Saprophytic Growth and Fate of Frankia Strains in Soil
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Frankia are nitrogen fixing bacteria that form root nodules with more than 200 actinorhizal plant species. In nature, Frankia are found in soil and in root nodules of specific host plants. Due to their low abundance in soil and difficulties to isolate them, most studies on Frankia focus on populations in root nodules, which are natural locale of enrichment for Frankia. As a consequence, little is known about the ecology of Frankia in soil. The work presented in this PhD. dissertation focused on two basic objectives:1) to elucidate the effects of specific environmental conditions on the fate of introduced Frankia strains in soil microcosms, and 2) to highlight the limitations of plant bioassay analyses in describing the diversity of Frankia in soil. <br />Work on the first objective provided evidence that Frankia strains differ with respect to their ability to utilize specific organic compounds (chapter II, and III), and that -within the Alnus host infection group- the utilization of leaf litter is a specific trait of few Frankia strains, and reflected in their taxonomic position (chapter III). Results for the second objective demonstrated that the host plant species has a large effect in the selection of Frankia strains from soil for potential nodule formation (chapter IV), and that this effect results in large differences between Frankia populations detected directly in soil and those in root nodules (chapter V). Consequently, the choice of the capture plant species has a significant effect in bioassays on diversity estimates of frankiae in soil. <br />Specific results for the first objective were that Frankia strains have the potential to grow saprophytically, with the majority of strains belonging the Elaeagnus and Alnus host infection groups growing in the rhizosphere of a non-host plant, Betula pendula, but not in the surrounding bulk soil. Casuarina-infective strains that are generally assigned to the Alnus host infection group, however, did not grow in the rhizosphere of B. pendula, even though these same strains did grow in the rhizosphere of Casuarina cunninghamiana. In contrast to results obtained for the rhizosphere of B. pendula, saprophytic growth on leaf litter as a C source was restricted to a small fraction of Frankia strains that all belonged to a distinct phylogenetic cluster within the Alnus host infection group. These results demonstrated that saprophytic growth of frankiae was a common trait for most members of the genus, and the supporting factors for growth (i.e. carbon utilization capabilities) varied with host infection group and phylogenetic affiliation of the strains. These studies also provided information on the usefulness of comparative nifH gene sequences analyses to distinguish Frankia clusters within the Elaeagnus and Alnus host infection group, with comparable assignments of strains but better resolution than the previously used insertion in the 23S rRNA gene. <p>Specific results for the second objective highlighted the potential role of host plant species in the selection of nodule-forming frankiae from soil in bioassays with two Morella, three Elaeagnus and one Shepherdia species as capture plants. Diversity of frankiae was larger in nodules on both Morella species than in nodules formed on the other plant species, and none of the plants captured the entire diversity of nodule-forming frankiae. The distribution of clusters of Frankia populations and their abundance in nodules was unique for each of the plant species with only one cluster being ubiquitous and most abundant while the remaining clusters were only present in nodules of one (six clusters) or two (two clusters) host plant species. These results demonstrated large effects of the host plant species in the selection of Frankia strains from soil for potential nodule formation, and thus the significant effect of the choice of capture plant species in bioassays on diversity estimates in soil. Meta-analysis including sequences previously published for cultures, for uncultured frankiae in root nodules of Morella pensylvanica formed in bioassays, and gene clone libraries for the respective soils displayed large differences in cluster assignments between sequences retrieved from clone libraries and those obtained from nodules, with assignments to the same cluster only rarely encountered for individual soils. These results demonstrated large differences between detectable Frankia populations in soil and those in root nodules indicating the inadequacy of bioassays for the analysis of frankiae in soil and the role of plants in the selection of frankiae from soil for root nodule formation.