Diversity of cultured isolates and field populations of the arbuscular mycorrhizal fungus 'Glomus intraradices' : development and application of molecular detection methods for mitochondrial haplotypes

摘要

Today’s plant communities have evolved together with arbuscular mycorrhizal fungi (AMF, Glomeromycota) for millions of years. In “arbuscular mycorrhiza”, a mutualistic symbiosis, plants provide carbohydrates to the fungi, which in turn make mineral nutrients like phosphate or nitrogen available to the plants. AMF species diversity is generally higher in natural sites than in agroecosystems, where it can be strongly reduced. The detection of AMF is either based on morphotyping of soil-borne spores or on molecular markers, which can be directly applied using colonized roots of the host plant.udUntil recently, studies of AMF diversity on the population level were impossible, as no suitable marker genes were available. The first population studies on AMF had to rely on DNA from spores or root organ cultures (ROCs) and the molecular markers used could not be applied for the detection of AMF genotypes directly in colonized plant roots from the field. Previous work from our laboratory had shown that mitochondrial ribosomal RNA large subunit gene (mtLSU) sequences are homogeneous within several isolates of Glomus species and that the mitochondrial gene region is a promising marker for distinguishing strains of G. intraradices. The phylotype GLOM A-1 of this morphospecies which was defined in previous studies of our laboratory based on nuclear-encoded rDNA internal transcribed spacers (ITS) sequences seems to occur ubiquitously, showing a high ecological versatility. It is frequently used as model organism and its genome is being sequenced.udThe aim of this thesis was to develop and apply detection methods based on the mtLSU in order to investigate the diversity of G. intraradices isolates and field populations. The main question was whether this marker is suitable to resolve the genetic structure of this morphospecies which might allow shedding light on the ecological role of strains within the species.udIn the first part of this thesis, the diversity of the mtLSU was investigated in a set of 16 G. intraradices isolates originating from five continents, either obtained as soil inoculum or as ROC. Among these isolates, 12 different mtLSU haplotypes could be distinguished, whereas homogeneity of the marker within the isolates was confirmed. Several mtLSU haplotypes were already distinguishable by size differences of the PCR products, mainly based on the presence or absence of length-variable introns. The reliability of the marker is dependent on evolutionary intron stability, which was confirmed for some introns by comparisons of multiple culture lineages of the same isolate obtained from different culture collections. In phylogenetic analyses of mtLSU exon sequences from isolates and root-colonizing G. intraradices, several clades could be distinguished. Comparison with ITS sequences from the isolates showed a higher resolution of mtLSU exon sequences which was increased by intron sequences.udIn order to increase the specificity for G. intraradices and to optimize amplification of the mtLSU fragment from colonized plant roots, a new nested PCR approach was developed and tested using field root samples from a semi-natural grassland and a mine spoil in Hungary. A RFLP approach was developed to reduce time-consuming and expensive cloning and sequencing procedures.udIn the second part of this thesis, the population structure of an AMF in roots from the environment was analyzed for the first time. Two agricultural field experiments in Switzerland, including different tillage treatments, and two semi-natural grasslands in Switzerland and France were chosen for the investigation of the genetic structure of G. intraradices phylotype GLOM A-1 using the PCR-RFLP approach. Each field site was dominated by one or two frequently found RFLP patterns of G. intraradices GLOM A-1, which were defined as Intra types. The composition of Intra types differed strongly between the agricultural sites and the semi-natural grasslands, but also between the two agricultural sites. In contrast to the situation often found in AMF species community studies, RFLP type richness was higher in the agricultural sites compared to the grasslands. Four Intra types, shared by different sites, were further resolved by sequence analyses, but only the two grasslands were found to share mtLSU sequence haplotypes. In phylogenetic analyses of completely sequenced examples of each Intra type, almost all haplotypes from the grassland sites fell within a separate “grassland clade”.udIf a single mtLSU haplotype could be specifically detected in a pool of others, such a molecular tool could be used for tracing single strains inoculated in a field site. Nested PCR primers were developed specifically for one single mtLSU haplotype, which dominated one of the agricultural sites and was known from previous studies analyzing ROCs. By applying this approach to all samples from the four study sites, it could be shown that the respective haplotype was only detected in samples previously tested positive for this type using the general approach. In other words, both methods confirmed each other.udTwo further specific nested PCR approaches were developed for two mtLSU haplotypes representing the G. intraradices isolate BEG140. These approaches were designed to be applied for tracing this isolate inoculated in a field experiment performed in a mine spoil bank of the Czech Republic in the context of a reclamation project.udBesides the considerable genetic structure of this fungus among the isolates studied and in the roots of the field sites, evidence of specialized mtLSU haplotypes was reported, which might represent ecotypes or even different (“cryptic”) species. It could be shown that world-wide mtLSU haplotype diversity of G. intraradices is considerably higher than previously assumed. More investigations of different ecosystems are required for the determination of adapted ecotypes.udThe approaches developed here will be furthermore useful for instance in inoculation experiments and functional tests, e.g. in greenhouse experiments. By presenting first insights into the genetic structure of the most widespread species of arbuscular mycorrhizal fungi, the findings presented here will have major implications on our views of processes of adaptation and specialization in these plant ⁄ fungus associations.udud