Molecular and biological investigation of Pseudomonas aureofaciens PA147-2 as a potential biocontrol agent of phytopathogenic fungi
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Pseudomonas aureofaciens PA 147-2 can inhibit the in vitro growth of phytopathogenic fungi, and suppress Phytophthora rot of asparagus in the glasshouse and in the field. Three antifungal defective mutants of PA147-2 were examined in detail. Two mutants, PA109 and PA138, were shown to have mutations in global regulator genes. The mutated gene in PA109 (designated finT) has similarity to members of the two-component regulator family, while PA138 has an insertion in a gene encoding a putative LysR-type transcriptional regulator (designated finR). FinR was found to be required in cis for the regulation of an adjacent gene (tinA), and a mutation infinA resulted in a reduction of antifungal activity. Protein analysis indicated that FinR and FinT have both positive and negative regulatory effects, and the two regulators have common targets for regulation. The N-terminal sequence of a protein that is not expressed by finR and finT mutants was not similar to any known protein. P. aureofaciens mutant PAH26 was shown to have an insertion in pstA. Consistent with findings in other organisms, the insertion in pstA conferred defects in high affinity phosphate uptake and interfered with regulation of the phosphate regulon. Results suggest that the inability of PAH26 to inhibit fungal growth is not directly related to known functions of PstA. Both PAH26 and PA109 are capable of fungal inhibition in phosphate excess conditions, implicating FinT in the phosphate-mediated regulation of antifungal activity. The recA gene of PA147-2 was cloned, and a simple system to construct recA deletions was developed. Creation of recA mutants using this system will facilitate trans complementation experiments. A preliminary field trial demonstrated that P. aureofaciens was able to protect asparagus from Phytophthora rot, and revealed that a high population of PA147-2 was not maintained in the field. In vitro experiments indicated that some antifungal-defective mutants have increased fitness relative to PA147 -2, and such mutants in laboratory culture conditions can outcompete the parental strain.