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 50 isolates based on partial sequences of recA and gyrB grouped strains into seven clusters, five of which belong to the 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. We carried out an in-depth phylogenetic study with representatives of the seven clusters using sequences from housekeeping genes (rrs, recA, glnII, gyrB, and dnaK) and obtained consistent results. A phylogeny based on the sequence of the symbiotic gene nodC identified four groups, three formed by Bradyrhizobium isolates and one by the Microvirga and Phyllobacterium isolates. Symbiotic behaviors of the representative strains were 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 with those of the rhizobial populations nodulating lupines in the Old World, including L. micranthus from other Mediterranean areas, which are nodulated mostly by Bradyrhizobium strains.IMPORTANCELupinus micranthus is a legume broadly distributed in the Mediterranean region and plays an important role in soil fertility and vegetation coverage by fixing nitrogen and solubilizing phosphate in semiarid areas. Direct sowing to extend the distribution of this indigenous legume can contribute to the prevention of soil erosion in pre-Saharan lands of Tunisia. However, rhizobial populations associated with L. micranthus are poorly understood. 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 Tunisian soils were in fact Bradyrhizobium symbionts, but the other half were found unexpectedly to be bacteria within the genera Microvirga and Phyllobacterium These unusual endosymbionts may 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 semiarid areas of Tunisia.

Microvirga tunisiensis sp. nov., a root nodule symbiotic bacterium isolated from Lupinus micranthus and L. luteus grown in Northern Tunisia.

Three bacterial strains, LmiM8T, LmiE10 and LluTb3, isolated from nitrogen-fixing nodules of Lupinus micranthus (Lmi strains) and L. luteus (Llu strain) growing in Northern Tunisia were analysed using genetic, phenotypic and symbiotic approaches. Phylogenetic analyses based on rrs and concatenated gyrB and dnaK genes suggested that these Lupinus strains constitute a new Microvirga species with identities ranging from 95 to 83% to its closest relatives Microvirga makkahensis, M. vignae, M. zambiensis, M. ossetica, and M. lotononidis. The genome sequences of strains LmiM8T and LmiE10 exhibited pairwise Average Nucleotide Identities (ANIb) above 99.5%, significantly distant (73-89% pairwise ANIb) from other Microvirga species sequenced (M. zambiensis and M. ossetica). A phylogenetic analysis based on the symbiosis-related gene nodA placed the sequences of the new species in a divergent clade close to Mesorhizobium, Microvirga and Bradyrhizobium strains, suggesting that the M. tunisiensis strains represent a new symbiovar different from the Bradyrhizobium symbiovars defined to date. In contrast, the phylogeny derived from another symbiosis-related gene, nifH, reproduced the housekeeping genes phylogenies. The study of morphological, phenotypical and physiological features, including cellular fatty acid composition of the novel isolates demonstrated their unique profile regarding close reference Microvirga strains. Strains LmiM8T, LmiE10 and LluTb3 were able to nodulate several Lupinus spp. Based on genetic, genomic and phenotypic data presented in this study, these strains should be grouped within a new species for which the name Microvirga tunisiensis sp. nov. is proposed (type strain LmiM8T=CECT 9163T, LMG 29689T).

Definition of two new symbiovars, sv. lupini and sv. mediterranense, within the genera Bradyrhizobium and Phyllobacterium efficiently nodulating Lupinus micranthus in Tunisia.

In this study, a polyphasic approach was used to analyze three representative strains (LmiH4, LmiM2 and LmiT21) from a collection of six previously described strains isolated in Tunisia from root nodules of Lupinus micranthus. The phylogenetic analysis of the concatenated rrs, recA and glnII genes showed that strain LmiH4 had 100% concatenated gene sequence identity with the type strain Bradyrhizobium retamae Ro19T. Similarly, strain LmiM2 shared 100% concatenated gene sequence identity with the species Bradyrhizobium valentinum LmjM3T. However, strain LmiT21 showed an identical concatenated gene sequence with reference strain Phyllobacterium sophorae CCBAU03422T. The recA-glnII concatenated protein-coding genes used produced incongruent phylogenies compared with 16S rDNA phylogeny. The nodC gene analysis showed that the strains were phylogenetically divergent to the Bradyrhizobium symbiovars defined to date, and represented two new symbiovars. Plant infection analysis revealed that the three strains showed moderate host range and symbiotic specificities. Based on their symbiotic characteristics, we propose that the three strains isolated from Lupinus micranthus nodules belong to two new symbiovars, with the first denominated lupini within the two species Bradyrhizobium valentinum (type strain LmiM2) and B. retamae (type strain LmiH4), and the second denominated mediterranense within the species P. sophorae (type strain LmiT21).

Diversity of Bradyrhizobium strains nodulating Lupinus micranthus on both sides of the Western Mediterranean: Algeria and Spain.

Lupinus micranthus is a lupine distributed in the Mediterranean basin whose nitrogen fixing symbiosis has not been described in detail. In this study, 101 slow-growing nodule isolates were obtained from L. micranthus thriving in soils on both sides of the Western Mediterranean. The diversity of the isolates, 60 from Algeria and 41 from Spain, was addressed by multilocus sequence analysis of housekeeping genes (16S rRNA, atpD, glnII and recA) and one symbiotic gene (nodC). Using genomic fingerprints from BOX elements, 37 different profiles were obtained (22 from Algeria and 15 from Spain). Phylogenetic analysis based on 16S rRNA and concatenated atpD, glnII and recA sequences of a representative isolate of each BOX profile displayed a homogeneous distribution of profiles in six different phylogenetic clusters. All isolates were taxonomically ascribed to the genus Bradyrhizobium. Three clusters comprising 24, 6, and 4 isolates, respectively, accounted for most of the profiles. The largest cluster was close to the Bradyrhizobium canariense lineage, while the other two were related to B. cytisi/B. rifense. The three remaining clusters included only one isolate each, and were close to B. canariense, B. japonicum and B. elkanii species, respectively. In contrast, phylogenetic clustering of BOX profiles based on nodC sequences yielded only two phylogenetic groups. One of them included all the profiles except one, and belonged to symbiovar genistearum. The remaining profile, constituted by a strain related to B. elkanii, was not related to any well-defined symbiotic lineage, and may constitute both a new symbiovar and a new genospecies.