Development of a qPCR assay and a LAMP assay forVerticillium longisporumdetection and a triplex qPCR assay for simultaneous detection ofV. longisporum,Leptosphaeria biglobosaandL. maculansfrom canola samples

Abstract

AbstractVerticillium wilt, Verticillium stem striping, and Verticillium stripe, are common disease names that all denote infection caused byVerticillium longisporum, on canola, or other Brassica crops. In this study, a quantitative PCR (qPCR) assay and a loop-mediated isothermal amplification (LAMP) assay were developed for the detection ofV. longisporumfrom canola stem samples. Both assays are specific toV. longisporumat the species level and ubiquitous at the strain level. The low limit for positive detection of the two assays is 1 pg fungal DNA in a 20-µ L reaction or 1,400 fungal cells in 100-mg plant tissue. The qPCR assay was combined with the duplex qPCR assay for the two blackleg pathogens,Leptosphaeria biglobosaandL. maculansto 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. longisporumwas 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 ofV. longisporum, especially when its presence is masked or obscured by symptoms of blackleg.