The Rapid Detection Method Of Tilletia Horrida And Ustilaginoidea Virens

Kernel smut of rice is a kind of fungal disease leading to pathological changes of rice grain caused by Tilletia horrida Tak.In recent years,with the promotion of hybrid rice and the increase of hybrid rice seed yield,the harmful increasingly serious,especially in hybrid rice farming,which gradually becomes one of the major diseases of rice.It affects the rice seed yield and hybrid rice seed quality seriously.It is a fundamental measure to establish and implement a more stringent plant quarantine system so as to prevent the rice seeds with disease from entering into the disease-free rice areas.Therefore,it is of very important significance to control the disease from the source and strengthen the detection of pathogenic bacteria,in order to predict the disease,and give scientific guidance on the prevention and cure of the disease.Rise false smut is rice grain fungous disease caused by Ustilaginoidea virens(Cooke)Tanaka.In recent years,with the promotion of high-yielding hybrid rice varieties and the level of fertilization,the occurrence of rise false smut is increasing year by year.Rise false smut not only reduces rice quality,affects rice yield,but also produces harmful toxins that cause harm to human beings and animals.The prevention and treatment of rise false smut mainly relies on chemical control.Therefore the prevention and cure of rise false smut relies heavily on the monitoring and prediction of pathogenic bacteria.In this study,real-time PCR,dd PCR and MALDI-TOF-MS identification technologies were used respectively to study the rapid detection technology of the teliospore of T.horrida and U.virens.It establishes Real-time PCR rapid detection system of T.horrida and U.virens spores;it assesses the application in the detection of T.horrida spores and U.virens spores;it evaluates the intracellular macromolecular substance using MALDI-TOF-MS technology,to get the characteristic fingerprints of T.horrida spores and U.virens spores.The results are as follows:1.The establishment of Real-time PCR rapid detection system for T.horrida spores.Based on the Real-time PCR technology,the standard curve of T.horrida spores quantity(y)and corresponding amplification Ct value(x)was built:Y=-0.2966x+10.529,(R~2=0.9974,P<0.0001).Quantitative seeds were mixed into rice of T.horrida spores of known concentration,the Real-time PCR test results showed significant positive correlation with the spores amount,and there was no significant difference(R~2=0.9329,P<0.0001).It conducted reliability demonstration of the Real-time PCR standard curve,the variation coefficient of technical repetition was between 0.46%and0.95%,which indicated that the method had high sensibility and good repeatability.2.The establishment of Real-time PCR rapid detection system of U.virens spores.The standard curve of U.virens spores quantity(y)and corresponding amplification Ct value(x)was built based on the Real-time PCR technology:Y=-0.2689x+10.508,(R~2=0.9937,P<0.0001).Quantitative seeds were mixed into U.virens spores of known concentration,the Real-time PCR test results showed significant positive correlation with the spores amount,and there was no significant difference(R~2=0.9329,P<0.0001).It conducted reliability demonstration of the Real-time PCR standard curve,the variation coefficient of technical repetition was between 0.45%and 0.83%,which indicated that the system had high sensibility and good repeatability.3.The establishment of normalization condition for the MALDI-TOF-MS appraisal of plant pathogenic fungi spores.In this study,comparison was made from pre-processing methods,matrix and point method,to optimize the main factors influencing MALDI-TOF-MS analysis.The results showed that the pre-processing methods of T.horrida spores and U.virens spores had the greatest influence on the test results.The determinatio that more complete biological information can be obtained using heat treatment in fungus sample treatment of thick cytoderm.The standard method of MALDI-TOF-MS appraisal of T.horrida spores and U.virens spores was established.Simple,fast and accurate appraisal of the fungus sample with thickened cytoderm can be made through expanding MALDI-TOF-MS fingerprint database.