Exploring Phytophthora agathidicida Oospore Viability and Germination

Abstract

<p><b>Kauri trees are a species native to the northern parts of Aotearoa/New Zealand. They are a crucial part of the ecosystem and are taonga (sacred/treasured) to the indigenous people. However, kauri are threatened by kauri dieback disease caused by the oomycete Phytophthora agathidicida. P. agathidicida has a complex lifecycle involving several important spore types. Arguably the oospore is the most important as these spores are essential for the long-range transmission of disease. In the field, oospores can survive extreme physical and chemical stresses and are capable of persisting dormant in the soil for years before germinating and continuing the disease cycle. However, these spores are difficult to produce and study in the laboratory, with few techniques available for P. agathidicida. The overall goal of my thesis was to explore the chemical signals that trigger or suppress oospore germination. The ability to modulate oospore germination has potential applications in controlling and mitigating disease outbreaks. </b></p> <p>The first specific aim of this research was to develop a protocol for producing and isolating viable P. agathidicida oospores in vitro. Incubation time, media type and the addition of β-sitosterol were explored as variables to optimise oospore growth. The optimal growth conditions were clarified 10% (w/v) carrot broth with 30 mg/mL β-sitosterol, grown for >3 weeks at 22 °C, in the dark. The optimised protocol produced approximately 5-fold more oospores than existing methods. I quantified the viability of laboratory-produced oospores using two different techniques: (i) germination in the presence of kauri extract and (ii) a colourimetric viability assay that utilises methylthiazolyldiphenyl tetrazolium bromide (MTT) to distinguish between viable and non-viable oospores. Overall, a method for producing and isolating oospores in vitro was successfully developed, with yields, viability and germination rates comparable to those reported in the literature for other Phytophthora species.</p> <p>During the development of the oospore production protocol, it became clear that an improved assay for quantifying oospore viability was needed: the MTT-based viability assays are based on subjective assessment of colour and therefore are low-throughput and potentially unreliable. Therefore, the second specific aim of this research was to develop a high-throughput fluorescence-based method for quantifying oospore viability. I selected and tested five different fluorescent dyes (fluorescein diacetate, FUN-1, SYTO 9, propidium iodide, and TOTO-3 iodide) for their effectiveness at staining viable and/or non-viable oospores. First, I developed a viability staining assay using fluorescein diacetate and TOTO 3 iodide that effectively differentiated viable, non-viable, and damaged oospores. I then developed a workflow to automate the detection of oospore viability using Ilastik, R, and CellProfiler. This pipeline increased the throughput of the assay and removed user bias. </p> <p>The third specific aim of this research was to develop a germination assay and apply it to find modulators of oospore germination. Due to the asynchronous germination of oospores, the germination assay was scored qualitatively. A 96-well plate-based assay utilising kauri root extract was developed and used to screen a compound library containing 379 compounds. This yielded 11 compounds that either activated or inhibited the germination of oospores. Four of these chemicals (decanoic acid, D-glucosamine, hydroxylamine, and alloxan) were validated as inhibitors of oospore germination in subsequent germination assays. These four chemicals were also tested using the fluorescence-based viability assay, killing between 25–94% of oospores. A further two chemicals (L phenylalanine and myo-inositol) were validated as activators of oospore germination. This screening assay has not only led to the discovery of several compounds that could be explored for use as control agents but has also shown that this method is a valuable method for discovering chemicals that are lethal to oospores.</p> <p>The final aim of this research was to transform P. agathidicida protoplasts and utilise RNA interference to understand the molecular triggers that cause oospores to germinate. I optimised a protoplasting protocol for use with P. agathidicida and succeeded in producing large numbers of protoplasts. However, I could not generate a stable transformant of P. agathidicida. </p> <p>Overall, this research has provided new tools for the study of oospores, as well as insights into the chemicals that modulate oospore germination. These results will hopefully facilitate further studies on this key part of the Phytophthora disease cycle and ultimately support the development of tools to manage kauri dieback disease.</p>