Diverse Bacteria Affiliated with the Genera Microvirga, Phyllobacterium, and Bradyrhizobium Nodulate Lupinus micranthus Growing in Soils of Northern Tunisia

The genetic diversity of bacterial populations nodulating Lupinus micranthus in five geographical sites from Northern Tunisia was examined. Phylogenetic analyses of fifty isolates based on recA and gyrB gene partial sequences grouped strains in seven clusters, five of which belong to genus Bradyrhizobium (28 isolates), one to Phyllobacterium (2 isolates), and one, remarkably, to Microvirga (20 isolates). The largest Bradyrhizobium cluster (17 isolates) grouped with the B. lupini species and the other five clusters were close to different, recently defined Bradyrhizobium species. Isolates close to Microvirga were obtained from nodules of plants from four of the five sites sampled. An in-depth phylogenetic study was carried out with representatives of the seven clusters using sequences from housekeeping genes ( rrs, recA, glnII , gyrB and dnaK ) and consistent results were obtained. A phylogeny based on the sequence of symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behavior of the representative strains was tested and some congruence between symbiovars and symbiotic performance was observed. These data indicate a remarkable diversity of L. micranthus root nodule symbionts in Northern Tunisia, including strains from the Bradyrhizobiaceae , Methylobacteriaceae and Phyllobacteriaceae families in contrast to the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, that are nodulated mostly by Bradyrhizobium strains. IMPORTANCE Lupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetal coverage by its ability to fix nitrogen and solubilize phosphate in semi-arid areas. Extension of this indigenous legume by direct sowing can contribute to prevent soil erosion in Presaharian lands of Tunisia. However, rhizobial populations associated to L. micranthus are poorly known. In this context, the diversity of endosymbionts of this legume was investigated. Most Lupinus species are nodulated by Bradyrhizobium strains. This work showed that about half of the isolates from Northern Tunisia soils are in fact Bradyrhizobium symbionts, but the other half unexpectedly were found to be bacteria within the genera Microvirga and Phyllobacterium . These unusual endosymbionts can have a great ecological relevance. Inoculation with the appropriate selected symbiotic bacterial partners will increase L. micranthus survival with consequent advantages for the environment in semi-arid areas of Tunisia.