Pseudomonas chlororaphis PA23 is a biocontrol agent (BCA) that is able to protect canola against the pathogenic fungus Sclerotinia sclerotiorum. PA23 secretes a number of metabolites that contribute to fungal antagonism, including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN) and degradative enzymes. Beyond pathogen suppression, the success of a BCA is dependent upon its ability to persist in the environment and avoid the threat of grazing predators, including protozoa. The first part of this thesis investigated whether PA23 is able to resist predation by Acanthamoeba castellanii (Ac) and defined the role of antifungal (AF) compounds in the bacterial-protozoan interaction. We discovered that PRN, PHZ and HCN contribute to PA23 toxicity towards Ac trophozoites. Ac preferentially migrated towards regulatory mutants devoid of AF metabolites as well as a PRN biosynthetic mutant, indicating that AF metabolites act to repel Ac. We also discovered that toxin-producing strains were able to survive inside trophozoites for up to 24 h. Collectively, our findings indicate that PRN, PHZ and HCN are involved in amoebicidal activity, and through the production of these molecules, PA23 is able to avoid the threat of predation.

PA23 accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. The second part of this thesis aimed to elucidate the role of PHAs in PA23 stress resistance and interaction with Ac. Three PHA biosynthesis mutants were created, PA23phaC, PA23phaC1ZC2 and PA23phaC1ZC2D, which no longer accumulated PHA. We observed that PA23phaC1ZC2D produced less PHZ compared to wild type (WT). All three mutants exhibited enhanced sensitivity to UV radiation, starvation, heat and cold stress, and exposure to hydrogen peroxide. Moreover, a lack of PHA production resulted in increased motility, biofilm formation, exopolysaccharide production and root attachment. Interaction studies with the protozoan predator Ac revealed that the WT, PA23phaC1 and PA23phaC1ZC2 mutants were less palatable compared to the PA23phaC1ZC2D mutant, which produced less PHZ. Taken together, the accumulation of PHA enhances bacterial resistance to various stress conditions, which could improve the environmental fitness of this bacterium in hostile environments.