Associations between microbes and macroalgae: Host, epiphyte and environmental effects Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. Franz Goecke Saavedra Kiel 2011 I II Vorsitz : Prof. Dr. Arne Körtzinger Referent : Prof. Dr. Johannes F. Imhoff Korreferent : Prof. Dr. Peter Schönheit Prüfer : Prof. Dr. Rüdiger Schulz Tag der mündlichen Prüfung: 09. Dez. 2011 Zum Druck genehmigt: 09. Dez. 2011 Der Dekan III Die vorliegende Arbeit wurde am Kieler Wirkstoff-Zentrum am Leibniz-Institut für Meereswissenschaften an der Christian-Albrechts-Universität zu Kiel von Juni 2008 bis September 2011 unter Anleitung von Prof. Dr. Johannes F. Imhoff durchgeführt. IV Para mi abuelo Helmuth… V LIST OF PUBLICATIONS The work of this thesis was published by means of the following publications. Paper I Goecke, F., Labes A., Wiese J., & Imhoff, J.F. (2010) Chemical interactions between marine macroalgae and bacteria. Marine Ecology Progress Series 409: 267-300 Paper II Goecke, F., Thiel V., Wiese J., Labes A., & Imhoff, J.F. Phylogenetic relationships among bacteria described from algae: A distinct source of new taxa. Microbial Ecology submitted 31 October 2011 Paper III Goecke, F., Labes A., Wiese J., & Imhoff, J.F. Phylogenetic analysis and antibiotic activity of epibiotic bacteria associated with two co-occurring macroalgae from the Baltic Sea. (under preparation) Paper IV Goecke, F., Labes A., Wiese J., & Imhoff, J.F. (2011) Dual effect of macroalgal extracts on growth of bacteria in Western Baltic Sea. Revista de Biologia Marina & Oceanografia submitted 01 September 2011 Paper V Goecke, F., Núñez A., Labes A., Wiese J., Neuhauser S., & Imhoff, J.F. Gall formation in the bull-kelp Durvillaea antarctica (Chamisso) Hariot in the central coast of Chile is associated with two different endophytes. (under preparation) Short note Goecke, F., Labes A., Wiese J., Schmaljohann R., & Imhoff, J.F. Observation of bacteria over the surface of released oogonia from Fucus vesiculosus L (Phaeophyceae). Gayana Botánica submitted 31 October 2011 VI TABLE OF CONTENTS 1.0. Zusammenfassung 01 2.0. Abstract 02 3.0. Introduction 03 3.1. Marine microorganisms in their interaction with macroalga 03 3.1.1. Algae as source of new bacterial taxa 04 3.1.2. Algae-microbial interactions 05 3.1.2.1. Microbial communities associated with macroalgae: Bacteria 05 3.1.2.2. Epibiosis 06 3.1.2.3. Macroalgal diseases 07 3.1.3. Chemical defense 08 3.1.4. Beneficial relationships 09 3.1.5. Goals of the investigation 10 3.2. Species under study 13 3.2.1. Fucus vesiculosus 13 3.2.2. Delesseria sanguinea 15 3.2.3. Durvillaea antarctica 17 4.0. Chapter I. Phylogenetic relationships among bacteria described from algae 20 4.1. Abstract 21 4.2. Introduction 21 4.3. Material and methods 22 4.3.1. Sequence handling 22 4.3.2. Data mining 23 4.4. Results 23 4.4.1. Phylogenetic analysis 24 4.5. Discussion 30 4.6. Appendix 36 5.0. Chapter II. Phylogenetic analysis and antibiotic activity of epibiotic bacteria associated with two co-occurring macroalgae from the Baltic Sea. 40 5.1. Abstract 41 5.2. Introduction 41 5.3. Material and methods 43 5.3.1. Sampling of the macroalgae 43 5.3.2. Isolation and cultivation of bacteria 43 5.3.3. 16S rRNA gene sequence analysis 44 5.3.4. Phylogenetic analysis 44 5.3.5. Preparation of cell extracts 45 5.3.6. Antimicrobial assay 45 5.4. Results 46 5.4.1. Isolation and phylogenetic classification of bacteria 46 5.4.2. Antimicrobial activities 47 5.5. Discussion 48 5.6. Appendix I Chapter II 59 6.0. Chapter III. Observation of bacteria over the surface of released oogonia from Fucus vesiculosus L (Phaeophyceae) 62 6.1. Abstract 63 6.2. Introduction 63 VII 6.3. Material and methods 64 6.4. Results 65 6.5. Discussion 66 7.0. Chapter IV. Biological activity of macroalgal extracts on selected bacterial strains 68 7.1. Abstract 69 7.2. Introduction 69 7.3. Material and methods 70 7.3.1. Sampling of the macroalgae 70 7.3.2. Macroalgal extract preparation 71 7.3.3. Antimicrobial testing 71 7.4. Results 72 7.5. Discussion 77 8.0. Chapter V. Potential pathogens of a Chilean macroalga 83 8.1. Abstract 84 8.2. Introduction 84 8.3. Material and methods 87 8.3.1. Field sampling 87 8.3.2. Light microscopy and characterization of the microorganisms 88 8.3.3. Scanning electron microscopy 88 8.4. Results 88 8.4.1. Healthy individuals, both sampling sites 88 8.4.2. Diseased individuals, macroscopic appearance 89 8.4.2.1. Diseased individuals from Coliumo bay samples 89 8.4.2.2. Diseased individuals from Valparaiso bay samples 89 8.5. Discussion 94 8.5.1. Coliumo bay samples 94 8.5.2. Valparaiso bay samples 95 9.0. General Discussion 98 10.0. Experimental Sections 102 10.1. Nutrient media composition 102 10.2. Molecular Biology Methods 103 10.3. Laboratory Equipment 104 10.4. Chemicals 105 11. References 106 12. List of Abbreviations 126 13. Acknowledgements 128 14. Declaration 129 15. Supporting Information 130 VIII List of the Supporting Information provided in the present investigation. S01. Paper I: Goecke F, Labes A, Wiese J, Imhoff JF. 2010. Chemical interactions between marine macroalgae and bacteria. Marine Ecology Progress Series 409:267-300. 130 S02. Figure of the life cycle of Fucales adapted from Lee 2008. 131 S03. Figure of a typical Polysiphonia-type life cycle for Delesseria sanguinea. 132 S04. Life cycle of Durvillaea antarctica (Fucales) after Collantes et al. (2002). 133 S05. Figure summarizing the research activities. 134 S06. List of congress and scientific meetings where part of the present work has been presented. 135 S07. Table of the different bacterial taxa affiliated to type strains previously described as associated with algae have been detected by molecular (16S rRNA clone libraries, DGGE), cultivation (culti) or optical methods from other algal sources (supporting information of Chapter I). 136 S08. Classification of bacterial strains from the two macroalgae based on 16S rRNA gene sequences analysis (supporting information of Chapter II). 138 S09. Number of different phylotypes exhibiting antibiotic active members with respect to their origin obtained in four sampling periods in 2 years (supporting information Chapter II). 141 S10. Antibacterial activity reported for the studied algal species in previously investigations around the world (supporting information of Chapter III). 142 S11. Supporting information of scanning electron microscopy (SEM). 144 S12. Individual scientific contribution to multiple author publication. 149 IX List of Figures General Fig. 01. Macroalgae in the marine environment 04 Fig. 02. Microorganisms associated with macroalgal surfaces 07 Fig. 03. Diseases, parasitism, epibiosis and biofouling over algae 08 Fig. 04. Schematic diagram shows the influence of secondary metabolites on algal surfaces 10 Fig. 05. The brown alga Fucus vesiculosus 13 Fig. 06. Morphological detail of Fucus vesiculosus 14 Fig. 07. Reproduction structures in Fucus vesiculosus 15 Fig. 08. The red alga Delesseria sanguinea 16 Fig. 09. Morphological detail of Delesseria sanguinea 16 Fig. 10. Seasonal growth of Delesseria sanguinea 17 Fig. 11. The brown alga Durvillaea antarctica 18 Fig. 12. Morphological detail of Durvillaea antarctica 19 Chapter I Fig. 01. Percentage by origin of newly described species of bacteria from algae 24 Fig. 02. Phylogenetic tree of the 16S rRNA sequences from bacteria described as new after isolation from algal sources 26 Fig. 03a. Phylogenetic tree, subtree Proteobacteria 27 Fig. 03b. Phylogenetic tree, subtree Verrucomicrobia, Planctomycetes, Actinobacteria and Firmicutes 28 Fig. 03c. Phylogenetic tree, subtree Bacteroidetes 29 Chapter II Fig. 01.a Phylogenetic tree of the 16S rRNA sequences belonged to Bacteroidetes 54 Fig. 01.b Phylogenetic tree of the 16S rRNA sequences belonged to Proteobacteria 55 Fig. 01.c Phylogenetic tree of the 16S rRNA sequences belonged to Firmicutes 56 Fig. 01.d Phylogenetic tree of the 16S rRNA sequences belonged to Actinobacteria 57 Fig. 02. Relative abundance of the strains belonging to different bacterial classes 58 Chapter III Fig. 01 Scanning electron microphotograph of cross section of conceptacle in Fucus vesiculosus 65 Fig. 02 SEM microphotograph of an oogonium in Fucus vesiculosus 66 Chapter IV Fig. 01a. Biological activity of macroalgal extracts against standard strains 74 Fig. 01b. Biological activity of macroalgal extracts against macroalga-associated strains 75 Fig. 02. Summary by bacterial strains of effects on growth performed by algal extracts 76 Fig. 03. Proportion of biological activity according the different macroalgal species 76 Chapter V Fig. 01. Map of the study area in Chile 87 Fig. 02. Healthy populations of Durvillaea antarctica in Chile 91 Fig. 03. Macroalgal samples of Coliumo bay, Chile 92 Fig. 04. Macroalgal samples of Valparaíso bay, Chile 93 X List of Tables Chapter I Table 01. Number of bacterial strains which have presented the ability to degrade polysaccharides and are described from algal sources 30 Table 02. List of newly described bacterial species 36 Table 03. List of the algal source of bacteria, and detail of the ability to degrade polysaccharides 38 Chapter II Table 01. Isolates previously described associated with algae 51 Table 02. Different phylotypes associated exclusively with Fucus or Delesseria 52 Table 03. Number of active phylotypes by algae 53 Table 04. Classification of the bacterial strains of the 2 macroalgae 59 Chapter IV Table 01. List of the macroalgae sampled from the Kiel Fjord 81 Table 02. Antibacterial activity and growth stimulation effects of macroalgal extracts 82 Chapter V Table 01. Macroalgae that have showed gall-formations in the Chilean coast before 86 XI 1 Zusammenfassung Während der letzten drei Jahre stand die Assoziation zwischen Makroalgen und Bakterien im Fokus vieler Untersuchungen.