| Bacterial canker of tomato(Lycopersicon esculentum Mill.), caused by Clavibacter michiganensis subsp. michiganensis(Cmm), is one of the most severe bacterial diseases on tomato crops. Since its discovery in a Michigan greenhouse of America in 1910, bacterial canker of tomato occurred frequently and spread to all tomato growing regions of the world. Epidemic of the disease is increasing in recent years. Cmm could be disseminated through seeds, transplants, infested debris and soil, thus being the primary sources of infection. No effective way to control the disease is available currently both in China and abroad. Control methods reported before could only manage the occurence and spread of the disease to some extent. Sensitive and reliable detection techniques of Cmm and biocontrol of this disease are important measures to reduce the incidence and spread of the disease. A loop-mediated isothermal amplification(LAMP) was developed for the detection of Cmm in this research,and the control effect of the strain Streptomyces setonii Z-L-22 against bacterial canker of t omato was studied.1. A visual LAMP detection of Cmm was developed with SYBR Green I as a fluorescence indicator in this study. Based on the special internal transcribed spacer(ITS)sequence of Cmm, a set of four LAMP primers was designed. The components in the system were optimized. The final reaction system is: template DNA 200 ng, the optimum concentration of Mg SO4 was 3.0 mmol/L, Bst DNA polymerase 2 U, optimal concentration of d NTPs for 0.3 mmol/L, and the ratio of outer primers and internal primers was 1:3 for optimum amplification in the total 25 μL reaction system. The results showed that the constant temperature of 65 ℃ for 40 min could acquire good amplification results. Thedetection sensitivity of LAMP was 50 cfu/m L by naked eyes after SYBR Green I dying,which was better than detection by agarose gel with the sensitivity of 500 cfu/m L. And the primers were high specific for Cmm detecting. The LAMP assay developed in this study was simple, fast, sensitive and specific, with good practicability.2. Based on the strain of Streptomyces setonii Z-L-22 which has strong antagonism effect on bacterial canker of tomato, we developed three types of field application formulations – water agent, granules and tablets, and determined the best components. The control effect against bacterial canker of tomato in the greenhouse and field were tested.The results showed that control effect can be over 80 percent when 10 times diluted fermentation, 5 g solid, and 1g tablet were used when plant in the greenhouse. Based on these dosage, filed test were employed and the incidence and severity were significantly lower than those Strepto Mycin sulfate treatments and blank control after 75 days which treated by the three formulations in the field. Among those formulations, the tablets showed they were easy in storage and transportation, and they had the advantages of low cost and long effective period, so it had a high development value. |
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