Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

Saccharomyces species assignment by long range ribotyping

Type strains of 10 genotypically distinctSaccharomyces species are differentiated by ribosomal DNA restriction fragment analysis (ribotyping). The full length of the chromosomal ribosomal repeat was amplified in two parts, the 18SrDNA including both ITS regions (2600 bp) and the 25SrDNA (3300 bp). Restriction fragments generated by 9 enzymes from these two products yield characteristic patterns, by which unknownSaccharomyces isolates are assigned to the type strains. For convenient separation and detection only fragments longer than 200 bp were monitored. In contrast to molecular differentiation methods of highest resolution as RAPD-PCR or fingerprinting, the results from ribotyping are absolutely reproducible and thereby suitable for databases. The phylogeny computed from the discrete character matrix for presence/absence of fragments by the PHYLIP program package is in complete accordance to the phylogeny derived from ribosomal RNA sequence analysis. By this the field of application of the long range ribotyping can be regarded basically as equal to DNA sequence analysis of the same locus. Because distant relationships are recognized, missidentified genera were detected upon the species assignment. This cannot be done by methods of higher resolution like RAPD-PCR or fingerprinting.     

Species and Strain Identification of the Predatory Mite Euseius Finlandicus by RAPD-PCR and ITS Sequences

The variation within and between Finnish Euseius finlandicus populations was investigated by RAPD-PCR and ITS sequence analyses. Resin DNA extraction was found to be a suitable method for samples of single mites used in PCR. The banding patterns from 24 RAPD primers and 10 primer pairs were very similar and reproducible in all specimens of the predatory mite studied. However, the E. finlandicus K-strain could be distinguished from organophosphate-resistant predatory mites (R-strain), since almost all of them produced a 1,400 bp RAPD-PCR product, which was missing or very rare in other strains studied. Another RAPD band of ca. 680 bp was in turn much more common in other mites of E. finlandicus than in the K-strain mites. Mite specific primers were designed and used to follow the survival of the R-strain released on apple trees. The 680 bp band obtained with specific primers was specific to the species E. finlandicus mites studied, including those that had been negative with RAPD primers. The 1,400 bp specific primers could be used as a marker for following the survival of R-strain mites on apple trees. At the species level it was possible to distinguish adults and eggs of E. finlandicus from Anthoseius rhenanus and Phytonemus pallidus by RAPD-PCR. In addition, a band at 480bp was found to correspond to DNA of the predatory mite Phytoseius macropilis, when both specific primer pairs were used together. It was not possible to amplify the ITS region of E. finlandicus rDNA using several primer pairs that work in other mites and aphids. However, a basidiomycete rDNA sequence was amplified with one of these ITS primer pairs in K-strain mites. Finally, it was found that fungal rDNA-specific primers amplified an ITS region of ca. 650 bp in several strains of E. finlandicus. Internal primers, designed to amplify the central part of the 650 bp product, successfully amplified this product from all the mites.     

Identification of yeast species from orange fruit and juice by RFLP and sequence analysis of the 5.8S rRNA gene and the two internal transcribed spacers

Yeast isolates from orange fruit and juice in a spontaneous fermentation were identified and classified by two molecular techniques. The first was analysis of the restriction pattern generated from the polymerase chain reaction (PCR)-amplified 5.8S rRNA gene and the two internal transcribed spacers (ITS) using specific primers. The second technique was sequence analysis of the ITS regions using the same two primers. Nine different restriction profiles were obtained from the size of the PCR products and the restriction analyses with three endonucleases (CfoI, HaeIII and HinfI). These groups were identified as Candida tropicalis, Clavispora lusitaniae, Hanseniaspora uvarum, Pichia anomala, Pichia fermentans, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Saccharomyces unisporus, and Trichosporon asahii. Checking against identification according to morphological, physiological and biochemical traits corroborated this molecular identification. A total concordance was found in the identification with PCR-restriction fragment length polymorphism of the ITS region after analysing certified yeast strains from two different culture collections. Consequently, a rapid and reliable identification of the yeast populations was achieved by using molecular techniques.     

Mannose-containing Polysaccharides of the Apiculate Yeasts Nadsonia, Hanseniaspora, Kloeckera, and Saccharomycodes, and Their Use as an Aid in Classification

The mannose-containing polysaccharides formed by species of Nadsonia, Hanseniaspora, Kloeckera, and Saccharomycodes were extracted with hot aqueous alkali and purified by precipitation as their copper complexes. N. fulvescens and N. elongata formed galactomannans, while Hanseniaspora and Kloeckera species and S. ludwigii formed mannans. H. valbyensis, H. uvarum, and K. apiculata were a group which formed mannans which had identical H-1 regions in their proton magnetic resonance (PMR) spectra, and H. osmophila, K. africana, and K. magna mannas formed another group based on similar spectra. K. javanica formed a mannan with an H-1 spectral region which resembled that of the H. valbyensis group in some respects and that of the H. osmophila group in others. The H-1 portion of the PMR spectrum of S. lugwigii mannan was very complex and was unlike that of any other apiculate yeast studied.     

Molecular genetic characterization of the yeast Lachancea kluyveri

A comparative study of Lachancea kluyveri strains isolated in Europe, North America, Japan, and the Russian Far East was performed using restriction analysis, sequencing of non-coding rDNA regions, molecular karyotyping, and the phylogenetic analysis of the alpha- galactosidase MEL genes. This study showed a close genetic relatedness of these L. kluyveri strains. The chromosomal DNAs of the L. kluyveri strains were found to range in size from 980 to 3100 kb. The haploid number of chromosomes is equal to eight. The IGS2 restriction patterns and single nucleotide substitutions in the ITS1/ITS2 rDNA region correlate neither with geographic origin nor with the source of the strains. The L. kluyveri strains isolated from different sources have a high degree of homology (79-100%) of their MEL genes. The phylogenetic analysis of all of the known alpha-galactosidases in the "Saccharomyces" clade showed that the MEL genes of the yeasts L. kluyveri. L. cidri, Saccharomyces cerevisiae, S. paradoxus, S. bayanus, and S. mikatae are species specific.     

Intraspecific diversity of the marine fish pathogen Tenacibaculum maritimum as determined by randomly amplified polymorphic DNA-PCR

AIM: The aim of the present study was to evaluate the intraspecific genetic variability within Tenacibaculum maritimum strains isolated from different species of marine fish. METHODS AND RESULTS: Twenty-nine strains isolated from five different fish species and three reference strains were characterized by randomly amplified polymorphic DNA (RAPD) method. Cluster analysis of RAPD-PCR profiles showed that the strains, regardless of the oligonucleotide primer employed (P2 and P6), were separated into two main groups that strongly correlated with the host species and/or O-serotypes described for this pathogen. One group composed all strains isolated from sole (Solea senegalensis and S. solea) and gilthead seabream (Sparus aurata), and the other compiled the T. maritimum isolates from yellowtail (Seriola quinqueradiata), Atlantic salmon (Salmo salar) and turbot (Scophthalmus maximus). An important exception was observed in the RAPD patterns of the reference strains, which were included in different genetic groups depending on the primer employed. CONCLUSIONS: The results obtained demonstrated genetic variability within the T. maritimum isolated from different marine fish. Such genetic variability proved to be strongly associated with the host and/or serogroups described for this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD analysis constitutes a valuable molecular technique for epidemiological studies of T. maritimum. Interestingly, this is the first report of intraspecific differentiation and characterization of T. maritimum strains isolated from cultured fish.     

Molecular divergence of the soil yeasts Williopsis sensu stricto

Fifty-three strains of saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA which comprised the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, Komagataea pratensis, and the Williopsis sensu stricto complex. Minisatellite primer M13 was proposed for the differentiation between twin species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.     

Application of molecular techniques for identification of fungal communities colonising paper material

Archives and libraries all over the world suffer from biodeterioration of writings caused by microorganisms, especially fungi. With traditionally used culture-dependent methods, only a small amount of effectively colonising organisms is detected. Restoration and maintenance of written cultural heritage is therefore problematic due to incomplete knowledge of the deterioration agents.In the present study, culture-independent molecular methods were applied to identify fungal communities colonising paper samples of different composition and age. Nucleic-acid-based strategies targeting the internally transcribed spacer (ITS) regions, which are nested in the nuclear rDNA repeats, were selected to investigate the fungal diversity on paper. The ITS regions possess a high variation among taxonomically distinct fungal species and even within the species.With this aim, several molecular biological methods were optimised for working with paper materials. Here, we introduce a DNA extraction protocol, which allowed the direct extraction of PCR-amplifiable DNA from samples derived from different kinds of paper. The DNA extracts were used to amplify either the ITS1 or ITS2 region by using different fungi-specific primer sets. The ITS-amplified regions were subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Conditions for DGGE analysis, gradient, voltage, and running time, were established to accurately discriminate different fungal species in complex communities. Pure fungal strains were used to constitute a marker for further comparative investigations of historic papers.     

An assessment of the genetic diversity within Ganoderma strains with AFLP and ITS PCR-RFLP

Ganoderma lucidum is one of the most important medicinal materials and plant pathogens. Because of its specific interhybridization, the genetic background, however, is relatively unclear. It made identification of Ganoderma strains, especially closely related strains difficulty. Amplified fragment length polymorphism (AFLP) using 14 primer combinations and internal transcribed spacer (ITS) PCR-RFLP were used in a comparative study which was designed to investigate the closely related Ganoderma strains genetic relations at molecular level. The analysis of 37 Ganoderma strains showed there were 177 polymorphic AFLP markers and 12 ITS PCR-RFLP markers, and all accessions could be uniquely identified. Among the Ganoderma accessions, similarity coefficients ranged from 0.07692 to 0.99194 in AFLP. The Ganoderma strains formed a tight cluster in nine groups in AFLP whereas seven groups in ITS PCR-RFLP. The cluster analysis revealed that the taxonomical system of subgenus Ganoderma is composed of Sect. Ganoderma and Sect. Phaeonema, and the strain 22 should be a variant form of strain 21. All methods delineated the Ganoderma strains from the different regions seeming to show a greater level of genetic diversity. It indicated that the genotype study at molecular level is a useful complement method to the current classification system of Ganoderma strains based on morphological traits. The congruency of the experiments was analyzed using the biostatistical software DPS V3.01.     

Rapid differentiation of phenotypically and genotypically similar <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> strains by PCR fingerprinting.

PCR amplification techniques viz., repetitive DNA element PCR (REP-PCR), short tandemly repeated repetitive PCR (STRR-PCR) and arbitrarily primed PCR (RAPD-PCR) were used for the taxonomic discrimination among the strains of the unicellular cyanobacterium Synechococcus elongatus collected across the coastal regions of the Indian subcontinent. These strains showed similar phenotypic and genotypic characteristics. Data obtained from genomic fingerprinting were used to perform cluster analysis and demonstrated ability to differentiate strains at intra-specific level. Polymorphisms of different PCR amplification products can serve as strain-specific molecular fingerprints. In comparison with the STRR and RAPD, the REP primer set generates fingerprints of lower complexity, but still the phenogram clearly differentiated the strains. In conclusion, described PCR fingerprinting methods can be considered as promising tools for the differentiation at the strain level of cyanobacteria from the same species.     

Glycerol and other fermentation products of apiculate wine yeasts

Ninety-six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata , whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.     

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.     

Biometric Study of Acetoin Production in Hanseniaspora guilliermondii and Kloeckera apiculata

Gas chromatographic analysis by direct injection of samples yielded quantitative data on acetoin content. Ninety-six strains of Hanseniaspora guilliermondii and Kloeckera apiculata were investigated for the ability to produce acetoin in synthetic medium and in must. High-level production of acetoin was found to be a characteristic of both species. In synthetic medium, the two species were not significantly different with respect to sugar utilization and ethanol or acetoin production. In grape must, the two species were significantly different (P = 0.001) in acetoin production and K. apiculata exhibited a significantly negative correlation between acetoin production and either sugar consumption or ethanol production. Use of selected apiculate yeasts in mixed cultures with Saccharomyces cerevisiae seems promising for optimization of wine bouquet.     

ITS1: a DNA barcode better than ITS2 in eukaryotes?

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer (ITS/ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large‐scale meta‐analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity‐based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample‐rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species.     

Limitations on the use of polymerase chain reaction--restriction fragment length polymorphism analysis of the rDNA NTS2 region for the taxonomic classification of the species Saccharomyces cerevisiae

Different molecular techniques were tested to determine which was the most effective in the identification of Saccharomyces cerevisiae strains. In particular, polymerase chain reaction--restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions and the nontranscribed spacer 2 (NTS2) region, sequencing of the D1/D2 domain, and electrophoretic karyotyping were applied to 123 yeast strains isolated from different sourdoughs and tentatively attributed to the species S. cerevisiae. All of the strains tested showed an identical PCR-RFLP pattern for the ITS regions, an identical nucleotide sequence of the D1/D2 domain, and the typical electrophoretic karyo type of S. cerevisiae. In contrast, 14 out of the 123 strains tested showed some polymorphism with BanI restriction analysis of the NTS2 region. Our results indicate that while the sequencing of the D1/D2 domain, the PCR-RFLP analysis of the ITS regions, and the electrophoretic karyotype can be employed successfully to identify S. cerevisiae strains, PCR-RFLP analysis of the NTS2 region does not allow a consistent and accurate grouping for S. cerevisiae strains. The fact that the NTS2 region of a small number of strains (8.78% of the total strains tested) is different from that of the other S. cerevisiae strains confirms that molecular methods should always be tested on a great number of strains.     

Phylogenetic relationships among yeasts of the 'Saccharomyces complex' determined from multigene sequence analyses

Species of Saccharomyces, Arxiozyma, Eremothecium, Hanseniaspora (anamorph Kloeckera), Kazachstania, Kluyveromyces, Pachytichospora, Saccharomycodes, Tetrapisispora, Torulaspora, and Zygosaccharomyces, as well as three related anamorphic species assigned to Candida (C. castellii, C. glabrata, C. humilis), were phylogenetically analyzed from divergence in genes of the rDNA repeat (18S, 26S, ITS), single copy nuclear genes (translation elongation factor 1alpha, actin-1, RNA polymerase II) and mitochondrially encoded genes (small-subunit rDNA, cytochrome oxidase II). Single-gene phylogenies were congruent for well-supported terminal lineages but deeper branches were not well resolved. Analysis of combined gene sequences resolved the 75 species compared into 14 clades, many of which differ from currently circumscribed genera.     

Molecular and technological approaches to evaluate strain biodiversity in Hanseniaspora uvarum of wine origin

AIMS: The characterization by molecular and physiological methods of wild apiculate strains, isolated from 'Aglianico del Vulture' grape must. METHODS AND RESULTS: The restriction analysis of 18S rDNA allowed the identification of strains at the species level, which were predominantly Hanseniaspora uvarum. The RAPD analysis and the evaluation of technological traits, such as the metabolic and enzymatic activities, were useful to evaluate the polymorphism of this species. CONCLUSIONS: The RAPD analysis clustered the wild H. uvarum strains in four main genetic groups and a very high phenotypic variability confirmed this genetic polymorphism. The technological variables, which determined the strain biodiversity differed significantly, demonstrating that these technological traits are strain dependent. A certain correlation was found between the strain behaviour and its isolation zone, indicating the influence of the environment on the genetic patrimony of the population. SIGNIFICANCE AND IMPACT OF THE STUDY: The genetic and technological biodiversity recorded among H. uvarum wild strains represents the basis for organizing a collection of apiculate strains exhibiting oenological characteristics at different levels, such as high/low production of secondary compounds, and, therefore, potentially useful for a selection programme.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Genetic diversity of the natural strains of Streptococcus thermophilus

The method of RAPD-PCR and comparative analysis of the PCR fingerprinting profiles similarity was used to characterize interspecific diversity of natural isolates of the lactic acid bacteria Streptococcus thermophilus. The strain genetic diversity was demonstrated using three primer variants, designed for different bacterial genome regions. The resolution of RAPD-PCR technique with different primers for identification at the species level and for certification at the strain level, was examined relative to the commercially important cultures of S. thermophilus. The results provided conclusion on preferable usage of RAPD-PCR with the primer ERIC-1 for specific identification of S. thermophilus, and with the primer M13 for certification of natural isolates of this species at the strain level.