An ISSR-based Approach for the Molecular Detection and Diagnosis of Dwarf Bunt of Wheat, Caused by Tilletia controversa Kuhn

Dwarf bunt of wheat, caused by Tilletia controversa Kuhn, is an important international quarantine disease in many countries. The objective of this investigation was to develop a diagnostic molecular marker generated from intersimple sequence repeat (ISSR) for rapid identification of T. controversa. A total of 60 primers were tested by ISSR to detect DNA polymorphisms between T. controversa and related species. The primer ISSR818 generated a polymorphic pattern displaying a 952- bp DNA fragment specific for T. controversa. The marker was converted into a sequence characterized amplified region (SCAR), and specific primers (TCKSF2/TCKSR2) were designed for use in a PCR detection assay. Its detection limit was 1 ng of DNA, which could be yielded by 1.1 mu g of teliospores in a 25- mu l PCR. Conclusively, a method to distinguish T. controversa from similar pathogenic fungi has been successfully developed based on the use of a SCAR marker.