Ultra-Sensitive Detection of Phytophthora pluvialis by Real-Time PCR Targeting a Mitochondrial Gene

Phytophthora pluvialis is a pathogen present in the United States, New Zealand, the United Kingdom, and Belgian forests. Reported hosts include Douglas-fir in the United States, New Zealand, the United Kingdom, and Belgium, as well as tanoak in the United States, radiata pine in New Zealand, and Japanese larch and western hemlock in the United Kingdom. Disease symptoms range from needle lesions and casting on radiata pine through to twig and stem cankers and crown dieback on western hemlock, Douglas-fir, and Japanese larch. Current detection methods rely on isolation and culture or PCR targeting a single-copy gene (ypt1). A qPCR assay targeting a multiple-copy mitochondrial gene (Cytochrome c oxidase subunit 2 [cox2]) was designed to increase the sensitivity of P. pluvialis detection, critical for early diagnostics. The resulting assay has a detection limit of 12.8 fg of mycelial DNA, detecting the pathogen on average 6.12 qPCR cycles before the ypt1 assay. In New Zealand samples, the assay was found to consistently detect P. pluvialis in all stages of needle disease symptoms, from early asymptomatic infection to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in pine needles 4 weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has enabled diagnostic support of the biosecurity response in the United Kingdom during recent detection of P. pluvialis. The assay has been used in applications requiring detection at low pathogen titer levels, including asymptomatic infection, stream baiting, cast needles, and biosecurity response, demonstrating its efficacy for early detection of P. pluvialis in affected forests.