Identification of the root nodule symbiont of the rare legume species Hedysarum confertum Desf. (Hedysarum humile L.) in its Italian relictual site

Hedysarum confertum Desf. (Fabaceae) represents a particular rarity in the Italian flora, signalled as late as in 1931 and nowadays still occurring only in few isolated stations in the central Apennine mountains. Within a framework of loss prevention of relevant botanical resources and landscape protection, we aimed at investigating possible factors limiting the distribution of this taxon and its potential to colonize suitable habitats. For these reasons we verified the existence of symbiotic relationships between H. confertum and soil bacteria assessing their identity and physiology. The plant was found to form histologically complete root nodules which are regularly invaded by bacteria. However, bacterial isolation in pure culture and cultivation ex planta appears prevented by a state of non-culturability. To assess the taxonomical position of nodule occupants the problem was circumvented by direct PCR amplification of 16S ribosomal RNA gene and nucleotide sequencing which revealed that H. confertum hosts bacteria of the genus Mesorhizobium, and that their ribosomal sequence has undergone a higher-than-average degree of divergence from the mesorhizobia nodulating other legumes in different countries. These differences suggest a possible link between the non-culturability of the symbiont and the isolated relictual status of its host plant.