Development of a Triplex Quantitative PCR System for the Detection of <i>Parastagonospora nodorum</i> , <i>Zymoseptoria tritici</i> , and <i>Pyrenophora tritici-repentis</i> from Wheat

A triplex quantitative PCR (qPCR) system was developed for the simultaneous detection of the three most prevalent wheat leaf spot diseases: Septoria nodorum blotch caused by <i>Parastagonospora nodorum</i>, Septoria tritici blotch caused by <i>Zymoseptoria tritici</i>, and tan spot caused by <i>Pyrenophora tritici-repentis</i>. In this system, the primer set for <i>P. tritici-repentis</i> targets a species-specific multicopy genomic region, whereas the primer sets for the other two pathogens target the ribosomal DNA (rDNA) region. The specificity of the system was validated through sequence analysis using the currently available database and by testing against 24 DNA samples from nontarget species. Sensitivity testing on serial DNA dilutions from the three target species demonstrated that the system can detect as little as 2 fg of DNA of each species in a 20-μl reaction. For <i>P. nodorum</i>, the system was capable of detecting DNA extracted from a conidia suspension containing as few as 100 conidia. The system was further evaluated on 145 wheat leaf samples (45 symptomatic and 100 asymptomatic) collected from various fields in Alberta, Canada. At least one of the three pathogens was detected in 112 out of the 145 samples, with <i>P. nodorum</i> and/or <i>P. tritici-repentis</i> identified in 74 of the 100 asymptomatic samples. This triplex qPCR system offers a powerful tool for the diagnosis of wheat leaf spot diseases, surveillance, breeding for disease resistance, and research in epidemiology, population genetics, and host-pathogen interactions.