Detection Of Tilletia Controversa Kühn By PCR And Real Time Quantitative PCR

Tilletia controversa Kühn (TCK), one of the important international quarantine fungal pathogens, causes wheat dwarf bunt disease (DB) which distribute in America, Europ, Northern Africa and West Asia, especially in 7 Northwest states of USA. Tilletia tritici Tu(lTCT)is the most relative classification taxa to TCK in teliospore morphology and genomic structure and it is so difficult to distinguish TCK from TCT. Although many researchers in abroad and domestic tryed to identify and analyze species-specific sequences among Tilletia species by analyzing internal transcribed spacer (ITS) region sequences, random amplified polymorphism DNA (RAPD) and repetitive-sequence- based polymerase chain reaction (rep-PCR) genomic fingerprinting, no one find specific fragment for differentiating TCK from TCT. The teliospore morphology of TCK and TCT is so siminar that it is difficult to distinguish, and then accurate identification methodology of the TCK and TCT is high priority for desease quarantine. With the introduction of infected wheat and lack of rapid detection technology, it can not satisfy with the agricultural products and foodstuff quickly entering a contry and efficiently controlling the ebb and flow of quarantine pathogen from infected or contanminated wheat. So it is necessary to develop quick, accuracy and reliable molecular detection technology to identification TCK and TCT. In this reseach, we designed specific primer pairs based on the species-specific DNA fragment of TCK (1322 bp) from telomere associate gene of TCK and successfully established the routine PCR and real-time PCR detection system for teliospore or mycelium of TCK, which will facilitate accurate detection and identification of TCK teliospore on seeds, soil, flower and feeds and infected mycelium inside of wheat tissue for development scientific strateges of desease risk assessment and management. The main results were as follows:①The genomic DNA extraction method from TCK/TCT teliospore was established, which include cell wall lysis of teliospore by alkali solution, destroying teliospore cell wall for releasing nucleic acid from cytoplasma membrain and elimination of the PCR inhibitors such as pigments or polyphenols component. This method resoved the difficult extraction the DNA of TCK/TCT teliospore due to its rigid cell wall and pigments and the extracted DNA capable to applying to PCRamplification. In addition, the genomic DNA of mycelia of TCK/TCT was availably extracted by CTAB method or benzyl chloride method and the field samples of DB were prepared by FTA card to mail and preserve safely.②Three routine PCR detection systems were developed. The specific primer pairs CQUTCK2/CQUTCK3,CQUTCK4/CQUTCK5 and CQUTCK6/CQUTCK7 were designed based on the species-specific DNA fragment of TCK (1322 bp) from telomere associated sequences (TAS) and the predicted amplification bands were 747bp, 644bp and 200bp respectively. In PCR detection systems, the DNA of recombinant plamid of TCK was used for safely positive reference and genomic DNA of the healthy wheat and rye was used for negative reference. The internal reference was established by the universal primer pair CQUTC6/CQUTC7 for Tilletia genera, which could be used to determinate the reality of PCR system and exclude both false positive and false negative. Using the primers CQUTCK2/CQUTCK3, CQUTCK4/CQUTCK5 and CQUTCK6/CQUTCK7, the DNA fragments of 18 physiological races of TCK from USA could be amplifyed, while those of all the stains of TCT could not be detected and the sesentivity of PCR could reach to100fg per reaction. It showed that the PCR detection system was specific and could be used for detection of the teliospore and myceliums of TCK from pure culture.③The SYBR Green I and TaqMan quantitative PCR detection systems were established. The specific primers pair CQUTCK4/CQUTCK5 and probe CQUP1 used for the two systems were selected by the primer design software and the reaction conditions were optimized. In the two systems, the standand cuve were constructed by the ten fold diluted DNA of recombinant plasmid and the sensentivity for DNA of recombinant plasmid was 1-2fg and that for genomic DNA of TCK was 5-10fg, which was higher 102-103 fold than PCR detection system. It showed that the RTi-PCR detection system was specific and capable to earlier diagnosis of mycelia of TCK from infected wheat plant tissue.④Using the established PCR system, the contaminated 4000 TCK spores per 50 grams of grain were sucessully detected, which was ten fold lower than the standard level (30000 TCK spores per 50 grams of grain), and DNA of the contaminated TCT could not be amplifyed. Moreover, the 196 samples of fungous gall of Tilletia or infected tissue from domestic during 2006-2007 were detected and the detection results were almost the same as disease situation in wheat field. It showed that the PCR sytems were stable and reliable. On the other side, the asymptomatic wheat plant samples were detected by PCR and real-time PCR. No bands were produced by routine PCR system, but the wheat samples at 3-leaf stage to turning green stage were positive by real-time PCR. By the calculated results of quantitative PCR, the TCK DNA of positive samples was so little that it is under the limit of detection. It showed that the sensentivity of real-time PCR was higher than routine PCR and the real-time PCR was more adaptive for detectiong asymptomatic samples.⑤The dryness reagent kits of routine PCR and real-time PCR were devolped by stabilization reagents of PCR with with the freezing air drier of vacum. The prepared reagent kits could be preserved for 4-6 months at room temperature and effectively used for TCK detection instantly.