Genetic diversity of indigenous rhizobial symbionts of the Lupinus mariae-josephae endemism from alkaline-limed soils within its area of distribution in Eastern Spain.

The genomic diversity of a collection of 103 indigenous rhizobia isolates from Lupinus mariae-josephae (Lmj), a recently described Lupinus species endemic to alkaline-limed soils from a restricted habitat in Eastern Spain, was investigated by molecular methods. Isolates were obtained from soils of four geographic locations in the Valencia province that harbored the known Lmj plant populations. Using an M13 RAPD fingerprinting technique, 19 distinct RAPD profiles were identified. Phylogenetic analysis based on 16S rDNA and the housekeeping genes glnII, recA and atpD showed a high diversity of native Bradyrhizobium strains that were able to establish symbiosis with Lmj. All the strains grouped in a clade unrelated to strains of the B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. The phylogenetic tree based on concatenated glnII, recA and atpD gene sequences grouped the Lmj isolates in six different operational taxonomic units (OTUs) at the 93% similarity level. These OTUs were not associated to any specific geographical location, and their observed divergence predicted the existence of different Bradyrhizobium genomic species. In contrast, phylogenetic analysis of symbiotic genes based on nodC and nodA gene sequences, defined only two distinct clusters among the Lmj strains. These two Lmj nod gene types were largely distinct from nod genes of bradyrhizobia nodulating other Old World lupine species. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host.

Durán D ，Rey L ，Sánchez-Cañizares C ，Navarro A ，Imperial J ，Ruiz-Argueso T ... -  《-》

Endosymbiotic bacteria nodulating a new endemic lupine Lupinus mariae-josephi from alkaline soils in Eastern Spain represent a new lineage within the Bradyrhizobium genus.

Lupinus mariae-josephi is a recently described endemic Lupinus species from a small area in Eastern Spain where it thrives in soils with active lime and high pH. The L. mariae-josephi root symbionts were shown to be very slow-growing bacteria with different phenotypic and symbiotic characteristics from those of Bradyrhizobium strains nodulating other Lupinus. Their phylogenetic status was examined by multilocus sequence analyses of four housekeeping genes (16S rRNA, glnII, recA, and atpD) and showed the existence of a distinct evolutionary lineage for L. mariae-josephi that also included Bradyrhizobium jicamae. Within this lineage, the tested isolates clustered in three different sub-groups that might correspond to novel sister Bradyrhizobium species. These core gene analyses consistently showed that all the endosymbiotic bacteria isolated from other Lupinus species of the Iberian Peninsula were related to strains of the B. canariense or B. japonicum lineages and were separate from the L. mariae-josephi isolates. Phylogenetic analysis based on nodC symbiotic gene sequences showed that L. mariae-josephi bacteria also constituted a new symbiotic lineage distant from those previously defined in the genus Bradyrhizobium. In contrast, the nodC genes of isolates from other Lupinus spp. from the Iberian Peninsula were again clearly related to the B. canariense and B. japonicum bv. genistearum lineages. Speciation of L. mariae-josephi bradyrhizobia may result from the colonization of a singular habitat by their unique legume host.

Sánchez-Cañizares C ，Rey L ，Durán D ，Temprano F ，Sánchez-Jiménez P ，Navarro A ，Polajnar M ，Imperial J ，Ruiz-Argüeso T ... -  《-》

Bradyrhizobium valentinum sp. nov., isolated from effective nodules of Lupinus mariae-josephae, a lupine endemic of basic-lime soils in Eastern Spain.

Bacterial strains isolated from nitrogen-fixing nodules of Lupinus mariae-josephae have been characterized following genetic, phenotypic and symbiotic approaches. Analysis of 16S rRNA genes placed them in a group together with Bradyrhizobium elkanii USDA 76(T), B. pachyrhizi PAC48(T), B. jicamae PAC68(T), 'B. retamae' Ro19(T) and B. lablabi CCBAU 23086(T) with over 99.0% identity. Phylogenetic analysis of concatenated housekeeping genes, recA, atpD and glnII, suggested that L. mariae-josephae strains represent a new Bradyrhizobium species, closely related to B. lablabi CCBAU 23086(T), B. jicamae PAC68(T) and 'B. retamae' Ro19(T) with 92.1, 91.9 and 90.8% identity, respectively. These results are consistent with overall genomic identities calculated as Average Nucleotide Identity (ANIm) using draft genomic sequences obtained for relevant strains. While L. mariae-josephae strains LmjM3(T)/LmjM6 exhibited a 99.2% ANIm value, they were significantly distant (<93% ANIm) from type strains of their closest species ('B. retamae' Ro19(T), B. lablabi CCBAU 23086(T) and B. jicamae PAC68(T)). Whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (WC-MALDI-TOF-MS) analysis of proteomic patterns of the same strains was consistent with these results. The symbiosis-related genes nodC, nodA and nifH genes from strains nodulating L. mariae-josephae were phylogenetically related to those from 'B. retamae' Ro19(T), but divergent from those of strains that nodulate other lupine species. Based on genetic, genomic, proteomic and phenotypic data presented in this study, L. mariae-josephae nodulating strains LmjM3(T), LmjM6 and LmjM2 should be grouped within a new species for which the name Bradyrhizobium valentinum sp. nov. is proposed (type strain LmjM3(T)=CECT 8364(T), LMG 2761(T)).

Durán D ，Rey L ，Navarro A ，Busquets A ，Imperial J ，Ruiz-Argüeso T ... -  《-》

Cytisus villosus from Northeastern Algeria is nodulated by genetically diverse Bradyrhizobium strains.

Ahnia H ，Boulila F ，Boulila A ，Boucheffa K ，Durán D ，Bourebaba Y ，Salmi A ，Imperial J ，Ruiz-Argüeso T ，Rey L ... -  《-》

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.

Bourebaba Y ，Durán D ，Boulila F ，Ahnia H ，Boulila A ，Temprano F ，Palacios JM ，Imperial J ，Ruiz-Argüeso T ，Rey L ... -  《-》