The Analysis Of The Bioactive Substance Produced By Coniothyrium Minitans Against Xanthomonas Oryzae Pv. Oryzae

Rice leaf blight is a danger disease in many parts of the world, the disease pathogen was Gram-negative bacteria (Xanthomonas spp), currently the world is in efforts to combat this disease. Coniothyrium minitans is an important biocontrol fungus. Current research is mainly focused on its biological control of Sclerotinia disease rather than Gram-negative bacteria disease. Jiang Daohong found Coniothyrium minitans strain chy-1 can metabolite some substance which can inhibit a variety of diseases. The metabolites has especially good inhibitory activity against the Xanthomonas oryzae pv.oryzae. The overall activity of metabolites can remain stable in acid but alkaline and can withstand the temperature of 121℃treatment. After Wang Hong (Wang Hong, etc.; 2009) studied the overall stability of metabolites, the result shows that the overall activity in the time, temperature and light treatment can still be maintained in good agreement, and later the research explored the effective extraction of target substances methods and the good choice of solvent. Later research combine Ultraviolet Spectrophotometry with biological activity of the substance to get the establishment of a rapid detective method of active substance of C. minitans (Hu Xianwen, etc.; 2011), and at last the research made a preliminary estimate of the unknown target substance. Given the special nature of the activity of metabolic products, to obtain their pure and specific information on the molecular structure is very urgent.In this study, based on the study before, we first obtain large number of metabolites of Coniothyrium minitans strain chy-1, then use proper solvent extraction to obtain crude extracts with activity against Xanthomonas oryzae pv.oryzae, then combine the use of chromatography with biological activity tests to get the crude extract purified. After that the physical and chemical properties of the target substance was discussed, and finally the structural testing and the elucidation of the structure of the active substance was accomplished. Main content and the results are as follows: 1. PDB liquid medium was fermented in the shaker under the conditions of 20℃180r/min for two weeks to get the culture broth 10L of Coniothyrium minitans, the difference between PDB liquid fermentation with grain solid fermentation was discussed. The results showed that liquid fermentation has a faster, smaller amount of work and can make the follow-up operation of the advantages of solvent extraction easier than the solid fermentation.2. Anhydrous ether was selected as solvent to obtain a mixture with activity. Petroleum ether and ethyl acetate gradient elution was selected by thin layer chromatography. With the use of column chromatography crude extracts were divided and subdivided by selected solvent. And then preparation of thin layer chromatography was used to purify the target product to get the final portion of high purity. Anhydrous ether and n-butanol, methanol was selected in previous studies, the differences among the three solvent was discussed to get the result that anhydrous ether is relatively easy to handle, less toxic and will not interfere with the activity of the test results.3. The purity of the target products were analyzed. Thin layer chromatography was used to determine the purity of the substance, and then the substance was analyzed by HPLC method to confirm the purity of the substance, the results show that the target product is of high purity. The stability of the substance was discussed to find that the substance may react with some material in the air or its structure changes under the room temperature. If the substance was involved in the reaction, the active site of the substance was not involved or the reaction was in a certain balance to maintain the activity of the substance.4. The structural analysis of the IR, UV, MS and NMR was carried. The results showed that the information of the target substance is very similar to macrosphelide A, but in the hydrogen NMR spectrum, it is different that the peak of hydroxy-8-C of the target substance was disappeared. the result shows that the product is the produt of macrosphelide A which is oxidated on the position 8-C. the target product was different from macrosphelide B which is the product of macrosphelide A by oxidation on 14-C.