| Bacterial leaf blight was one of "the top three diseases" at one time on rice production in our country. Although it was controlled in a certain degree by using and extending resistant varieties,it is a obstacle factor on rice production in different rice planting regions at present,especially in the coastal regions of our country. Supervising and knowing the virulence differentiation, population structure and genetic diversity of Xanthomonas oryzae pv.Oryzae(Xoo) wll in the regions where disease emerged, can help to guide breeding and using the resistant cultivar properly, which would be a valid means to controll the occurence of disease and lower the yield loss.Our reseach had seperated 71 Xoo strains from the rice sample infected Bacterial leaf blight in different regions of Hubei province. Then we identified the race and genetic diversity of these strains. Underlying this study, we established a Real-Time PCR detection system aiming at Xoo and tested it’s specificity and sensitivity. Then we tracked and measured the change of Xoo number on rice leaf after being inoculated Xoo. The main results we obtained are as follows:1.We collected and isolated 71 strains of Xoo from various regions in Hubei Province between 2013 and 2014,which were then identified by differential rice cultivar. The results showed that the physiological races of bacterial leaf blight of rice in Hubei consisted of R1,R2, R3, R4, R5, R6, R9. The dominant pathogenic race was R3, whose occurrence frequency of was 33.80%. Compared with the period between 2013 and 2014, the dominant pathogenic race of Xoo in Hubei province had been replaced by R3.In addition,the dominant pathogenic race in various regions also had changed a lot.In Wuhan it was R4 all the same, but it’s occurrence frequency had increased 28.0%; the dominant pathogenic race changed from R4,R5, R9 to R3, R5 in Xiaogan; R4, R5 to R1, R5 in Yichang; R3, R4 to R3 in Huanggang; R5 to R4 in Jingzhou; R3, R5 to R3 in Xianning; R2, R4 to R1 in Xiangyang.2.We amplificated these genom DNA of 71 strains by Rep-PCR.The analysis of fingerprint type show that primers ERIC and BOX present 16 and 15 types of pattern separately, the resolution ratio of primer REP was too low so that can’t be used for clusteranalysis. Fringed by bands similar rate of 70% between each other, UPGMA cluster analysis divided these 71 strains into 13clusters(ERIC),8 clusters(BOX) separately. But we didn’t find obvious correlation between geographic origins, races and genetic diversity.3.We then selected primers with high specificity and high sensitivity and established the real-time PCR detection system of Xoo in this study. Compared with conventional PCR detection, the results of test on Xoo suppension, simulated rice seed incorporated Xoo,simulated soil incorporated Xoo showed that the sensitivity of Real-time PCR was 10 to 1000 times higher than conventional PCR, by which can detect standard plasmid with a concentration of 100 copies/μL, Xoo suppension with a concentration of 103cfu/m L. In the simulated seed and soil incorporated Xoo, the pathogen can be detected by Real-Time PCR detection, and the sensitivity is still higher than conventional PCR. The study also synthesised standard plasmid used for precise quantitative test, then we detected the variation of Xoo quantity on rice leaveas inoculated Xoo and found the threshold value of symptomatic appearance which is 108 Xoo copies on rice leaves with a length of 10 centimeter, the study can provide a support for disease forecast. |
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