Development of a PCR-based diagnostic tool specific to wheat dwarf bunt, caused by Tilletia controversa

Wheat dwarf bunt, one of the important international quarantine diseases, is caused by Tilletia controversa. Tilletia caries is a close relative species of T. controversa and the teliospore morphology and genomic structure of T. caries are very similar to those of T. controversa. In order to distinguish between them, a random amplified polymorphic DNA (RAPD) primer-mediated asymmetric-PCR (RM-PCR) was developed to screen differential sequences between the two pathogens. By RM-PCR, a 1,322 bp DNA fragment (PR32) was selected from 18 T. controversa and 29 T. caries strains. The PR32 genes were specific for T. controversa and almost had no homology to T. caries or other fungi in the present database. With primers designed from PR32, all 18 T. controversa strains were amplified, but no bands appeared in 29 T. caries strains by classical PCR. To identify T. controversa rapidly and accurately, SYBR Green I and TaqMan probe real-time PCR were established based on PR32. With TaqMan Real-Time PCR, different T. controversa strains and T. caries strains were detected. The results showed that all T. controversa strains were amplified with Ct from 19–29 and amplified curves were obtained. In contrast, the amplification of all T. caries strains did not show any signals, without Ct and amplified curves. Moreover, the developed TaqMan real-time PCR was used to detect T. controversa from asymptomatic wheat tissues successfully.