Hansjörg Prillinger

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 taxonomy and biodiversity of rock fungal communities in an urban environment (Vienna, Austria)

The diversity of fungal communities on three different historical monuments in the city of Vienna (Austria) was analyzed and compared to the fungal diversity of microfungi on rock in the original quarry located in a rural area (Zogelsdorf, Austria). The fungal strains isolated were characterized by morphology and the complete rock fungal community was identified based on molecular data, that is, by sequencing parts of the small ribosomal subunit (18S) and internal transcribed spacer region 1 (ITS1). The genera Coniothyrium, Epicoccum and Phoma were found to be dominant {on} monument and rock surfaces. Additionally, black yeasts such as Exophiala species and microcolonial fungi like Sarcinomyces and Coniosporium which hitherto were regarded as typical rock inhabitants in semi-arid environments are frequently found on all rock surfaces in Vienna. The biodiversity of the fungi in the urban environment was much higher than on the same rock type in a rural environment, this difference can be attributed to the elevated organic pollution in the city.

Differentiation of Ophiostoma piceae and Ophiostoma quercus by Morphology and RAPD Analysis.

Summary Ophiostoma piceae (Munch) H. and G. Sydow was found to comprise two distinct species by means of morphological investigation on synnematal size as well as by RAPD technique for genomic analysis. The two species are separated undoubtedly by the means of morphological investigations on synnematal size, the onset of a respective yeast stage, and genomic analysis by the PCR based RAPD technique. Different mean values of synnematal size were corroborated statistically by the Kolmogorov — Smirnov Test. RAPD analysis is ensured by phylogenetic computation of the discrete state matrix arising from the arbitrary PCR fragment patterns. The two Ophiostoma species separated harbour isolates predominantly grown on spruce on the one hand ( O. piceae ) and gathered isolates from oak and probably other hardwoods on the other hand ( O. quercus ). For the latter species the name O. quercus (Georgev.) Nannf. must be reinstated.

Species pattern and genetic diversity of Trichoderma in a mid-European, primeval floodplain-forest

We investigated the occurrence and genetic diversity of Trichoderma in the river Danube national park, a primeval, riparian forest area located south-east of Vienna (Austria) which represents one of the last cases of an original European river-floodplain landscape. Forty-six strains were isolated and identified at the species level by analysis of morphological characters, by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS 1 and 2) of the rDNA cluster and--in some cases--a fragment of the translation elongation factor 1alpha (tef1) gene, and RAPD-analysis. Twenty-one strains were positively identified as T. harzianum, thirteen as T. rossicum, four as T. cerinum, two as T. hamatum, and one each as T. atroviride and T. koningii: four strains yielded two different ITS1 and 2 as well as tef1 sequence types, which were not alignable with any known species. Our studies show that they represent two new taxa of Trichoderma.

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.

Trichoderma brevicompactum sp. nov.

Trichoderma brevicompactum, a new species, was isolated from soil or tree bark in North, Central and South America, including the Caribbean Islands, and southwestern and southeastern Asia. Morphological and physiological characters, the internal transcribed spacer regions of the rDNA cluster (ITS1-5.8SrDNA-ITS2) and partial sequences of translation elongation factor 1-alpha (tef1) are described. Trichoderma brevicompactum is characterized by a pachybasium-type morphology, morphologically resembling other small-spored species referable to Trichoderma section Pachybasium but with essentially subglobose conidia. It is most closely related phylogenetically to Hypocrea lutea, from which it differs in morphological and physiological characters.

Trichoderma populations from alkaline agricultural soil in the Nile valley, Egypt, consist of only two species

The biodiversity of Trichoderma was studied in the Northern half of the Nile valley in Egypt. 20 strains were isolated from 9 different geographic locations, representing 19 different habitats, all with a pH between 7.3 and 8.4. Only T. harzianum (three ITS1/2 haplotypes and three RAPD-genotypes) and the anamorph of Hypocrea orientalis were found. One of the T. harzianum haplotypes (4 strains) is new. The occurrence of T. harzianum haplotypes and of H. orientalis appeared to be essentially independent of the habitat (pH, plant, soil type), and also did not correlate with biochemical properties (cellulase and chitinase activity) of the individual strains. These two taxa seem to be indigenous to the Nile valley, their presence not being influenced by the agricultural history of the soils.