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Development and evaluation of rRNA targeted in situ probes and phylogenetic relationships of freshwater fungi
Zitatschlüssel Baschien2003
Autor Baschien, Christiane
Jahr 2003
Schule Technische Universität Berlin, FG Umweltmikrobiologie
Zusammenfassung The aim of the study was the molecular phylogenetic characterisation of aquatic hyphomycetes, the evaluation of the factors influencing their accessibility for fluorescence in situ hybridisation and the application of newly developed taxon specific rRNA-targeted oligonucleotide probes in the German lowland river Elbe and the Austrian alpine 2nd order stream Oberer Seebach. During 24 sampling campaigns in three years performed in the Elbe river, with only eleven different species a remarkable low diversity of aquatic hyphomycetes could be distinguished while the majority of fungi observed were terrestrial and airborne fungi. Conidial numbers in fixed foam samples of 3.75 10 ml -1 (autumn) were extremely low in the Elbe river compared to conidial numbers of 5635 10 ml -1 (autumn) and 2654 10 ml -1 (summer) in foam samples from the Oberer Seebach. During two short term, three week campaigns 36 different species of aquatic hyphomycetes could be observed and isolated from different substrates in the Oberer Seebach. Phylogenetic analyses of sequences of different rDNA regions of 11 species of aquatic hyphomycetes confirmed the assumption of a polyphyletic origin of aquatic hyphomycetes. On the basis of 18S rDNA data, nine species (Tetracladium marchalianum, Lemonniera terrestris, L. aquatica, Varicosporium elodeae, Tricladium angulatum, T. splendens, Alatospora acuminata, Anguillospora crassa, and A. furtiva) were placed in the Ascomycota class Leotiomycetes. The species Heliscus lugdunensis and Anguillospora longissima were placed in the classes Sordariomycetes and Dothideomycetes, respectively. In ITS and LSU sequence analyses T. marchalianum showed most likely phylogenetic affinities to the Helotiales. On the basis of ITS sequence data, two morphotypes of A. acuminata were separated into two clades containing the sensu lato and the sensu stricto type, respectively. T. splendens showed close relationships to A. crassa and to the marine hyphomycete Zalerion varium (Helotiales) in ITS analyses. T. angulatum is related to the Helotiales and showed close relation to members of the family Hyaloscyphaceae as revealed from ITS analyses. A. crassa was placed most likely within the Helotiales. In contrast A. longissima is a member of the Pleosporales with closer relation to the genus Lophiostoma than to the suspected teleomorph genus Massarina. Thus the genus Anguillospora has to be revised. For the phylogenetic analyses and subsequent probe design, the ARB software environment for sequence data was adapted to properly work with fungi by incorporating 18S rRNA and 28S rRNA secondary structure information and extending and generating 18S rRNA, ITS and 28S rRNA sequence databases. Newly developed 18S rRNA-targeted taxon-specific probes termed FUN1429, MY1574 are specific for a wide range of Eumycota. The genus specific probe TCLAD1395 (Tetracladium) as well as the species specific probes ALacumi1698 (A. acuminata), TRIang322 (T. angulatum), Alongi340 (A. longissima) and Hlug1698 (H. lugdunensis) are targeted against the 18S rRNA. The probes TmarchB10, TmarchC1_1, TmarchC1_2 and AlongiB16, specific for T. marchalianum and A. longissima, respectively, are targeted on the 28S rRNA. The specificity of all newly designed probes was successfully tested in whole fungal hybridisations against target and non-target organisms. The fixation method, the fungal inherent and substrate mediated autofluorescence, the imaging technique, the age of the mycelium, the different mycelial parts, and the permeability of fungal cell walls were shown to be the most obvious influencing factors for the detection of fungal FISH signals. Autofluorescence scans revealed that many freshwater fungi showed inherent fluorescence in the green visible light spectrum. This could be successfully overcome by use of probe labels emitting red fluorescence. Generally, autofluorescence intensities increased with the age of the fungal mycelium. Bleaching of autofluorescing organisms and substrates with sodiumborohydrid was useful for enhanced probe signal detection. Employing epifluorescence microscopy, fungal FISH signals were often insufficient to detect due to high background noise. In contrast, confocal laser scanning microscopy significantly enhanced the detection of fungal FISH signals. Additionally, the spatial distribution of freshwater fungi on leaves could be documented more clearly by CLSM. The FISH signal intensity and rRNA content resembling the metabolical status of different structures of the mycelium were positively correlated. The cell wall permeability could be enhanced by chitinase treatment. However, the most reliable results for FISH signal detection were yielded by employing the Electroporation-FISH method newly developed in this study. Application of the probes MY1574 and Hlug1698 yielded FISH signals of metabolically active hyphae in the Elbe river. In the Oberer Seebach aquatic hyphomycetes were successfully detected on PE slides, leaves and in germination experiments using the set of newly developed fungal FISH probes.
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