Development of a Rapid, Field‐Based Method and a Real‐Time Multiplex PCR for Simultaneous Detection of Multiple Forestry Phytophthora Species

ABSTRACT

There are currently several Phytophthora species that are of major concern for forestry. Three of these species: P. pluvialis , P. pseudosyringae and P. ramorum, share a similar host range across the British Isles and display similar symptoms and disease progression. As P. pluvialis and P. ramorum are regulated pathogens in Great Britain, early and accurate detection is fundamental for appropriate management of these potentially invasive and highly destructive pathogens. We developed two diagnostic tests, both targeting the mitochondrial gene Cytochrome c oxidase subunit 2 (cox2): a multiplex real‐time polymerase chain (RT‐PCR) reaction, which enables simultaneous detection of these three Phytophthora species, and an isothermal recombinase polymerase amplification (RPA) assay for detection of P ramorum and P. pluvialis . The RT‐PCR is highly specific and sensitive, with a limit of detection (LOD) ranging from 7.4 to 36 fg of mycelial DNA and is suitable for detection from infected plant material, with > 90% diagnostic accuracy. The RPA can be used at the point of sampling and provides results within 20 min. This assay showed high specificity at LODs between 1 and 10 pg of mycelial DNA, depending on species and lineage. Although less sensitive than the RT‐PCR assay, this rapid, frontline test is suitable for plant matrices, with positive detections from samples with a RT‐PCR positive of < 30 cycles. These new diagnostic tools improve accuracy and speed of detection, reducing cost without compromising sensitivity. The RPA assays provide rapid results in the field, borders and ports, and enable effective triage of samples for laboratory analysis.