Detection and prevention: Improving techniques to manage Phytophthora agathidicida, the causal agent of kauri dieback : A thesis submitted in partial fulfilment of the requirements for the Degree of Master of Science at Lincoln University

Abstract

Phytophthora agathidicida is the causal agent of kauri dieback, which threatens fragmented, remnant kauri (Agathis australis) forests in Aotearoa|New Zealand. Presently, there are persistent knowledge gaps in relation to detection and prevention of kauri dieback, which this study aims to address. Molecular detection methods for P. agathidicida using real-time polymerase chain reaction (qPCR) and loop-mediated isothermal amplification (LAMP) have been published, however, they are not yet optimised for testing environmental samples, such as soils. We modified the DNA extraction methods from the qPCR protocol, with a focus on improving both cell lysis and extract purity. We then compared the efficacy of DNA recovery between a manual DNA extraction method and two commercial DNA extraction kits. Soil shaking with a mechanical apparatus that contained ball bearings improved the yield of extracted DNA over handshaking, although there was no significant difference in yield when comparing glass beads to steel ball bearings of different sizes. Despite efforts to reduce the co-precipitation of humic acids with DNA, extract purity remained too compromised for downstream analysis without the use of a commercial clean-up kit. P. agathidicida was detected in all manually extracted samples, whereas detection with commercial extraction kits was inconsistent. The second part of this study addressed dieback prevention by testing the efficacy of four anti-oomycete fungicides along with five essential oils (EOs) on P. agathidicida mycelial growth. The sensitivity range and average values (in parentheses) of fungicide concentrations that reduced mycelial growth by 50% (EC50) for ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin were 0.072 to 0.104 µg/ml (0.087), 0.303 to 0.414 µg/ml (0.369), 0.018 to 0.022 µg/ml (0.020), and 1.30 x 10-4 to 1.70 x 10-4 µg/ml (1.55 x 10-4), respectively. Exotic plant EOs (Thymus vulgaris and Pelargonium graveolens) more effectively inhibited mycelial growth than indigenous plant EOs (kānuka and mānuka), although essential oils as a whole were significantly less effective at reducing mycelial growth when compared to fungicides. Several compounds identified in these essential oils by gas chromatography-mass spectrometry warrant further screening against P. agathidicida due to their known antimicrobial properties. The findings of these chemical trials may offer alternative or supplementary treatments to phosphite injections, which are the only know treatment, but not cure, for kauri dieback. Together, these experiments aim to improve P. agathidicida detection and prevention techniques, which are critical components of disease management and essential for the long-term survival of kauri.