Mitochondrial DNA Restriction Fragment Length Research Articles

Preliminary Study on Yeasts Associated with the Production of "Tostado"-a Traditional Sweet Wine from Galicia (NW Spain).

Tostado is a traditional sweet wine from the Designations of Origins (DOs) of Ribeiro and Valdeorras in Galicia (NW Spain). The harvested grapes are air-dried and pressed to increase the concentrations of sugars, acids, and flavour compounds. Therefore, knowledge of the yeasts involved in fermentation under these conditions is essential to guarantee the quality and uniqueness of the valuable, distinctive, and expensive Tostado wines. Saccharomyces and non-Saccharomyces yeasts were identified using Wallerstein Laboratory (WL) Nutrient Agar and lysine plating, followed by polymerase chain reaction (PCR) amplification, enzymatic digestion, and sequencing. Saccharomyces cerevisiae isolates were further characterised at the strain level using mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP). Statistical analyses were also performed, including different diversity indices, Similarity Percentage (SIMPER) analysis, principal component analysis (PCA), neighbor-joining clustering, parsimony-phylogram, and network plot. In addition, the total acidity, volatile acidity, reducing sugars, and alcoholic strength by volume of the Tostado wines were analysed. A wide diversity of autochthonous yeasts was found, which were predominantly species of oenological relevance, such as Lachancea thermotolerans, Starmerella bacillaris, Hanseniaspora uvarum, Debaryomyces hansenii, Torulaspora delbrueckii, Pichia spp., and Saccharomyces cerevisiae from the must and paste stages of Tostado wine. In addition, 19 different S. cerevisiae strains were identified. This high yeast diversity, which changed from the early stages of fermentation, could contribute to the distinctive characteristics observed in Tostado wine. Characteristic and differentiating chemical and microbiological profiles were found as early as the pre-fermentation stages, which adds value to these special wines that have rarely been studied.

Diversity of Saccharomyces cerevisiae strains isolated of the spontaneous fermentation of cachaça from northeastern Brazil

Cachaca is a beverage obtained by distilling fermented sugar cane juice. The state of Bahia in northeastern Brazil is the second-largest producer of traditional cachaca, and this region has the potential to improve the quality and quantity of its beverage production. The aim of this study was to analyze the genetic diversity of Saccharomyces cerevisiae populations isolated from must in six distilleries in Bahia using mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP). Among the three hundred and thirty S. cerevisiae strains isolated, mtDNA-RFLP analysis identified a total of 30 molecular patterns. Analysis of molecular variance (AMOVA) revealed that the greatest genetic variation was found among, rather than within, the populations. Population structure analyses showed the presence of three distinct gene pools, thereby corroborating the AMOVA results. This study represents an important contribution to better understanding the molecular characterization and genetic variability of S. cerevisiae strains during the fermentation of cachaca. The dominant molecular patterns identified here may be used to select S. cerevisiae strains that could improve the quality and volume of traditional cachaca production in Bahia.

No native trout on Cyprus

No native trout on Cyprus

Isolation and identification of Acanthamoeba strains from soil and tap water in Yanji, China

BackgroundMembers of the genus Acanthamoeba are widely distributed throughout the world, and some of them are considered pathogenic, as they are capable of causing corneal and central nervous system diseases. In this study, we isolated Acanthamoeba strains from soil and tap water in Yanji, China.MethodsWe identified four strains of Acanthamoeba (CJY/S1, CJY/S2, CJY/S3, and CJY/W1) using mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) analysis. Nuclear 18S rDNA sequences were used for phylogenetic analysis and species identification.ResultsGenotypic characterization of the isolates showed that they belonged to genotypes T4 (CJY/S1 and CJY/S2), T5 (CJY/S3), and T16 (CJY/W1). Sequence differences between CJY/S1 and Acanthamoeba castellanii Neff, CJY/S2 and Acanthamoeba KA/E7, and CJY/S3 and Acanthamoeba lenticulata 68–2 were 0.31, 0.2, and 0.26%, respectively. 18S ribosomal deoxyribonucleic acid (rDNA) of CJY/W1 had 99% sequence identity to that of Acanthamoeba sp. U/H-C1. Strains CJY/S1 and CJY/S2, isolated from soil, had similar mtDNA RFLP patterns, whereas strain CJY/W1, isolated from tap water, displayed a different pattern.ConclusionsTo the best of our knowledge, this is the first report on the identification of genotypes T4, T5, and T16 from environmental sources in Yanji, China.

Phylogenetic analysis of the Australian rosella parrots (Platycercus) reveals discordance among molecules and plumage

Relationships and species limits among the colourful Australian parrots known as rosellas (Platycercus) are contentious because of poorly understood patterns of parapatry, sympatry and hybridization as well as complex patterns of geographical replacement of phenotypic forms. Two subgenera are, however, conventionally recognised: Platycercus comprises the blue-cheeked crimson rosella complex (Crimson Rosella P. elegans and Green Rosella P. caledonicus), and Violania contains the remaining four currently recognised species (Pale-headed Rosella P. adscitus, Eastern Rosella P. eximius, Northern Rosella P. venustus, and Western Rosella P. icterotis). We used phylogenetic analysis of ten loci (one mitochondrial, eight autosomal and one z-linked) and several individuals per nominal species primarily to examine relationships within the subgenera, especially the relationships and species limits within Violania. Of these, P. adscitus and P. eximius have long been considered sister species or conspecific due to a morphology-based hybrid zone and an early phylogenetic analysis of mitochondrial DNA restriction fragment length polymorphisms. The multilocus phylogenetic analysis presented here supports an alternative hypothesis aligning P. adscitus and P. venustus as sister species. Using divergence rates published in other avian studies, we estimated the divergence between P. venustus and P. adscitus at 0.0148–0.6124MYA and that between the P. adscitus/P. venustus ancestor and P. eximius earlier at 0.1617–1.0816MYA, both within the Pleistocene. Discordant topologies among gene and species trees are discussed and proposed to be the result of historical gene flow and/or incomplete lineage sorting (ILS). In particular, we suggest that discordance between mitochondrial and nuclear data may be the result of asymmetrical mitochondrial introgression from P. adscitus into P. eximius. The biogeographical implications of our findings are discussed relative to similarly distributed groups of birds.

Rapid genetic identification of brown bullhead (Ameiurus nebulosus), black bullhead (Ameiurus melas) and their hybrids

Hybridization can complicate the effective conservation of cryptic and morphologically similar species. Here we provide a rapid genetic method for the identification of pure-strain brown bullhead (Ameiurus nebulosus) and black bullhead (Ameiurus melas) using nuclear and mitochondrial DNA restriction fragment length polymorphism assays. We confirm earlier reports of introgressive hybridization among A. nebulosus and A. melas in Lake Erie, and reveal the presence of introgressed hybrids in Detroit River. Our method provides a prime example of the utility of internet-based genetic databases for the development of molecular tools in biological and environmental research, and suggests hybridization plays a larger role in the genetic structure of bullheads.

Modulation of chemical and sensory characteristics of red wine from Mencía by using indigenous <em>Saccharomyces cerevisiae</em> yeast strains

<p style="text-align: justify;"><strong>Aim</strong>: To evaluate the influence of native <em>Saccharomyces cerevisiae </em>strains in red wines from <em>Vitis vinifera</em> cv. Mencía: fermentative ability, inoculation success, and sensory and chemical characteristics of wines.</p><p style="text-align: justify;"><strong>Methods and results</strong>: Indigenous yeast strains (Sc5, Sc11, Sc21 and Sc24) were inoculated in grape musts and their inoculation success was followed by mtDNA-RFLP (mitochondrial DNA-restriction fragment length polymorphism) at different stages of fermentation. The results showed that the added yeast strains fermented in co-dominance with a resident strain, which also controlled the spontaneous processes. Chemical analysis of basic wine parameters using official methodologies showed significant differences among wines for alcohol degree and volatile acidity. Fermentative aroma compounds were determined by gas chromatography. Wines made with different yeast strains varied in higher alcohols, ethyl ester, 2-phenylethanol, ethyl lactate and acetoin content. Sensory analysis indicated that wine from strain Sc24 had the best overall score, whereas that from strain Sc11 achieved the highest scores for colour intensity, structure and fruity character.</p><p style="text-align: justify;"><strong>Conclusions</strong>: The application of selected <em>S. cerevisiae</em> strains allowed us to obtain differentiated wines from both the chemical and sensory points of view.</p><p style="text-align: justify;"><strong>Significance and impact of the study</strong>: The results confirmed that indigenous yeasts can be used to elaborate singular wines and may constitute a useful tool to diversify Mencía wines.</p>

Convergent evolutionary processes driven by foraging opportunity in two sympatric morph pairs of Arctic charr with contrasting post-glacial origins

The expression of two or more discrete phenotypes amongst individuals within a species (morphs) provides multiple modes upon which selection can act semi-independently, and thus may be an important stage in speciation. In the present study, we compared two sympatric morph systems aiming to address hypotheses related to their evolutionary origin. Arctic charr in sympatry in Loch Tay, Scotland, exhibit one of two discrete, alternative body size phenotypes at maturity (large or small body size). Arctic charr in Loch Awe segregate into two temporally segregated spawning groups (breeding in either spring or autumn). Mitochondrial DNA restriction fragment length polymorphism analysis showed that the morph pairs in both lakes comprise separate gene pools, although segregation of the Loch Awe morphs is more subtle than that of Loch Tay. We conclude that the Loch Awe morphs diverged in situ (within the lake), whereas Loch Tay morphs most likely arose through multiple invasions by different ancestral groups that segregated before post-glacial invasion (i.e. in allopatry). Both morph pairs showed clear trophic segregation between planktonic and benthic resources (measured by stable isotope analysis) but this was significantly less distinct in Loch Tay than in Loch Awe. By contrast, both inter-morph morphological and life-history differences were more subtle in Loch Awe than in Loch Tay. The strong ecological but relatively weak morphological and life-history divergence of the in situ derived morphs compared to morphs with allopatric origins indicates a strong link between early ecological and subsequent genetic divergence of sympatric origin emerging species pairs. The emergence of parallel specialisms despite distinct genetic origins of these morph pairs suggests that the effect of available foraging opportunities may be at least as important as genetic origin in structuring sympatric divergence in post-glacial fishes with high levels of phenotypic plasticity. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ••, ••–••.

Neither philopatric nor panmictic: microsatellite and mtDNA evidence suggests lack of natal homing but limits to dispersal in Pacific lamprey

Most species with lengthy migrations display some degree of natal homing; some (e.g. migratory birds and anadromous salmonids) show spectacular feats of homing. However, studies of the sea lamprey (Petromyzon marinus) indicate that this anadromous species locates spawning habitat based on pheromonal cues from larvae rather than through philopatry. Previous genetic studies in the anadromous Pacific lamprey (Entosphenus tridentatus) have both supported and rejected the hypothesis of natal homing. To resolve this, we used nine microsatellite loci to examine the population structure in 965 Pacific lamprey from 20 locations from central British Columbia to southern California and supplemented this analysis with mitochondrial DNA restriction fragment length polymorphism analysis on a subset of 530 lamprey. Microsatellite analysis revealed (i) relatively low but often statistically significant genetic differentiation among locations (97% pairwise F(ST) values were <0.04 but 73.7% were significant); and (ii) weak but significant isolation by distance (r(2) = 0.0565, P = 0.0450) but no geographic clustering of samples. The few moderate F(ST) values involved comparisons with sites that were geographically distant or far upstream. The mtDNA analysis--although providing less resolution among sites (only 4.7%F(ST) values were significant)--was broadly consistent with the microsatellite results: (i) the southernmost site and some sites tributary to the Salish Sea were genetically distinct; and (ii) southern sites showed higher haplotype and private haplotype richness. These results are inconsistent with philopatry, suggesting that anadromous lampreys are unusual among species with long migrations, but suggest that limited dispersal at sea precludes panmixia in this species.

Genetic Identity of Walleye in the Cumberland River

Walleye from the upper Cumberland River drainage were examined by means of mitochondrial DNA restriction fragment length polymorphism (RFLP) analysis, microsatellite analysis of nuclear DNA, and sequencing of the mitochondrial DNA control region. The objectives of this study were to identify native Eastern Highland and introduced Great Lakes walleye and determine if introgression occurs between the two stocks. Additional collections were made from rivers of the Eastern Highlands to establish evolutionary relationships. Native and introduced walleye appear reproductively partitioned in the upper Cumberland River drainage, with natives persisting in the tributary Rockcastle River and introduced Great Lakes walleye reproducing in the Big South Fork of the Cumberland River. Additional partitioning between native and introduced stocks may occur in the Kanawha River, in the Ohio River drainage. Native walleye from the Cumberland River share a common ancestor with others from Eastern Highland rivers but are distantly related to populations from the southern Mobile River basin and the northern Great Lakes.

Natural and Anthropogenic Forces Shape the Population Genetics and Recent Evolutionary History of Eastern United States Bay Scallops (Argopecten irradians)

ABSTRACT Bay scallops (Argopecten irradians Lamarck) are ecologically important in U.S. Atlantic waters off northeastern states and in the Florida Gulf of Mexico, and have been intensely harvested from both of those regions for decades. However, a detailed study comparing their basic population genetic structures using more than a single type of genetic marker has not been conducted. Through such a study, key phylogeographic, taxonomic, and fisheries issues can be addressed. We used variation in allozyme loci and mitochondrial DNA restriction fragment length polymorphisms to evaluate and compare the population genetic structures of bay scallops from those two regions, to propose a new interpretation for the composition of the North Carolina bay scallop population, to resolve the taxonomic quandary of Argopecten irradians taylorae, and to evaluate the apparent and potential genetic effects of the common fishery practice of hatchery-based stock enhancement on the genetic diversity and relatedness of Atlanti...

Intraspecific Diversity of Yeast Associated to Vitis vinifera Albariño Must from Different Vineyard Ecosystems

The genetic diversity of Saccharomyces cerevisiae associated to the must (Vitis vinifera Albarino) from different geographic areas and spontaneous fermentation was studied by analysis of mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP). Colonies were isolated in musts at different stages of fermentation from three different geographic areas, O Val do Salnes, O Rosal and O Condado do Tea from Galicia (NW Spain). A total of 17 different patterns out of 273 S. cerevisiae colonies were identified using mitochondrial DNA restriction analysis. Pattern I from O Rosal, VI from O Val do Salnes and XII from O Condado do Tea were the predominant ones. Only pattern II was common to all fermentations. This molecular study provides useful information about distribution and genetic relationships among several S. cerevisiae strains associated to Vitis vinifera Albarino from different vineyard ecosystems and their influence on certain oenological properties.

Genetic Structure of Phytophthora infestans Populations in China Indicates Multiple Migration Events

One hundred isolates of Phytophthora infestans collected from 10 provinces in China between 1998 and 2004 were analyzed for mating type, metalaxyl resistance, mitochondrial DNA (mtDNA) haplotype, allozyme genotype, and restriction fragment length polymorphism (RFLP) with the RG-57 probe. In addition, herbarium samples collected in China, Russia, Australia, and other Asian countries were also typed for mtDNA haplotype. The Ia haplotype was found during the first outbreaks of the disease in China (1938 and 1940), Japan (1901, 1930, and 1931), India (1913), Peninsular Malaysia (1950), Nepal (1954), The Philippines (1910), Australia (1917), Russia (1917), and Latvia (1935). In contrast, the Ib haplotype was found after 1950 in China on both potato and tomato (1952, 1954, 1956, and 1982) and in India (1968 and 1974). Another migration of a genotype found in Siberia called SIB-1 (Glucose-6-phosphate isomerase [Gpi] 100/100, Peptidase [Pep] 100/100, IIa mtDNA haplotype) was identified using RFLP fingerprints among 72% of the isolates and was widely distributed in the north and south of China and has also been reported in Japan. A new genotype named CN-11 (Gpi 100/111, Pep 100/100, IIb mtDNA haplotype), found only in the south of China, and two additional genotypes (Gpi 100/100, Pep 100/100, Ia mtDNA haplotype) named CN-9 and CN-10 were identified. There were more diverse genotypes among isolates from Yunnan province than elsewhere. The SIB-1 (IIa) genotype is identical to those from Siberia, suggesting later migration of this genotype from either Russia or Japan into China. The widespread predominance of SIB-1 suggests that this genotype has enhanced fitness compared with other genotypes found. Movement of the pathogen into China via infected seed from several sources most likely accounts for the distribution of pathogen genotypes observed. MtDNA haplotype evidence and RFLP data suggest multiple migrations of the pathogen into China after the initial introduction of the Ia haplotype in the 1930s.

Allochronic isolation and incipient hybrid speciation in tiger swallowtail butterflies

Hybridization leading to reproductively isolated, novel genotypes is poorly understood as a means of speciation and few empirical examples have been studied. In 1999, a previously non-existent delayed flight of what appeared to be the Canadian tiger swallowtail butterfly, Papilio canadensis, was observed in the Battenkill River Valley, USA. Allozyme frequencies and morphology suggest that this delayed flight was the product of hybridization between Papilio canadensis and its sibling species Papilio glaucus. The mitochondrial DNA (mtDNA) restriction fragment length polymorphisms presented here indicate that only P. canadensis-like mtDNA occurs in this population, suggesting that introgression likely occurred from hybrid males mating with P. canadensis females. Preliminary studies of this population indicated that delayed post-diapause pupal emergence in this hybrid genotype was the root cause behind the observed delayed flight, which suggests a potential empirical example of a mechanism leading to reproductive isolation. Here we provide further evidence of the role of adult pupal emergence as a reproductive barrier likely leading to reproductive isolation. In particular, we present results from pupal emergence studies using four different spring and two different winter temperature treatments. The results indicate a clear separation of adult emergences between the hybrid population and both parental species. However, our results indicate that exceptionally hot springs are likely to lead to greater potential for overlap between the local parental species, P. canadensis, and this delayed population with hybrid origins. Conversely, our results also show that warmer winters are likely to increase the temporal separation of the hybrid population and the parental species. Finally, we report recently collected evidence that this hybrid population remains morphologically distinct.

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaca fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaca fermentation process. Cachaca S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.

Endosymbionts of Acanthamoeba Isolated from Domestic Tap Water in Korea

In a previous study, we reported our discovery of Acanthamoeba contamination in domestic tap water; in that study, we determined that some Acanthamoeba strains harbor endosymbiotic bacteria, via our molecular characterization by mitochondrial DNA restriction fragment length polymorphism (Mt DNA RFLP). Five (29.4%) among 17 Acanthamoeba isolates contained endosymbionts in their cytoplasm, as demonstrated via orcein staining. In order to estimate their pathogenicity, we conducted a genetic characterization of the endosymbionts in Acanthamoeba isolated from domestic tap water via 16S rDNA sequencing. The endosymbionts of Acanthamoeba sp. KA/WP3 and KA/WP4 evidenced the highest level of similarity, at 97% of the recently published 16S rDNA sequence of the bacterium, Candidatus Amoebophilus asiaticus. The endosymbionts of Acanthamoeba sp. KA/WP8 and KA/WP12 shared a 97% sequence similarity with each other, and were also highly similar to Candidatus Odyssella thessalonicensis, a member of the alpha-proteobacteria. The endosymbiont of Acanthamoeba sp. KA/WP9 exhibits a high degree of similarity (85-95%) with genus Methylophilus, which is not yet known to harbor any endosymbionts. This is the first report, to the best of our knowledge, to show that Methylophilus spp. can live in the cytoplasm of Acanthamoeba.

Populational analysis of Saccharomyces cerevisiae strains from different appellations of origin and grape varieties by microsatellite analysis.

The objective of the present study was to evaluate populational relationships among Saccharomyces cerevisiae strains isolated from some of the Portuguese most important grapevine varieties in different appellations of origin, using polymorphic microsatellites. One hundred ninety two grape samples were collected during the 2006 and 2007 harvest season in the Vinho Verde (grape varieties: Arinto, Alvarinho, Avesso, Loureiro, Touriga Nacional) Bairrada (grape varieties: Arinto, Baga, Castelão Francês, Maria Gomes, Touriga Nacional) Alentejo (grape varieties, Aragonês, Trincadeira, Touriga Nacional), Terras do Sado (grape variety Castelão) Bucelas (grape variety Arinto) and Estremadura (grape varieties: Arinto, Aragonês, Castelão, Trincadeira, Touriga Nacional) appellations of origin. From the final stage of spontaneous fermentations, 2820 yeast isolates were obtained, mainly belonging to the species S. cerevisiae. An initial genetic screen, based on mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and/or interdelta sequence analysis was followed by microsatellite analysis of strains with unique genetic profiles, using 10 highly polymorphic microsatellites. Our results showed that microsatellite analysis revealed a high resolution populational screen, showing that genetic differences and populational structures among S. cerevisiae populations derived from both “diagnostic” vineyard-, specific alleles and the accumulation of small allele-frequency differences across ten microsatellite loci. Heterozygosity was three to four times lower than the expected value, confirming the strong populational substructuring. The presented large-scale approach shows that each vineyard contains differentiated S. cerevisiae populations, showing the occurrence of specific native strains that can be associated with a terroir. Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762).

Populational analysis of Saccharomyces cerevisiae strains from different appellations of origin and grape varieties by microsatellite analysis.

AbstractThe objective of the present study was to evaluate populational relationships among Saccharomyces cerevisiae strains isolated from some of the Portuguese most important grapevine varieties in different appellations of origin, using polymorphic microsatellites. One hundred ninety two grape samples were collected during the 2006 and 2007 harvest season in the Vinho Verde (grape varieties: Arinto, Alvarinho, Avesso, Loureiro, Touriga Nacional) Bairrada (grape varieties: Arinto, Baga, Castelão Francês, Maria Gomes, Touriga Nacional) Alentejo (grape varieties, Aragonês, Trincadeira, Touriga Nacional), Terras do Sado (grape variety Castelão) Bucelas (grape variety Arinto) and Estremadura (grape varieties: Arinto, Aragonês, Castelão, Trincadeira, Touriga Nacional) appellations of origin. From the final stage of spontaneous fermentations, 2820 yeast isolates were obtained, mainly belonging to the species S. cerevisiae. An initial genetic screen, based on mitochondrial DNA restriction fragment length polymorphism (mtDNA RFLP) and/or interdelta sequence analysis was followed by microsatellite analysis of strains with unique genetic profiles, using 10 highly polymorphic microsatellites. Our results showed that microsatellite analysis revealed a high resolution populational screen, showing that genetic differences and populational structures among S. cerevisiae populations derived from both “diagnostic” vineyard-, specific alleles and the accumulation of small allele-frequency differences across ten microsatellite loci. Heterozygosity was three to four times lower than the expected value, confirming the strong populational substructuring. The presented large-scale approach shows that each vineyard contains differentiated S. cerevisiae populations, showing the occurrence of specific native strains that can be associated with a terroir. Financially supported by the programs POCI 2010 (FEDER/FCT, POCTI/AGR/56102/2004) and AGRO (ENOSAFE, Nº 762).

Commercial Ripening Starter Microorganisms Inoculated into Cheese Milk Do Not Successfully Establish Themselves in the Resident Microbial Ripening Consortia of a South German Red Smear Cheese

Production of smear-ripened cheese critically depends on the surface growth of multispecies microbial consortia comprising bacteria and yeasts. These microorganisms often originate from the cheese-making facility and, over many years, have developed into rather stable, dairy-specific associations. While commercial smear starters are frequently used, it is unclear to what degree these are able to establish successfully within the resident microbial consortia. Thus, the fate of the smear starters of a German Limburger cheese subjected to the "old-young" smearing technique was investigated during ripening. The cheese milk was supplemented with a commercial smear starter culture containing Debaryomyces hansenii, Galactomyces geotrichum, Arthrobacter arilaitensis, and Brevibacterium aurantiacum. Additionally, the cheese surface was inoculated with an extremely stable in-house microbial consortium. A total of 1,114 yeast and 1,201 bacterial isolates were identified and differentiated by Fourier transform infrared spectroscopy. Furthermore, mitochondrial DNA restriction fragment length polymorphism, random amplified polymorphic DNA, repetitive PCR, and pulsed field gel electrophoresis analyses were used to type selected isolates below the species level. The D. hansenii starter strain was primarily found early in the ripening process. The G. geotrichum starter strain in particular established itself after relocation to a new ripening room. Otherwise, it occurred at low frequencies. The bacterial smear starters could not be reisolated from the cheese surface at all. It is concluded that none of the smear starter strains were able to compete significantly and in a stable fashion against the resident microbial consortia, a result which might have been linked to the method of application. This finding raises the issue of whether addition of starter microorganisms during production of this type of cheese is actually necessary.

Genetic and phenotypic diversity of autochthonous Saccharomyces spp. strains associated to natural fermentation of ‘Malvasia delle Lipari’

Characterize from both genetic and phenotypic standpoints the indigenous strains of Saccharomyces spp. associated with natural fermentation of 'Malvasia delle Lipari'. A total of 192 yeast isolates were obtained from completed fermentation of a mix of 'Malvasia delle Lipari' (92%) and 'Corinto nero' (8%) grapes in two wineries in Salina Island (Sicily, Italy). Fifty-one Saccharomyces spp. isolates were characterized using ITS-PCR, random amplified polymorphic DNA-PCR and mitochondrial DNA restriction fragment length polymorphism and 12 biotypes were identified. Representative strains of each biotype, tested for their physiological traits, exhibit different killer activity, fermentation vigour, production of hydrogen sulphide and show similar beta-glucosidase and proteolytic activity. It is possible to cluster in different groups naturally occurring indigenous biotypes of Saccharomyces cerevisiae from 'Malvasia delle Lipari' on the basis of molecular profiles. Deeper insight on indigenous wine yeast of a conserved environment. The knowledge gained might offer a contribution to the selection of autochthonous wine yeast as starters for controlled fermentations.