Development of a qPCR Assay for the Detection and Quantification of the Fungal Pathogen Calonectria canadiana on Conifers

Abstract

A real-time PCR TaqMan assay was developed for the detection of Calonectria canadiana, a fungal pathogen responsible for damping off, root rot and seedling blight in conifer forest nurseries in central and eastern North America. While highly significant in Quebec Forest nurseries, coniferous seedling mortality decreased significantly when nurseries transitioned from bare root to container seedling production. However, over the past few years, this pathogen has re-emerged as a threat and millions of container white spruce seedlings were culled in two nurseries in eastern Quebec. A sensitive detection and quantification assay for C. canadiana was essential to investigate the biological and environmental factors driving this new epidemic. We designed primers and a TaqMan probe targeting the internal transcribed spacer (ITS) of C. canadiana. The resulting Ccan TaqMan assay successfully differentiated C. canadiana from other soil-borne pathogens of the Nectriaceae encountered in Quebec Forest nurseries. The limit of detection of the assay was established at eight copies of C. canadiana ITS. The Ccan TaqMan assay quickly identified the presence of the pathogen in both symptomatic and asymptomatic white spruce (Picea glauca) seedlings. Furthermore, we demonstrated that the pathogen was more easily detected when DNA was extracted from necrotic needles at the base of the stem rather than from necrotic roots. This molecular tool will greatly aid in understanding the biology and epidemiology of C. canadiana.