Thesis: Improving techniques to manage ...

Thurston A. 2021. Detection and prevention: Improving techniques to manage Phytophthora agathidicida, the causal agent of kauri dieback. Unpublished Masters of Science thesis, Lincoln University. 202 p.

Real-time polymerase chain reaction (qPCR) and loop-mediated isothermal amplification (LAMP) have been published as molecular detection techniques for P. agathidicida; however, they are not yet optimized for testing environmental samples, such as soils.

In this research two experiments were conducted.

1. DNA extraction methods from the qPCR protocol were modified, 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. 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.   

2. The efficacy of four anti-oomycete fungicides along with five essential oils (EOs) were tested 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.