Three bacterial strains, 1AS11^T, 1AS12 and 1AS13, members of the new symbiovar salignae and isolated from root nodules of Acacia saligna grown in Tunisia, were characterized using a polyphasic approach. All three strains were assigned to the Rhizobium leguminosarum complex on the basis of rrs gene analysis. Phylogenetic analysis based on 1734 nucleotides of four concatenated housekeeping genes (recA, atpD, glnII and gyrB) showed that the three strains were distinct from known rhizobia species of the R. leguminosarum complex and clustered as a separate clade within this complex. Phylogenomic analysis of 92 up-to-date bacterial core genes confirmed the unique clade. The digital DNA–DNA hybridization and blast-based average nucleotide identity values for the three strains and phylogenetically related Rhizobium species ranged from 35.9 to 60.0% and 87.16 to 94.58 %, which were lower than the 70 and 96% species delineation thresholds, respectively. The G+C contents of the strains were 60.82–60.92 mol% and the major fatty acids (>4 %) were summed feature 8 (57.81 %; C_18 : 1 ω7c) and C_18 : 1 ω7c 11-methyl (13.24%). Strains 1AS11^T, 1AS12 and 1AS13 could also be differentiated from their closest described species (Rhizobium indicum, Rhizobium laguerreae and Rhizobium changzhiense) by phenotypic and physiological properties as well as fatty acid content. Based on the phylogenetic, genomic, physiological, genotypic and chemotaxonomic data presented in this study, strains 1AS11^T, 1AS12 and 1AS13 represent a new species within the genus Rhizobium and we propose the name Rhizobium acaciae sp. nov. The type strain is 1AS11^T (=DSM 113913^T=ACCC 62388^T).

Three slow-growing rhizobial strains, designated as 1AS2L^T, 1AS20L and 1AS5L, were isolated from nodules of Acacia saligna in Borj Cedria, northern Tunisia. These strains, which belong to the symbiovar cyanophyllae, were characterized using a polyphasic approach. Phylogenetic analysis of the 16S rRNA (rrs) gene placed these strains within the genus Bradyrhizobium, specifically in the superclade associated with Bradyrhizobium japonicum. Further phylogenetic analysis using concatenated sequences of the recA, atpD, glnII and gyrB genes (totalling 1,734 bp) positioned the strains in a distinct lineage, with Bradyrhizobium shewense identified as their closest related species, sharing a sequence identity of 95.2%. The type strain, 1AS2L^T, exhibited average nucleotide identity values of 89.10%, 89.08% and 89.00% with the type strains of the closest valid species: Bradyrhizobium frederickii, B. shewense and Bradyrhizobium ottawaense, respectively. Additionally, digital DNA–DNA hybridization values confirmed the novelty of strain 1AS2L^T, showing low similarity (38.0%–38.3%) with the type strains of the closest known species. Phylogenomic analyses based on up-to-date bacterial core genes, Type (Strain) Genome Server and the Genome Taxonomy Database (GTDB) pipelines further supported the uniqueness of the 1AS2L^T, 1AS20L and 1AS5L strains. The GTDB analysis also robustly clustered two strains (SZCCT0449 and NSD-1) with our strains, suggesting putative members of the proposed novel species. The differentiation of these novel strains from their closest phylogenetic neighbours was also corroborated by phenotypic, physiological and fatty acid content analyses. Based on genomic, phenotypic and biochemical data, we propose the establishment of a novel species, Bradyrhizobium tunisiense sp. nov., with strain 1AS2L^T (=LMG 33170^T=DSM 114401^T) as the type strain.

Acacia saligna , a leguminous plant known for its ability to thrive under challenging environmental conditions, plays an important role in bioremediation by enhancing soil fertility and facilitating pollutant degradation. In this study, three slow-growing rhizobial strains, designated 1.29L, 1.27L and 5.13L, were isolated from root nodules of A. saligna growing naturally in two contaminated sites in Tunisia. A polyphasic taxonomic approach was applied. Phylogenetic analysis based on the 16S rRNA gene placed the isolates within the genus Bradyrhizobium , clustering within the Bradyrhizobium japonicum superclade. Multilocus sequence analysis using concatenated housekeeping genes ( rec A, atp D, gln II and gyr B: 1,734 bp) positioned the strains as a distinct lineage, with Bradyrhizobium arachidis , Bradyrhizobium stylosanthis and Bradyrhizobium shewense as the closest relatives, sharing 94.5–94.8% sequence identity. Comparative genomic analyses further supported their taxonomic distinctiveness. The proposed type strain, 1.29Lᵀ, exhibited average nucleotide identity (ANI) values of 90.6%, 90.1% and 89.1% with Bradyrhizobium liaoningense , Bradyrhizobium diversitatis and Bradyrhizobium forestalis , respectively, and digital DNA–DNA hybridization (dDDH) values ranging from 34.4 to 42.5%. In contrast, strains 1.29Lᵀ, 1.27L and 5.13L shared >99.9% ANI and 100% dDDH, confirming their conspecificity. Phylogenomic analyses conducted using the Type Strain Genome Server and GTDB-Tk pipelines further confirmed their placement within the B. japonicum supergroup while clearly separating them from all described species. Phenotypically, the isolates tolerated high concentrations of heavy metals and were capable of producing indole-3-acetic acid, solubilizing phosphate and producing siderophores. Based on the combined genomic, phylogenetic and phenotypic evidence, these strains represent a novel species, for which the name Bradyrhizobium cedriense sp. nov. is proposed, with strain 1.29Lᵀ (=LMG 33169ᵀ=DSM 116455ᵀ) designated as the type strain.

This study investigates nitrogen-fixing rhizobia associated with Pisum sativum, a member of the tribe Vicieae (Fabaceae), whose species establish symbioses with bacteria belonging predominantly to the symbiovar viciae within the Rhizobium leguminosarum complex (Rlc). Based on a comprehensive taxonomic revision of the F-clade within this complex, we report the identification and characterization of a novel rhizobial subspecies, Rhizobium laguerreae subsp. mediterraneum subsp. nov., isolated from pea nodules in Tunisia. Phylogenetic analyses based on 16S rRNA and multilocus sequence analysis (recA, atpD, dnaK, and glnII) placed strains 25PS6 and 10PS4 within the Rlc, while whole-genome phylogenomics using 2960 single-copy orthologues supported their assignment to a distinct monophyletic clade (Q-II). Subspecies-level clustering consistency was maximized using an optimized ANIm criterion of 97.40%, corresponding to 76.65% dDDH. Both strains belong to symbiovar viciae and exhibited improved symbiotic performance on pea plants compared to the reference strain, indicating strong symbiotic performance and potential relevance for biological nitrogen fixation. Cluster-specific SNP analysis identified 63 exclusive non-synonymous mutations with putative functional effects predicted in silico. These results suggest that cluster-specific nsSNPs may contribute to genomic differentiation within the Q-II lineage. Phenotypic and chemotaxonomic analyses further distinguished the novel subspecies based on carbon utilization, enzymatic activity, antibiotic resistance, and fatty acid profiles. Together, these findings highlight the genomic diversity within nitrogen-fixing rhizobia associated with legumes and identify a novel subspecies with potential agronomic relevance for improving symbiotic nitrogen fixation in pea cultivation. The proposed subspecies, Rhizobium laguerreae subsp. mediterraneum, is represented by strains 10PS4 and 25PS6, with strain 25PS6^T (=DSM 116212^T = LMG 33205^T) designated as the type strain.