Diversity of cultured isolates and field populations of the arbuscular mycorrhizal fungus Glomus intraradices: Development and application of molecular detection methods for mitochondrial haplotypes Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Boris Börstler aus Braunschweig, Deutschland Basel, 2010 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Thomas Boller, Prof. Dr. Dirk Redecker, Prof. Dr. Andres Wiemken Basel, den 22.06.2010 Prof. Dr. Eberhard Parlow, Dekan Table of contents Summary .............................................................................................................................. 1 Chapter 1: General introduction........................................................................................ 3 1.1 Mycorrhizas – origin, evolution and types ............................................................................... 3 1.2 The arbuscular mycorrhiza (AM) ............................................................................................. 7 1.2.1 Phylogeny and taxonomy of the Glomeromycota ............................................................. 7 1.2.2 Species concept and genetics of AM fungi (AMF) ......................................................... 12 1.2.3 Structural features of the AM and life cycle of AMF ..................................................... 13 1.2.4 AM partnerships: benefits and ecology ........................................................................... 17 1.2.4.1 Extent of plant – AMF associations in the plant kingdom ....................................... 17 1.2.4.2 Functional aspects of AM associations .................................................................... 19 1.2.4.3 Multitrophic interactions .......................................................................................... 22 1.2.4.4 Other ecological aspects: AMF and ecosystem functioning .................................... 23 1.2.5 Biodiversity studies of AMF communities and populations ........................................... 25 1.2.5.1 Different strategies and methods for the detection of AMF in the field................... 25 1.2.5.2 Marker genes for AMF community studies ............................................................. 26 1.2.5.3 Fingerprinting methods and quantitative approaches ............................................... 28 1.2.5.4 Marker genes available for the “species” or intraspecific level of AMF ................. 29 1.2.5.5 AMF communities in different environments .......................................................... 30 1.2.5.6 The first studies of intraspecific gene structure of AMF species in field settings .... 33 1.3 Mitochondria in AMF ............................................................................................................ 34 1.3.1 Mitochondrial genome structure and inheritance ............................................................ 34 1.3.2 The use of mitochondrial genes in phylogenetic analyses or as molecular markers ....... 36 1.4 Aims of this thesis .................................................................................................................. 38 Chapter 2: Genetic diversity of the arbuscular mycorrhizal fungus Glomus intraradices as determined by mitochondrial large subunit rRNA gene sequences is considerably higher than previously expected ................................................................ 41 2.1 Summary ................................................................................................................................ 42 2.2 Introduction ............................................................................................................................ 42 2.3 Materials and Methods ........................................................................................................... 45 2.3.1 Root organ cultures of G. intraradices ............................................................................ 45 2.3.2 Inocula and pot cultures of G. intraradices ..................................................................... 45 2.3.3 Field-collected root samples ............................................................................................ 46 2.3.4 DNA extraction ............................................................................................................... 46 2.3.5 PCR amplification of mtLSU .......................................................................................... 47 i Table of contents 2.3.6 PCR amplification of nuclear-encoded ITS rDNA ......................................................... 49 2.3.7 Cloning, sequencing and sequence analyses ................................................................... 50 2.3.8 Phylogenetic analyses ..................................................................................................... 50 2.3.9 RFLP analyses ................................................................................................................. 51 2.4 Results .................................................................................................................................... 52 2.4.1 Diversity of ITS sequences and mtLSU haplotypes in G. intraradices isolates ............. 52 2.4.2 MtLSU exon and intron phylogeny ................................................................................. 54 2.4.3 Intron stability in the mtLSU of G. intraradices ............................................................. 58 2.4.4 Detection of haplotypes in field-collected roots .............................................................. 58 2.4.5 RFLP analyses ................................................................................................................. 58 2.5 Discussion .............................................................................................................................. 60 2.6 Acknowledgements ................................................................................................................ 63 Chapter 3: Diversity of mitochondrial large subunit rDNA haplotypes of Glomus intraradices in two agricultural field experiments and two semi-natural grasslands . 65 3.1 Abstract .................................................................................................................................. 66 3.2 Introduction ............................................................................................................................ 66 3.3 Materials and methods ........................................................................................................... 69 3.3.1 Field sites and sampling .................................................................................................. 69 3.3.2 DNA extraction ............................................................................................................... 71 3.3.3 PCR amplification of mtLSU .......................................................................................... 71 3.3.4 Specific PCR amplification of mtLSU haplotype I ......................................................... 72 3.3.5 RFLP analyses and definition of RFLP types ................................................................. 73 3.3.6 Sequencing, cloning and sequence analyses ................................................................... 74 3.3.7 Phylogenetic analyses ..................................................................................................... 77 3.3.8 Population genetics ......................................................................................................... 77 3.3.9 Statistical analyses ........................................................................................................... 77 3.4 Results .................................................................................................................................... 78 3.4.1 Amplification success ..................................................................................................... 78 3.4.2 RFLP analyses ................................................................................................................. 80 3.4.3 Sequence-based analyses ................................................................................................. 85 3.4.4 Phylogenetic analyses ..................................................................................................... 89 3.4.5 The specific approach to detect haplotype I .................................................................... 89 3.5 Discussion .............................................................................................................................. 90 3.6 Acknowledgements ................................................................................................................ 93 ii Table of contents Chapter 4: Development of specific mitochondral markers for the Glomus intraradices isolate BEG140 .............................................................................................. 95 4.1 Introduction ............................................................................................................................ 95 4.2 Characterization of mitochondrial haplotypes of BEG140 .................................................... 95 4.2.1 Materials and Methods ...................................................................................................