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 ...................................................................................................