Phytophthora species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, P. agathidicida, infects and ultimately kills the treasured kauri trees (Agathis australis) that are endemic to New Zealand. Currently there are few options for managing kauri dieback disease. In this study, we sought to assess the toxicity of the oomycide oxathiapiprolin against several life cycle stages of two geographically distinct P. agathidicida isolates. The effective concentration to inhibit 50% of mycelial growth (EC50) was determined to be approximately 0.1 ng/ml, indicating that P. agathidicida mycelia are more sensitive to oxathiapiprolin than those from most other Phytophthora species that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50 = 2–9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

Phytophthora species cause disease and devastation of plants in ecological and horticultural settings worldwide. A recently identified species, P. agathidicida, infects and ultimately kills the treasured kauri trees that are endemic to New Zealand. Currently there are few options for controlling or treating P. agathidicida. In this study, we sought to assess the toxicity of the oomycide oxathiapiprolin against several lifecycle stages of two geographically distinct P. agathidicida isolates. Half maximal effective concentration (EC50) values were determined to be approximately 0.1 ng/ml for inhibiting mycelial growth, indicating that P. agathidicida mycelia are more sensitive to oxathiapiprolin than those from most other Phytophthora species that have been studied. Oxathiapiprolin was also highly effective at inhibiting the germination of zoospores (EC50 = 2-9 ng/ml for the two isolates) and oospores (complete inhibition at 100 ng/ml). In addition, oxathiapiprolin delayed the onset of detached kauri leaf infection in a dose-dependent manner. Collectively, the results presented here highlight the significant potential of oxathiapiprolin as a tool to aid in the control of kauri dieback disease.

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. <br>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.

Microbiomes play critical roles in host functioning and therefore there is increasing interest in the microbiome assembly of plants. However, sampling strategies for long-lived perennial trees need to be standardised to produce robust data that accurately represents the microbiome over time. This issue is currently unresolved because there is little evidence indicating which portion of perennial tree species (e.g., root region or surrounding soil) is the best to sample to produce the most accurate measure of microbiome communities. Our aim was to sample different compartments of a plant’s belowground microbiome to identify the optimal sampling strategy to account for the microbial community present. We found that the structure of the microbial community depends most strongly on the environment (site) and compartment of sample collected (bulk soil, rhizosphere, or rhizoplane), rather than the depth or cardinal direction of the sample. We also found that the microbial community increased in diversity with increased distance from the tree within the rhizoplane and rhizosphere. The data presented here provides systematic evidence for a pragmatic and robust sampling regime that was tested and validated across different environments and soil types while controlling for host genotype. This sampling regime will enable effective partitioning of root compartments when studying the microbiome associated with perennial tree species, allowing targeted questions about the microbiome to be explored with greater accuracy.

Understanding the interaction of endophytic microbiomes and their tree hosts may provide insights into wood formation and quality. Given the role of wood in carbon and nutrient cycling, this will provide valuable insights for forest growth and carbon cycling globally. Furthermore, the management of these interactions may add new value to wood- and fibre-based forest products. We assessed the microbiome of outer and inner bark, cambium tissue, year 2-8 wood increments, and the pith of 11Pinus radiatatrees, a widely planted, model conifer species. Diverse prokaryotic and fungal microbiomes were present in all trees, with communities structured by tissue type (ρ<0.001). Inner and outer bark tissues had high richness and the most distinct communities. Microbiome richness was lowest in year 2 through to year 8 wood, and the communities in these samples had similar composition. Prokaryote communities were dominated by Alpha- Beta-, and Gamma-proteobacteria, Actinobacteria, Firmicutes (Clostridia and Bacilli). Within fungal communities, Sordariomycetes comprised over 90% of the taxa present. Microbiomes of cambial and pith tissues were distinct to those niches. Overall, we provide further support that the wood of conifers is host to distinct microbiome communities. Microbiomes in these niches are profoundly placed to impact tree physiology, health, and fitness, through to ecosystem function and global carbon cycles.