Comparison of vineyard-associated Saccharomyces cerevisiae populations by microsatellite analysis

The aim of the present study was to evaluate populational structures among fermenting S. cerevisiae populations. Analysis of six polymorphic microsatellite loci was performed in 468 S. cerevisiae isolates derived from a previous screening (using mtDNA RFLP or electrophoretic karyotyoing) of 2490 yeast strains obtained from spontaneous fermentations of grapes collected in three vineyards of the Vinho Verde Region (northwest Portugal), and one vineyard of the Languedoc Region (South France) during the 2001 – 2003 harvest seasons. Among the 93 alleles obtained, 52 new alleles were identified. For all loci analyzed, observed heterozygosity was three to four times lower than the expected value, probably due to a strong populational substructuring. Populational structures were identified based on the accumulation of small allele-frequency differences across six loci in groups of strains. The present work is the first large-scale approach showing that microsatellite typing reveals a very fine population resolution of indigenous S. cerevisiae strains isolated from vineyards.This study was financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762)

Estudo ecológico das estirpes Saccharomyces cerevisiae numa vinha da Região dos Vinhos Verdes em Portugal

ENOSAFE - (Nº 762, Programa AGRO, medida 8).Instituto Português para a Cooperação Científica e Tecnológica Internacional (ICCTI)/Embaixada Francesa em Lisboa - concessão/subvenção nº 657 C2

The genetic characterization of Saccharomyces cerevisiae commercial enological strains : a survey of molecular typing techniques

Resumo e poster da comunicação apresentada no "23rd International Specialized Symposium on Yeasts", em 2003, Budapeste, Hungria.Wine production by the addition of active dry wine yeast is today widely accepted, being about 50% of the wine produced in Europe in this way. This enological practice required the development of techniques that were able to distinguish the inoculated strain from the rest of the wild yeast strains present in the must. In the last years, several methodologies based on DNA polymorphisms, have become widely accepted for the discrimination between closely related wine strains. The aim of this study is to validate the usefulness of each typing method (karyotype analysis [1], ∂ sequence typing [2][5], mtDNA restriction analysis [3], and microsatellite genotyping [4]) by studying the degree of polymorphism generated by each of them in 23 commercially available winery yeasts from 5 companies. The amplification of delta sequence interspersed DNA regions generated only 8 patterns for primer pair A [2] and 20 for primer pair B [5] respectively. The discriminative power of karyotype analysis and mtDNA RFLP (using the restriction enzyme HinfI) was very similar, and generated 21 patterns for the 23 strains. The results obtained by both methods indicated the occurrence of one strain that is commercialized by 3 different active dry yeast producers. Microsatellite typing, using multiplex reactions for six markers on different chromosomes, unequivocally confirmed the results obtained by karyotyping and by mtDNA RFLP. In global terms, the results show that microsatellite analysis is a very precise and fast method for the typing of S. cerevisiae strains. Studies are now underway to type a yeast strain collection and to perform biodiversity studies by this method. [1] Blondin, B. and Vezinhet, F. 1988. Rev. Fr. Oenol. 28: 7-11. [2] Ness, F., Lavallée, F., Dubourdieu, D., and Aigle, M. 1993. J. Sci. Food Agric. 62: 89-94. [3] Querol, A., Barrio, E., and Ramón, D. 1992. System. Appl. Microbiol. 15: 439-446. [4] Pérez, M.A., Gallego, F.J., Martinez, I. and Hidalgo, P. 2001. Lett. Appl. Microbiol. 33, 461-466. [5] Legras, J.L. and Karst, F. 2003. Submitte

Recensement ecologique des souches de Saccharomyces cerevisiae presentes dans des vignobles de la region du Vinho Verde (Portugal)

ENOSAFE (Nº 762, Programa AGRO, medida 8).Portuguese Institute for International Scientific and Technological Cooperation (ICCTI)/French Embassy in Lisbon - grant nº 657 C2

Avaliação da dispersão da levedura starter - Zymaflore Vl1 de Laffort Oenologie, em ambiente natural na proximidade de adegas da Região dos Vinhos Verdes

Resumo da comunicação oral apresentada no encontro científico "10as Jornadas de Biologia de Leveduras Professor Nicolau van Uden", em Maio 2002, Faro, Portugal.Nos últimos 20 anos, foi seleccionado um elevado número de estirpes da levedura Saccharomyces cerevisiae tendo em vista a sua aplicação em enologia. Hoje em dia, cerca de 50% da produção total de vinho na Europa é obtida através do uso destas leveduras industriais, comercializadas por várias empresas. Do ponto de vista ecológico tratam-se de estirpes não-indígenas de S. cerevisiae, que são introduzidas anualmente no ecossistema na zona da adega. Conhecer a capacidade de adaptação e/ou dispersão destas estirpes no ambiente natural, e em zonas geográficas distintas, constituiu o objectivo do presente trabalho. A duração prevista destes estudos é 3 anos. Seleccionaram-se três vinhas localizadas na proximidade de adegas da região demarcada dos Vinhos Verdes: Quinta de Covela (S. Tomé de Covela/Baião), Quinta do Ameal (Ponte de Lima) e Provam (Monção). O critério de selecção das adegas assentou no facto de estas distanciarem mais de 10 km entre si e de utilizarem continuamente a levedura starter (Zymaflore VL1 de Laffort Oenologie) nos últimos 5 anos. A colheitas das uvas foi realizadas em seis pontos de amostragem em campanhas pré-vindima e pós-vindima. Foram realizadas 36 vinificações à escala laboratorial (0,5 l de mosto proveniente de cerca de 2 Kg de uvas), à temperatura de 20ºC. Em cada microvinificação recolheram-se 30 colónias nos pontos da fermentação alcoólica correspondentes à perda de 30g/l (fase intermédia da fermentação) e de 70g/l (fase final da fermentação). A identificação das estirpes isoladas foi realizada por análise dos padrões de amplificação de sequências ∂ [1, 2] e por análise dos padrões de restrição de DNA mitocondrial [3]. Na Quinta de Covela, e apenas no ponto mais próximo da adega se detectaram colónias com padrões idênticos à VL, tanto na amostra pré-vindima (3%), como na amostra pós-vindima (97%)

Genetic instability of commercial Saccharomyces cerevisiae strains recovered from vineyard’s microflora

From spontaneous fermentations carried out with grapes collected in vineyards from the Vinho Verde Region, 101 isolates were recovered whose mitochondrial DNA restriction length polymorphism (mtDNA RFLP) were identical to strain Zymaflore VL1, a commercial strain (Lallemand) that has been used by the wineries in the last years. Genetic polymorphisms were evaluated by microsatellite analysis, interdelta sequence analysis and chromosomal karyotyping comparatively to those of 30 isolates of the original commercialized strain. Our data show that the recovered commercial yeast isolates present a considerable genetic instability that can be assessed by distinct methods. Microsatellite allelic polymorphisms were found in 12 natural isolates, two of them were characterized by complete loss of heterozygosity, whereas their chromosomal constitution showed a loss of structural heteromorphism. Major changes of chromosomal patterns were found among the natural isolates, apparent by the absence or changed position of bands in the presumable region of chromosomes VI and III. Interdelta amplification patterns depended on the primer pair used, and changes were apparent by additional bands. Whether these changes are associated with the yeast’s permanence in natural environments is subject of current investigations.Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762)

Selected yeast utilization and biodiversity

The use of commercial wine yeast strains as starters has been extensively generalised over the past two decades. Wine yeast strains are annually released in wineries environment and on an annual basis. However, little is known about the fate of these strains in the vineyard. To evaluate the industrial starter yeasts’ ability to survive and spread in nature, and become part of the natural microflora of musts, we have devised a large-scale sampling plan over a period of three years in six different vineyards (3 in Portugal and 3 in France). Each vineyard has used the same industrial yeast strain(s) continuously in the last 5 years. A total of 198 grape samples were collected at various distances from the wineries, before and after harvest. Towards the end of the spontaneous fermentations, the composition of the yeast flora was determined by different typing methods (PCR-amplification of ∂-sequences, pulse field electrophoresis, RFLP of mitochondrial DNA, and microsatellite typing). Among 3780 yeast strains identified, 296 isolates had a genetic profile identical to that of commercial yeast strains. For a large majority (94%), these strains were recovered at very close proximity to the winery (10-200m). Commercial strains were mostly found in the post harvest samples, reflecting immediate dissemination. Analysis of population variations from year to year indicated that permanent implantation of commercial strains in the vineyard did not occur, but instead that these strains were subject to natural fluctuations of periodical appearance/disappearance like autochthonous strains. Overall the data show that dissemination of commercial yeast in the vineyard is restricted to short distances and limited periods of times and is largely favoured by the presence of water runoff

Commercial yeast strains do not significantly disseminate in vineyard ecosystems

Resumo e poster da comunicação apresentada no Congresso Nacional de Microbiologia, em 2003, Tomar, Portugal.Industrial yeasts are traditionally used in winemaking and subsequently released into the environment. Nevertheless, there is very little data showing what becomes of them in the natural environment. To evaluate the dynamics of industrial yeast strains in the vineyard, a large-scale sampling plan was devised over a period of three years. This includes 36 sampling sites in 6 different vineyards (3 in France, 3 in Portugal) that used industrial starter yeast for at least 5 years. In each vineyard samples were taken before harvest (annual remanence) and at late harvest (immediate release), at three distances from the winery and from opposite directions. Small-scale fermentations were realised. Must samples were plated when 70 g/L of the CO2 had been released, and 30 colonies were randomly selected. By using selective media non-Saccharomyces strains were eliminated, and the remainder were characterised by karyotyping [1] and RFLP of mitochondrial DNA [2]. During the year 2001 the genetic patterns of only 2% of 720 isolates in France (collected at 100 to 1000m from the winery) were identical to those of the industrial yeast. In Portugal, the typing patterns of about 10% of the 570 isolates were identical to those of the commercial yeast, only in post-harvest samples close to the winery (20-40 m). The identification of the S. cerevisiae strains isolated during the year 2002 is now underway. These first data sets show that dissemination and remanence of commercial yeast in the vineyard is very limited. Next year’s data will complete this study and allow us to confirm these results. [1] Blondin, B. and Vezinhet, F. 1988. Rev. Fr. Oenol. 28: 7-11. [2] Querol, A., Barrio, E., and Ramón, D. 1992. System. Appl. Microbiol. 15: 439-446

Assessment of environmental impact of commercial wine yeast in vineyard ecosystems

Poster e resumo apresentados no congresso "Intrafood 2005 - Innovations in traditional foods", em Valencia, Espanha, em 2005.Modern winemaking practices and diversification of wine products involve an increasing quest for specialised wine yeasts. During the last two decades considerable efforts have been made to improve wine yeast strains through the use of new biotechnologies. In the present study we used commercial wine yeast currently used in wineries as a model to assess the potential environmental risks associated with the utilisation of genetically modified wine yeast strains in the wine industry. To evaluate the dynamics of industrial yeast strains in the vineyard, a large-scale sampling plan was devised over a period of 3 years, in 6 different vineyards that have used the same starter yeast for at least 5 years. Among the 3780 yeast strains identified after spontaneous fermentation, 296 had a genetic profile identical to that of commercial yeast strains. In 4 of the 6 vineyards, where the samples were taken at distances from wineries higher than 100 m, only 0-2% of the fermentative microflore had a genetic profile identical to that of commercial yeast. In the other 2 vineyards, where the samples were taken at very close proximity to the winery and the water rill with the liquid effluents, the proportion of commercial yeasts increased to 10-43%. The data show that dissemination of commercial yeast in the vineyard is limited to short distances and periods of times and largely favoured by the presence of liquid effluents. If some of these strains are able to remain in the vineyard, they don’t become implanted systematically in the ecosystem and are not able to dominate the natural microflore but they are subject to natural fluctuations of periodical appearance/disappearance as autochthonous strains

Dissémination des levures œnologiques dans le vignoble

Comunicação apresentada "Premières Rencontres Internationales du Rosé" Toulon, França, 2004.Depuis une quinzaine d’années des efforts considérables ont été réalisés dans le domaine de l’amélioration des levures œnologiques par ingénierie génétique. L’utilisation future de ces souches est prévisible et doit s’accompagner d’une évaluation des risques potentiellement associés à l’introduction de ces nouvelles technologies dans la filière œnologie. Dans ce contexte, une question essentielle concerne l’impact de l’utilisation de ces souches sur l’environnement. Les levures sèches actives (LSA) sont classiquement utilisées en cave en milieu ouvert, et peuvent être disséminées dans le vignoble en même temps que différents effluents et sous-produits. Leur devenir dans l’environnement naturel est pratiquement inconnu ainsi que leur impact dans l’écosystème. L’objectif de cette étude est d’évaluer l’importance de la dissémination des souches oenologiques dans le vignoble ainsi que leur survie, en utilisant comme modèle les levures commerciales. Nous avons pour cela défini un large plan d’échantillonnage, sur une durée de trois années (2001-2003). Il inclut 36 lieux de prélèvement repartis sur 6 vignobles (3 en France et 3 au Portugal) et utilisant une même LSA depuis plus de cinq années. Les baies de raisin ont été collectées avant (rémanence annuelle) et après (dissémination immédiate) les vendanges, à trois distances de la cave (20-1000 m), et à deux orientations opposées en fonction du vent dominant. Des fermentations à échelle laboratoire (0.25 l) ont été réalisées. Les levures ont été isolées après dégagement de 70 g/l de CO2 et 30 colonies choisies au hasard ont été identifiées. Après élimination des non-Saccharomyces, les souches restantes ont été analysées par électrophorèse en champ pulsé et techniques moléculaires (ADN mitochondrial, séquences delta, microsatellites). En France, 2% des S. cerevisiae isolées (17 souches sur 735), correspondant à 0.8% de la microflore capable de fermenter, ont un profil identique à celui d’une souche commerciale. Le profil de 16 de ces souches est identique à celui de la souche commerciale ICV D254, ce qui pourrait indiquer une dissémination antérieure. Cependant, cette souche ayant été initialement isolée dans la région, il n´est pas possible conclure sur ce point. La souche restante a un profil identique à la souche ICV-INRA K1 marquée, utilisée majoritairement dans les trois vignobles français depuis 5 à 15 ans. On peut noter que cette levure, la plus massivement utilisée et depuis longtemps n´a pas été retrouvée, excepté en ce cas, dans le vignoble, ce qui témoigne d’un très faible niveau de dissémination dans l’écosystème. Au Portugal, la situation est similaire à ce observé en France dans un des trois vignobles étudiés, mais très différente dans les deux autres ou des prélèvements ont été effectués à proximité immédiate des caves. Ainsi, 22% des S. cerevisiae isolées au Portugal ont un profil identique à celui de souches commerciales, avec une majorité de ces souches (78%) retrouvées dans un rayon de 10 à 50 m de la cave, avant et après vendanges en 2001 et seulement après vendanges dans les années suivantes. L´analyse globale des résultats montre que la dissémination des levures commerciales dans le vignoble est limitée dans l’espace et le temps et, de plus, largement favorisée par la présence d’effluents liquides. Si certaines des souches peuvent survivre dans le vignoble, elles ne s´implantent pas de manière systématique dans l´écosystème et ne sont pas capables de dominer la flore naturell