Tilletia controversa Kühn (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.

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.