Antimicrobial Resistance and Sensitivity of Phytophthora agathidicida

Members of the oomycete genus Phytophthora are highly infectious plant pathogens. P. agathidicida affects the New Zealand native keystone species Agathis australis(kauri) and is the cause of kauri dieback. The complex oomycete lifecycle makes Phytophthora infections hard to manage. The current management of kauri dieback has been limited and antimicrobial resistance is a concern. Phosphite agrichemical preparations are commonly used in the control of Phytophthora diseases, including kauri dieback. However, phosphite is not the only option; the agrichemicals oxathiapiprolin, and the plant-derived natural products polygodial and falcarindiol, have also been shown to have activity against P. agathidicida. The overall goal of this thesis was to further explore aspects of sensitivity and resistance of P. agathidicidatowards these four compounds.In New Zealand, there are three commercially available phosphite preparations, Agri-Fos 600, Phosgard, and Foschek. All previous studies have used Agri-Fos 600, so the first aim was to determine whether the particular formulation altered anti-oomycete activity. No significant difference was found between the 50% inhibitory concentrations (EC50 values) for the three formulations. Interestingly, however, formulating polygodial and falcarindiol with the surfactants and other non-phosphite ingredients of Foschek led to a significant increase in their inhibitory effects. The second aim of this thesis was to implement a serial passaging protocol for P. agathidicida and attempt to isolate mutants with increased resistance to phosphite, polygodial or falcarindiol. Serial passaging was carried out on amended agar plated with increasing concentrations of each chemical. However, even after 7 passages, over 16-18 weeks of growth, no mutants with increased resistance were isolated. This could be due to the complicated modes of action of the polygodial, falcarindioland phosphite, which makes it likely that several specific mutations are required to effect resistance.
IIOxathiapiprolin is a highly potent, new anti-oomycete agrichemical. It targets the Phytophthora oxysterol binding protein (OSBP) related protein (ORP1). Mutations in this protein are known to give oxathiapiprolin resistance in other species of Phytophthora; however, the P. agathidicida protein (PaORP1) has never been studied. In this work, the gene for PaORP1 was partially sequenced from five P. agathidicida isolates. None contained any of the known resistance mutations. A new protocol for expressing PaORP1 in E. coli and purifying it using immobilised metal affinity chromatography was also developed. After optimisation, this protocol yielded up to 30 mg of purified protein per litre of E. coli culture and is the first successful example of heterologously expressing and purifying any P. agathidicida protein. In future, this will allow the biomolecular interaction between PaORP1 and oxathiapiprolin to be studied in more detail. Overall, the work presented in this thesis assessed commercial formulations of phosphite, established a directed evolution protocol for studying resistance in P. agathidicida, and reported the first in vitro characterisation of a P. agathidicidaprotein. This research suggests that commercial formulation of plant-derived natural products may be a powerful new approach for combatting kauri dieback and, promisingly, also suggests that the risk of developing resistance to these compounds might be low.