| Recent years, rice bacterial foot rot has become one of the most serious diseases on rice in some provinces or areas of our country. The disease was first discovered in Hubei Province in2008, and caused devastating damage in a few rice fields. To avoid the wide spread of the pathogen of the disease and the resulting epidemic, more attention should be paid to the detection of Dickeya zeae(Dz), ie the pathogen of the disease. In the past, people applied some traditional methods in the detection and identification of the plant pathogenic bacterium. These methods include the observation of the symptoms, isolation and cultivation of the pathogen, microscopic observation, bacteria staining technique, phage specificity and test of pathogenicity. Now these methods seems time-consuming and low in efficiency, accuracy and sensitivity. In recent years, PCR has been widely applied to the detection of the phytopathogen, the technique is more rapid and efficient in contrast to the traditional ones. Moreover a technique named Real-time fluorescent PCR developed based on PCR has greatly improved in efficiency and accuracy. The SYBR Green protocol is easy to design, needless to design probe and share universal property in series of experiment. Meanwhile the method is low in cost and analysis of solubility curve and the test for specificity could be achieved in the process. The main results are as following:1. Based on the previous research of pathogens, Gram staining,morphological characteristics and pathogenic characteristics were further tested. The results showed that the bacteria was gram-negative, short rods in the electron microscope, flagellum around the bacteria and the size was (0.5—0.7)um×(1.0—2.5)um. Cultivating bacteria liquid, rice can show the typical symptoms by injecting and cutting root, bacteria liquid can make the bud Browning, reduce the emergence of bud. The resistance of the conventional cultivars was explored, six species was moderate resistance, nine species was moderate susceptible, nine species was highly susceptible, the results in this study has practical value. 2. Based on the specific ITS site in the ribosomal DNA sequence of Erwinia chrysanthemi pv. zeae(Ecz), a pair of specific primers named X1and X2were designed(X1:GAGTAGAAGTGCCTGCGTG, X2:AGGTTAGCGTTGACCGTGC), thus the detection system of real-time fluorescent PCR was established. Soon afterwards the specificity and sensitivity of the method was tested using different bacterial genomic DNA (gDNA), the positive band can only be amplified from the gDNA of Ecz using the primer X1/X2. Meanwhile the detection threshold value proved to be5×10-3ng/μl. When the experiment was conducted using bacteria liquid the detection limit was102cful/mL. The conclusion is that the sensitivity was100times higher than the conventional PCR. Further research proved that pathogen incorporated into soil and seeds both could be detected. Moreover, the method was confirmed to be applicable to the detection of the pathogen in live rice plant, q-PCR could reveal the existence of the pathogen2dpi. Due to its high sensitivity, rapid and reliable outcome, the detection method established in this study has practical use in disease forecast.3. The transmission pathway of the disease were explored, the results showed that infected plants emerged in both experiments of soil and sick rice bag transmission of disease, infected plant was not found in seedling stage, the typical symptoms was found in tillering stage and heading stage. Combine the sympotom observation and detection, a conclusion could be safely drawn that the pathogen could survive in the soil and sick rice bag, which constitute the major source of primary infection. while not a single infected plant was found in the experiment of seed transmission of disease using pathogen infested seeds of susceptible cultivar. |
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