Orsolya Molnár

Trichosporon mycotoxinivorans sp. nov., A New Yeast Species Useful in Biological Detoxification of Various Mycotoxins

A yeast strain isolated from the hindgut of the lower termite Mastotermes darwiniensis (Mastotermitidae) was found to represent a new member of the genus Trichosporon. Trichosporon mycotoxinivorans is closely related to T. loubieri on the basis of the phylogenetic trees based on the D1/D2 region of 26S rDNA, an approx. 600 bp fragment of the 18S rDNA and both ITS regions. However, the two species differ at nine positions in the D1/D2 region of 26S rDNA. The IGS1 region of T. mycotoxinivorans is 401 bp long. T. mycotoxinivorans is distinguished from T. loubieri by its ability to assimilate inulin and galactitol, and its inability to grow at 40 degrees C. The name of this newly isolated strain refers to an important characteristics of T. mycotoxinivorans to detoxify mycotoxins such as ochratoxin A and zearalenone. Therefore this strain can be used for the deactivation of the respective mycotoxins in animal feeds.

Phenotypic and genotypic identification of yeasts from cheese.

Eighty-five yeast strains isolated from different cheeses of Austria, Denmark, France, Germany, and Italy were identified using physiological methods and genotypically using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Good congruence was found between the phenotypic and genotypic data for 39 of the isolates. However, 26 isolates of Geotrichum could only be identified to the species level using the genotypic methods and 7 isolates were correctly identified to the genus level only using phenotypic identification methods. The phenotypic identification did not agree with the genotypic data for 14 yeast isolates. Using ubiquinone analysis, yeast cell wall sugars and the diazonium blue B test 5 incorrectly identified isolates with phenotypic methods could be identified genotypically. In addition the 7 isolates identified only to the genus level by the phenotypic methods and the 26 Geotrichum strains were identified to the species level using the polyphasic molecular approach mentioned above. Eleven strains remained unidentified. The 76 identified yeast isolates were assigned to 39 species, the most frequent assignments were made to Debaryomyces hansenii, Geotrichum candidum, Issatchenkia orientalis, Kluyveromyces lactis, K. marxianus, Saccharomyces cerevisiae, Yarrowia lipolytica, andCandida catenulata. It is proposed that Debaryomyces hansenii (Zopf) Lodder et Kreger-van Rij and Debaryomyces fabryi Ota should be reinstated. The RAPD-PCR data reinforced the view that the species Galactomyces geotrichum is heterogeneous with all of the Geotrichum isolates from cheese products being assigned G. geotrichum group A sensu M.T. Smith. It is suggested that the name Geotrichum candidum be conserved for this rather common species.

Genotypic Identification of Saccharomyces Species using Random Amplified Polymorphic DNA Analysis

Summary According to different molecular approaches the genus Saccharomyces was divided recently into 10 genotypically distinct species ( S. bayanus, S. castellii, S. cerevisiae, S. dairensis, S. exiguus, S. kluyveri, S. paradoxus, S. pastorianus, S. servazzii, S. unisporus ). This was corroborated by Random Amplified Polymorphic DNA — Polymerase Chain Reaction (RAPD-PCR) analysis in the present paper. Thirtytwo strains including the type strains of 20 Saccharomyces species defined originally by phenotypic characteristics (e.g. S. chevalieri, S. diastaticus, S. ellipsoideus ) clustered with the pattern of S. cerevisiae , fourteen (e.g. type strains of S. globosus, S. heterogenicus, S. inustiatus ) with the pattern of S. bayanus , six including the type strains of S. carlsbergensis and S. monacensis with the pattern of S. pastorianus and two with the pattern of S. paradoxus . Two further strains isolated newly were identified to belong to S. paradoxus . In comparison with nuclear DNA/DNA hybridization or electrophoretic karyotyping, RAPD-PCR anaylsis turned out to be a simple and reliable method to separate Saccharomyces species at the genotypic level. In contrast to phenotypic characters genotypic identification using RAPD-PCR analysis guarantees species specificity if type strains are included in the investigation. The ten Saccharomyces species arising from RAPD-PCR analysis are differentiated from each other to the maximal extent with exception of the relationship between S. bayanus and S. pastorianus . In this case, the estimated similarity value of 45% is significantly higher than the background noise (0–20%), but less than the values within species (83 to 100%).

Molecular characterization and application of random amplified polymorphic DNA analysis of Mrakia and Sterigmatomyces species

The qualitative and quantitative monosaccharide spectra of purified yeast cell walls revealed that there are three phylogenetically distinct lineages of sterigma-forming basidiomycetous yeasts: (i) Kurtzmanomyces and Sterigmatomyces species, which contain high levels of mannose; (ii) Tilletiopsis species, which contain glucose, galactose, and small amounts of mannose; and (iii) Fellomyces, Kockovaella, Sterigmatosporidium, and Tsuchiyaea species, which appear to be closely related on the basis of their high levels of glucose and the presence of xylose. The yeast cell wall neutral sugars of Sporobolomyces antarcticus and Sterigmatomyces aphidis were similar to those of members of the genus Tilletiopsis. However, the possibility that these taxa are conspecific was eliminated by the results of a random amplified polymorphic DNA (RAPD) analysis. The conspecificity of Mrakia frigida and Mrakia nivalis, the conspecificity of Mrakia gelida and Mrakia stokesii, and the conspecificity of Sterigmatomyces halophilus and Sterigmatomyces indicus were confirmed by RAPD analysis results. RAPD analysis was found to be a simple and highly sensitive method which can be used to differentiate species at the DNA level; it can replace nuclear DNA-nuclear DNA hybridization experiments for species identification, characterization, and delimitation.

Yeasts associated with termites: a phenotypic and genotypic characterization and use of coevolution for dating evolutionary radiations in asco- and basidiomycetes

Summary Thirtynine yeast isolates or dimorphic fungi were obtained from the hindgut of the lower termites Mastotermis darwiniensis (Mastotermitidae), Zootermopsis angusticollis, Z. nevadensis (Hodotermitidae), Neotermes jouteli (Kalotermitidae), Reticulitermes santonensis, Heterotermes indicola (Rhinotermitidae) and the roach Cryptocercus punctulatus. Using RAPD-PCR the 39 yeast isolates were assigned to 13 different species. Commonly yeast species were specific to the termite species isolates from. There were only two yeast species which were found in different species of lower termites. Based on phenotypic characters Debaryomyces hansenii showed a high score in four species. The qualitative and quantitative yeast cell wall monosaccharide composition, the ubiquinone system, partial sequencing of 18S ribosomal DNA (bases 1273 to 948; numbering according to the gene of Saccharomyces cerevisiae), and the ultrastructure of septal pores indicate that 11 yeast species belong to the Endomycetales. Although ascospores were lacking, two of these species were identified to belong to the genus Debaryomyces. One remaining yeast isolate was identified as a Sporothrix anamorph representative for the filamentous Ascomycetes (Ophiostomataceae s. str.); the second species showed affinities to the Basidiomycetes in particular to the genus Trichosporon. Comparing an additional 18S rDNA fragment (bases 595 to 993) and RAPD-PCR data using different species type strains of the genus Sporothrix, the filamentous ascomycete was genotypically identified as Sporothrix albicans. Sporothrix. albicans, although phylogenetically closely related to S. schenckii var. schenckii and Ophiostoma stenoceras remains genotypically distinct. An emended species description of S. albicans is presented. Evidence is provided that the yeasts isolated from the hindgut can be considered symbionts.

Phytopathogenic Filamentous (Ashbya, Eremothecium) and Dimorphic Fungi (Holleya, Nematospora) with Needle‐shaped Ascospores as New Members Within the Saccharomycetaceae

Phylogenetic relationships between species from the genera Kluyveromyces and Saccharomyces and representatives of the Metschnikowiaceae (Holleya, Metschnikowia, Nematospora) including the two filamentous phytopathogenic fungi Ashbya gossypii and Eremothecium ashbyii were studied by comparing the monosaccharide pattern of purified cell walls, the ubiquinone system, the presence of dityrosine in ascospore walls, and nucleotide sequences of ribosomal DNA (complete 18S rDNA, ITS1 and ITS2 region). Based on sequence information from both ITS regions, the genera Ashbya, Eremothecium, Holleya and Nematospora are closely related and may be placed in a single genus as suggested by Kurtzman (1995; J. Industr. Microbiol. 14, 523–530). In a phylogenetic tree derived from the ITS1 and ITS2 region as well as in a tree derived from the complete 18S rDNA gene, the genus Metschnikowia remains distinct. The molecular evidence from ribosomal sequences suggests that morphology and ornamentation of ascospores as well as mycelium formation and fermentation should not be used as differentiating characters in family delimitation. Our data on cell wall sugars, ubiquinone side chains, dityrosine, and ribosomal DNA sequences support the inclusion of plant pathogenic, predominantly filamentous genera like Ashbya and Eremothecium or dimorphic genera like Holleya and Nematospora with needle‐shaped ascospores within the family Saccharomycetaceae. After comparison of sequences from the complete genes of the 18S rDNA the genus Kluyveromyces appears heterogeneous. The type species of the genus, K. polysporus is congeneric with the genus Saccharomyces. The data of Cai et al. (1996; Int. J. Syst. Bacteriol. 46, 542–549) and our own data suggest to conserve the genus Kluyveromyces for a clade containing K. marxianus, K. dobzhanskii, K. wickerhamii and K. aestuarii, which again can be included in the family Saccharomycetaceae. The phylogenetic age of the Metschnikowiaceae and Saccharomycetaceae will be discussed in the light of coevolution.

Molecular Characterization and Genotypic Identification of Metschnikowia Species

Summary Ten species currently described in the genus Metschnikowia (M. agaveae, M. australis, M. bicuspidata, M. gruessii, M. hawaiiensis, M. krissii, M. lunata, M. pulcherrima, M. reukaufii, M. zobellii) were examined for their cell wall carbohydrate composition and ubiquinone type. Glucose and mannose are the only carbohydrate components identified, and Q-9 is the main coenzyme-Q system. The RAPD-PCR fingerprinting supported the separation of the genus in the species. According to the molecular features a proper position for the genus Metschnikowia among ascomycetous yeasts is proposed.

New species of Fellomyces Isolated from Epiphytic Lichen Species

Summary Five new species of Fellomyces, F. borneensis, F. chinensis, F. lichenicola, F. sichuanensis , and F. thailandicus isolated from lichens growing on tree barks are described. Species delimitation was performed using RAPD-PCR and partial sequencing of ribosomal DNA. A fragment of 385 nucleotides, corresponding to the gene position 547 through 931 of the 18S rDNA in Saccharomyces cerevisiae was used. Based on phylogenetic data it was not possible to separate the ballistosporous genus Kockovaella from the sterigma forming genus Fellomyces . Species reproduce by enteroblastic budding or by forming one or more conidia on sterigmata per cell. Sterigmatoconidia are delivered by an end break in distal position of the sterigmata. Ubiquinone Q-10 is the major ubiquinone component in all species. The presence of xylose in purified yeast cell walls was chracteristic for all strains investigated. It was not possible to separate the five different Fellomyces species by phenotypic criteria of the physiological standard characterization only.

Yeast biodiversity in the guts of several pests on maize; comparison of three methods: classical isolation, cloning and DGGE

The yeast biodiversity in the guts of several pests (Diabrotica virgifera, Helicoverpa armigera, Ostrinia nubilalis) on maize from two isolation sources was assessed by cultivation-dependent and cultivation-independent methods. These yeasts are considered to bear a potentially high biotechnological relevance due to their potential ability to degrade several mycotoxins incorporated by their hosts. The 97 isolated yeast strains showed 21 different partial sequence types of the 26S rRNA gene which could be assigned to 10 different genera. The determined genera and species are discussed in terms of the meaning of their taxonomic status or their occurrence in nature. Two cultivation-independent methods, cloning and DGGE, were compared. We propose the combination of these methods as well as the combination of both cultivation-independent and cultivation-dependent approaches, for gaining better insights into fungal biodiversity.

Analysis of coenzyme Q systems, monosaccharide patterns of purified cell walls, and RAPD-PCR patterns in the genus Kluyveromyces.

Analysis of the coenzyme Q system and the monosaccharide pattern of purified cell walls were used for species characterization in the genus Kluyveromyces. All the type strains of the genus possess coenzyme Q-6 and the mannose-glucose (‘Saccharomyces type’) cell wall sugar pattern. With the help of Random Amplified Polymorphic DNA-Polymerase Chain Reaction analysis 17 species were separated: K. aestuarii, K. africanus, K. bacillisporus, K. blattae, K. delphensis, K. dobzhanski, K. lactis (anamorph Candida sphaerica), K. lodderae, K. marxianus (syn. K. fragilis, K. bulgaricus, K. cicerisporus anamorphs Candida macedoniensis, C. pseudotropicalis, C. kefyr), K. phaffii, K. piceae, K. polysporus, K. sinensis, K. thermotolerans (syn. K. veronae, anamorph Candida dattila), K. waltii, K. wickerhamii, K. yarrowii (anamorph Candida tannotolerans). A strain of K. drosophilarum showed with the type strain of K. lactis only 63% similarity. The strain originally described as the type strain of K. cellobiovorus nom. nud. was excluded from the genus (Q-9), and found to be conspecific with the type strain of Candida intermedia.