Most phytopathogenic bacteria with a free-living life cycle stage can be transmitted through water, suggesting phytopathogens may have shared or convergently evolved mechanisms to persist in water. Understanding bacterial physiology during starvation can inform development of improved irrigation water treatments that will be effective against starved phytopathogenic bacteria.In objective 1, we use identify fitness factors that allow bacterial pathogens to persist in water. Ralstonia spreads predominantly through irrigation water, so we will use functional genomics, targeted mutagenesis, and biological assays to investigate how Ralstonia persists in water.In objective 2, we will characterize how bacterial pathogens evolve after prolonged starvation outside of host plants. Plant pests alternate between transmission and pathogenesis stages of their life cycles. Selection from plant hostsand environmental forces during transmission both shape the evolution of pathogen populations. Ralstonia isolate collections provide a unique opportunity to determine whether and how fitness factors and other pathogen genes evolve during environmental persistence between infection cycles. Many Ralstonia and other phytopathogen isolate collections are stored as "waterstocks", which are vials of isolates in sterile water. Although Ralstonia can survive and maintain virulence in waterstocks stored at room temperature for decades, waterstocks are living populations that accumulate mutations and slowly evolve. We will use genetic and phenotypic assays to analyze the evolution of decades-old Ralstonia waterstocks to determine whether certain mutations are selected or whether the evolution is random. In parallel, we will optimize a method to rescue virulent phytopathogen variants from the living collections.