Development of a triplex qPCR system for the detection of and from wheat.
Heting Fu, Junye Jiang, Michael Wayne Harding, Kher Zahr, Yalong Yang, Shiming Xue, Ronald Nyandoro, Maria Antonia Henriquez, Lipu Wang, David Feindel, Jie Feng
Author Information
Heting Fu: Alberta Plant Health Lab, Edmonton, Alberta, Canada; Heting.Fu@gov.ab.ca.
Junye Jiang: Alberta Plant Health Lab, Edmonton, Alberta, Canada; junye.jiang@gov.ab.ca.
Michael Wayne Harding: Alberta Agriculture and Rural Development, Plant Pathology, 301 Horticultural Station Road East, Brooks, Alberta, Canada, T1R 1E6; michael.harding@gov.ab.ca.
Kher Zahr: Alberta Plant Health Lab, Edmonton, Alberta, Canada; kher.zahr@gov.ab.ca.
Yalong Yang: Alberta Plant Health Lab, Edmonton, Alberta, Canada; yalong.yang@gov.ab.ca.
Shiming Xue: Alberta Plant Health Lab, Edmonton, Alberta, Canada; shiming.xue@gov.ab.ca.
Ronald Nyandoro: Alberta Plant Health Lab, Edmonton, Alberta, Canada; ronald.nyandoro@gov.ab.ca.
Maria Antonia Henriquez: Agriculture and Agri-Food Canada, Science and Technology Branch, 2701 Grand Valley Road, Box 1000A, R.R. #3, Brandon, Manitoba, Canada, R7A 5Y3; MariaAntonia.Henriquez@agr.gc.ca.
Lipu Wang: University of Saskatchewan, Plant Sciences, 51 Campus Drive, Saskatoon, Saskatchewan, Canada, S7N 5A8; lipu.wang@usask.ca.
David Feindel: Alberta Plant Health Lab, Edmonton, Alberta, Canada; david.feindel@gov.ab.ca.
Jie Feng: Alberta Plant Health Lab, Edmonton, Alberta, Canada; Jie.Feng@gov.ab.ca.
A triplex qPCR system was developed for the simultaneous detection of the three most prevalent wheat leaf spot diseases: septoria nodorum blotch caused by Parastagonospora nodorum, septoria tritici blotch caused by Zymoseptoria tritici, and tan spot caused by Pyrenophora tritici-repentis. In this system, the primer set for P. tritici-repentis targets a species-specific multicopy genomic region, while 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 non-target 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-��L reaction. For P. nodorum, 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 P. nodorum and/or P. tritici-repentis 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.
