High degree of correlation between molecular polymorphism and geographic origin of wine yeast strains

AIMS: To guarantee the endemic genetic background of the isolates obtained in yeast isolation programs, it is necessary to differentiate between endemic and commercial strains because the progressive use of commercial yeast in wine areas around the world would affect the autochthonous yeast populations. METHODS AND RESULTS: Mitochondrial DNA restriction analysis, electrophoretic karyotyping and random amplification of polymorphic DNA (RAPD) were evaluated as experimental approaches to correlate genomic polymorphism and geographic origin of native wine yeast strains. The three molecular methods were capable of detecting a European commercial strain among native Chilean strains; however, RAPD proved to have the best performance. CONCLUSIONS: The molecular polymorphism analysis is useful to evaluate the geographical origin of native yeast isolates and confirms or refutes the genetic background of currently marketed strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits a genetic characterization of native yeast populations and confirms its utility as a tool for evaluating if a native isolate derives from the region where it was collected, permitting, furthermore, to develop studies on the evolution of native yeast populations and to evaluate the effect of introduced yeasts on these populations.     

Comparative study on the identification of food-borne yeasts

Morphologically distinct yeast colonies from partially and fully processed fruits and vegetables were isolated over a 3-year period. Identification of 239 strains was achieved by using standard methods, commercial identification kits (API 20C and API YEAST-IDENT), and a simplified system for food-borne yeasts. The identified strains of fruit origin represented 36 species belonging to 19 genera. Among strains of vegetable origin, 34 species representing 17 genera were identified. The simplified identification system and the conventional method provided the same results in 80% of the cases. The commercial identification kits were easy to use but were not appropriate for food-borne yeast species. Computer-assisted identification was helpful.     

Comparative study on the identification of food-borne yeasts.

Morphologically distinct yeast colonies from partially and fully processed fruits and vegetables were isolated over a 3-year period. Identification of 239 strains was achieved by using standard methods, commercial identification kits (API 20C and API YEAST-IDENT), and a simplified system for food-borne yeasts. The identified strains of fruit origin represented 36 species belonging to 19 genera. Among strains of vegetable origin, 34 species representing 17 genera were identified. The simplified identification system and the conventional method provided the same results in 80% of the cases. The commercial identification kits were easy to use but were not appropriate for food-borne yeast species. Computer-assisted identification was helpful.     

The value of electrophoretic fingerprinting and karyotyping in wine yeast breeding programmes

Electrophoretic banding pattens of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains ofSaccharomyces 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.     

Evaluation of yeast diversity during wine fermentations with direct inoculation and pied de cuve method at an industrial scale

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.     

New PCR-based methods for yeast identification

AIMS: To characterize reference yeast strains and identify indigenous strains isolated from wine fermentations by PCR methods. METHODS AND RESULTS: We compared several PCR techniques for yeast identification. We used oligonucleotide primers that are complementary to (i) intron splice sites, (ii) REP and (iii) ERIC elements to produce PCR fingerprints that display specific patterns between the different yeast species. These three techniques were used to characterize 41 reference yeast strains belonging to 15 different species and to identify 40 indigenous strains isolated from grape must and wine fermentations. Species-specific banding patterns were obtained with the three PCR-techniques with different degrees of intraspecific differentiation depending on the method. By comparing the PCR fingerprints of unknown isolates with those produced by reference strains, we identified yeast strains isolated from an industrial wine fermentation. CONCLUSIONS: All three PCR techniques are rapid, reliable and simple methods of yeast identification. As far as we know, this is the first time that the primers designed for amplifying repetitive elements in bacteria have been successfully used in yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Industry needs rapid, reliable and simple methods of yeast identification. The proposed PCR techniques will allow to achieve this objective.     

Dissemination and survival of commercial wine yeast in the vineyard: a large-scale, three-years study

The use of commercial wine yeast strains as starters has been extensively generalised over the past two decades. In this study, a large-scale sampling plan was devised over a period of three years in six different vineyards to evaluate the dynamics and survival of industrial yeast strains in the vineyard. A total of 198 grape samples were collected at various distances from the wineries, before and after harvest, and yeast strains isolated after spontaneous fermentation were subsequently identified by molecular methods. 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 (10-200 m). 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. Our data show that dissemination of commercial yeast in the vineyard is restricted to short distances and limited periods of time and is largely favoured by the presence of water run-off.     

Application of genotypic and phenotypic analyses to commercial probiotic strain identity and relatedness

AIMS: The objective of this study was to generate strain-specific genomic patterns of a bank of 67 commercial and reference probiotic strains, with a focus on probiotic lactobacilli. METHODS AND RESULTS: Pulsed-field gel electrophoresis (PFGE) was used as the primary method for strain differentiation. This method was compared with carbohydrate fermentation analysis. To supplement visual comparison, PFGE patterns were analysed quantitatively by cluster analysis using unweighted pair group method with arithmetic averages. SmaI, NotI and XbaI were found to effectively generate clear and easy-to-interpret PFGE patterns of a range of probiotic strains. Some probiotic strains from different sources shared highly similar PFGE patterns. CONCLUSIONS: Results document the value of genotypic strain identification methods, combined with phenotypic methods, for determining probiotic strain identity and relatedness. No correlation was found between relatedness determined by carbohydrate fermentation profiles alone compared with PFGE analysis alone. Some commercial strains are probably derived from similar sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is valuable to the probiotic industry to develop commercial strain identification patterns, to provide quality control of strain manufacturing production runs, to track use of protected strains and to determine the relatedness among different research and commercial probiotic strains.     

Biodiversity of Saccharomyces yeast strains from grape berries of wine-producing areas using starter commercial yeasts

The use of commercial wine yeast strains as starters has grown extensively over the past two decades. In this study, a large-scale sampling plan was devised over a period of 3 years in three different vineyards in the south of France, to evaluate autochthonous wine yeast biodiversity in vineyards around wineries where active dry yeasts have been used as fermentation starters for more than 5 years. Seventy-two spontaneous fermentations were completed from a total of 106 grape samples, and 2160 colonies were isolated. Among these, 608 Saccharomyces strains were identified and 104 different chromosomal patterns found. The large majority of these (91) were found as unique patterns, indicating great biodiversity. There were differences in biodiversity according to the vineyard and year, showing that the biodiversity of Saccharomyces strains is influenced by climatic conditions and specific factors associated with the vineyards, such as age and size. Strains that were terroir yeast candidates were not found. The biodiversity of S. cerevisiae strains after harvest was similar to that in the early campaign; moreover, a temporal succession of S. cerevisiae strains is shown. This fact, together with the differences in biodiversity levels verifies that other factors were more important than commercial yeast utilization in the biodiversity of the vineyard.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Genomic characterization of Saccharomyces cerevisiae strains isolated from wine-producing areas in South America

AIMS: The wide use of yeast inoculum for wine fermentations permit the spreading of commercial Saccharomyces strains in wine areas all over the world. To study the impact of this practice on the autochthonous yeast populations it is necessary to have tools that permit the evaluation of the geographical origin of native isolates and differentiate them from commercial strains. METHODS AND RESULTS: Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to characterize the genome of native S. cerevisiae isolates associated to wine from three countries in South America. Both methods revealed differences in the genomic structure between these populations, in addition to differences between sub-populations collected in wine-producing areas in Chile. CONCLUSIONS: Our data support that molecular polymorphism analysis may be useful to evaluate the geographical origin of native isolates of yeast strains for industrial use. Furthermore, these findings are in agreement with the idea of a clonal mode of reproduction of wine yeasts in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study permits the characterization of native yeast isolates in relation to their geographical origin. This procedure could be used as a tool for evaluating if a native isolate derives from the region were it was collected or if it is a strain derived from a commercial strain by microevolution.     

石河子不同树龄蟠桃土壤中可培养酵母多样性分析及功能酵母的筛选

为了探究新疆本土蟠桃园可培养酵母菌多样性,并挖掘功能酵母资源,本研究以新疆石河子蟠桃园3年、8年、15年树龄的根际和非根际土壤以及桃树叶片为材料,经过传统的分离培养方法获得可培养的酵母菌菌株,并进行形态学、生理生化以及26S r DNA的D1/D2区序列分析,共获得可培养酵母菌129株,从属于12个属17个种,其中子囊菌酵母为优势菌群,占分离属的88%,分布于威克汉姆酵母属(Wickerhamomyces),Vanrija属,Barnettozyma属和有孢圆酵母属(Torulaspora)等11个属的15个种。担子菌占分离属的12%,分布于隐球酵母属(Cryptococcus)的2个种。其中优势属威克汉姆酵母属,包括异常威克汉姆酵母(W. anomalus)和W. pijperi两个种,占总比例的33%,优势种异常威克汉姆酵母所占总株数比例为17%。从可培养酵母中共筛选出23株功能酵母,其中富硒酵母21株,优势种为白地霉(Galactomyces candidum),产蛋白酶酵母2株均属于隐球酵母属的Cryptococcus albidus。结果表明,新疆桃园中蕴含丰富的酵母菌资源,非根际土壤中的酵母多样性大于根际及叶片酵母多样性,且分离得到富硒酵母及产蛋白酶酵母。本研究挖掘了新疆本土可培养酵母菌资源,同时也为功能酵母的开发和利用提供理论指导。     

Genetic diversity of Agrobacterium vitis as determined by DNA fingerprints of the 5'-end of the 23S rRNA gene and Random Amplified Polymorphic DNA

Sixty-nine strains of Agrobacterium vitis , the causal agent of grape crown gall, originating from different geographical regions of the USA and Europe, were characterized by fingerprint analysis of the 5'-end of the 23S rRNA gene and by Random Amplified Polymorphic DNA (RAPD) markers. For 5'-end 23S fingerprinting, amplicons were digested with Taq I, Rsa I, Ava I, Cfo I and Alu I. For RAPD analysis, three 10-mer primers were used to generate PCR products. There was a high degree of correlation between strain groupings generated by the two methods. However, more diversity was identified when groupings were based on RAPDs. For example, 28 of 29 strains having nopaline type Ti plasmids generated identical 5'-end 23S patterns but formed two distinct RAPD groups that separated strains originating from the USA and Hungary. Similarly by RAPDs, one cluster of strains carrying vitopine-type Ti plasmids could be separated into those originating in the USA and Europe. The composition of strain groups generated by 5'-end 23S and RAPDs were highly correlated with a previous fingerprint analysis of the intergenic spacer region (located between the 16S and 23S rRNA genes) and with RFLP analysis for characterizing Ti plasmids. These findings show that among Ag. vitis strains there is a high level of correlation between two regions of the rRNA operon, total genomic DNA (as determined by RAPDs) and the type of Ti plasmid they carry.     

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.     

Effect of the use of commercial Saccharomyces strains in a newly established winery in Ronda (Málaga, Spain)

An ecological study of the yeasts present in a spontaneous and an inoculated fermentation in red wine was carried out in 2005 vintage in a winery located in the Denomination of Origin "Sierras de Málaga" (Málaga, southern of Spain). The winery operated by the first time with the 2003 vintage and since then, has used commercial yeast inocula to start alcoholic fermentation. Yeast isolates were identified by PCR-RFLP analysis of the 5.8S-ITS region from the ribosomal DNA and by mitochondrial DNA RFLP analysis. Except for non-Saccharomyces yeasts found in the fresh must before fermentation, all the isolates were found to be commercial Saccharomyces cerevisiae strains employed by the winery during the successive vintages; thus, no indigenous Saccharomyces yeasts were isolated during fermentation. The same four restriction patterns were found in non inoculated and inoculated vats, although with different frequencies. The use of commercial yeast starter in a new established winery seems to have prevented the development of a resident indigenous Saccharomyces flora.     

A homozygous diploid subset of commercial wine yeast strains

Genetic analysis was performed on 45 commercial yeasts which are used in winemaking because of their superior fermentation properties. Genome sizes were estimated by propidium iodide fluorescence and flow cytometry. Forty strains had genome sizes consistent with their being diploid, while five had a range of aneuploid genome sizes that ranged from 1.2 to 1.8 times larger. The diploid strains are all Saccharomyces cerevisiae, based on genetic analysis of microsatellite and minisatellite markers and on DNA sequence analysis of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA of four strains. Four of the five aneuploid strains appeared to be interspecific hybrids between Saccharomyces kudriavzevii and Saccharomyces cerevisiae, with the fifth a hybrid between two S. cerevisiae strains. An identification fingerprint was constructed for the commercial yeast strains using 17 molecular markers. These included six published trinucleotide microsatellites, seven new dinucleotide microsatellites, and four published minisatellite markers. The markers provided unambiguous identification of the majority of strains; however, several had identical or similar patterns, and likely represent the same strain or mutants derived from it. The combined use of all 17 polymorphic loci allowed us to identify a set of eleven commercial wine yeast strains that appear to be genetically homozygous. These strains are presumed to have undergone inbreeding to maintain their homozygosity, a process referred to previously as ‘genome renewal’.     

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90.6% of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.     

The genome sequence of the wine yeast VIN7 reveals an allotriploid hybrid genome with Saccharomyces cerevisiae and Saccharomyces kudriavzevii origins

The vast majority of wine fermentations are performed principally by Saccharomyces cerevisiae. However, there are a growing number of instances in which other species of Saccharomyces play a predominant role. Interestingly, the presence of these other yeast species generally occurs via the formation of interspecific hybrids that contain genomic contributions from both S.?cerevisiae and non-S.?cerevisiae species. However, despite the large number of wine strains that are characterized at the genomic level, there remains limited information regarding the detailed genomic structure of hybrids used in winemaking. To address this, we describe the genome sequence of the thiol-releasing commercial wine yeast hybrid VIN7. VIN7 is shown to be an almost complete allotriploid interspecific hybrid that is comprised of a heterozygous diploid complement of S.?cerevisiae chromosomes and a haploid Saccharomyces kudriavzevii genomic contribution. Both parental strains appear to be of European origin, with the S.?cerevisiae parent being closely related to, but distinct from, the commercial wine yeasts QA23 and EC1118. In addition, several instances of chromosomal rearrangement between S.?cerevisiae and S.?kudriavzevii sequences were observed that may mark the early stages of hybrid genome consolidation.     

Ecological survey of Saccharomyces cerevisiae strains from vineyards in the Vinho Verde Region of Portugal

One thousand six hundred and twenty yeast isolates were obtained from 54 spontaneous fermentations performed from grapes collected in 18 sampling sites of three vineyards (Vinho Verde Wine Region in northwest Portugal) during the 2001-2003 harvest seasons. All isolates were analyzed by mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and a pattern profile was verified for each isolate, resulting in a total of 297 different profiles, that all belonged to the species Saccharomyces cerevisiae. The strains corresponding to seventeen profiles showed a wider temporal and geographical distribution, being characterized by a generalized pattern of sporadic presence, absence and reappearance. One strain (ACP10) showed a more regional distribution with a perennial behavior. In different fermentations ACP10 was either dominant or not, showing that the final outcome of fermentation was dependent on the specific composition of the yeast community in the must. Few of the grape samples collected before harvest initiated a spontaneous fermentation, compared to the samples collected after harvest, in a time frame of about 2 weeks. The associated strains were also much more diversified: 267 patterns among 1260 isolates compared to 30 patterns among 360 isolates in the post- and pre-harvest samples, respectively. Fermenting yeast populations have never been characterized before in this region and the present work reports the presence of commercial yeast strains used by the wineries. The present study aims at the development of strategies for the preservation of biodiversity and genetic resources as a basis for further strain development.