Hinton, K. G.; Vinchira-Villarraga, D.; Dhaouadi, S.; Thomas, G. B.; Rabiey, M.; McCann, H. C.; McDonald, M. C.; Jackson, R. W.

Long term chronic infections of plants by bacterial pathogens are largely unknown. Understanding how pathogens adapt during chronic infection provides a key insight to pathogen evolutionary strategy both for persistence and survival, but also for potential future outbreaks. Pseudomonas savastanoi pv. fraxini (Psf), a member of phylogroup 3 within the Pseudomonas syringae species complex, causes canker disease in European ash (Fraxinus excelsior). Infections persist for years within the bark parenchyma, where bacteria are enclosed in cavities that contribute to the gradual expansion of host periderm. This pathosystem therefore provides an opportunity to examine pathogen evolution in a long-lived, largely unmanaged host. We combined population genomics and phenotypic analysis of 124 Psf strains collected from six sites across the UK. Phylogenetic analysis revealed a highly clonal population, with only 833 core genome SNPs across a 5.3 Mb genome, and a relatively small accessory genome largely shaped by gain and loss of large mobile genetic elements. Despite this limited genomic diversity, mutations were enriched in regulatory genes, including two-component systems, chemotaxis proteins, and cell envelope-associated loci. Notably, the global regulator gacA/S was independently mutated multiple times within the same clonal lineage. These mutations, typically small deletions, were associated with changes in motility, nutrient utilisation, stress tolerance, and virulence across genetic backgrounds. As a result, phenotypic heterogeneity was observed within otherwise clonal populations, including within individual lesions. These findings indicate that repeated mutation of regulatory systems represents a key mechanism of adaptation in this chronic plant-pathogen interaction, enabling phenotypic diversification despite limited sequence divergence. This study provides a microevolutionary perspective on P. syringae populations in the phyllosphere and highlights the role of regulatory variation in the evolution of low-virulence, ecologically restricted pathogens.