烟草角斑病菌拮抗细菌筛选及抗菌活性物质研究

Screening of antagonistic bacterial strain against Pseudomonas syringae pv. tabaci and studies on antagonistic active substances

研究从黑龙江省健康烟草植株及其根际土壤和海南三亚采集的20种杂草中分别分离到287和98个细菌分离物，筛选出对烟草角斑病菌有拮抗活性的菌株22个，其中来自三亚稗草叶片中的一株细菌PA-2的抑菌效果最好，抑菌效果稳定。经形态学、生理生化及分子生物学鉴定， PA-2为铜绿假单胞杆菌（Pseudomonas aeruginosa）。筛选出最佳实验室发酵配方：氮源1%（牛肉膏？？蛋白胨=5？？3），葡萄糖0.3%， NaCl 0.5%， KNO30.3%；最佳培养条件：发酵温度28℃，培养基pH 7.0~7.5，接种量4%，装液量为250 mL三角瓶中装液50 mL。PA-2优化发酵液的田间防效达69.77%，比农用链霉素防效提高24.47%。采用硫酸铵分级沉淀法分离抑菌活性物质，结果表明，在（NH4）2SO4浓度为30%~90%各浓度梯度下均有抑菌活性物质沉淀，其中60%~70%（不含60%）是抑菌活性物质的最适盐析饱和度。抑菌活性物质具有较强的热稳定性、较好的蛋白酶耐受性、较好的耐碱性、较差的耐酸性，能溶于部分有机溶剂，经乙醇、乙醚、氯仿、乙酸乙酯、正丁醇和丙酮萃取后，该抑菌活性物质抑菌活性下降范围为1.4%~4%。确定该抑菌活性物质为小分子碱性、水溶性脂肽类物质。

In the present study, 287 and 98 bacterial strains were isolated from healthy tobacco plants and their rhizospheric soil samples as wel as 20 kinds of weeds from Hainan Province, in which 22 strains had antibacterial activity against Pseudomonas syingae pv.tabaci. The strain PA-2 that was identified as Pseudomonas aeruginosa by morphological, physiological and biochemical as wel as molecular biology tests was isolated from the leaf of Echinochloa crusgal i in Sanya and had the best inhibiting and stable effect. The optimal fermentation medium components for PA-2 constituted of 1%of nitrogen （beef extract：peptone=5：3）, 0.1% of glucose, 0.5% of sodium chloride, and 0.3% of potassium nitrate. The optimal fermentation conditions： cultural temperature 28 ℃, optimum initial pH 7.0-7.5, inoculation seed liquid 4%and 50 mL of loading liquid in a 250 flask. The field effect of PA-2 fermentation liquid against tobacco angular leaf spot was 69.77%, 24.47% increase over the streptomycin. We used the ammonium sulfate precipitation classification method to separate the antibacterial active substances, the results showed that each 30%-90%saturation of （NH4）2SO4 had precipitated antibacterial active substances, in which 60%-70%（exclusive 60%） was the most appropriate saturation. The antibacterial active substances had stronger heat stability, better tolerance to proteinase K, better resistance to alkali, but poor tolerance to acid, dissolved in parts of the organic solvents. The decreasing range of the antibacterial activity of extractants of Ethyl alcohol, Ethyl ether, chloroform, ethyl acetate, n-butanol and acetone was 1.4%-4.0%. So they might be a kind of alkaline and water-solubility antibacterial substance.