Yeast biodiversity in the vineyards of the Azores Archipelago (Portugal)

This study aims to identify the fermentative yeasts communities of the vineyards from the mid-Atlantic islands ecosystems of the Azores Archipelago and to evaluate whether the populations vary according to the islands geography, grape variety and type of vineyard. From an ecological point of view, vineyards of these isolated volcanic islands terroirs correspond to very particular ecosystems, whose yeast flora is completely unknown. During the harvests of 2009 and 2010, 88 and 75 grape samples were collected from eight islands of the archipelago, respectively. They were obtained from traditional grape varieties (Arinto, Verdelho and Terrantez) and hybrid varieties. The sampling plan covered 36 locations, including vineyards in appellations of origin and vineyards that were abandoned for at least 5 years. A total of 4890 yeast isolates was obtained from the crushed grapes. Species identification was performed by restriction fragment length polymorphism analysis (RFLP) and sequencing of ITS regions. Twenty-seven yeast species were identified, being Hanseniaspora uvarum, Issatchenkia terricola, Candida zemplinina and Metschnikowia pulcherrima the most representative ones. Three putative new species were also found. In 2009, differences in the microflora composition were apparent between islands, grape varieties and type of vineyard. A high percentage of C. zemplinina differentiated the yeast microflora of the eastern islands Santa Maria and São Miguel (25% and 36%, respectively). The species M. pulcherrima appeared associated with the traditional grape varieties (20% and 2% in traditional and hybrid varieties, respectively). H. uvarum was found in higher proportions in abandoned vineyards (53% and 43% in abandoned and non-abandoned vineyards, respectively). In the following sampling year, characterized by unfavorable climatic conditions and very low grape production, the yeast species richness decreased considerably from 25 species in 2009 to 14 species in 2010 and H. uvarum represented 86% of the total isolates, 40% more than in the previous year.João Drumonde-Neves is recipient of a fellowship supported by the program PRO-EMPREGO and the Azores Government. This study was financially supported by FP7 (nº 232454). Financial support was also obtained from FEDER funds through the program COMPETE and by national funds through FCT by the project FCOMP-01-0124-008775

Biogeographical survey and characterization of indigenous yeasts from the vineyards of the azores archipelago

This study aims at the characterization of the fermentative yeasts flora of the vineyards from Azores, for biodiversity preservation and the constitution of a Saccharomyces cerevisiae strain collection. During the harvest of 2009, 88 grape samples were collected from eight islands of the archipelago, that belonged to traditional (Arinto, Verdelho and Terrantez) and hybrid grape varieties. The sampling plan covered 36 locations, including vineyards in appellations of origin and abandoned vineyards. Forty nine spontaneous fermentations were achieved. About 83% of the musts obtained from traditional varieties finished fermentation, while this value was 47% and 33% for musts from hybrid varieties in non-abandoned and abandoned vineyards, respectively. From the final phase of the fermentations, a total of 1470 isolates was obtained. The identification was performed by restriction fragment length polymorphism analysis (RFLP) of ITS sequences. The S. cerevisiae strains were differentiated by the comparison of polymorphic patterns that were obtained from interdelta sequences amplification by PCR. We obtained 660 isolates of non-Saccharomyces species and 810 isolates of S. cerevisiae, classified in 168 strains. Surprisingly, the percentage of S. cerevisiae in fermentations performed with grapes from abandoned vineyards was significantly higher (75%) compared to the values determined for non-abandoned vineyards (56%, for both traditional and hybrid varieties). Contrarily, the number of S. cerevisiae strains per fermentation was lower (3-11) in samples from abandoned vineyards compared to non-abandoned vineyards (1-23). The relative proportion of S. cerevisiae and non-Saccharomyces species was not associated with the grape variety, but rather the type of vineyard. The highest number of S. cerevisiae strains per fermentation was observed in samples collected from the Graciosa Island. Our results show that Azorean vineyards have a high genetic diversity of S. cerevisiae, even in locations where no human intervention occurs.Fundação para a Ciência e a Tecnologia (FCT) - Programa Operacional Ciência e Inovação POCI2010 , POCI/AGR/56102/2004), PTDC(AGR-ALI/103392/2008)Direcção Regional da Ciência e Tecnologia -Açores (DRCT

A biogeografia de populações de leveduras fermentativas das vinhas do arquipélago dos Açores

Nas vindimas de 2009 e 2010 foram obtidos 163 mostos de amostras de uvas colhidas em oitos ilhas do Arquipélago dos Açores. A amostragem incluiu castas tradicionais, e híbridas a partir de vinhas em zonas demarcadas e não demarcadas e vinhas abandonadas. Em 2009 obtiveram-se 2640 isolados de levedura da fase inicial da fermentação, que pertenceram a mais de 20 espécies. Relativamente às espécies mais representativas, foram observadas diferenças entre ilhas, castas e tipos de vinha. Da fase final das fermentações obteve-se um total de 2850 isolados (1470 e 1380 em 2009 e 2010, respectivamente). Destes, 1770 foram identificados como pertencentes à espécie Saccharomyces cerevisiae e foram classificados em 275 estirpes (168 em 2009 e 107 em 2010). A percentagem de S. cerevisiae e o número de estirpes revelou diferenças entre ilhas, tipos de vinha e anos de amostragem.Direcção Regional da Ciência e Tecnologia e Comunicações (DRCTC) (M3.1.2/F/006/2008)Fundação para a Ciência e a Tecnologia (FCT

The biogeography of fermentative yeast populations from the vineyards of the Azores Archipelago

This study aims at the characterization of the fermentative yeasts flora of the vineyards from Azores, for biodiversity preservation and the constitution of a Saccharomyces cerevisiae strain collection. During the harvest of 2009, 88 grape samples were collected from eight islands of the archipelago, that belonged to traditional (Arinto, Verdelho and Terrantez) and hybrid grape varieties. The sampling plan covered 36 locations, including vineyards in appellations of origin and abandoned vineyards. Forty nine spontaneous fermentations were achieved. About 83% of the musts obtained from traditional varieties finished fermentation, while this value was 47% and 33% for musts from hybrid varieties in non-abandoned and abandoned vineyards, respectively. From the final phase of the fermentations, a total of 1470 isolates was obtained. The identification was performed by restriction fragment length polymorphism analysis (RFLP) of ITS sequences. The S. cerevisiae strains were differentiated by the comparison of polymorphic patterns that were obtained from interdelta sequences amplification by PCR. We obtained 660 isolates of non-Saccharomyces species and 810 isolates of S. cerevisiae, classified in 168 strains. Surprisingly, the percentage of S. cerevisiae in fermentations performed with grapes from abandoned vineyards was significantly higher (75%) compared to the values determined for non-abandoned vineyards (56%, for both traditional and hybrid varieties). Contrarily, the number of S. cerevisiae strains per fermentation was lower (3-11) in samples from abandoned vineyards compared to non-abandoned vineyards (1-23). The relative proportion of S. cerevisiae and non-Saccharomyces species was not associated with the grape variety, but rather the type of vineyard. The highest number of S. cerevisiae strains per fermentation was observed in samples collected from the Graciosa Island. Our results show that Azorean vineyards have a high genetic diversity of S. cerevisiae, even in locations where no human intervention occurs

The phenotypic landscape of a Saccharomyces cerevisiae strain collection

Within our previous work [1] we developed computational models to predict strains with specific phenotypes (e.g. low ethanol resistance, growth at 30ºC and growth in media containing galactose, raffinose or urea) from microsatellite allelic patterns. The objective of the present work was to gain deeper understanding of the phenotypic diversity of a heterogeneous Saccharomyces cerevisiae strain collection, using a large battery of tests with biotechnological relevance, and apply computational data mining algorithms to predict a strain´s potential to be used as a winemaking strain from a few selected phenotypic data. A S. cerevisiae collection was constituted, comprising 172 strains of different geographical origins and technological uses (winemaking, brewing, bakery, distillery, etc.). Phenotypic screening was performed considering 30 physiological traits that are important from an oenological point of view, such as ethanol tolerance, growth in synthetic must media at various temperatures or resistance to fungicides. Data was analyzed using Principal Component Analysis and some phenotypes were identified (growth in the presence of potassium bisulfite, growth at 40˚C, and resistance to ethanol) as being responsible for the highest strain variability. Statistical analysis revealed relevant associations between several phenotypes and the strains technological use. Based on the phenotypic data, naїve Bayesian classifier, as implemented in the software Orange [2], correctly assigned (AUC=0.70) most of strains from vineyards (73%) and commercial strains (77%) to the respective group. Data mining approaches identified, for the group of commercial strains, 18 phenotypic tests with the highest weight. Globally, the growth patterns of this group of strains in must containing iprodion (0,05mg/mL) or cycloheximide (0,1µg/mL) revealed to have the highest predictive score for the assignment of a strain as a commercial strain. The results obtained herein demonstrate the potential of computational approaches to explore phenotypic variability and to predict the probability of a S. cerevisiae strain to be used as a commercial strain.Fundação para a Ciência e a Tecnologia (FCT

A new high-throughput method for the rapid extraction of yeast DNA

Ecological, evolutionary and population genetic studies of yeasts require the processing of a high numbers of isolates. The current molecular methods for yeast identification and characterization require time-consuming and labor intensive DNA extraction protocols. Within this research, we aimed to develop a high-throughput method for yeast DNA extraction. Total yeast DNA extraction was based on a previously described standard miniprep method (Lopez et al., 2001; Schuller et al., 2004). Several steps were simplified or skipped. Microplates containing 30, 50, 100 or 150 μL yeast cell suspensions per well were used instead of microtubes. All reagents volumes were reduced in proportion to the volumes of cell suspensions. The most suitable yeast cell suspensions were 100 and 150 μL. The quality of the obtained DNA was not affected by the modifications that were introduced. The final DNA concentration obtained with the modified protocol was in the range of 20-50 ng/μL, being most suitable for the usual DNA amplification protocols. DNA concentrations using the conventional method were much higher (800 ng/μL) and required time-consuming dilutions prior to PCR amplifications. To validate the modified method, a group of 12 yeast species and 12 Saccharomyces cerevisiae strains were processed. DNA restriction fragment length polymorphism (RFLP) of ITS sequences and interdelta sequence analysis were performed using the DNA obtained by the conventional and the herein presented method and the results were identical. This high-throughput method for yeast DNA extraction allows the processing of 1600 yeast isolates per day, which is a ten-fold increase compared to the conventional method. The required reagents volumes were reduced by 90%.Fundação para a Ciência e a Tecnologia (FCT

Genotypic and pheno-metabolomic characterization of a Saccharomyces cerevisiae strain collection

The objective of the present work was to gain a deeper understanding of the phenotypic diversity of natural isolates of Saccharomyces cerevisiae, using high-throughput methods in combination with bioanalytical data. A S. cerevisiae strain collection was constituted comprising 187 strains from different geographical origins and technological uses. Some microsatellite alleles and phenotypes were identified as responsible for the highest strain variability. All strains were used for fermentation with white grapes must, and final fermentation products were analysed using fiber optics spectroscopy and bioanalytical quantification. Inter-strain aromatic profiles (primary fermentation products, higher alcohols, esters, fatty acids) were discriminated by HPLC and GC-MS. Relating the metabolic signature of the strains with phenotypic and genetic data by computational analysis is a pre-requisite to obtain a holistic overview of yeast pheno-metabolomics.O objectivo do presente trabalho consistiu em explorar a diversidade fenotípica de isolados naturais de Saccharomyces cerevisiae, através de métodos de alto débito combinados com dados bioanalíticos. Uma colecção de 187 estirpes de S. cerevisiae foi constituída com isolados de origens geográficas e aplicações tecnológicas diferentes. Alguns alelos de microssatélites e alguns fenótipos foram identificados como responsáveis pela grande variabilidade entre estirpes. Todas as estirpes foram usadas para fermentações em mosto de uvas brancas, e os produtos finais de fermentação foram analisados usando espectroscopia de fibra óptica e quantificação bioanalítica. Os perfis aromáticos entre estirpes (produtos de fermentação primários, álcoois superiores, esteres, ácidos gordos) foram descriminados por HPLC e GC-MS. A relação, através de abordagens computacionais, entre a assinatura metabólica das estirpes e os dados fenotípicos e genotípicos, é um pré-requisito para obter uma visão holística do feno-metaboloma da levedura.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/48591/2008, SFRH/BD/46737/2008, SFRH/BD/48616/2008, SFRH/BD/74798/2010

Association between Grape Yeast Communities and the Vineyard Ecosystems

The grape yeast biota from several wine-producing areas, with distinct soil types and grapevine training systems, was assessed on five islands of Azores Archipelago, and differences in yeast communities composition associated with the geographic origin of the grapes were explored. Fifty-seven grape samples belonging to the Vitis vinifera grapevine cultivars Verdelho dos Acores (Verdelho), Arinto da Terceira (Arinto) and Terrantez do Pico (Terrantez) were collected in two consecutive years and 40 spontaneous fermentations were achieved. A total of 1710 yeast isolates were obtained from freshly crushed grapes and 1200 from final stage of fermentations. Twenty-eight species were identified, Hanseniaspura uvarum, Pichia terricola and Metschnikowia pulcherrima being the three most representative species isolated. Candida carpophila was encountered for the first time as an inhabitant of grape or wine-associated environments. In both sampling years, a higher proportion of H. uvarum in fresh grapes from Verdelho cultivar was observed, in comparison with Arinto cultivar. Qualitatively significant differences were found among yeast communities from several locations on five islands of the Archipelago, particularly in locations with distinctive agro-ecological compositions. Our results are in agreement with the statement that grape-associated microbial biogeography is non-randomly associated with interactions of climate, soil, cultivar, and vine training systems in vineyard ecosystems. Our observations strongly support a possible linkage between grape yeast and wine typicality, reinforcing the statement that different viti-cultural terroirs harbor distinctive yeast biota, in particular in vineyards with very distinctive environmental conditions.Joao Drumonde Neves is the recipient of a fellowship of the Azorean Government (M321/006/F/2008) and PROEMPREGO. This work was supported by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI), and by national funds through FCT by the projects FCOMP-01-0124-008775, PTDC/AGR-ALI/103392/2008 and PTDC/AGR-ALI/121062/2010.info:eu-repo/semantics/publishedVersio

A collection of indigenous saccharomyces cerevisiae strains from appellations of origin in Portugal and France

Apresentação efectuada nas "Jornadas Portuguesas de Genética, 35, Braga, 2010"The model organism Saccharomyces cerevisiae stands today at the forefront of molecular biology and functional analysis in genetics and genomics. However, understanding of the ecological, evolutionary and population genetic features that shaped the biology of this species is underscored by a wealth of knowledge on molecular and cellular biology obtained from a very limited number of laboratory strains. In this reasoning, we constituted one of the largest bio-databanks of S. cerevisiae that were obtained from winemaking environments in Portugal and France. During the harvest time of 2001 to 2009, 604 grape samples were collected in appellations of origin in Portugal (Vinho Verde, Dão, Douro, Bairrada, Estremadura, Palmela, Ribatejo, Açores) and France (Languedoc). The grape samples belonged to the varieties Alvarinho, Aragonez, Arinto, Avesso, Baga, Bical, Castelão, Carignan, Loureiro, Maria Gomes, Terrantez, Touriga Nacional and Verdelho. Yeast populations, in particular S. cerevisiae, were isolated after spontaneous fermentation of the extracted grape juice. From the final stage of 258 fermentations, 7740 yeast isolates were obtained, belonging mainly (5496 isolates) to the species S. cerevisiae. An initial genetic screen, based on mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP), electrophoretic karyotyping or interdelta sequence analysis, was followed by microsatellite analysis of strains with unique genetic profiles. Isolates were assigned to 752 different strains, based on their microsatellite allelic distribution. The resulting web-based autochthonous strain collection is one of the largest S. cerevisiae bio-databanks and is a resource for sustainable biodiversity preservation, equitable sharing of genetic data and winemaking strain selection.Fundação para a Ciência e Tecnologia (FCT)Direcção Regional da Ciência e Tecnologia (DRCT