Diversity of root nodule endophytes associated with native legumes in Flanders (Belgium)

The association between legume plants and rhizobia is quite old and enables the plant to survive nutrient poor conditions because of the nitrogen fixation capacity of the rhizobia that reside inside their root nodules. Legumes are a large family which occurs almost everywhere on earth and is divided into three subfamilies, the Caesalpinioideae, Mimosoideae and Faboideae. In Belgium only legumes occur from this last subfamily. The aim of this study was to make an in-depth analysis from the diversity of root nodule endosymbionts present in indigenous and exotic legumes in Flanders. The nodulation and nitrogen fixation capacity was analysed to obtain insights in the function and host specificity of the endosymbionts. In total 655 nodules were selected for further research. First of all, the nodules were surface sterilised, squashed and plated on YMA isolation medium. In total 4464 isolates were obtained which were analysed using rep-PCR clustering and partial 16S rRNA and recA gene sequencing. This revealed the presence of on the one hand traditional rhizobia (85%), Bradyrhizobium, Ensifer (Sinorhizobium), Mesorhizobium and Rhizobium and on the other hand non-rhizobial endophytes belonging to several genera in the Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmibacteria, Flavobacteria and Sphingobacteria. The rhizobial endosymbionts were dominated by Rhizobium and especially the species R. leguminosarum. The non-rhizobial endophyte population was dominated by Bacillus, Paenibacillus and Pseudomonas species. However, in most cases, the non-rhizobial endophytes, coexisted with traditional rhizobia accounting for approximately 80% of the nodule isolates. Several new groups were found within the genera Bradyrhizobium, Mesorhizobium and Bosea. The Bosea isolates were further characterised using a polyphasic approach and three new species have been proposed. Additionally, 24 strains showing low sequence similarity with existing genera in the family Bradyrhizobiaceae were further characterised and described as Tardiphaga robiniae. The symbiosis genes nodC and nifH were investigated for a selection of strains which revealed the presence of different symbiovars in traditional rhizobia including genistearum, glycinearum, loti, meliloti, officinalis, trifolii and viciae. Additionally, three new nodC types were assigned to strains originating from Ononis, Robinia and Wisteria. Within the non-rhizobial endophytes, rhizobial symbiovars genistearum, trifolii and viciae were present. This indicates that horizontal gene transfer could have occurred between rhizobia and non-rhizobial endophytes. Additionally, the majority of the isolates showed a unique nodC gene sequence type distinct from any previously described symbiovar. This nodC type was associated with a broad variety of host plant and we refer to this type as the sablum nodC type since it was present in strains originating from legume root nodules harvested in sandy soils