U.F.R. Sciences et Techniques Biologiques Georg-August-Universität Göttingen Ecole Doctorale Ressources Procédés Produits Environnement Binational doctoral thesis submitted to obtain the Ph.D. degree of the « Université Henri Poincaré » of Nancy 1, France, and the « Georg-August-Universität » of Göttingen, Germany. Presented by Marlis Reich Using high-throughput genotyping for monitoring communities of soil fungi The public defence was held on the 28th of May 2009. Defence committee: Reviewers: Gertrud LOHAUS Georg-August-Universität Göttingen, Germany M. Roland MARMEISSE Université Claude Bernard, Lyon, France Evaluators: Andrea POLLE Georg-August-Universität Göttingen, Germany Xavier NESME Université Claude Bernard, Lyon, France Jean-Pierre JACQUOT Université Henri Poincaré Nancy, France Francis MARTIN INRA-Nancy, France (supervisor) Marc BUEE INRA-Nancy, France (Co-supervisor) Laboratoire Interactions Arbres/Micro-organismes UMR 1136 INRA/UHP ; INRA Nancy Büsgen Institut, Forstbotanik und Baumphysiologie, Georg-August-Universität Göttingen 1 To a european spirit and an international exchange…. 2 When I the starry courses know, And Nature's wise instruction seek, With light of power my soul shall glow… On y voit le cours des étoiles ; Ton âme, échappant à la nuit, Pourra voguer à pleines voiles… Erkennest dann der Sterne Lauf, Und wenn Natur dich unterweist, Dann geht die Seelenkraft dir auf… (Faust I, Night, Johann Wolfgang Goethe) 3 Table of contents 1. PUBLICATION LIST 5 2. PREFACE 7 2.1. SUMMARY 7 2.2. OBJECTIVES OF MY THESIS 8 2.3. OVERVIEW ON THE CHAPTERS 9 3. CHAPTER I: INTRODUCTION 11 3.1. MYCORRHIZA IN THE FOCUS OF RESEARCH 11 3.1.1. MYCORRHIZA, A MUTUALISTIC SYMBIOSIS 11 3.1.2. DIFFERENT APPROACHES TO STUDY MYCORRHIZAL FUNGI 12 3.2. VARIETY OF MYCORRHIZAL TYPES 15 3.2.1. SEVEN FORMS OF MYCORRHIZA 15 3.2.2. ARBUSCULAR MYCORRHIZA 15 3.2.3. ECTOMYCORRHIZA 18 3.3. ECOLOGY OF MYCORRHIZAL FUNGI 21 3.3.1. ENVIRONMENTAL FACTORS 21 3.3.2. ECOLOGY OF ARBUSCULAR MYCORRHIZAL FUNGI 22 3.3.3. THE ECOLOGY OF ECTOMYCORRHIZAL COMMUNITIES 26 3.4. TECHNIQUES FOR IDENTIFYING MYCORRHIZAL FUNGAL SPECIES 35 3.4.1. MORPHOTYPING 35 3.4.2. MOLECULAR TECHNIQUES TO STUDY FUNGAL DIVERSITY 39 3.4.3. CLOSING WORDS ABOUT DETECTION TECHNIQUES 60 4. CHAPTER II: DEVELOPMENT AND VALIDATION OF AN OLIGONUCLEOTIDE MICROARRAY TO CHARACTERIZE ECTOMYCORRHIZAL COMMUNITIES 61 5. CHAPTER III: DIAGNOSTIC RIBOSOMAL ITS PHYLOCHIP FOR IDENTIFICATION OF HOST INFLUENCE ON ECTOMYCORRHIZAL COMMUNITIES 62 6. CHAPTER IV: 454 PYROSEQUENCING ANALYSES OF FOREST SOIL REVEAL AN UNEXPECTED HIGH FUNGAL DIVERSITY 63 4 7. CHAPTER V: COMPARISON OF THE CAPACITY TO DESCRIBE FULLY IDENTIFIED FUNGAL SPECIES USING THE TWO HIGH­THROUGHPUT TECHNIQUES 454 PYROSEQUENCING AND NIMBLEGEN PHYLOCHIP 72 8. CHAPTER VI: QUANTITATIVE TRACEABILITY OF ECTOMYCORRHIZAL SAMPLES USING ARISA 73 9. CHAPTER VII: SYMBIOSIS INSIGHTS FROM THE GENOME OF THE MYCORRHIZAL BASIDIOMYCETE LACCARIA BICOLOR. 86 10. CHAPTER VIII: FATTY ACID METABOLISM IN THE ECTOMYCORRHIZAL FUNGUS LACCARIA BICOLOR 87 11. CHAPTER X: CONCLUSIONS 103 12. REFERENCES OF INTRODUCTION AND CONCLUSIONS 114 5 1. Publication list • Fatty acid metabolism in the ectomycorrhizal fungus Laccaria bicolor Marlis Reich, Cornelia Goebel, Annegret Kohler, Marc Buée, Francis Martin, Ivo Feussner, Andrea Polle (published in New Phytologist (2009) 182: 950-964) • Symbiosis insights from the genome of the mycorrhizal basidiomycete Laccaria bicolor. Martin F, Aerts A, Ahrén D, Brun A, Danchin EGJ, Duchaussoy F, Gibon J, Kohler A, Lindquist E, Pereda V, Salamov A, Shapiro HJ, Wuyts J, Blaudez D, Buée M, Brokstein P, Canbäck B, Cohen D, Courty PE, Coutinho PM, Delaruelle C, Detter JC, Deveau A, DiFazio S, Duplessis S, Fraissinet-Tachet L, Lucic E, Frey-Klett P, Fourrey C, Feussner I, Gay G, Grimwood J, Hoegger PJ, Jain P, Kilaru S, Labbé J, Lin YC, Legué V, Le Tacon F, Marmeisse R, Melayah D, Montanini B, Muratet M, Nehls U, Niculita-Hirzel N, Oudot-Le MP, Peter M, Quesneville H, Rajashekar B, Reich M, Rouhier N, Schmutz J, Yin T, Chalot M, Henrissat B, Kües U, Lucas S, Van de Peer Y, Podila G, Polle A, Pukkila PJ, Richardson PM, Rouzé P, Sanders IR, Stajich JE, Tunlid A, Tuskan G, Grigoriev IV (published in Nature (2008) 452: 88-92) • 454 pyrosequencing analyses of forest soil reveal an unexpected high fungal diversity Buée M*, Reich M*, Murat C, Morin E, Nilsson RH, Uroz S, Martin F * These authors contributed equally to this work. (published in New Phytologist (2009) 184: 449-456) • Development and validation of an oligonucleotide microarray to characterize ectomycorrhizal communities Marlis Reich, Annegret Kohler, Francis Martin and Marc Buée 6 (submitted to BMC Microbiology) • Diagnostic ribosomal ITS phylochip for identification of host influence on ectomycorrhizal community Marlis Reich, Marc Buée, Henrik Nilsson, Benoit Hilseberger, Annegret Kohler, Emilie Tisseran, Francis Martin (submitted to ISME Journal) • Quantitative traceability of ectomycorrhizal samples using ARISA Marlis Reich, Christine Delaruelle, Jean Garbaye, Marc Buée (submitted to Forest Ecology and Management) 7 2. Preface 2.1.Summary Forests are highly complex ecosystems and harbor a rich biodiversity above-ground and below-ground. In the forest soil a multilayer array of microorganisms can be found occupying various niches. They play essential roles in the mineralization of organic compounds and nutrient cycling (Fitter et al., 2005). Mycorrhizal fungi are a highly abundant and functionally very important group of soil micro-organisms. They live in mutualistic symbiosis with the plants and deliver their plant hosts with nutrients and water and receive in return carbohydrates. Many environmental factors influence the richness and the composition of mycorrhizal communities. They can be grouped into biotic and abiotic factors. As mycorrhizal fungi are nearly wholly dependent on plant-derived carbohydrates it is not astonishing, that plant communities have a main impact on structure and function of mycorrhizal communities. This is achieved in a number of ways, e.g. by the host plant species, the age or successional status of the host tree/forest or physiological features such as litter fall, root turnover or root exudations of carbons (Johnson et al., 2005). Recent research focused e.g. on the effect of host taxonomic distance on ectomycorrhizal (ECM) communities. It was reported that communities sharing host trees of similar taxonomic status showed similar structure compared to communities associated to more taxonomical distinct host trees (Ishida et al., 2003). Anyhow, differences in ECM communities were observed when they were associated to two congeneric host trees with different leaf physiology (Morris et al., 2008). An important abiotic factor in boreal and temperate forests is the climate change over seasons. Temporal patterns of ECM fungi occur during a year and can be explained by ecological preferences of fungal species and enzymatic adaptation to changing weather and changing resource conditions (Buée et al., 2005; Courty et al., 2008). All these studies reveal the diversity of factors and their impact on mycorrhizal communities. As mycorrhizal communities are playing an important role in forest ecosystems, the dynamics of mycorrhizal communities as response to environmental factors have to be studied to understand the global dynamic and biodiversity of forest ecosystems. But how can we study and describe in detail such complex communities? In the last decades molecular biological detection techniques were developed and used alongside with classical morphological and anatomical-based methods. Especially the determination of ITS as DNA barcode for fungi and the adjustment of PCR conditions for the amplification of the total 8 fungal community opened the way for more detailed community studies (Horton & Bruns, 2001). Traditional molecular techniques such as ITS-fingerprinting or Sanger-sequencing were widely applied (reviewed in Anderson, 2006). However, these techniques are limited by the number of samples, which can be processed in a realistic time frame (Mitchell & Zuccharo, 2006). Identification of fungal taxa can nowadays be expanded to high-throughput molecular diagnostic tools, such as phylochips (a microarray to detect species) and 454 sequencing. The ongoing implementation of array technique led to its high-throughput capacity, as thousands of features can be fixed to the carrier glass. In the case of phylochips, features are oligonucleotides targeting barcode genes of the species of interest. So far, phylochips were used for the identification of bacterial species from complex environmental samples (Brodie et al., 2006) or for few genera of pathogenic (Lievens et al., 2003, 2005) and composting fungi (Hultman et al., 2008). 454 sequencing is a newly developed sequencing technique combining the complete sequence process covering all subsequent steps from the barcode region of interest to the finished sequence (Margulies et al., 2005). In first experiments 454 sequencing technique was used to sequence genomes (Andrie et al., 2005) or transcriptomes (Bainbridge et al., 2006). With the ongoing development metagenomic analysis were carried out. Bacterial community structures of different ecosystems were described with more than over 10,000 sequences (Huber et al., 2007). So far, no studies were published on fungal communities by using 454 sequencing. Phylochip and 454 sequencing analysis started to revolutionize the understanding of bacterial community structure and