A molecular method to assess viability of Phytophthora in infected wood following heat treatment

Abstract

International trade in wood products is an important component of the global economy. However, wood and wood products may have pests associated with them that could be introduced into importing countries, posing phytosanitary risks, and leading to the implementation of regulatory restrictions that affect wood trade. The application of heat to kill wood-associated pests has been a successful phytosanitary method to reduce their spread. To evaluate the efficacy of wood heat treatment to kill fungal and fungus-like pathogens, the method of choice has been to grow organisms in cultures for subsequent identification. However, some plant pathogens can be difficult or impossible to grow in axenic cultures and a molecular method can still be useful for assessing pathogen viability after heat-treatment. RNA is a single stranded molecule that is responsible for the transcription of genes. Since it becomes rapidly unstable after cell death, it provides a measure of viability. We therefore designed and tested RNA-based molecular diagnostic assays targeting essential genes and assessed their presence after heat treatment in wood colonized by four Phytophthora species of phytosanitary concern (P. xmultiformis, P. cinnamomi, P. lateralis and P. ramorum) through reverse transcription and real-time polymerase chain reaction (RT-qPCR). Our assays differentiate between genomic and mRNA as the TaqMan probes span exon-intron junctions. We validated these RT-qPCR assays to assess heat treatment efficacy of Phytophthora-inoculated wood. These assays can be very useful tools to assess effectiveness of current and emerging phytosanitary wood treatments.