The genetic diversity of bacterial populations nodulating Lupinus luteus (yellow lupine) in Northern Tunisia was examined. Phylogenetic analyses of 43 isolates based on recA and gyrB partial sequences grouped them in three clusters, two of which belong to genus Bradyrhizobium (41 isolates) and one, remarkably, to Microvirga (2 isolates), a genus never previously described as microsymbiont of this lupine species. Representatives of the three clusters were analysed in-depth by multilocus sequence analysis of five housekeeping genes (rrs, recA, glnII, gyrB and dnaK). Surprisingly, the Bradyrhizobium cluster with the two isolates LluI4 and LluTb2 may constitute a new species defined by a separate position between Bradyrhizobium manausense and B. denitrificans. A nodC-based phylogeny identified only two groups: one formed by Bradyrhizobium strains included in the symbiovar genistearum and the other by the Microvirga strains. Symbiotic behaviour of representative isolates was tested, and among the seven legumes inoculated only a difference was observed i.e. the Bradyrhizobium strains nodulated Ornithopus compressus unlike the two strains of Microvirga. On the basis of these data, we conclude that L. luteus root nodule symbionts in Northern Tunisia are mostly strains within the B. canariense/B. lupini lineages, and the remaining strains belong to two groups not previously identified as L. luteus endosymbionts: one corresponding to a new clade of Bradyrhizobium and the other to the genus Microvirga.

Strain aSej3^T was isolated from a root nodule of a Lupinus angustifolius plant growing in Bizerte, Tunisia. 16S rRNA gene analysis placed this strain within the genus Bradyrhizobium . Multilocus sequence analysis (MLSA) including three housekeeping genes (glnII, gyrB and recA) grouped aSej3^T together with Bradyrhizobium rifense CTAW71^T, Bradyrhizobium cytisi CTAW11^T, Bradyrhizobium ganzhouense RITF806^T, Bradyrhizobium lupini USDA 3051^T and Bradyrhizobium canariense BTA-1^T. MLSA with five housekeeping genes (dnaK, glnII, gyrB, recA and rpoB) revealed that this strain shares less than 93.5 % nucleotide identity with other type strains. Genome sequencing and inspection revealed a genome size of 8.83 Mbp with a G+C content of 62.8 mol%. Genome-wide average nucleotide identity and digital DNA–DNA hybridization values were below 87.5 and 36.2 %, respectively, when compared to described Bradyrhizobium species. Strain aSej3^T nodulated L. angustifolius plants under axenic conditions and its nodC gene clustered within the genistearum symbiovar. Altogether, the phylogenetic data and the chemotaxonomic characteristics of this strain support that aSej3^T represents a new species for which we propose the name Bradyrhizobium hipponense sp. nov. with the type strain aSej3^T (=DSM 108913^T=LMG 31020^T).

Thirty-one rhizobial isolates nodulating native Lupinus angustifolius (blue lupine) plants growing in Northern Tunisian soils were isolated and analysed using different chromosomal and symbiotic gene markers. Phylogenetic analyses based on recA partial sequences grouped them into at least five groups: four of them within the genus Bradyrhizobium (26 isolates) and one into the genus Microvirga (5 isolates). Representative strains were analysed by multilocus sequence analysis of three housekeeping genes rrs-recA-glnII and rrs-gyrB-dnaK for Bradyrhizobium and Microvirga isolates, respectively. Based on this analysis, eight isolates clustered with the previously described strains Bradyrhizobium lupini USDA3051 and Bradyrhizobium canariense BTA-1. However, five of the isolates clustered separately and may constitute a new species within the Bradyrhizobium genus. The remaining five isolates were closely related to the strain Microvirga sp. LmiM8 and may constitute a new Microvirga species. The analysis of the nodC gene showed that all Bradyrhizobium strains nodulating blue lupine belong to the symbiovar genistearum, whereas the Microvirga isolates are associated with the symbiovar mediterranense. The results of this study support that the L. angustifolius root nodule symbionts isolated in Northern Tunisia belong mostly to the B. canariense/B. lupini lineages. However, new clades of Bradyrhizobium and Microvirga have been identified as L. angustifolius endosymbionts.