| Tobacco bacterial wilt, caused by Ralstonia solanacearum, is destructive to tobacco production throughout the world of tropical, subtropical and warm regions, and serious to some of the world tobacco production, especially caused more serious loss in continuous-cropping tobacco fields. Ralstonia solanacearum lived in diseased plant residue in soil cause the primary infection when entering into roots and then colonizing in vascular tissues. Bacterial wilt symptoms always outbreak under a condition of high-temperature and high humidity, the infected plants showed rapid and systemic wilting, death and roots necrosis. Lack of high-resistant varieties and effective chemicals, the current control to bacterial wilt was mainly rely on cultural method and integrated pest management, but it is still difficult to control this disease. However, a sensitive and specific detection on soil-borne pathogenic Ralstonia solanacearum is crucial for disease forecast, control design, evaluation of control efficacy and resistance of variety resistance. The traditional detection has many disadvantages, such as time-consuming, multifarious operation and low sensitivity, and therefore do not meet the requirement of the rapid detection. With the development of molecular biological technology, Polymerase Chain Reaction (PCR) has been used widely for detection of pathogenic microorganism with its time-saving, high specificity and sensitivity.The goals of this study were to establish the PCR-based molecular detection system of Ralstonia solanacearum in soil, and try to apply it in detection of the number of Ralstonia solanacearum overwintered in continuous-cropping soil, and in assay of the early infection in tobacco growing season, and disease-risk assessment, and evaluating control effect of different control measures and so on. The main study results of the thesis are as follows:1. Ralstonia solanacearum in continuous-cropping fields was isolated and identified by using conventional bacteriological methods.2. The primers for PCR detection of Ralstonia solanacearum were screened, a new sensitive and specific PCR detection system was established. The results showed that both of the PCR primers RaITS-1/2 based on the conserved sequence of Ralstonia solanacearum 16SrDNA ITS and the specific PCR primers Ralflic-F/R based on the pathogenicity-related gene of Ralstonia solanacearum had the sensitive and stable detection. By utilizing the optimized PCR condition, the 145bp and 724bp PCR products be obtained respectively, which were completely consistent with the expected size. This detecting system has high specificity and sensitivity, and the detection limitation could reach 10 fg DNA/μl, equivalent to 100cfu/mL, which is suitable for the detection of Ralstonia solanacearum in soil and other materials.3. Six protocols for soil DNA extraction was compared and a method for preparing high quality soil DNA was explored which could effectively solve the technical puzzles caused by soil humic acid, humic acid-like material, phenolic compounds, heavy metals, sediment unknown, cell lysis components and RNA etc. inhibiting subsequent PCR reaction.4. A semi-quantitative molecular detection system for Ralstonia solanacearum in soil was constructed. Its stability and reliability was verified with different soil samples. The detection results of 54 soil samples from Xinhua, Qianjiang, Chongqing were consistent with field disease survey. Using this technology to analyze and evaluate the Ralstonia solanacearum overwintering, early infection and control effect of agricultural measures is the first report. |
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