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Single-strand conformation polymorphism (SSCP) analysis of ribosomal DNA (rDNA) was investigated for rapid differentiation of phenotypically similar yeast species. Sensitive tests indicated that some yeast strains with one, most strains with two, and all strains with three or more nucleotide differences in the internal transcribed spacer 1 (ITS1) or ITS2 region could be distinguished by PCR SSCP analysis. The discriminative power of SSCP in yeast species differentiation was demonstrated by comparative studies of representative groups of yeast species from ascomycetes and basidiomycetes, including Saccharomyces species, medically important Candida species, and phylloplane basidiomycetous yeast species. Though the species within each group selected are closely related and have relatively similar rDNA sequences, they were clearly differentiated by PCR-SSCP analysis of the ITS1 region, given the amplified fragments were less than 350 bp in sizes. By using SSCP analysis for rapid screening of yeast strains with different rDNA sequences, species diversity existing in a large collection of yeast strains from natural sources was effectively and thoroughly investigated with substantially reduced time and cost in subsequent DNA sequencing.

This work presents DNA sequence motifs from the internal transcribed spacer (ITS) of the nuclear rRNA repeat unit which are useful for the identification of five European and Asiatic truffles (Tuber magnatum, T. melanosporum, T. indicum, T. aestivum, and T. mesentericum). Truffles are edible mycorrhizal ascomycetes that show similar morphological characteristics but that have distinct organoleptic and economic values. A total of 36 out of 46 ITS1 or ITS2 sequence motifs have allowed an accurate in silico distinction of the five truffles to be made (i.e., by pattern matching and/or BLAST analysis on downloaded GenBank sequences and directly against GenBank databases). The motifs considered the intraspecific genetic variability of each species, including rare haplotypes, and assigned their respective species from either the ascocarps or ectomycorrhizas. The data indicate that short ITS1 or ITS2 motifs (< or = 50 bp in size) can be considered promising tools for truffle species identification. A dot blot hybridization analysis of T. magnatum and T. melanosporum compared with other close relatives or distant lineages allowed at least one highly specific motif to be identified for each species. These results were confirmed in a blind test which included new field isolates. The current work has provided a reliable new tool for a truffle oligonucleotide bar code and identification in ecological and evolutionary studies.

Infections caused by yeasts have increased in previous decades due primarily to the increasing population of immunocompromised patients. In addition, infections caused by less common species such as Pichia, Rhodotorula, Trichosporon, and Saccharomyces spp. have been widely reported. This study extensively evaluated the feasibility of sequence analysis of the rRNA gene internal transcribed spacer (ITS) regions for the identification of yeasts of clinical relevance. Both the ITS1 and ITS2 regions of 373 strains (86 species), including 299 reference strains and 74 clinical isolates, were amplified by PCR and sequenced. The sequences were compared to reference data available at the GenBank database by using BLAST (basic local alignment search tool) to determine if species identification was possible by ITS sequencing. Since the GenBank database currently lacks ITS sequence entries for some yeasts, the ITS sequences of type (or reference) strains of 15 species were submitted to GenBank to facilitate identification of these species. Strains producing discrepant identifications between the conventional methods and ITS sequence analysis were further analyzed by sequencing of the D1-D2 domain of the large-subunit rRNA gene for species clarification. The rates of correct identification by ITS1 and ITS2 sequence analysis were 96.8% (361/373) and 99.7% (372/373), respectively. Of the 373 strains tested, only 1 strain (Rhodotorula glutinis BCRC 20576) could not be identified by ITS2 sequence analysis. In conclusion, identification of medically important yeasts by ITS sequencing, especially using the ITS2 region, is reliable and can be used as an accurate alternative to conventional identification methods.

The quality and market price of truffles vary with the species and, traditionally, the place of origin. The premium species Tuber magnatum produces white truffles and has a patchy distribution restricted to Italy and some Balkan areas. We used polymorphic microsatellites to evaluate 316 specimens grouped into 26 populations sampled across the species' geographic range to determine if natural populations of T. magnatum are genetically differentiated. We found that the southernmost and the northwesternmost populations were significantly differentiated from the rest of the populations. The simple sequence repeat data also could be used to make inferences about the postglacial T. magnatum expansion pattern. This study is the first to identify a genetic and phylogeographic structure in T. magnatum. The presence of a genetic structure can be of practical interest in tracing truffle populations according to their geographic origin for marketing strategies. Evidence for extensive outcrossing in field populations of T. magnatum also is provided for the first time.

Four ascomycete species of the genus Gaeumannomyces infect roots of monocotyledons. Gaeumannomyces graminis contains four varieties, var. tritici, var. avenae, var. graminis, and var. maydis. G. graminis varieties tritici, avenae, and graminis have Phialophora-like anamorphs and, together with the other Gaeumannomyces and Phialophora species found on cereal roots, constitute the Gaeumannomyces-Phialophora complex. Relatedness of a number of Gaeumannomyces and Phialophora isolates was assessed by comparison of DNA sequences of the 18S rRNA gene, the 5.8S rRNA gene, and the internal transcribed spacers (ITS). G. graminis var. tritici, G. graminis var. avenae, and G. graminis var. graminis isolates can be distinguished from each other by nucleotide sequence differences in the ITS regions. The G. graminis var. tritici isolates can be further subdivided into R and N isolates (correlating with ability [R] or inability [N] to infect rye). Phylogenetic analysis of the ITS regions of several oat-infecting G. graminis var. tritici isolates suggests that these isolates are actually more closely related to G. graminis var. avenae. The isolates of Magnaporthe grisea included in the analysis showed a surprising degree of relatedness to members of the Gaeumannomyces-Phialophora complex. G. graminis variety-specific oligonucleotide primers were used in PCRs to amplify DNA from cereal seedlings infected with G. graminis var. tritici or G. graminis var. avenae, and these should be valuable for sensitive detection of pathogenic isolates and for diagnosis of take-all.