Abstract

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 25PS6T (=DSM 116212T = LMG 33205T) designated as the type strain.

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