Agricultural productivity in Australian soils is constrained by nutrient deficiencies, with insufficient soil nitrogen the most common cause of reduced crop and pasture yields. One way to increase soil nitrogen is incorporating grain and pasture legumes into agricultural systems, as these plants can form a nitrogen-fixing symbiosis with soil bacteria known as rhizobia. Nitrogen fixed by rhizobia in symbiosis with legumes provides Australian agriculture with a cost-effective and low carbon-emissions approach to boosting soil nitrogen in our nutrient-challenged soils. All of Australia’s agricultural legumes are introduced species that do not form compatible symbioses with native rhizobia. Thus, compatible exotic rhizobia are introduced as inoculants for pasture and grain legumes, following dedicated strain selection programs. There is considerable specificity in rhizobia-legume symbioses, with very few host-strain combinations giving rise to efficient nitrogen-fixing associations. However, we do not understand the genetic determinants of host-strain specificity that underpin these elite associations, which increases screening time to select new inoculant rhizobial strains. The aims of this project are to address this knowledge gap with the annual pasture legume Biserrula pelecinus, a robust pasture tolerant of limited rainfall introduced from Morocco in the mid-1990’s.Mesorhizobium ciceri WSM1497 is the commercial inoculant strain for B. pelecinus and this strain only effectively nodulates this host. By contrast, M. ciceri WSM1284 has a much broader host range, nodulating species across six genera of legumes, although not always effectively. Whole genome sequences of both strains were interrogated to investigate the genetic determinants of the host-range. This showed significant differences between these two strains in their core genomes, symbiosis accessory gene complements and accessory plasmids. To date, 14 mutants of WSM1284 have been constructed, targeting both putative symbiosis transcriptional regulators and genes involved in host-strain nodulation signalling. To assess the role of the core genome in modulating host range, symbiosis genes encoded on chromosomal Integrative and Conjugative Elements have been transferred from both WSM1284 and WSM1497 into diverse non-symbiotic Mesorhizobium species. In addition, potential host-range factors encoded on accessory plasmids are being functionally assessed by curing the repABC-type plasmid from both WSM1284 and WSM1497, using a plasmid incompatibility approach. The effect of these mutations and genetic alterations on host-range is currently being evaluated in planta across a wide spectrum of legume hosts. Ultimately, this study will provide genetic data critical to improving our new inoculant strain selection program and assist in optimising the benefits of nitrogen fixation in Australian agriculture.