Using a GFP-labeled Stagonospora nodorum strain as a DNA extraction efficiency standard in plant disease diagnosis

Abstract

Quantitative polymerase chain reaction (qPCR) is a commonly used technique for plant disease diagnosis, which generally requires DNA extraction from the plant samples. The efficiency of DNA extraction has a significant influence on the accuracy of qPCR. Thus, including a DNA extraction efficiency control is important in qPCR analyses. To develop and evaluate a DNA extraction efficiency control, a Stagonospora nodorum (syn. Phaeosphaeria nodorum, synonym and correct taxonomic name: Parastagonospora nodorum) strain named HUA-22 was created by transferring a green florescent protein (GFP) gene into the genome of the S. nodorum strain Sn15. HUA-22 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 HUA-22 conidia, and confirmed that HUA-22 could be used to characterize the variation in replicated DNA extractions. Conidia of HUA-22 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 HUA-22 could be used as a standard for DNA extraction efficiency in qPCR-based plant disease diagnosis. Adding HUA-22 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.