Abstract
A Stagonospora nodorum strain named DPGZL-2023 was created by transferring a green florescent protein (GFP) gene into the genome of the S. nodorum strain Sn15. DPGZL-2023 showed a similar pathogenicity as Sn15 but carried a strong GFP activity. A qPCR primers/probe set named P-GFP, targeting the GFP sequence, was designed. Using P-GFP, qPCR analysis was conducted on DNA extracted from replicated samples of DPGZL-2023 conidia, and confirmed that DPGZL-2023 could be used to characterize the variation in replicated DNA extractions. Conidia of DPGZL-2023 were used to spike soil samples inoculated with the canola clubroot pathogen Plasmodiophora brassicae, canola stem samples infected with the blackleg pathogens Leptosphaeria biglobosa and/or L. maculans and wheat/barley samples infected with Xanthomonas translucens pv. translucens (Xtt) or X. translucens pv. undulosa (Xtu). Duplex qPCR using P-GFP and a primers/probe set specific to P. brassicae, triplex qPCR using P-GFP and primers/probe sets specific to L. biglobosa and L. maculans, and triplex qPCR using P-GFP and primers/probe sets specific to Xtt and Xtu were conducted. The results indicated that DPGZL-2023 could be used as a standard for DNA extraction efficiency in qPCR-based plant disease diagnosis. Adding DPGZL-2023 conidia to plant or soil samples prior to DNA extraction, and subsequent use of the P-GFP detection control, provided an added control that could distinguish truly negative from false-negative qPCR results.