Development of a SCAR marker for molecular detection and diagnosis of Tilletia controversa Kuhn, the causal fungus of wheat dwarf bunt

Tilletia controversa Kuhn (TCK) is an important quarantine pathogen that causes wheat dwarf bunt and results in devastating damage to wheat production. The fungus is difficult to be distinguished from T. caries and T. laevis, which cause wheat common bunt, based on morphological, physiological and symptomatological characteristics of the pathogens. The traditional detection of the fungus can be a long and tedious process with poor accuracy. The inter-simple sequence repeat (ISSR) technique has been used for identifying molecular markers for detection of TCK. Of 28 ISSR primers screened, ISSR-859 amplified a specific 678 bp DNA fragment from all TCK isolates but not from any isolates of the common bunt fungi or other pathogenic fungi tested. Based on the fragment sequence, a pair of sequence characterized amplified region (SCAR) primers was designed, which amplified a 372 bp DNA fragment specifically in TCK. The SCAR marker was detected using as low as 1 ng template DNA of TCK, and was also detected using broken teliospores and DNA from asymptomatic wheat samples. We developed the SYBR Green I and TaqMan Green I and TaqMan real-time polymorphism chain reaction methods to detect TCK with the detection limit of 0.1 fg with asymptomatic wheat samples. Further work is needed to develop a rapid test kit for this pathogenic fungus using the designed specific primers.