Studies On Pest Risk Analysis, Identification, Detection And Antagonistic Bacteria Of Burkholderia Glumae

Burkholderia glumae induced seedling rot and grain rot symptom of rice is theating to rice production in distributed countries. There is no occurrence report except Taiwan in China. Because of the quarantine significance, it would be listed as a quarantine pest before this study by Ministry of Agriculture, however, little information is available for the research of B. glumae in China.The quantitative PRA of B. glumae in China was processed; The resistant analysis of major rice varieties, grown in Zhejiang and Heilongjiang, was evaluated; The identification of B. glumae from so-called "health looking seeds" was performed. In order to increase the detecting efficiency, the present study combined the ISE method with classical PCR; the antagonistic bacteria against B. glumae were screened from rice seeds in virtro, and their effect evaluation and mensuration against B. glumae were undertaken.The PRA system of B. glumae was constructed based on our experiments, the selected data from other countries, ISPM rules, the examples of PRA in China and combined with the situation of China. General formula on biosafety evaluation, R =f (S, P) was simplified as R=S×P, could express objectively the relationship among the risk related factors of B. glumae. To simplify the PRA system and save time, only the main risk related factors were selected. The R(8.4) value of B. glumae to China was calculated, consequently B. glumae was belonged to high-risk pest. Using the published data related to PRA, constructed system was validated primarily. The result also showed B. glumae was high-risk pest, and demonstrated the constructed PRA system of B. glumae was reasonable and workable. Therefore, B. glumae was belonged to the high risk pest in China, and should be managed by quarantine actions. It was firstly established the quantitative PRA of B. glumae in China.Eighteen commercial varieties were inoculated with 4 isolates of B. glumae for testing their resistance to bacterial grain rot at heading stage. The results indicated no variety was higher resistant. The susceptibility of varieties and pathogenicity of 4 isolates of B. glumae were significantly different (P< 0.05). Among 18 rice varieties tested, Kongyu 131 was the most susceptible, Bing 9904 was the middle-resistent. The isolate B2012 was the most virulent while the isolate B.2012b was the least. The results showed that the pathogen didn't affect the germination of rice seeds but caused seedlings to die about 98% when rice seeds were dipped in a bacterial suspension of B. glumae (10~8cfu/ml) for 30 minutes prior to sowing. Rice plants inoculated with the suspension of B. glumae (10~8cfu/ml) at booting stage by injection method, the panicles and sheaths of rice plants were all infected and discolored. The pathogen didn't infect the sheaths but the panicles when rice plants were inoculated at either booting or heading stage by spraying method. It was estimated that more than 60% of unfull grains were recorded when rice plants inoculated at booting stage. The Kongyu 131 was more susceptible to 4 isolates of B. glumae 2 days before or after heading if inoculation at heading stage.In order to monitor whether there is the infection of B. glumae in rice in China and prevent it, a survey on examination of the casual organism from 623 seed samples was conducted during 1997-2006. Six pathogenic bacterial strains were isolated from two samples of rice ( Oryza sativa L.) from Hainan in 2006, They were identified as B. glumae which is the causal organism of bacterial grain rot of rice by bacteriology, colony morphology, pathogenicity test, Biolog, Fatty acid methyl ester (FAME) analysis (computer aided numeric taxonomic methods) and RAPD-PCR compared with the 4 standard reference strains. It was firstly confirmed in mainland China that B. glumae was existed in so-called "health looking seeds".The present study combined the ISE method with classical PCR to increase the detecting efficiency. The 4 antisera of rabbit anti whole cell of B. glumae have been successfully obtained by common serological method, they are ASBg14, ASBg32, ASBg26 and ASBg04. The antibody of 4 antisera could be effective when diluted by 5120 times and reached 1:32 by ODD test. The specificity of antibody of ASBg14 and ASBg32 was very high, however, the specificity of ASBg26 and ASBg04 was not very satisfied. The results showed that all the strains of B. glumae tested produced 500bp specific fragments by the ISE and the direct PCR method, while others showed negative PCR result. Comparing the sensitivity of these 2 detecting methods, about 1×10~5cfu/ml of the bacterial suspension were detected by direct PCR and below 1×10~3cfu/ml only can be detected by immunocapture PCR method. In the detecting experiments of the artificial inoculated seeds, at least 2 seeds should be required by direct PCR, however, only 1 seed was enough for detection by immunocapture PCR.About 4253 bacterial strains were isolated from 432 random rice samples, and tested for antagonists against B. glumae in virtro. It was showed that 23 strains were antagonists. In antagonism on Petri dish testing, 40% of 23 strains also antagonized Rhizoctonia solani AG2, Fusarium moniliform and Xanthomonas oryza pv. Oryzicola, and showed some broad spectrum. Five antagonistic bacteria were selected to investigate for antagonistic activities of their culture filtrates. It showed that culture filtrate of T021 and S087 had certain inhibitory effect on B. glumae.The inhibition percentage of T021 is 50.2% and 51.4% against B. glumae and Rhizoctonia solani AG2, respectively. Detached leaf and screenhouse experiments indicated that only strain T021 could promote the growth of rice seeling roots. The identification of strain T021 by morphological characterization, cultural characteristics, physiological and biochemical characteristice, BIOLOG, FAMEs and 16S rDNA determination and analysis was done. It was identified as Pseudomonas aeruginosa.