Don’t go out there! How interaction outside the host can affect the Rhizobia-Legume Symbiosis

The interaction between rhizobia and their bacteriophage predators represents an understudied yet critical component of soil microbial ecosystems, with potential implications for the agriculturally important rhizobia-legume symbiosis. Little is understood about how phage-driven evolution of rhizobia in the soil influences their symbiotic relationships with legumes. Using experimental and mathematical modelling, this thesis aims to bridge this knowledge gap by investigating the evolutionary consequences of phage predation on rhizobia and the resulting impacts on symbiosis. We demonstrate that prolonged exposure of rhizobia to phage predation in the soil rapidly selects for phage-resistant bacterial strains. Although these resistant rhizobia did not exhibit direct symbiosis-specific trade-offs, phage-driven selection did induce genetic mutations that indirectly influenced symbiotic quality of evolved rhizobia. Genome sequencing revealed that phage-exposed rhizobia accumulated mutations predominantly affecting metabolic pathways and biofilm formation. Consequently, some of these mutations indirectly affected rhizobia's symbiotic quality, highlighting that phage coevolution can shape the functionality of the rhizobia-legume mutualism. Additionally, we show that the ability of rhizobia to escape phage exposure by colonizing root nodules creates an important ecological trade-off. Early nodule colonization allows rhizobia to evade phage-driven selection, preserving symbiotic efficiency, but renders them more susceptible to phage predation upon their return to the soil. Highlighting the interplay between symbiosis and coevolution, where the timing of host association can influence bacterial fitness and evolutionary trajectories. Mathematical modelling showed that plant-associated nodules serve as spatial refuges, influencing rhizobia-phage coevolution. Refuges promote stable coexistence between rhizobia and free-living phages, while trade-offs in phage resistance, particularly those tied to refuge use, are crucial for maintaining both resistant and susceptible strains in the soil. Collectively, this research emphasizes that bacteriophage predation not only shapes rhizobial evolution in the soil but has downstream consequences for their ecological interactions with legumes.