Understanding the intraspecies genetic and phenotypic diversity of the clover symbiont Rhizobium leguminosarum

Rhizobia are agriculturally important bacteria capable of forming symbiosis with legumes and fixing atmospheric nitrogen which sustainably improves plant productivity and soil fertility. The Rhizobium leguminosarum species complex is highly genetically diverse and contains five genetically distinct genospecies. Significant phenotypic diversity is also displayed within Rhizobium leguminosarum; however, no phenotypes are genospecies-exclusive. The importance of the broad genetic diversity of Rhizobium leguminosarum and its influence on phenotypic diversity and rhizosphere-associated interactions are unclear. In this thesis, Rhizobium leguminosarum symbiovar trifolii (Rlt) intraspecies diversity was investigated by assessing the genetic and phenotypic variation of white clover nodule Rlt from agricultural field managements across Europe. This thesis identified that the significant genetic diversity of Rlt can manifest in substantial transcriptional and phenotypic variation across strains, and this diversity can influence plant-mediated symbiont selectivity and competitive strain interactions. A novel multiplexed high-throughput amplicon sequencing approach, MAUI-seq, was developed to improve detection of chimeras and other erroneous sequences for confident determination of intraspecies diversity from environmental samples. Using this method, significant Rlt nodule population diversity was identified between clover genotypes due to the combined effects of plant-host filtering and geospatial variation in allele frequencies of individual genes. Investigation of multiple Rlt strain transcriptomes demonstrated that genospecies displayed differences in core genome expression which was associated with phenotypic growth traits and putative differences in bacterial metabolism. Genomic and transcriptomic variation was utilised to identify transcriptional units conserved across strains. Pairwise growth competition experiments between Rlt strains further showed that significant competitive variation is evident and potentially associated with genospecies differences. This research demonstrates that utilising multiple strains can aid identification of species-specific traits by considering the representative variation within a species. The work presented here has laid the groundwork for future investigation into the implications of intraspecies diversity for symbiotic effectiveness in the rhizobia-legume symbiosis.