| Tilletia controversa Kühn (TCK), one of the important international quarantine fungal pathogens, causes wheat dwarf bunt disease (DB) which distributes in America, Europe, Northern Africa and West Asia, especially in Northwest states of USA. It is a typical soil borne disease and lack of effient control measurement. TCK teliospores were found in the imported wheat frequency which came from wetern countries such as America, Canada and European Union. Establishment of risk analysis of TCK indicates that acreage of areas with high risk possibility is 19.3% of the whole winter wheat regions of the country and lack of resistant varieties of wheat.A lot of methods have been used to detect wheat pathogen, including visual method based on symptoms and pathogen morphology, serology detection, and several polymerase chain reaction assays. The application of conventional and real-time PCR, specially, provides for the detection and quarantine of plant pathogen a more rapid, more sensitive,more precise and more convenient method. All these methods, can only detect single target and have differnt disadvantages, for instance time-consuming, troublesome, and subjectivity. Therefore, it can not satisfy with the agricultural products and foodstuff quickly entering customs and efficiently controlling flow of quarantine pathogen from infected or contanminated wheat. Though mulriple PCR can can be used to detect several targets simutaniusly, which are difficult to be optimized due to the competition and restrain among the different primer pairs influence directly the specificity and st??ity of the detection. Padlock probes are long single-strand oligonucleotides, whose ends are complementary to adjacent target sequences. The link sequence of the padlock probe is not necessary for target detection and so can design the universal primers for amplification. The special structure of the padlock probe, which can be amplified and detected by many means, thus meets the need of specificity and multi analysis of detection.In this research, Tilletia controversa Kühn strains are used for research object. Padlock probe for TCK was designed based on the species-specific DNA fragment (1322 bp) of TCK. And amplification primers of padlock probe were designed according to the universal linking sequence of padlock probe which came from Xist gene of mice; the ligation, digest condition, and rolling circle amplification system were all optimized, and then hyper-branched rolling cycle amplification (HRCA) detection method for TCK was, whose specificity and sensitivity were tested compared with conventional PCR and SYBR Green I RTi-PCR, was developed; Specificity, sensitivity and stability of RCA method were then tested by detecting the different samples of CB. Fosmid genomic DNA library of TCK was constructed for further determination of TCK genetic diversity. The main results were as follows:①Padlock probe was designed based on the unique gene sequence of TCK and tested the specificity. Experiment result indicated that the padlock probe can discriminate the single base mismatch. Mismatches positioned at the 3′end of PLP were strongly discriminating, while those at the 5′end provided much less specificity. This showed satisfactory base specialization of PLP.②This research established a optimized detection system of TCK with HRCA method, which provided a stable, reliable and novel technique for early diagnosis of CB disease. HRCA is capable of amplifiying teliosporal and mycelial DNA of TCK, while not detecting related smut species of Tilletia spp and other plant pathogen, which is accordant with conventional PCR and RTi-PCR. The detection sensitivity of HRCA are 1 fg/μL for plasmid DNA and 10 pg/μL for genomic DNA of TCK respectively, which is 10-fold higher than that of conventional PCR. The detection sensitivity of HRCA is 10-fold lower than that of RTi-PCR. The results also show HRCA method to be specific, sensitive and accurate. HRCA provided a new approach to the regulation and prevention of wheat dwarf bunt disease and identification of TCK.③The HRCA detection s??m of can apply to detecting the teliospore and mycelia of TCK of varied samples from wheat field. The detection result of field sample with HRCA method was almost the same as conventional PCR and SYBR Green I RTi-PCR. It showed that the HRCA method was stable and reliable and was fit for detetcting TCK sample.④The large fragment fosmid genomic DNA library of TCK was constructed with an average insert DNA fragment of 40kb. The library titre was 1.8×104cfu/ml and library capacity was 3.6×104cfu. This indicates the quality of library was very well.
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