypes were scored using information gain ratio, and significant findings were confirmed by permutation tests and estimation of false discovery rate. The phenotypes associated with higher number of alleles were the capacity to resist to sulphur dioxide (tested by the capacity to grow in the presence of potassium bisulphite) and the presence of galactosidase activity. Our study demonstrates the utility of computational modelling to estimate a strain technological group and phenotype from microsatellite allelic combinations as tools for preliminary yeast strain selection. This article is protected by copyright. All rights reserved.
... To ensure a successful fermentation, the presence of S. cerevisiae is essential for winegrowers. For a reliable and predictable fermentation process, the must fermentation can be carried out by adding a sufficient amount of a pure culture of a selected commercially available S. cerevisiae yeast strain [10]. Consequently, this will prevent stuck fermentation and off-flavors that may be generated by other microorganisms naturally present on grapes [11]. ...
... The fragment lengths obtained by the primers are in the same range as the values reported in the literature [10,26,27,32,33]. ...
Considering that many Saccharomyces cerevisiae strains exist and that they have different fermentation capacities, the challenge is to select the yeast strain that generates the most interesting wine character and wine flavor for the winemaker. A method based on simple sequence repeats (SSRs) markers, occurring in the yeast genome, was developed to differentiate the collected S.cerevisiae strains. For the amplification of the polymorphic SSR markers performed by polymerase chain reaction (PCR), two primer sets showing different size products for different S. cerevisiae strains were designed. The PCR-method with gel electrophoresis was validated using capillary sequencing and then used as a service for winegrowers combined with a sensory analysis via napping. This approach can be used for the preservation of the yeast diversity associated with given terroirs and as an option for an increased safety of fermentations. The application of S. cerevisiae strains collected in spontaneous fermentations and used for fermentation sustains the initial character of the wine and ensures a secure fermentation at the same time.
... This manuscript reports, for the first time, automated measurement of cell viability using a machine learning algorithm implemented on lens-free reconstructed images of colour-stained cells and demonstrates the success of this computational approach in measuring the viability and concentration of Saccharomyces cerevisiaethe most common yeast species used in the food, alcoholic beverage, and biofuel industries. 10,22,23 There exist many strains within this species with very similar morphology and size. 22,24,25 Among these, we selected the distillers active dry yeast of the Saccharomyces cerevisiae due to its wide-scale use in various applications and industries. ...
... 10,22,23 There exist many strains within this species with very similar morphology and size. 22,24,25 Among these, we selected the distillers active dry yeast of the Saccharomyces cerevisiae due to its wide-scale use in various applications and industries. By varying the viability and concentration of these yeast cells in our experiments, we compared AYAP's performance with fluorescence exclusion staining using regression analysis. ...
Monitoring yeast cell viability and concentration is important in brewing, baking and biofuel production. However, existing methods of measuring viability and concentration are relatively bulky, tedious and expensive. Here we demonstrate a compact and cost-effective automatic yeast analysis platform (AYAP), which can rapidly measure cell concentration and viability. AYAP is based on digital in-line holography and on-chip microscopy and rapidly images a large field-of-view of 22.5 mm(2). This lens-free microscope weighs 70 g and utilizes a partially-coherent illumination source and an opto-electronic image sensor chip. A touch-screen user interface based on a tablet-PC is developed to reconstruct the holographic shadows captured by the image sensor chip and use a support vector machine (SVM) model to automatically classify live and dead cells in a yeast sample stained with methylene blue. In order to quantify its accuracy, we varied the viability and concentration of the cells and compared AYAP's performance with a fluorescence exclusion staining based gold-standard using regression analysis. The results agree very well with this gold-standard method and no significant difference was observed between the two methods within a concentration range of 1.4 × 10(5) to 1.4 × 10(6) cells per mL, providing a dynamic range suitable for various applications. This lensfree computational imaging technology that is coupled with machine learning algorithms would be useful for cost-effective and rapid quantification of cell viability and density even in field and resource-poor settings.
... The complete sequence of S. cerevisiae genome allows for the identification of these regions. Some of the most frequently utilized loci include YOR267C, SC8132X and SCPTSY7 (Techera et al., 2001). These loci can also be used for multiplex-PCR reactions where two or more loci are amplified (Vaudano & Garcia-Moruno, 2008;Richards et al., 2009). ...
... Results are expressed as a number of repeats of the loci. These loci have been identified and used in studies to successfully discriminate between S. cerevisiae strains (Techera et al., 2001) and evaluated to distinguish between commercially available yeast strains (Vaudano & Garcia-Moruno, 2008). INTRODUCTION ...
“Natural wines”, nowadays, could appear an evocative recall to what wine was like before. During the ages, science allowed winemakers to produce wines with the full control of
vinification condition, obtained mainly by addition of exogenous components, supplements
and microorganisms. Contrarily, “natural wine” come back to basics and simplicity as invasive operations are avoided; but, on the other hand, a deeper knowledge and monitoring of winemaking process is required, in order to obtain high quality products
without exogenous substances. Natural wine has been produced by spontaneous
fermentation of must by yeasts originated from grapes and winery equipment. The wide variety of natural yeasts reflects the biodiversity, which is still under-exploited despite the
large use of commercial Saccharomyces cerevisiae in most grape musts. During
fermentation, several strains compete in the same fermenting must, and the dominance of Saccharomyces cerevisiae takes place when it overcomes all the others. The aim was to investigate Saccharomyces cerevisiae diversity and its technological behavior in two
biodynamic wine farms in Tuscany. Autochthonous S. cerevisiae from Syrah fermentations
were isolated and molecularly characterized in 2009 and 2013 harvests. Saccharomyces cerevisiae strains were isolated starting from Syrah musts with zero or low levels of
sulphite added, according with the tendency of natural wines producers to abait every
invasive operation during wine making. Samples were collected in different steps of winemaking. ITS-PCR method confirmed isolates belong to Saccharomyces sensu stricto complex. The multiplex PCR amplification of microsatellite loci (SC8132X, YOR267C and
SCPTSY7) discriminated S. cerevisiae strains. Genetically diverse S. cerevisiae strains
were subsequently subjected to technological characterization. Micro-fermentations were set up to study fitness and quality traits of biotypes characterized. Weight loss kinetics
were measured and chemical analyses were performed. Collected Saccharomyces
cerevisiae strains were tested also in order to verify the effect of low sulphite additions on biodiversity pursuing information about stress-adaptation mechanisms that allow the
survival in the challenging environment of fermenting must. Sulphite addition may act as a
selective factor to induce the presence and the activity of strains with diverse features about sulphite metabolism. The addition of different dose of sulphite induced a considerable variability among strains. However, some biotypes exhibited tolerance to
certain concentration and only one biotype was classified as resistant to a determinate
concentration. In addition, biotypes were subjected to an other environmental stress factor such as ethanol. The increasing of levels of ethanol limited considerably the activity of selected strains, but some of them highlighted a positive tolerance to various ethanol
concentrations. During the harvest of 2013, the alcoholic fermentation was conducted
inoculating a biotype characterized previously, which expressed good fermentative performance and a multi-strains culture of other S. cerevisiae strains characterized in the
same harvest (2009) in a biodynamic farm from a workgroup of Edmund Mach Foundation
of San Michele all'Adige (TN). The results of 2009 highlighted peculiar behaviors of 13 biotypes (characterized from four hundred isolates) that dominate the spontaneous fermentations. Not surprisingly, some biotypes were also found in fermentations of 2013,
demonstrating yeasts are ubiquitous in that environment. Sensorial analyses of wine,
originated by biotypes fermentation, revealed certain diversity in aroma and flavor traits, but also in the structure. Overall, this work suggests the consolidate relationship existing
between autochthonous yeasts and the terroir, where yeasts are ubiquitous. Furthermore,
autochthonous yeasts surely contribute to the unique identity of wine. In addition, this work suggests that the use of isolated and characterized autochthonous yeast strains could help the winemaking process, where the vintage could be inadequate to develop a particular product such as the wine of a specific wine farm.
... The same six SSR loci mentioned by Duarte et al. [1] were used, namely ScAAT1, ScAAT2, ScAAT3, ScAAT5, SCYOR267C and SC8132X [19,20]. SSR amplifications were conducted in two multiplex reactions as previously described [1]. ...
Wine production has developed from spontaneous to controlled fermentations using commercial active dry yeasts (ADY). In this study, S. cerevisiae commercial ADY were tested, and yeast community dynamics were monitored at different fermentation stages in three winery-based trials with volumes ranging from 60 L to 250 hL. The differentiation of S. cerevisiae strains was achieved using microsatellite markers. In Experiment 1, results showed that both ADY strains revealed similar profiles, despite being described by the producer as having different properties. In Experiment 2, higher genetic diversity was detected when co-inoculation was tested, while in sequential inoculation, the initial ADY seemed to dominate throughout all fermentation. Pilot-scale red wine fermentations were performed in Experiment 3, where one single ADY strain was tested along with different oenological additives. Surprisingly, these trials showed an increase in distinct profiles towards the end of fermentation, indicating that the dominance of the ADY was lower than in the blank modality. The use of ADY is envisaged to promote a controlled and efficient alcoholic fermentation, and their purchase represents an important cost for wineries. Therefore, it is most relevant to survey commercial ADY during wine fermentation to understand if their use is effective.
... The preference for specific fermentation techniques appears to be a result of cultural histories, market demand, and the degree and timing of industrialization of food processing more broadly (Tamang et al. 2017). Often the same food item will be fermented a different way depending on local norms (Birch et al. 2013;Di Cagno et al. 2008;González Techera et al. 2001;Pretorius 2000;Sun et al. 2009;Xu et al. 2018). For example, China and India rely heavily on spontaneous fermentation to produce many foods including rice wine, vinegar, and traditional fermented fish and vegetable dishes (Devi, Deka, and Jeyaram 2015;Satish Kumar et al. 2013;Wang et al. 2016;Xu et al. 2018), while populations such as Japan and Italy tend to favor a more selective, industrial process for foods including soy sauce, beans, meat, and cheese (Gambelli et al. 1999;Luh 1995;Tabanelli et al. 2012;Yamada 1977). ...
Fermented foods are an important part of the human diet. While the types of fermented foods consumed as well as the processes used to create them vary regionally, the majority of human populations globally deliberately produce and consume fermented foods as a central part of their diets. This pattern is in contrast to that of other vertebrates, including nonhuman primates. However, it remains unclear when and why humans began to include high amounts of fermented foods in their diets. Here, we review existing knowledge regarding the timing and impetus for the emergence of fermented food use by humans and put forth a new hypothesis that fermented food use began as a “predigestion” strategy to increase nutrient availability in harsh, terrestrial environments with physically and chemically defended food resources. We explore support for this hypothesis in comparative behavioral and physiological data from extant nonhuman primates. Together the data presented in this paper suggest that food fermentation may have had an impact on human evolutionary trajectories via interactions with human biology and health. Future work should build on this foundation to interrogate these processes in more detail. © 2021 The Wenner-Gren Foundation for Anthropological Research. All rights reserved.
... Yeasts were genetically identified as Saccharomyces cerevisiae through DNA microsatellite markers (González Techera et al., 2001); the non-Saccharomyces were identified by sequencing the variable D1/D2 region of the 26S rDNA gene. Nucleotide differences of ≤1% in the D1/ D2 region were considered to be yeast strains of the same species (Kurtzman and Robnett, 1998). ...
Recently, the increase in microbreweries and the consequent production of craft beers have reached exponential growth. The interest in non-conventional yeasts for innovation and a unique selling feature in beer fermentation is increasing. This work studied the autochthonous Saccharomyces and non-Saccharomyces yeasts, isolated from various food sources, with the ability to modify and improve the fermentative and aromatic profiles during alcoholic fermentation. The ability to ferment maltose and produce desirable aroma compounds were considered as the key characters for the screening selection. A synthetic beer wort was developed for this purpose, to simulate beer wort composition.
A total of forty-seven yeast strains belonging to different genera were analysed according to their fermentation profile, volatile compounds production and sensory analysis. Three native strains of Saccharomyces cerevisiae, Zygoascus meyerae and Pichia anomala were selected to evaluate their aromatic profile in single and mixed fermentations. The strains produced 4-vinylguaiacol, β-phenylethyl alcohol, and isoamyl alcohol at levels significantly above the sensory threshold, making them interesting for wheat and blond craft beer styles. The native Hanseniaspora vineae was also included in a co-fermentation treatment, resulting in a promising yeast to produce fruity beers.
... 25,[62][63][64][65][66] . The 2 multiplex PCR contained (for 8 samples) a total of 15.5 μl multiplexed primers, 50 µl of QIAGEN Multiplex PCR kit Master Mix and 18.5 μl water MilliQ. ...
Saccharomyces cerevisiae is the main actor of wine fermentation but at present, still little is known about the factors impacting its distribution in the vineyards. In this study, 23 vineyards and 7 cellars were sampled over 2 consecutive years in the Bordeaux and Bergerac regions. The impact of geography and farming system and the relation between grape and vat populations were evaluated using a collection of 1374 S. cerevisiae merlot grape isolates and 289 vat isolates analyzed at 17 microsatellites loci. A very high genetic diversity of S. cerevisiae strains was obtained from grape samples, higher in conventional farming system than in organic one. The geographic appellation and the wine estate significantly impact the S. cerevisiae population structure, whereas the type of farming system has a weak global effect. When comparing cellar and vineyard populations, we evidenced the tight connection between the two compartments, based on the high proportion of grape isolates (25%) related to the commercial starters used in the cellar and on the estimation of bidirectional geneflows between the vineyard and the cellar compartments.
... AFLP has been used in few studies (de Barros Lopes et al. 1999;Azumi and Goto-Yamamoto 2001). In contrast, the analysis of the polymorphism of microsatellite loci has progressively expanded because of their ease to use (González Techera et al. 2001;Hennequin et al. 2001;Pérez et al. 2001;Howell et al. 2004;. Microsatellite loci are regions A peak is equivalent to a band on one of the gels shown on the left containing a variable number of repeats of motifs of two, three, or four bases, and provide co-dominant data: in case of a diploid heterozygote the two alleles are observed and taken into account for further genetic analysis. ...
Multiple studies have revealed the richness of the fungal microbial community of grape must and wine which are under the influence of environmental and anthropogenic factors. However, until recently, we had no evidence of how historical processes may have impacted the diversity of the yeasts found on grapes and developing during wine alcoholic fermentation. The different generations of molecular methods have provided progressively more and more information for Saccharomyces cerevisiae, and recently for other yeast species of the grape must microbial communities, gradually revealing how the development of wine technology has shaped the diversities of wine yeasts. This chapter presents the recent advances made on the knowledge of the diversities of yeast species of the grape must and wine microbial community. For the model species S. cerevisiae, the successive generations of genotyping methods and population genomics have offered the most comprehensive view of its evolution including population structure, genomic specificities, and demographic history. The development of molecular methods revealed as well the importance of other Saccharomyces species and their related interspecies hybrids with S. cerevisiae. More recently, the exploration of the diversities of non-Saccharomyces yeast species members of the grape must and wine community such as Hanseniaspora uvarum, Torulaspora delbrueckii, Brettanomyces bruxellensis, Lachancea thermotolerans and Starmerella bacillaris revealed specific population structures.
... AFLP has been used in few studies (de Barros Lopes et al. 1999;Azumi and Goto-Yamamoto 2001). In contrast, the analysis of the polymorphism of microsatellite loci has progressively expanded because of their ease to use (González Techera et al. 2001;Hennequin et al. 2001;Pérez et al. 2001;Howell et al. 2004;. Microsatellite loci are regions A peak is equivalent to a band on one of the gels shown on the left containing a variable number of repeats of motifs of two, three, or four bases, and provide co-dominant data: in case of a diploid heterozygote the two alleles are observed and taken into account for further genetic analysis. ...
Multiple studies have revealed the richness of the fungal microbial community of grape must and wine which are under the influence of environmental and anthropogenic factors. However, until recently, we had no evidence of how historical processes may have impacted the diversity of the yeasts found on grapes and developing during wine alcoholic fermentation. The different generations of molecular methods have provided progressively more and more information for Saccharomyces cerevisiae, and recently for other yeast species of the grape must microbial communities, gradually revealing how the development of wine technology has shaped the diversities of wine yeasts. This chapter presents the recent advances made on the knowledge of the diversities of yeast species of the grape must and wine microbial community. For the model species S. cerevisiae, the successive generations of genotyping methods and population genomics have offered the most comprehensive view of its evolution including population structure, genomic specificities, and demographic history. The development of molecular methods revealed as well the importance of other Saccharomyces species and their related interspecies hybrids with S. cerevisiae. More recently, the exploration of the diversities of non-Saccharomyces yeast species members of the grape must and wine community such as Hanseniaspora uvarum, Torulaspora delbrueckii, Brettanomyces bruxellensis, Lachancea thermotolerans and Starmerella bacillaris revealed specific population structures.
... These last decades, several studies have been focused on genetic diversity and biogeographical distribution of diverse S. cerevisiae strains. Molecular techniques used for this purpose among many others are pulsed field gel electrophoresis (PFGE) (Vezinhet et al., 1990), restriction analysis of the mitochondrial DNA (mtDNA-RFLP) (Aigle et al., 1984;Querol et al., 1992), amplified fragment length polymorphism (AFLP) (Azumi and Goto-Yamamoto, 2001), amplification of interdelta regions by PCR (Ness et al., 1993;Legras and Karst, 2003) and microsatellite markers (Techera et al., 2001;Legras et al., 2005). Microsatellite analyses have offered significant advances in understanding distribution and dynamics of S. cerevisiae population from different terroirs. ...
... Differences in the number of repeats are identified as length polymorphisms in PCR products by using high-resolution electrophoresis, including automatic DNA sequencers. Microsatellite codominant polymorphisms have proven very useful for strain discrimination (González-Techera et al., 2001;Pérez et al., 2001;Legras et al., 2005;Masneuf-Pomarède et al., 2007), for the genetic analysis of yeast populations (Ayoub et al., 2006;Legras et al., 2007), and to also determine levels of heterozygosity and ploidy (Bradbury et al., 2006;Katz Ezov et al., 2006). ...
The processes of yeast selection for using as wine fermentation starters have revealed a great phenotypic diversity both at interspecific and intraspecific level, which is explained by a corresponding genetic variation among different yeast isolates. Thus, the mechanisms involved in promoting these genetic changes are the main engine generating yeast biodiversity. Currently, an important task to understand biodiversity, population structure and evolutionary history of wine yeasts is the study of the molecular mechanisms involved in yeast adaptation to wine fermentation, and on remodeling the genomic features of wine yeast, unconsciously selected since the advent of winemaking. Moreover, the availability of rapid and simple molecular techniques that show genetic polymorphisms at species and strain levels have enabled the study of yeast diversity during wine fermentation. This review will summarize the mechanisms involved in generating genetic polymorphisms in yeasts, the molecular methods used to unveil genetic variation, and the utility of these polymorphisms to differentiate strains, populations, and species in order to infer the evolutionary history and the adaptive evolution of wine yeasts, and to identify their influence on their biotechnological and sensorial properties.
... A number of authors have previously identified microsatellite markers useful for distinguishing yeast strains (Field and Wills 1998;Gonzalez Techera et al. 2001;Hennequin et al. 2001;Perez et al. 2001;Legras et al. 2005). We identified seven new AT dinucleotide loci that proved to be polymorphic, although none were as informative as the trinucleotide locus, YML091c, which has been widely used (Field and Wills 1998;Perez et al. 2001;Howell et al. 2004;Legras et al. 2005). ...
... In spite of clear differences in colony morphology and cell arrangement, in depth analysis into genetic differences between smooth and rough-colony strains have failed to reveal the presence of any underlying variations at a DNA level so far. The PCR microsatellite methodology has been extensively used for S. cerevisiae strain identification especially when assessing the wine fermentation populations [15][16][17] ; more recently this technology has been used for assessing the biodiversity of native bioethanol yeast strains. 18 This technique has been revealed to be sensitive and robust enough to detect the extensive genetic diversity of the indigenous strains of S. cerevisiae in Brazilian ethanol-producing units. ...
Strains of Saccharomyces cerevisiae may display characteristics that are typical of rough-type colonies, made up of cells clustered in pseudohyphal structures and comprised of daughter buds that do not separate from the mother cell post-mitosis. These strains are known to occur frequently in fermentation tanks with significant lower ethanol yield when compared to fermentations carried out by smooth strains of S. cerevisiae that are composed of dispersed cells. In an attempt to delineate genetic and phenotypic differences underlying the two phenotypes, this study analysed 10 microsatellite loci of 22 S. cerevisiae strains as well as stress resistance towards high concentrations of ethanol and glucose, low pH and cell sedimentation rates. The results obtained from the phenotypic tests by Principal-Component Analysis revealed that unlike the smooth colonies, the rough colonies of S. cerevisiae exhibit an enhanced resistance to stressful conditions resulting from the presence of excessive glucose and ethanol and high sedimentation rate. The microsatellite analysis was not successful to distinguish between the colony phenotypes as phenotypic assays. The relevant industrial strain PE-2 was observed in close genetic proximity to rough-colony although it does not display this colony morphology. A unique genetic pattern specific to a particular phenotype remains elusive.
... For each of them, a small amount of fresh colony was suspended in 50 l of Milli-Q water, and 7 l of this suspension was dropped on an FTA card for DNA preservation. The samples were then genotyped using 2 multiplex PCRs of 8 and 7 microsatellite loci, respectively, for mixtures 1 and 2 (see Table S3 in the supplemental material) (32)(33)(34)(35)(36)(37). Mixtures were prepared in a total volume of 84 l for 8 samples, with 50 l of 2ϫ Qiagen Multiplex PCR master mix, 15.5 l primers, and 18.5 l water. ...
Three wine estates were sampled in Sauternes, a typical appellation of Bordeaux wine area producing sweet white wine. Over those wine estates, 551 yeasts strains were collected between 2012 and 2014 adding 102 older strains from 1992 to 2011 from wine estate C. All strains were analyzed through 15 microsatellites markers resulting in 503 unique
Saccharomyces cerevisiae
genotypes, thus revealing a high genetic diversity, and a low presence of commercial yeast starters. Population analysis performed from Fst statistics, or from ancestry profiles, revealed that the two closest wine estates, B and C which have juxtaposed vineyard plots and common seasonal staff, share more related isolates together than with wine estate A indicating exchange between estates. The characterization of isolates collected 23 years ago in wine estate C related to recent isolates obtained in wine estate B revealed the long-term persistence of isolates. Last, during the 2014 harvest period, a temporal succession of ancestral subpopulations related to the different batches associated to the selective picking of noble rotted grapes was highlighted.
Importance
A high genetic diversity of
S. cerevisiae
isolated from spontaneous fermentation in wine estates in the Sauternes appellation of Bordeaux were revealed. Only 7% of all Sauternes strains were considered as genetically related to specific commercial strains. The long-term persistence (over 20 years) of
S. cerevisiae
profils in a given wine estate was highlighted for the first time.
... In this medium H. vineae growth is identified by its intense brilliant green colour, whereas strain ALG804 is a matt creamy colour. Native S. cerevisiae were identified and differentiated using microsatellite markers and involves DNA extraction and PCR analysis as described previously (Gonzalez Techera, Jubany, Carrau, & Gaggero, 2001;Pérez et al., 2011). Identification and analysis of native isolates was done using previously developed methods and the Saccharomyces industrial strains data bank (Jubany et al., 2008). ...
... i) There is no requirement for sequence data or prior knowledge of genetics in the organism to be studied. The same AFLP primers can be used for any species, whereas for other high resolution methods such as Microsatellites new primers must be developed in each new species ( González Techera et al. 2001); ii) Large numbers of polymorphic loci from multiple regions of the genome are generated, giving the method high resolution. Some studies have suggested they are dispersed throughout the genome but not evenly ( Zhu et al. 1998), or that the polymorphic markers might favour naturally variable regions (van den Braak et al. 2003). ...
... DNA extraction was carried out with MasterPureTM Yeast Kit (Epicentre Technologies Corp., Madison, WI 53719, USA.) as described by the manufacturer. In order to identify the yeasts as the commercial yeast strains that were used for the inoculation, a microsatellite multiplex PCR analysis with the primers C5, C11 and SCYOR267X was carried out according to Legras et al. (2005) and a PCR SC8132X according to Gonzalez Techera et al. (2001) (Table 1) ...
Five different yeasts (X5, VL3, Delta, Revelation Thiols and Alchemy II) and one yeasts mixture (X5+Alchemy II) were tested for the ability to liberate volatile thiols during alcoholic fermentation. The chemical composition and the sensory attributes of the Sauvignon blanc wines were monitored. The yeasts applied provoked significant differences regarding the production of 4-mercapto-4-methylpentan-2-one (4MMP) and 3-mercaptohexan-1-ol (3MH). Fermentation aroma substances isoamyl acetate, phenylethyl acetate, 2-phenylethanol and ethyl hexanoate were as well significantly influenced by the yeasts used. Co-inoculated fermentation of different yeast strains can produce wines with different chemical composition and sensory profile, compared to wines produced from the same must with pure strains. In this experiment, the yeasts used for fermentation were not just for the aroma production a deciding factor, but as well for a successful alcoholic fermentation. Not all the yeasts applied fermented the grape juice to dryness.
... Mixed DNA samples of host and pathogen may be used, provided that stringent thermal cycling conditions are maintained (Weising, Atkinson & Gardner, 1995;Walczak et al., 2007). Microsatellite genotyping continues to play an important analytical role, particularly in the differentiation of phytopathogenic fungi (Kim et al., 2000;Mohali, Burgess & Wingfield, 2005), industrial fungal strains (González-Techera et al., 2001;Legras et al., 2005) and medically important species (Foulet et al., 2005). The utility of STR genotyping in other eukaryotes and mammals has resulted in the development of equipment and methods for high Application of the general tools available for STR genotyping can benefit analysis of fungi by this marker system. ...
Advanced molecular genetic techniques enhance our capabilities to identify and characterize microbial pathogens, resulting
in accurate testing for pathogen identification, sub-species-level DNA fingerprinting, pathogen-load testing and disease spread
monitoring. These applications are instrumental to the study of plant disease epidemiology, so that adequate control measures
can be accordingly implemented. In this chapter, a survey of the most popular DNA profiling techniques is presented together
with some of the newer and most discriminating molecular methods. Combinations of different analytical techniques are also
proposed as a useful approach for low throughput bioassays. Advantages and disadvantages of each single test are taken into
account and key issues (sampling, validation, large-scale testing, etc.), encountered in the practical application of these
assays, are discussed. An outline of emerging high-throughput molecular technologies expected to improve diagnostic approaches
and aid disease management is also provided.
... The yeast strains examined were isolated from fermentations conducted in different Uruguayan wineries. They were identified as Saccharomyces cerevisiae (strains CP881, CP882, CP863, CP874, CP873, and KU1) through DNA microsatellite markers (González Techera et al. 2001). Two commercial yeasts were used as controls: Montrachet 522 (University of California, Davis) and Lalvin 254 D (Lallemand, Montreal, Canada). ...
The capacity of yeast to modify anthocyanin concentration during red wine fermentation was studied with the objective of classifying yeast strains according to their capacity for color removal during vinification. A model red grape juice medium was used to better understand yeast-anthocyanin interactions. An anthocyanin extract of Vitis vinifera cv. Tannat was prepared from grape skins and diluted in a white must in order to obtain a red grape juice without solids. Anthocyanin removal was confirmed to be higher for compounds with higher polarity. Acylated anthocyanins (acetyl and p-coumaryl compounds) were differentially removed, and the percentage of removal for each compound was determined. Results showed no correlation between color intensity and total anthocyanin concentration after fermentation, possibly because of the presence of anthocyanin derivatives formed during the process. HPLC-MS analysis allowed identification of anthocyanin derivatives, while the quantification of several derivatives was performed by HPLC-DAD. The sum of these derivatives showed a direct correlation with the color intensity obtained with each strain, thus explaining the color variability observed. Copyright © 2005 by the American Society for Enology and Viticulture. All rights reserved.
... Beaucoup de méthodes ont été développées pour le typage des S. cerevisiae, dont les premières étaient l'analyse des caryotypes et la restriction de l'ADN mitochondrial et ces deux méthodes ont été très adoptées (Querol et al., 1992b ;, Nadal et al., 1996Codon et al., 1998;Nadal et al 1999;Lopez et al., 2001). L'amplification des régions entre deux LTR delta de transposons est aussi très largement appliquée alors que le typage des S. cerevisiae par l'analyse de marqueurs microsatellites est encore moins utilisé (Gallego et al., 1998;Hennequin et al., 2001, Techera et al., 2001. D'autres méthodes basées sur des amplifications par PCR ont aussi été développées comme les amplifications d'introns (de Barros Lopez, 1998 ;Lopez et al., 2003) ou la RAPD qui peut être associée à des analyses de restriction ou au séquençage (Quesada et al., 1995 ;McGrath et al., 1998 ;Perez et al., 2001a). ...
We explored in this study the biodiversity of the fermenting indigenous Lebanese flora
of Saccharomyces cerevisiae by collecting samples of natural fermentations of grape musts
from various localities dispersed within the Lebanese territory. A great molecular diversity of
the Lebanese flora was found in the natural fermentations, as well as in the wineries as a
whole and between different geographic localities. However, cases of dominance and
perenniallity were also recorded in wineries. Besides, fermentations in the same wineries
seemed to involve lineages of related strains. Relatedness of strains was also seen in narrow
geographical areas while it was lost as the areas enlarged. Despite its diversity, the Lebanese
flora could nevertheless be interrelated, which property was observed when this flora was
compared to other oenological floras of diverse origins. Finally, it seemed that the effect of
the thriving media was greater on the lineages of fermenting floras than the effect of the
geographical distance.
We also evaluated the usefulness of the molecular typing methods used for assessing the
biodiversity of the S. cerevisiae floras. Two of them, interdelta fingerprinting and
microsatellites analysis, were previously developed, while we tested a new method, the
MLST. The first two methods were very useful for typing due to the great variability of their
markers and thus to their great discriminatory power, while MLST showed a lower
discriminatory power. But our MLST scheme could probably be improved by using more
variable loci. MLST was nonetheless more useful in inferring strains relatedness, especially
for poorly related isolates.
We further examined the phenotypic diversity that accompanies the molecular one in
order to initiate a selection scheme for some oenological strains and also to verify the
correspondence between the molecular and the phenotypic diversity. The studied strains of S.
cerevisiae presented different fermenting capacities which could be exploited to push further
the selection scheme for potential commercial uses, in particular in the Lebanese winemaking
industry. On the other hand, a certain congruence between the molecular and the phenotypic
diversities was observed and it was mainly due to a correlation between high acetaldehyde
productions and particular molecular profiles. If this path is studied further, this congruence
might help predict acetaldehyde production for particular strains
... Although it is difficult to distinguish and identify S. cerevisiae at the strain level using classic biochemical methods, numerous molecular methods have been developed that provide new approaches to yeast strain differentiation. These methods include randomly amplified polymorphic DNA (RAPD) [1,5,25,35] mitochondrial DNA (mtDNA) restriction analysis [12,18,24,32], application of microsatellites [10,14,15,28,30], and PCR amplification of the mitochondrial gene COX1 [19]. Among these techniques, interdelta sequence typing has been proven to be a convenient method to monitor the diversity of S. cerevisiae strains because it is rapid, reproducible, and sensitive [29]. ...
The diversity and composition of yeast populations may greatly impact wine quality. This study investigated the yeast microbiota in two different types of wine fermentations: direct inoculation of a commercial starter versus pied de cuve method at an industrial scale. The pied de cuve fermentation entailed growth of the commercial inoculum used in the direct inoculation fermentation for further inoculation of additional fermentations. Yeast isolates were collected from different stages of wine fermentation and identified to the species level using Wallersterin Laboratory nutrient (WLN) agar followed by analysis of the 26S rDNA D1/D2 domain. Genetic characteristics of the Saccharomyces cerevisiae strains were assessed by a rapid PCR-based method, relying on the amplification of interdelta sequences. A total of 412 yeast colonies were obtained from all fermentations and eight different WL morphotypes were observed. Non-Saccharomyces yeast mainly appeared in the grape must and at the early stages of wine fermentation. S. cerevisiae was the dominant yeast species using both fermentation techniques. Seven distinguishing interdelta sequence patterns were found among S. cerevisiae strains, and the inoculated commercial starter, AWRI 796, dominated all stages in both direct inoculation and pied de cuve fermentations. This study revealed that S. cerevisiae was the dominant species and an inoculated starter could dominate fermentations with the pied de cuve method under controlled conditions.
... Saccharomyces sp. is the safest and most effective microorganism for fermenting sugars to ethanol and is used in the industry for bioalcohol production (Yoon et al., 2003). Besides, several alcoholic beverages such as wine or liqueurs are obtained from fruit juices fermented by Saccharomyces cerevisiae strains (González et al., 2001). Moreover, S. cerevisiae has been employed to remove undesired compounds by fermentation, such as monosaccharide and disaccharide by-products in carbohydrate preparations (Yoon et al., 2003) and benzoic acid in concentrated lingo berry juices (Visti et al., 2003). ...
The aim of the present study was to evaluate the effect of the inoculation of the heated aqueous red
beetroot extracts (30, 50 and 60°C) by different concentrations of Saccharomyces cerevisiae (0.50, 0.75 and 1.00%). The optimum experimental temperature value and S. cerevisiae concentration were found
to be 50°C and 0.50% for both sugar consumption and pigment preservation. The antioxidant capacity
measured by ABTS•+ assay (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical cation)
shows that this extract present an appreciable activity (EC50= 10.77 µg.mL-1
) above the tested samples.
The betalains compounds were analyzed and identified using UPLC LTQ Orbitrap MS method. Betalains
(vulgaxanthin I, betanin, isobetanin, betanidin and neobetanin) were determined in the fermented
extract.
... In this medium H. vineae growth is identified by its intense brilliant green colour, whereas strain ALG804 is a matt creamy colour. Native S. cerevisiae were identified and differentiated using microsatellite markers and involves DNA extraction and PCR analysis as described previously (Gonzalez Techera, Jubany, Carrau, & Gaggero, 2001;Pérez et al., 2011). Identification and analysis of native isolates was done using previously developed methods and the Saccharomyces industrial strains data bank (Jubany et al., 2008). ...
... Thus, microsatellites are considered mutational "hot spots". They show a substantial level of polymorphism between individuals of the same species and are extensively used for paternity exclusion tests (HELMINEN et al. 1988), forensic medicine (HAGELBERG et al. 1991) and for molecular typing of different organisms including cultivars of Vitis vinifera (BOWERS et al. 1999 a and b) and wine yeast strains (GONZÁLEZ TECHERA et al. 2001). While 6 polymorphic microsatellite loci are usually enough to identify a grapevine variety with a very low probability of error, finding differences among clones of the same variety requires the analysis of a larger number of SSR markers. ...
DNAs from 9 clones of cv. Tannat (Vitis vinifera) were analyzed at 89 microsatellite loci. Only one, VMCNg 1d12, showed a differential pattern that separated the clones in two groups. The statistical analysis of concentrations for aroma compounds from microvinifications also resulted in the same two groupings of clones. Many analyzed micro-satellite loci amplified only one allele, implying that Tannat is a highly homozygous variety. For a given set of 15 micro-satellites the level of homozygosity was 53 % for Tannat, in contrast to 6 % for Pinot, 20 % for both Cabernet Franc and Chardonnay and 33 % for Cabernet Sauvignon. We provide molecular data for Tannat, originating from south-western France and nowadays becoming the emblematic cultivar of Uruguayan fine red wines. We also report a cor-relation between aroma-related compounds and molecular markers within clones of a cultivar. K e y w o r d s : microsatellite loci, Tannat clones, Tannat wine aromas.
Reliable typing of yeast strains is of great importance to the alcoholic beverage industry to ensure a reliable fermentation process and high-quality products. Saccharomyces cerevisiae is the most used yeast species in wine, sake, and ale beer fermentation, whereas Saccharomyces uvarum is more commonly used for cider fermentation and, due to its cryotolerance, white wine production. We propose a promising method for species identification and strain discrimination of S. cerevisiae and S. uvarum using Raman spectroscopy in combination with convolutional neural networks (CNNs). Raman spectra collected from various S. cerevisiae and S. uvarum strains were accurately classified at the species level using random forest. Cultivation time and temperature did not significantly affect the spectral reproducibility and discrimination capability. An overall accuracy of 91.9% was achieved to discriminate 27 yeast isolates at the strain level using a CNN model. Raman-CNN further identified eight yeast isolates spiked in grape juice with an accuracy of 98.1%. Raman spectral signatures derived from diverse protein and lipid compositions may contribute to this discrimination. The proposed approach also precisely predicted the concentration of a specific yeast strain within a yeast mixture with an R ² of 0.9913 and an average error of 4.09%. The entire analysis can be completed within 1 hour following cultivation and only requires simple sample preparation and low consumable cost. Taken together, Raman spectroscopy coupled with CNN is a robust, accurate, and reliable approach for typing of fermentation yeast strains.
IMPORTANCE
The use of S. cerevisiae and S. uvarum yeast starter cultures is a common practice in the alcoholic beverage fermentation industry. As yeast strains from different or the same species have variable fermentation properties, rapid and reliable typing of yeast strains plays an important role in the final quality of the product. In this study, Raman spectroscopy combined with CNN achieved accurate identification of S. cerevisiae and S. uvarum isolates at both the species and strain levels in a rapid, non-destructive, and easy-to-operate manner. This approach can be utilized to test the identity of commercialized dry yeast products and to monitor the diversity of yeast strains during fermentation. It provides great benefits as a high-throughput screening method for agri-food and the alcoholic beverage fermentation industry. This proposed method has the potential to be a powerful tool to discriminate S. cerevisiae and S. uvarum strains in taxonomic, ecological studies and fermentation applications.
Yeasts can be identified according to morphological, physiological, and biochemical characteristics by means of fermentation and assimilation tests of various sources of sugars and nitrogen compounds, and morphology of the sexual and asexual states of yeasts. Phenotypic tests present a certain degree of ambiguity, are time consuming, and require considerable knowledge for the interpretation of the results. Although these tests can be used for yeast identification, they are not applied to yeast differentiation at the intraspecific level, as it is desirable in the fermentative environment. DNA-based methods of yeast identification and characterization are more discriminatory, sensitive, and independent of gene expression and environmental conditions than the conventional system based on phenotypic characteristics. In this chapter, the application and limitations of the electrophoretic karyotyping profile are discussed together with the use of other methods such as microsatellites, mitochondrial DNA and sequencing of rDNA genes and regions both for identification and characterization of yeast strains. The physiological characteristics common to native yeasts but absent in Saccharomyces cerevisiae yeasts are also pointed out to explain the use of differential and selective culture media employed for the isolation and quantification of contaminant yeasts.KeywordsSelective mediaEletrophoretic karyotypingMicrosatellitesMitochondrial DNArDNA sequencingYeast identificationYeast characterization
Most alcoholic fermentations in winemaking are nowadays performed by commercially produced yeast strains that are inoculated into the fermentation vessel. Although many yeast strains are available on the global market, the generation of new strains that display different and/or new traits continues to attract significant industrial interest. Many traditional breeding and selection, as well as genetic engineering, approaches have been used to achieve specific targets. These approaches can now be complemented with powerful systems biology tools which provide new insights and opportunities for the generation of new yeast strains while holistically improving our knowledge of wine microbial ecology.
In recent years an increasing trend towards terroir-emphasizing yeasts has emerged, and with that a need to distinguish local strains from commercial available wine yeasts. Therefore, in the present study, a database was set up with the genetic profi les of commercial wine yeasts used in Austria. This is an important tool for the selection and verifi cation of autochthonous wine yeasts from vineyards, wineries or must productions. Three diff erent molecular markers, namely microsatellites, interdelta analysis and M13 fingerprinting, were tested for their ability to differentiate 75 commercial wine yeast strains in a single approach and in combination. It turned out that at least two of the methods should be used for a meaningful comparison and distinct discrimination. Four autochthonous yeasts could be differentiated from their commercially propagated relatives. If a quick initial assessment has to be done, then microsatellite analysis is certainly the favored method.
Differenzierung von kommerziellen Weinhefestämmen mittels molekularer Marker.
In den letzten Jahren hat sich ein zunehmender Trend zu terroir-betonten Hefen herausgebildet, mit dem die Notwendigkeit verbunden ist, lokale Stämme von kommerziell erhältlichen Weinhefen zu unterscheiden. In der vorliegenden Studie wurde daher eine Datenbank mit den genetischen Profilen der in Österreich verwendeten kommerziellen Weinhefen erstellt. Dies ist ein wichtiges Instrument für die Auswahl und Überprüfung von autochthonen Weinhefen aus Weinbergen, Weingütern oder Mostproduktionen. Drei verschiedene molekulare Marker, nämlich Mikrosatelliten, Interdelta-Analyse und M13-Fingerprinting, wurden auf ihre Fähigkeit getestet, 75 kommerzielle Weinhefestämme in einem einzigen Ansatz und in Kombination zu unterscheiden. Es stellte sich heraus, dass mindestens zwei der Methoden für einen aussagekräft igen Vergleich und eine eindeutige Unterscheidung verwendet werden sollten. Vier autochthone Hefen konnten von ihren kommerziell vermehrten Verwandten unterschieden werden. Wenn eine schnelle Ersteinschätzung durchgeführt werden soll, ist die Mikrosatellitenanalyse mit Sicherheit die zu bevorzugende Methode.
Saccharomyces cerevisiae est l’acteur principal de la fermentation du moût de raisin, mais l’influence de facteurs sur sa distribution dans les vignobles est peu connue. La région bordelaise, par son histoire et ses appellations, est une région d’intérêt pour étudier la diversité de S.cerevisiae. Au total, 2422 isolats de S.cerevisiae provenant de prélèvements de raisins et de cuves en fermentation spontanées sur deux années consécutives ont été analysés par 15 à 17 marqueurs microsatellites. Une très grande diversité génétique est mise en évidence, supérieure en mode de conduite conventionnel par rapport au mode biologique. Le mode de conduite influence faiblement la structure de la population de S.cerevisiae au vignoble. L’appellation et le domaine impactent significativement la structure de population, sans que des gradients de diversité n‘apparaissent, mais nos analyses révèlent des connections importantes dans le sens Pessac-Léognan vers les autres appellations du Bordelais, en particulier le Médoc. Des flux importants bidirectionnels sont mis en évidence entre les compartiments vigne et chai, illustrés par la présence de 25% de souches apparentées à des levures commerciales au vignoble, retour des souches du chai au vignoble jusqu’alors sous-estimé, alors qu’un flux d’importance similaire est observé entre le vignoble et le chai. La présence de populations ancestrales communes dans des prélèvements anciens (plus de 20 ans) et récents révèle la stabilité des populations sur le long terme à l’échelle d’une appellation. Une succession temporelle des populations du chai pourrait être favorisée par la mise en œuvre de pied de cuve avec repiquages successifs.
The study of yeast biodiversity represents an important step in the preservation of the local heritage, and this work in particular has an innovative character since no further studies have investigated ‘Merwah’, one of the main grape varieties used in winemaking in Lebanon. To gain deeper knowledge of the genetic diversity and population structure of native Saccharomyces cerevisiae wine strains, 202 isolates were collected during spontaneous alcoholic fermentation of eight must/wine samples of cultivar ‘Merwah’, over two consecutive years (2016, 2017) in a traditional winery in Mount Lebanon (1400 m a.s.l.). The isolates were identified as S. cerevisiae on the basis of their morphology and preliminary sequence analysis of their internal transcribed spacer (ITS) PCR. They were then characterised at the strain level by interdelta PCR and genotyped using multiplex PCR reactions of 12 microsatellite markers. High genetic diversity was observed for the studied population. To select potential yeast starter strains from this population, micro-fermentations were carried out for 22 S. cerevisiae strains that were selected as representative of the ‘Merwah’ wine yeast population in order to determine their technological and oenological properties. Three indigenous yeast strains might represent candidates for pilot-scale fermentation in the winery, based on relevant features such as high fermentation vigour, low production of volatile acidity and H2S and low residual sugar content at the end of alcoholic fermentation.
Saccharomyces yeasts are emerging as model organisms for ecology and evolution, and researchers need environmental Saccharomyces isolates to test ecological and evolutionary hypotheses. However, methods for isolating Saccharomyces from nature have not been standardized and isolation methods may influence the genotypes and phenotypes of studied strains. We compared the effectiveness and potential biases of an established enrichment culturing method against a newly developed direct plating method for isolating forest floor Saccharomyces spp. In a European forest, enrichment culturing was both less successful at isolating S. paradoxus per sample collected and less labor intensive per isolated S. paradoxus colony than direct isolation. The two methods sampled similar S. paradoxus diversity: the number of unique genotypes sampled (i.e., genotypic diversity) per S. paradoxus isolate and average growth rates of S. paradoxus isolates did not differ between the two methods, and growth rate variances (i.e., phenotypic diversity) only differed in one of three tested environments. However, enrichment culturing did detect rare S. cerevisiae in the forest habitat, and also found two S. paradoxus isolates with outlier phenotypes. Our results validate the historically common method of using enrichment culturing to isolate representative collections of environmental Saccharomyces. We recommend that researchers choose a Saccharomyces sampling method based on resources available for sampling and isolate screening. Researchers interested in discovering new Saccharomyces phenotypes or rare Saccharomyces species from natural environments may also have more success using enrichment culturing. We include step‐by‐step sampling protocols in the supplemental materials.
The study was performed to genotype some commercial wine yeast strains with SSR-markers. Five polymorphic SSR-markers were tested in a selection of 15 yeast strains. Tested SSR-markers showed a high level of informativeness as well as polymorphism and can be used further to analyze the genetic diversity of wine yeast
A large variety of fermented foods and beverages with traditional and cultural value have been described in the world, including industrial products such as wine, cider, and beer as well as traditional ones. In contrast with the massive scientific information available about the microbiota responsible for winemaking, yeasts responsible for most traditional fermented beverages around the world remain undiscovered. Both industrial and traditional fermentation processes coexist in Patagonia, making this region an ideal scenario for fermentative yeast diversity studies. The most relevant feature of this area is the fact that most traditional processes are produced at low temperature (below 20 °C), which directly affects the microbial diversity. We identified and characterized fermentative yeasts present during industrial fermentations of wine and cider and traditional fermentations (chichas) obtained from wild apples and Araucaria araucana seeds, substrates typically used by aboriginal communities to prepare soft alcoholic beverages. As a general rule, only Saccharomyces cerevisiae strains were obtained from wines and ciders, and they showed a close genetic relationship with European strains of this species. In traditional fermentations, commercial bakery and European wine strains of S. cerevisiae were detected as pure or mixed cultures with Saccharomyces uvarum, a cryotolerant species. This last species was also isolated from A. araucana seeds in Patagonian forests together with Saccharomyces eubayanus, another cryotolerant species of the genus. Genetic information obtained from the analysis of S. uvarum from apple chichas evidenced a closer relationship to industrial (European) strains than to natural (Patagonian) strains of this species. North Patagonia is an interesting scenario to study cryotolerant (S. uvarum and S. eubayanus) yeast diversity studies, and a source of new strains with potential biotechnological interest.
Beyond their traditional use in food fermentative processes, yeasts are currently workhorses for the production of a diverse range of products, from proteins of pharmaceutical and industrial interest to small molecules such as ethanol and glycerol. For the purposes of this article, we will focus on yeasts that are relevant for food production. Fermented food production was carried out without an understanding of the microbiological nature of the process over thousands of years until the nineteenth century. Current yeast species used in fermented food productions are mainly Saccharomycetes of the Ascomycetes phylum. Among them, Saccharomyces cerevisiae has been widely used by humanity for 1000 years due to its ability to produce alcohol and flavors from rich sugar raw materials. Molecular methods have allowed our understanding of yeast metabolism under aerobic and fermentative conditions, as well as mixed strain dynamics during wine, beer, cider, and bread production to be improved. However, yeast diversity is still currently under investigation, and nonconventional species are being studied for their known contribution to flavor and aroma, increasing the sensory complexity of foods.
Benzyl alcohol and other benzenoid-derived metabolites of particular importance in plants confer floral and fruity flavors to wines. Amongst the volatile aroma components in Vitis vinifera grape varieties, benzyl alcohol is present in its free and glycosylated forms. These compounds are considered to originate from grapes only and not from fermentative processes. We have found increased levels of benzyl alcohol in red Tannat wine compared to grape juice, suggesting de novo formation of this metabolite during vinification. In this work, we show that benzyl alcohol, benzaldehyde, p-hydroxybenzaldehyde and p-hydroxybenzyl alcohol are synthesized de novo in the absence of grape-derived precursors by Hanseniaspora vineae. Levels of benzyl alcohol produced by 11 different H. vineae strains were twenty to two hundred times higher than those measured in fermentations with Saccharomyces cerevisiae strains. These results show that H. vineae contributes to flavor diversity by increasing grape variety aroma concentration in a chemically defined medium. Feeding experiments with phenylalanine, tryptophan, tyrosine, p-aminobenzoic acid and ammonium in an artificial medium were tested to evaluate the effect of these compounds either as precursors or as potential pathway regulators for the formation of benzenoid-derived aromas. Genomic analysis shows that the phenylalanine ammonia-lyase (PAL) and tyrosine ammonia lyase (TAL) pathways, used by plants to generate benzyl alcohols from aromatic amino acids, are absent in H. vineae genome. Consequently, alternative pathways derived from chorismate with mandelate as an intermediate are discussed.
Adult tephrids require sugar and protein for survival and for development of eggs, and volatile chemicals from these substances are the basis for food-based lures developed as baits for these pests. In this chapter, we discuss food-based lures that mimic food sources for adults other than host fruit. These have been primarily nitrogen sources that provide the protein needed by adult flies, although non-nitrogen-containing volatile chemicals are also included in this category. After male lures, food-based lures have been the predominant attractants used in traps for tephritid fruit flies. Although typically not as powerful as male lures, food-based lures have several advantages over male-specific attractants. They can be used for species for which there are no male lures known; they capture both females and males of target species; they tend to be female-biased, that is, they capture a higher percentage of females than males; and, at least for the Mediterranean fruit fly, traps baited with food-based lures tend to capture flies earlier than traps baited with male lure. There has been a long history of research on the development of food-based attractants for pest tephritids. Several review articles have documented the early history, which started with investigations of sugar-based food lures and lead to the development of the liquid protein baits and synthetic protein-based food lures, the standard food-lures that are currently in use. In this chapter, we discuss the development of and, as much as possible, the diversity of food-based lures that have been tested and/or are used in traps for pest tephritids. Future research directions are also discussed.
Mixed fruit wine (pineapple and watermelon) was produced using Saccharomyces cerevisiae isolated from palm wine. Primary and secondary fermentation of the fruits lasted for 7 and 28 days respectively, during which aliquot samples analysis of pH, titrable acidity, specific gravity, alcohol content and reducing sugar were carried out using standard procedures. Specific gravity of the wine was observed to reduce drastically as the fermentation progresses. The pH of the fruit must during the period of fermentation ranged from 3.0 to 4.46. During the fermentation period, consistent increase in alcohol content was observed with time. At the end of the 28 th day of fermentation, the alcohol content was observed to be 3.2%. The titrable acidity of the wine was observed to show steady trend with time throughout the period of fermentation. This study showed that acceptable wine can be produced from mixed fruits pineapple and watermelon using yeasts especially Saccharomyces cerevisiae isolated from palm wine.
This chapter discusses molecular identification and characterization of wine yeasts. Yeast species can be identified by comparison of nucleotide sequences from rDNA regions. The two most commonly used regions are the D1 and D2 regions at the 5' end of the genes encoding the 26S and 18S ribosomal subunits. The availability of sequences in DNA databases, particularly for the D1/D2 region of the 26S gene, makes this technique particularly useful for assigning unknown yeast to a specific species when the homology of the sequences is greater than 99%. Molecular characterization of commercial yeast strains is necessary for two reasons. First, it is needed for quality-control purposes to confirm that the obtained yeast is the one that was originally selected and not a contaminant, and, second, to detect fraud. Wine is a highly appropriate culture medium for the growth of a large number of microorganisms, in part due to its richness in organic acids, amino acids, residual sugars, growth factors, and mineral salts. Techniques for the detection of spoilage yeasts are essential. These techniques must be very sensitive and allow quantification of the number of microorganisms present. They must also be rapid to allow the application of corrective measures on the production line prior to release of the products onto the market.
Torulaspora delbrueckii yeast strains are being increasingly applied at the industrial level, such as in the winemaking process, and so their identification and characterisation require effective, fast, accurate, reproducible and reliable approaches. Therefore, the development of typing techniques that allow discrimination at the strain level will provide an essential tool for those working with T. delbrueckii strains. Here, 28 T. delbrueckii strains from various substrates were characterised using different PCR-fingerprinting molecular methods: random amplified polymorphic DNA with polymerase chain reaction (RAPD-PCR), minisatellites SED1, AGA1, DAN4 and the newly designed T. delbrueckii (Td)PIR, and microsatellites (GAC)5 and (GTG)5. The aim was to determine and compare the efficacies, reproducibilities and discriminating powers of these molecular methods. RAPD-PCR using the M13 primers and the newly designed TdPIR3 minisatellite primer pair provided discrimination of the greatest number of T. delbrueckii strains. TdPIR3 clustered the 28 strains into 16 different groups with an efficiency of 100%, while M13 clustered the strains into 17 different groups, although with a lower efficiency of 89%. Moreover, the TdPIR3 primers showed reproducible profiles when the stringency of the PCR protocol was varied, which highlighted the great robustness of this technique. In contrast, variation of the stringency of the M13 PCR protocol resulted in modification of the amplified profiles, which suggested low reproducibility of this technique.
Copyright © 2015 Elsevier B.V. All rights reserved.
Spontaneous fermentation is thought to provide conditions where diversity of indigenous Saccharomyces cerevisiae strains can flourish. A broad diversity of strains may provide unique characteristics to a wine and enhance its sensorial attributes. Population dynamics of S. cerevisiae strains were followed during a spontaneous fermentation of Vitis vinifera L. var. Pinot noir grapes at a winery in Kelowna, British Columbia, Canada, to determine the relative contribution of commercial active dry yeast (ADY) and indigenous strains during fermentation. Grapes were harvested, processed, and fermented separately. Yeasts from grapes were isolated and plated onto culture media. Fermentation tanks were sampled at early, mid, and late stages of fermentation. Yeast strains were isolated onto culture media at each stage. DNA was extracted from yeast cultures and amplified using primers defining six different microsatellite loci. Results were compared with a database comprising DNA fingerprints from commercially produced ADY strains used previously by the winery. Strains isolated from fermentation tanks were all ADY strains closely related or identical to those in the winery database. Strain richness tended to be greater in the mid and final stages than in the early stage. Lalvin ICV D254/Fermol Premier Cru and Red Star Premier Cuvee were the dominant yeasts during the mid and final stages. The former was the only strain found in all tanks in the mid and final stages. An ADY strain, Fermol Arome-Plus, was isolated from both grapes and spontaneous fermentation. This strain could represent a resident strain, where it persists year after year and cycles annually between the winery and vineyard.
The transformation of must into wine includes two sequential microbial processes. One of them is the degradation of the sugars during alcoholic fermentation (AF) conducted by yeast, where Saccharomyces cerevisiae is the dominant species. The other is the conversion of L-malic acid to L-lactic acid during the malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) and where Oenococcus oeni is the predominant species. Each of these transformations has its own characteristics that contribute to the final organoleptic composition of wine. The aim of this study was to optimize the specific oenological practices and improve the organoleptic profile of red wines produced in Mendoza through the selection and characterization of native microorganisms with different winemaking skills.
First, O. oeni native strains were characterized and their oenological ability to perform MLF in red wines. In addition, the possibility of simultaneous inoculation at the beginning of AF with different pairs of S. cerevisiae and O. oeni commercial strains were studied in different Malbec grapes during two vintages. Inoculation at the beginning of the AF allowed the development of both fermentations without negative effects on the viability of the inoculated microorganisms. This methodology allowed a reduction of total time of fermentation (AF+MLF) without producing a negative impact on the chemical composition and sensory characteristics of wines. In the simultaneous inoculation is essential the correct choice of strains used, as well as the effect exerted by the substrate on the microbial interactions.
Second, in order to select native strains of S. cerevisiae to ferment red musts at 15 ºC for the production of fruity young wines, yeast strains isolated from spontaneous fermentations were characterized oenological. Of the 14 native strains identified as S. cerevisiae, 8 presented the best oenological characteristics for red must fermentation 15 ºC and produced monoterpenes at that temperature. These strains were evaluated in red must fermentations at low temperatures at different scales (microvinification and pilot scale). Native strains MaB-2C and Bo-1C were selected because they produced wines at 15 °C with more aromatic complexity and better chemical characteristics.
The use of selected native microorganisms allow to the wineries optimize technological processes commonly used in the region and incorporate new techniques for the preparation of young red wines with better organoleptic characteristics.
An extensive survey of different methods of yeast strain identification (classical microbiological tests, whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction analysis show differences between strains. Both techniques are very complex to be used in bio technological industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on mtDNA restriction analysis.
The origins of the classic European wine grapes (Vitis vinifera) have been the subject of much speculation. In a search for parental relationships, microsatellite loci were analyzed in
more than 300 grape cultivars. Sixteen wine grapes that have long been grown in northeastern France, including ‘Chardonnay’,
‘Gamay noir’, ‘Aligoté’, and ‘Melon’, have microsatellite genotypes consistent with their being the progeny of a single pair
of parents, ‘Pinot’ and ‘Gouais blanc’, both of which were widespread in this region in the Middle Ages. Parentage analysis
at 32 microsatellite loci provides statistical support for these relationships.
A PCR-based method has been developed that permits both intraspecies differentiation and species identification of yeast isolates. Oligonucleotide primers that are complementary to intron splice sites were used to produce PCR fingerprints that display polymorphisms between different species of indigenous wine yeasts. Although polymorphisms existed between isolates of the same species, the banding patterns shared several amplification products that allowed species identification. Importantly, the method was able to distinguish between species of the closely related Saccharomyces sensu stricto yeasts. In two cases where isolates could not be positively identified there was discrepancy between the phenetic and phylogenetic species concept. The method has applications in yeast ecological studies, enabling the rapid grouping of isolates with related genomes and the investigation of population dynamics of strains of the same species.
The polymorphism of a TTC/TTTC microsatellite in the promoter sequence of the elongation factor 3 gene of Candida albicans was investigated by PCR. One primer was fluorescein labeled, and PCR signals were read with an automatic sequencer. Twenty-nine reference strains and 31 independent clinical isolates were studied. Eleven different alleles were identified, giving 16 different profiles among the 60 strains tested, with a discriminatory power of 0.88. This marker is stable upon subculture, and reproducibility was achieved by automated procedures. When several microsatellite markers are available, many isolates can be rapidly and reproducibly tested for epidemiological questions, such as the prevalence of a given strain in a hospital setting and transmission between patients.
We examined the distributions of short tandemly repeated DNAs (microsatellites) in nine complete microbial genomes (Saccharomyces cerevisiae, Archaeoglobus fulgidus, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanococcus jannaschii, Mycoplasma pneumoniae, M. genitalium, and Synechocystis PCC6803.) These repeats contribute differently to the global features of these genomes, and we explore the evolutionary implications of these differences by empirical examination of length polymorphisms at 20 long triplet-repeats repeats in S. cerevisiae, and by comparison of observed and expected repeat distributions. All of a sample of 20 microsatellites found in S. cerevisiae are highly polymorphic in length, suggesting that mutation pressure overcomes overall selection for small genome size that will tend to shorten or eliminate unnecessary DNA. By comparison, prokaryotes have fewer long repeats than expected, except for a few statistically improbable repeats that appear to function in gene regulation. Finally, we find that in all these genomes there is an excess of repeats shorter than those traditionally considered to be microsatellites. This finding suggests that even in prokaryotes these repeats are being generated by mutational pressures. These results have important potential implications for understanding genome stability and evolution in these microbial species.
Amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in commercial strains, type strains and winery isolates from a number of yeast species. AFLP was shown to be effective in discriminating closely related strains. Furthermore, sufficient similarity in the fingerprints produced by yeasts of a given species allowed classification of unknown isolates. The applicability of the method for determining genome similarities between yeasts was investigated by performing cluster analysis on the AFLP data. Results from two species, Saccharomyces cerevisiae and Dekkera bruxellensis, illustrate that AFLP is useful for the study of intraspecific genetic relatedness. The value of the technique in strain differentiation, species identification and the analysis of genetic similarity demonstrates the potential of AFLP in yeast ecology and evolutionary studies.
Seven strains of cryophilic wine yeasts and two strains of mesophilic wine yeasts were investigated for their utilization of sugars, viability of ascospores in hybrid obtained from between cryophilic wine yeast and mesophilic wine yeast, electrophoretic karyotype, G+C content of DNA and DNA similarity in photobiotin microplate-hybridization. Six strains of cryophilic wine yeasts were identified as Saccharomyces uvarum and one strain of cryophilic wine yeast was identified as S. chevalieri according to The Yeasts, 2nd ed. (Lodder, J., ed., North Holland Publ. Co., Amsterdam, 1970) on the basis of mainly sugar utilization. However, ascospores of hybrids obtained from between cryophilic wine yeast and mesophilic wine yeast did not germinate. These cryophilic wine yeasts were similar to the type strain of S. bayanus (IFO 1127) in electrophoretic karyotype and G+C content. The cryophilic wine yeasts also showed high DNA similarity values to the type strain of S. bayanus and low DNA similarity values to the type strain of S. cerevisiae (IFO 10217). Accordingly, these seven strains of cryophilic wine yeasts were classified to S. bayanus. Additionally, another eight strains of S. bayanus, classified on the basis of DNA similarity, showed good fermentability at low temperatures accompanied by production of low amounts of ethanol at intermediate temperatures, the same as cryophilic wine yeasts. These results suggest that the fermentation characteristics are specific for S. bayanus. Two strains of mesophilic wine yeasts showed high DNA similarity values to the type strain of S. cerevisiae.
THE genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome1. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair2-4. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA poly-merases slipping during replication, and that some types of colo-rectal cancer may reflect mutations in genes involved in DNA mismatch repair.
Electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains of Saccharomyces cerevisiae used for commercial production of wine. These fingerprinting procedures provided unique profiles for all the different yeast strains and can therefore be used to identify and control industrial strains. Furthermore, the protein profiles, restriction fragments banding patterns and electrophoretic karyotyping by contour clamped homogeneous electric field electrophoresis (CHEF), were valuable to differentiate hybrid and parental strains in yeast breeding programmes. Hybrid strains, with desirable oenological properties, were obtained by mass spore-cell mating between a heterothallic killer yeast and two homothallic sensitive strains and all were shown to have unique DNA fingerprints and electrophoretic karyotypes.
There is considerable anthropological and forensic interest in the possibility of DNA typing skeletal remains. Trace amounts of DNA can be recovered even from 5,500-year-old bones and multicopy human mitochondrial DNA sequences can frequently be amplified from such DNA using the polymerase chain reaction (PCR). But given the sensitivity of PCR, it is very difficult to exclude contaminating material. We now report the successful identification of the 8-year-old skeletal remains of a murder victim, by comparative typing of nuclear microsatellite markers in the remains and in the presumptive parents of the victim. This analysis establishes the authenticity of the bone DNA and the feasibility of bone DNA typing in forensic investigations.
Short simple sequence stretches occur as highly repetitive elements in all eukaryotic genomes and partially also in prokaryotes
and eubacteria (1–6). They are thought to arise by slippage like events working on randomly occuring internally repetitive
sequence stretches (3, 7, 8). This predicts that they should be generally hypervariable in length. I have used the polymerase
chain reaction (PCR) process to show that several randomly chosen simple sequence loci with different nucleotide composition
and from different species show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) may be usefully
exploited for identity testing, population studies, linkage analysis and genome mapping.
26 cases of disputed paternity were tested by the methods routinely used in Scandinavian countries and by the DNA "fingerprinting" technique. In all the studied cases the results of DNA analyses were similar to those obtained with the routine examinations based on protein polymorphisms; and, in the cases where even HLA typing did not distinguish between tentative fathers, the results obtained with DNA analyses were unambiguous.
Repetitive DNA sequences form a large portion of the genomes of eukaryotes. The 'selfish DNA' hypothesis proposes that they are maintained by their ability to replicate within the genome. The behaviour of repetitive sequences can result in mutations that cause genetic diseases, and confer significant fitness losses on the organism. Features of the organization of repetitive sequences in eukaryotic genomes, and their distribution in natural populations, reflect the evolutionary forces acting on selfish DNA.
The genomes of all eukaryotes contain tracts of DNA in which a single base or a small number of bases is repeated. Expansions of such tracts have been associated with several human disorders including the fragile X syndrome. In addition, simple repeats are unstable in certain forms of colorectal cancer, suggesting a defect in DNA replication or repair. We show here that mutations in any three yeast genes involved in DNA mismatch repair (PMS1, MLH1 and MSH2) lead to 100- to 700-fold increases in tract instability, whereas mutations that eliminate the proof-reading function of DNA polymerases have little effect. The meiotic stability of the tracts is similar to the mitotic stability. These results suggest that tract instability is associated with DNA polymerases slipping during replication, and that some types of colorectal cancer may reflect mutations in genes involved in DNA mismatch repair.
Destabilisation of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair Hypervariability of simple sequences as a general source for polymorphic DNA markers The value of electrophoretic ®ngerprinting and karyotyping in wine yeast breed-ing programmes
- M Strand
- T A Prolla
- R M Liskay
- T D Petes
Strand, M., Prolla, T.A., Liskay, R.M. and Petes, T.D. (1993) Destabilisation of tracts of simple repetitive DNA in yeast by mutations affecting DNA mismatch repair. Nature 365, 274±276. Tautz, D. (1989) Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Research 17, 6463±6471. Van Der Westhuizen, T.J. and Pretorius, I.S. (1992) The value of electrophoretic ®ngerprinting and karyotyping in wine yeast breed-ing programmes. Antoni Van Leeuwenhoek 61, 249±257.
- F M Ausubel
- R Brent
- R E Kingston
- D D Moore
- J G Seidman
- J A Smith
- K Struhl
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G., Smith, J.A. and Struhl, K. (1994) Current Protocols in Molecular
Biology Vol. 2. New York: Wiley.
The Yeasts, a Taxonomic Study 1st edn
- C P Kurtzman
- J W Fell
Kurtzman, C.P. and Fell, J.W. (1998) The Yeasts, a Taxonomic Study
1st edn. Amsterdam: North-Holland Publishers.