Development of a qPCR and a Lamp Assay for Verticillium longisporum Detection and a Triplex qPCR Assay for Simultaneous Detection of V. longisporum, Leptosphaeria biglobosa, and L. maculans from Canola Samples

Abstract

Verticillium wilt, Verticillium stem striping, and Verticillium stripe are common disease names that all denote infection caused by Verticillium longisporum on canola or other brassica crops. In this study, a quantitative PCR (qPCR) and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection of V. longisporum from canola stem samples. Both assays are specific to V. longisporum at the species level and ubiquitous at the strain level across all whole-genome sequenced strains. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µl reaction or 700 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens, Leptosphaeria biglobosa and L. maculans, to constitute a triplex qPCR system for simultaneous detection of all three pathogens. The usefulness of this triplex qPCR system was verified on canola samples collected from various locations in Alberta, Canada. Using this triplex qPCR system, V. longisporum was detected from one sample, while the two blackleg pathogens were detected at higher frequencies. Since it is sometimes difficult to differentiate Verticillium stripe and blackleg on Alberta canola samples based on visual symptoms, the triplex qPCR system is an important tool for the detection of V. longisporum, especially when its presence is masked or obscured by symptoms of blackleg. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .