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Development and Evaluation of Rrna Targeted in Situ Probes and Phylogenetic Relationships of Freshwater Fungi

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Development and Evaluation of Rrna Targeted in Situ Probes and Phylogenetic Relationships of Freshwater Fungi Development and evaluation of rRNA targeted in situ probes and phylogenetic relationships of freshwater fungi vorgelegt von Diplom-Biologin Christiane Baschien aus Berlin Von der Fakultät III - Prozesswissenschaften der Technischen Universität Berlin zur Erlangung des akademischen Grades Doktorin der Naturwissenschaften - Dr. rer. nat. - genehmigte Dissertation Promotionsausschuss: Vorsitzender: Prof. Dr. sc. techn. Lutz-Günter Fleischer Berichter: Prof. Dr. rer. nat. Ulrich Szewzyk Berichter: Prof. Dr. rer. nat. Felix Bärlocher Berichter: Dr. habil. Werner Manz Tag der wissenschaftlichen Aussprache: 19.05.2003 Berlin 2003 D83 Table of contents INTRODUCTION ..................................................................................................................................... 1 MATERIAL AND METHODS .................................................................................................................. 8 1. Used organisms ............................................................................................................................. 8 2. Media, culture conditions, maintenance of cultures and harvest procedure.................................. 9 2.1. Culture media........................................................................................................................... 9 2.2. Culture conditions .................................................................................................................. 10 2.3. Maintenance of cultures.........................................................................................................10 2.4. Harvest procedure.................................................................................................................. 10 2.4.1. Harvest of cultures ........................................................................................................... 10 2.4.2. Harvest of conidia ............................................................................................................ 11 2.4.3. Harvest of hyphal tips ...................................................................................................... 11 3. River sampling sites ..................................................................................................................... 11 3.1. Elbe river, Germany ............................................................................................................... 11 3.2. Oberer Seebach, Austria........................................................................................................ 12 4. Sampling and monoconidial isolation strategies .......................................................................... 13 4.1. Foam ...................................................................................................................................... 13 4.2. Leaves and wood ................................................................................................................... 13 4.3. Water and river snow ............................................................................................................. 14 4.4. Sporocarps............................................................................................................................. 14 4.5. Monoconidial isolations from cultures .................................................................................... 14 4.6. Trapping by surface exposure ............................................................................................... 14 4.6.1. Polyethylene slides .......................................................................................................... 14 4.6.2. Leaves.............................................................................................................................. 15 4.6.3. Cellulose .......................................................................................................................... 15 5. Germination experiments ............................................................................................................. 15 5.1. Exposure of conidia and spores in cages .............................................................................. 16 6. Phenotypic characterisation ......................................................................................................... 18 6.1. Morphological determination.................................................................................................. 18 6.2. Fluorescent stains .................................................................................................................. 18 6.2.1. Calcofluor white ............................................................................................................... 18 6.2.2. Live-Dead stain ................................................................................................................ 18 6.2.3. SYTO stains.....................................................................................................................18 6.2.4. Lectin stain....................................................................................................................... 19 7. Genotypic characterisation........................................................................................................... 20 7.1. Extraction of genomic DNA.................................................................................................... 20 7.2. Amplification of the 18S rRNA genes..................................................................................... 20 7.3. Amplification of the ITS1, 5.8S rRNA and ITS2 genes .......................................................... 20 7.4. Amplification of the 28S rRNA genes..................................................................................... 21 7.5. Ribosomal DNA sequencing .................................................................................................. 21 7.5.1. Nucleotide sequence accession numbers ....................................................................... 23 7.6. Alignment of rDNA sequences............................................................................................... 24 8. Modification of ARB software settings.......................................................................................... 24 8.1. Change of the reference organism and adjusting the 18S and the 28S rRNA secondary structure................................................................................................................................. 24 8.2. Enlargement of the ARB database with fungal sequences.................................................... 25 9. Reconstruction of phylogenetic trees ........................................................................................... 25 9.1. Neighbour Joining .................................................................................................................. 25 9.2. Maximum Parsimony.............................................................................................................. 25 9.3. Maximum Likelihood ..............................................................................................................25 9.4. Bayesian analysis .................................................................................................................. 25 10. Fluorescence in situ hybridisation ............................................................................................ 29 10.1. Fixation................................................................................................................................. 29 10.1.1. Paraformaldehyde fixation ............................................................................................ 29 10.1.1.1. Cultures, conidia, hyphal tips................................................................................ 29 10.1.1.2. Biofilms ................................................................................................................. 29 10.1.1.3. River snow ............................................................................................................ 29 10.1.1.4. Foam..................................................................................................................... 29 I Table of contents 10.1.2. Ethanol fixation ............................................................................................................. 29 10.1.3. Methanol fixation........................................................................................................... 29 10.2. Permeabilisation................................................................................................................... 29 10.2.1. Permeabilisation by chitinase treatment....................................................................... 29 10.2.2. Permeabilisation by electroporation.............................................................................. 30 10.3. Design and evaluation of specific oligonucleotide probes ................................................... 30 10.4. Hybridisation procedure ....................................................................................................... 31 10.4.1. Cultures, conidia, hyphal tips........................................................................................ 31 10.4.2. Biofilms.......................................................................................................................... 32 10.4.3. River snow and foam ...................................................................................................
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