Development Of A Molecular Detection Of Tilletia Controversa Kühn And Ultrastructural Observation Of The Infection Cycle

Dwarf bunt of wheat(DB), caused by Tilletia controversa Kühn(TCK) is an international quarantine disease on wheat in China. Tilletia tritici Tul(TCT) is the most relative classification taxa to TCK in teliospore morphology and genomic structure and it is so difficult to distinguish TCK from TCT. So it is necessary to develop quick, accuracy and reliable molecular detection technology to identification TCK and TCT. Fungi in host to complet e the form of mycelia growth cycle of infection for the teliospore, forming the interaction mode.In this reseach, we designed specific primer pairs based on the species-specific DNA fragment of TCK(661bp) from telomere associate gene of TCK and successfully established the routine PCR and real-time PCR detection system for teliospore or hyphostroma of TCK, which will facilitate accurate detection and identification of TCK teliospore on different infected mycelium inside tissue of wheat. Besides, The infection process of DB by TCK was investigated with electron microscopy. The results were as follows:1、Two routine PCR detection systems were developed. The specific primer pairs were designed based on the species-specific DNA fragment of TCK(661bp) and the predicted amplification bands were 372 bp and 112 bp respectively. Using the specific primers, the DNA fragments of 5 physiological races of TCK could be amplifyed, while all isolates of the other species could not be detected and the sesentivity of PCR could detect the SCAR fragment with one teliospore. 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.2、The SYBR Green I and Taq Man quantitative PCR detection systems were established. 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 0.1fg, which was higher 102-103 fold than PCR detection system. It showed that the RT-PCR detection system was specific and capable to earlier diagnosis of mycelia of TCK from infected wheat plant tissue.3 、 The asymptomatic wheat plant samples were detected by PCR an d real-time PCR. Both routine PCR system and real-time PCR could detect hyphostroma of TCK from infected wheat plant tissue. By the calculated results of quantitative PCR, it showed that the real-time PCR was more adaptive for detectiong asymptomatic samples.4 、 The infection process of wheat samples at different growth stages following artificial inoculation were observed by confocal laser scanning microscope. Following penetration of the coleoptiles in wheat, intercellular hyphae reached cells of growing point. Anther infected following ovary infected to fertilization; flag leaf, stem of mature period could be simultaneously observed teliospore and hyphostroma.5、In the early stage of pathogenesis, the ultrastructure of mesophyll cell were significantly changed after infection by SEM and TEM, such as plasma membrane invaginations and fracture 、 deformity、 from mesophyll cells, the nucleus structure was destroyed、cytoplasmic density deepen. The empty cells and fibrous membrane of intercellular space. The teliospore of root、stem、grain and flag leaf at mature stage can be observed with the TEM.