Tremblay, É. D., D. Shearlaw, H. D. T. Nguyen, G. J. Bilodeau and S. Hambleton. 2019. Laboratory testing of qPCR assays designed in silico reveal promising results to rapidly identify phytopathogenic Tilletia species. In Plant Canada 2019 meeting. July 7

Tilletia species are grass fungal pathogens of concern, especially for grain-exporting countries like Canada, because some cause diseases on wheat. Among them, Tilletia indica (Karnal bunt) and T. controversa (dwarf bunt) are subject to quarantine regulations and therefore are highly unwanted in areas that are free of those species. Both pathogens are closely related to un-regulated species: T. indica to T. walkeri and T. controversa to T. caries or T. laevis. There is a need for more rapid and sensitive diagnostic tools compared to the morphological methods currently used by regulatory agencies. Recent in silico comparative genomics analyses identified sets of candidate qPCR primers and probes targeting single copy genes that are species-specific. These markers have been tested in the laboratory against DNA from pure culture isolates for nine species: T. indica, T. walkeri, T. asperifolia, T. barclayana, T. fusca, T. rugispora, T. laevis, T. controversa, and T. caries. Although some assays showed differing levels of cross-reactivity, one for T. walkeri and one for T. indica were highly specific with promising sensitivity values (30-200 fg). The next steps will be to increase sensitivity by optimizing the parameters and confirm efficacy by testing against Tilletia-infected field samples. The validated assays will help improve diagnostic throughput, speed and accuracy for these agriculturally important pathogens.