Technology For Detection Of The 2 Plant Quarantine Bacteria And Preparation For The Detecting Kits

Bacterial fruit blotch of watermelon and bacterial canker of tomato are both international and national quarantine diseases of China. The former is one of new bacterial diseases in recent years and caused by Acidovorax avenae subsp. citrulli, which mainly infects cucurbitaceous plants and is threating to the agricultural production. The later caused by Clavibacter michiganense subsp. michiganense is one of the destructive diseases in tomato production. It not only affects the tomato yield but also the quality. These 2 bacterial diseases are seedbore and seed transmitted. Therefore, it is necessary to develop a set of accurate, rapid and sensitive methods for detection of the 2 bacterial pathogens to prevent the invasion and spread of the diseases in the country.The present study combined the ISE method with classical PCR to increase the detecting efficiency. The 3 antisera of rabbit anti whole cell of Acidovorax avenae subsp.citrulli ( Ab) and Clavibacter michiganense subsp. michiganense (cmmbj) have been successfully obtained by common serological method, they are 1001,1002 of anti Ab, and 1011 of anti cmmbj. The antibody of 1001and 1002 could be effective when diluted by 5210 times and reached 1:64 by ODD test with the high specificity. The antibody of 1011 was effective when diluted by 1280 times and reached 1:32 by ODD test, however, the specificity was not very satisfied.The results showed that all the strains of A. avenae subsp.citrulli tested produced 360bp specific fragments by the ISE and the direct PCR method, while others including 23 strains of Pseudomonas, Xanthomonas, Erwinia, Clavibacter, Burkholderia and Bacillus showed negative PCR result. Comparing the sensitivity of these 2 detecting methods, about 1×10~4cfu/ml of the bacterial suspension were detected by direct PCR and below 1×10~2cfu/ml only can be detected by immunocapture PCR method. In the detecting experiments of the artificial inoculated seeds, at least 2 seeds should be used by direct PCR, however, only 1 seed was enough for detecton by immunocapture PCR. Detecting 7 batches of melon seeds from the markets by immunocapture PCR showed that the cantaloupe seeds of 86-1 from Xinjiang carried Acidovorax avenae subsp.citrulli , which matched with the result of the growth-cheking of the melon seeds.Detecting bacterial canker of tomato pathogen by direct PCR and immunocapture PCR showed that 614bp specific fragments were produced in pathogenic C. michiganense subsp. michiganense(Cmm)stxains, while other 21 strains of non-pathogenic Cmm, Pseudomonas, Xanthomonas, Erwinia, Acidovorax, Burkholderia and Bacillus were not. Comparing the sensitivity of the 2 detection methods, about 1×10~5cfu/ml of the bacterial suspension of Cmmcould be detected by direct PCR while about lxl06cfu/ml were detected by immunocapture PCR. The seedborne pathogen of Cmm of tomato were not detected from the market seed batches.