A FIASCO-Based Approach for Detection and Diagnosis of Puccinia graminis f. sp. tritici in China

Stem or black rust of wheat, caused by the fungus Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn. (Pgt), has historically caused severe losses to wheat (Triticum aestivum L.) production worldwide. In the Fujian and Guangdong provinces of China, six moderate-to-severe epidemics of wheat stem rust have occurred, which caused destructive losses of wheat between 1949 and 1966, although these were brought under control by integrated management. A rapid and reliable detection of the pathogen will contribute to the accurate forecast and seasonal control of this disease. The objective of this study was to develop a diagnostic molecular marker generated from simple sequence repeats (SSR) for the early rapid identification of P. graminis. The genomic DNA of P. graminis, Puccinia striiformis, Puccinia triticina and seven other species was amplified by a pair of SSR primers generated by the FIASCO (fast isolation by AFLP sequences containing repeats) enrichment protocol. The primer set Pgtw (f)/Pgtw (r) generated a polymorphic pattern displaying a 330-bp DNA fragment specific for P. graminis whereas no DNA fragment was obtained from other non-target wheat fungal pathogens. The detection limit of the primer was 1 ng DNA in a 25-μL PCR reaction. The SSR markers of P. graminis can also be used to detect the presence of latent hyphae in Pgt-infected wheat leaves as early as 30 h post-inoculation. A rapid approach to distinguish P. graminis from similar pathogenic fungi would be anticipated in further study.