Ultra-sensitive detection of Phytophthora pluvialis by real-time PCR targeting a mitochondrial gene

Abstract

Phytophthora pluvialis is a forest tree pathogen present in the USA, New Zealand, UK and Belgium. Reported hosts include Douglas-fir in the USA, New Zealand, UK and Belgium, as well as tanoak in the USA, radiata pine in New Zealand, Japanese larch and western hemlock in the UK. 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 using a single-copy gene target with limited sensitivity at low pathogen titre. A qPCR assay targeting a multiple-copy mitochondrial gene was designed to increase sensitivity of P. pluvialis detection in forest samples, critical for informing biosecurity, long term disease management and ongoing research. The resulting assay has a detection limit of 12.8 fg mycelial DNA and can detect the pathogen on average 6.12 qPCR cycles before the single-copy target assay. In New Zealand forest samples, the assay was found to consistently detect P. pluvialis in all stages of radiata pine needle disease symptoms from early asymptomatic infection through to fully cast needles. The new assay allowed for asymptomatic detection of P. pluvialis in radiata pine needle samples four weeks before visual symptoms of disease were observed. The availability of a highly sensitive assay has also enabled rapid and confident diagnostic support of the biosecurity response in the UK during recent detection of P. pluvialis. The assay has been used in applications requiring detection at low pathogen titre levels including asymptomatic infection, stream baiting, cast needles and during biosecurity responses, making it a useful tool for effective early detection and management of P. pluvialis in affected forests.