Crop and pasture productivity in Australia is often constrained by insufficient soil nitrogen (N). Incorporating N2-fixing legumes that form a root nodule symbiosis with soil bacteria known as rhizobia provides a cost-effective and low carbon-emissions approach to boosting soil N. Australian agricultural legumes are introduced species and compatible exotic rhizobia are introduced as inoculants, following dedicated strain selection programs. There is considerable specificity in rhizobia-legume symbioses, with very few host-strain combinations giving rise to efficient N2-fixing associations. Understanding host specificity would facilitate better inoculation management practices in Australia. This project aims to address this knowledge gap by investigating the microsymbionts of the annual pasture legume Biserrula pelecinus. Thecommercial inoculant strain for B. pelecinus, Mesorhizobium ciceri WSM1497, only effectively nodulates this legume, and therefore has a narrow host range. By contrast, M. ciceri WSM1284 which also fixes N2 effectively with B. pelecinus has a broad host range being capable of nodulating species in seven other legume genera. WSM1497 and WSM1284 have highly similar genomes (ANI 98.3%), consisting of a single chromosome (~6.5 Mb) with a tripartite symbiosis Integrative and Conjugative Element (ICE), and a repABC type plasmid (~0.5 Mb). Importantly, WSM1284 contains 21 genes involved in the nodulation process, while WSM1497 has only 14 of these genes. Thirteen WSM1284 site-directed mutants constructed to target putative Nod factor transcriptional regulators (nodD) and biosynthesis genes (nodA, nodZ and nolL) have been evaluated in planta on B. pelecinus, Lotus ornithopodioides, Leucaena leucocephala and Ornithopus sativus. Deletion of genes abolishes nodulation on B. pelecinus, L. leucocephala and O. sativus but does not alter nodulation of L. ornithopodioides indicating complex factors are responsible for modulating host range. To assess the role of core genomes in modulating host range, symbiosis ICEs have been transferred from both strains into diverse non-symbiotic Mesorhizobium species. Finally, potential plasmid-encoded host-range factors are being functionally assessed using a plasmid incompatibility approach to cure plasmids.