Evolutionary relationships within 'pygmaeus' group microphallids using genetic analysis and scanning electron microscopy.

There are four species of 'pygmaeus' microphallids, namely Microphallus pygmaeus, M. piriformes, M. pseudopygmaeus and M. triangulatus (Trematoda: Microphallidae) which are parasites of marine birds and their sporocysts give rise to transmissible metacercariae inside littoral gastropods (mostly littorines). Universally primed polymerase chain reaction (UP-PCR) showed no apparent pattern between genetic diversity of the metacercariae as estimated by genomic banding profiles and their geographic region or molluscan host species. At the same time UP-PCR product cross-hybridization showed that M. pseudopygmaeus and M. triangulatus are genetically very similar, indicating that these taxa represent one species complex. In contrast, M. pygmaeus and M. piriformes are genetically well separated from each other and also from the pseudopygmaeus-triangulatus complex. Scanning electron microscopy of ventral spines, and analyses of spine angles and the number of teeth per spine, showed that all species differed significantly from one another. It was concluded that M. piriformes represents the original western member of the 'pygmaeus' group. Microphallus pygmaeus probably diverged from M. piriformes as it progressively specialized for sea duck final hosts. Microphallus pseudopygmaeus and M. triangulatus diverged from each other and the piriformes-pygmaeus ancestral line relatively recently. Microphallus pseudopygmaeus specialized for adoption of a wide range of gastropod host species and M. triangulatus developed morpho-functional specialization associated with final host exploitation.

Phylogenetic relationship of Fusarium langsethiae to Fusarium poae and Fusarium sporotrichioides as inferred by IGS, ITS, beta-tubulin sequences and UP-PCR hybridization analysis.

Fusarium langsethiae was recently described to accommodate "powdery" isolates of Fusarium poae, which morphologically resemble F. poae, but whose metabolite profile is similar to that of Fusarium sporotrichioides. In order to investigate the phylogenetic relationship of F. langsethiae to closely related species, we sequenced the internal transcribed spacer (ITS) regions 1 and 2 and part of the intergenic spacer (IGS) region of the rDNA cluster and part of the beta-tubulin gene from 109 strains of F. poae, F. sporotrichioides, F. langsethiae and Fusarium kyushuense from different geographic origin. Sequence analysis of ITS1 and 2 was unable to separate all F. sporotrichioides strains from F. langsethiae strains. Sequence analysis of beta-tubulin distinguished all four species, but it did not resolve the phylogenetic relationship between these two species. Sequence analysis of the IGS region distinguished the four species and led to a higher number of subgroups of the individual species, of which that of F. sporotrichioides var. minus isolates was even better supported than that of F. poae and F. langsethiae. Neighbor-joining and POY analyses of all combined sequences reliably separated all species studied, including F. langsethiae, clearly from F. sporotrichioides. The high intraspecific variability of the IGS sequences were found useful to group isolates according to their geographic origin. These results are in accordance with the results of the UP-PCR hybridization analysis. In summary, our data offer molecular support for the description of F. langsethiae as a new species in section Sporotrichiella.

Identification of a universally primed-PCR-derived sequence-characterized amplified region marker for an antagonistic strain of Clonostachys rosea and development of a strain-specific PCR detection assay.

We developed a PCR detection method that selectively recognizes a single biological control agent and demonstrated that universally primed PCR (UP-PCR) can identify strain-specific markers. Antagonistic strains of Clonostachys rosea (syn. Gliocladium roseum) were screened by UP-PCR, and a strain-specific marker was identified for strain GR5. No significant sequence homology was found between this marker and any other sequences in the databases. Southern blot analysis of the PCR product revealed that the marker represented a single-copy sequence specific for strain GR5. The marker was converted into a sequence-characterized amplified region (SCAR), and a specific PCR primer pair was designed. Eighty-two strains, isolated primarily from Danish soils, and 31 soil samples, originating from different localities, were tested, and this specificity was confirmed. Two strains responded to the SCAR primers under suboptimal PCR conditions, and the amplified sequences from these strains were similar, but not identical, to the GR5 marker. Soil assays in which total DNA was extracted from GR5-infested and noninoculated field soils showed that the SCAR primers could detect GR5 in a pool of mixed DNA and that no other soil microorganisms present contained sequences amplified by the primers. The assay developed will be useful for monitoring biological control agents released into natural field soil.

Intraspecific variation in gamma-radiation resistance and genomic structure in the filamentous fungus Alternaria alternata: a case study of strains inhabiting Chernobyl reactor no. 4.

This is probably the first report on intraspecific variation in radiation resistance for filamentous fungi. It was revealed that natural ("field") strains of the filamentous fungus Alternaria alternata are extremely variable in response to gamma-irradiation ranging from supersensitive to highly resistant to radiation. At the same time nearly all strains originating from the highly radiation-polluted reactor of the Chernobyl (Ukraine) Nuclear Power Plant possessed high radiation resistance. The genome structure of strains studied by universally primed polymerase chain reaction (UP-PCR) was found to be well conserved in "reactor" but not in "control" strains. The "reactor" strains appear to be genetically adapted to this high radiation habitat by means of selection, thus providing a natural source of genetically homogeneous fungal lineages.

[Serine proteinase from the higher basidiomycetes of Coprinus genus]. / Serinovaia proteinaza iz vysshego bazidiomitseta roda Coprinus.

A thiol-dependent serine proteinase has been isolated for the first time from a higher basidiomycete Coprinus 7N culture filtrate by affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on DEAE-Sepharose. This procedure resulted in a homogeneous enzyme with 32-fold purification and 55% yield. The enzyme has a molecular mass of 33,000 Da and pI of 8.5; its amino acid composition appears as follows: Lys7, His7, Arg10, Asx29, Thr24, Ser30, Glx19, Pro13, Gly39, Ala40, Cys2-3, Val23, Met1-2, Ile14, Leu13, Tyr6, Phe7. The enzyme shows the optimal activity towards Z-Ala-Ala-Leu-pNA at 8.5 and is stable at pH 6-9. The temperature optimum of the enzyme activity lies at 37 degrees C. The proteinase is completely inactivated by the specific inhibitors of serine proteinases, diisopropylfluorophosphate and phenylmethylsulfonylfluoride, as well as by the SH-group reagent, p-chloromercuribenzoate. The Coprinus 7N proteinase hydrolyzes, azocasein, azoalbumin, hemoglobin, fibrin and synthetic chromogenic peptide substrates, e. g., Z-Ala-Ala-Leu-pNA, Z-Gly-gly-Leu-pNA. Some properties of the Coprinus 7N proteinase are very similar to those of thiol-dependent serine proteinases from bacilli, actinomycetes, fungi and plants which form a subfamily of thiol-dependent serine proteinases within the family of subtilisins.