Hidden diversity: genetic characterization of non-symbiotic rhizobium endophytes across agro- forestry legume species in Southern Ethiopia

Legumes often harbor a complex community of non-rhizobial endophytes whose roles and taxonomic identities remain poorly understood in diverse ecological zones. We investigated the diversity of endophytic bacteria associated with the root nodules of four woody legumes ( Acacia abyssinica , Albizia gummifera , Indigofera arrecta , and Millettia ferruginea ) and seven herbaceous species ( Arachis hypogaea , Cajanus cajan , Cicer arietenum , Glycine max , Phaseolus vulgaris, Vigna radiata , and Vigna unguiculata ) in southern Ethiopia. A total of 135 bacterial strains were isolated from surface-sterilized root nodules collected from 49 field plants and 62 trap hosts. Classification via partial 16S rDNA sequencing identified Enterobacter (31%), Bacillus (18%), and Rhizobium (15%) as the most prevalent genera. To further resolve the taxonomy of the Rhizobium isolates, we conducted a multilocus sequence analysis (MLSA) by using three housekeeping genes: 16S rDNA, recA , and rpoB genes. Maximum likelihood and concatenated phylogenies consistently grouped the test isolates together, showing high similarity to the type strains of Rhizobium tropici and R. leucaenae . Despite this genetic proximity, none of the isolates exhibited rhizobial function during nodulation tests or possessed key symbiotic genes ( nifH , nodA , nodC ). Furthermore, AFLP fingerprinting suggested these non-symbiotic Rhizobium isolates likely represent at least three distinct novel genospecies. We discuss the potential causes for these findings, including the inherent absence of symbiotic loci or the loss of symbiotic genes during repeated sub-culturing.