A PCR, qPCR, and LAMP toolkit for the detection of the wheat blast pathogen in seeds

Wheat blast is a devastating disease caused by the pathogenic fungusPyricularia oryzae.Wheat blast first emerged in South America before more recently reaching Bangladesh. Even thoughthe pathogen can spread locally by air-dispersed spores, long-distance spread is likely to occur viainfected wheat seed or grain. Wheat blast epidemics are caused by a genetic lineage of the fungus,called theTriticumlineage, only differing from the otherP. oryzaelineages by less than 1% geneticdivergence. In order to prevent further spread of this pathogen to other wheat-growing areas inthe world, sensitive and specific detection tools are needed to test for contamination of traded seedlots by theP. oryzae Triticumlineage. In this study, we adopted a comparative genomics approach toidentify new loci specific to theP. oryzae Triticumlineage and used them to design a set of new markersthat can be used in conventional polymerase chain reaction (PCR), real-time PCR, or loop-mediatedisothermal amplification (LAMP) for the detection of the pathogen, with improved inclusivity andspecificity compared to currently available tests. A preliminary biological enrichment step of theseeds was shown to improve the sensitivity of the tests, which enabled the detection of the targetat an infection rate as low as 0.25%. Combined with others, this new toolkit may be particularlybeneficial in preventing the trade of contaminated seeds and in limiting the spread of the disease