Development of a Duplex qPCR System for Detection and Quantification of the Two Canola Blackleg Pathogens Leptosphaeria biglobosa and L. maculans

Two probe-based qPCR systems, namely P-Lb and P-Lm, specific to the canola blackleg pathogens Leptosphaeria biglobosa and L. maculans, respectively, were developed, and their efficiencies were tested. Each of the two systems targets a single-copy gene exclusively present in the corresponding species. The specificities of the two systems on the species level and their ubiquities on the subspecies level were confirmed by in silico sequence analyses and testing on L. biglobosa (17 strains), L. maculans (10 strains), and other plant pathogens (31 species). For sensitivities, the two systems were tested on synthesized DNA fragments (gBlock) of the targeted regions, from which a standard curve was generated for each system. In addition, standard curves were also generated on gBlocks for duplex qPCR in which the two systems were used in the same reaction. The two systems were further tested in both singleplex and duplex qPCR on DNA samples extracted from fungal spores, inoculated canola cotyledons, and naturally infected canola stubble samples collected from commercial fields. Our data indicated that the two systems are specific to L. biglobosa and L. maculans, respectively, and one reaction could detect as few as 200 spores of either species. When used in duplex qPCR on DNA samples with various origins, the two systems generated similar results as in singleplex qPCR. The duplex qPCR system, along with the sample preparation and DNA extraction specified in this study, constituted a first-reported duplex qPCR protocol for detection and quantification of the two blackleg pathogens from field samples.

Automated summary

Two probe-based qPCR systems, P-Lb and P-Lm, were developed for specific detection of Leptosphaeria biglobosa and L. maculans, the blackleg pathogens of canola. The systems showed species-specificity and broad subspecies-level applicability. Sensitivity tests demonstrated the ability to detect as few as 200 spores of either species. When used in duplex qPCR, the systems yielded similar results as in singleplex qPCR. This study presents the first-reported duplex qPCR protocol for detection and quantification of these blackleg pathogens from field samples.