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【目的】植物寄生线虫是危害植物的重要病原物,为了筛选到能在植物体内稳定定殖并且对植物寄生线虫具有较高杀线虫活性的植物内生细菌生防菌。【方法】以松材线虫为靶标,用直接触杀法进行筛选。对高活性菌株采用正交实验优化发酵条件,测定发酵液杀线虫活性的稳定性,并对菌株进行鉴定。【结果】从6种植物中分离筛选出13株对松材线虫具有较高杀线虫活性的植物内生细菌菌株,这些菌株的发酵上清液对松材线虫处理24h杀线虫率均达到了100%;其中BCM2、SZ5、CCM7和DP1这4个菌株的杀线虫活性较高,发酵上清液稀释3倍处理24h杀线虫率均达到95%以上,DP1和SZ5菌株达到了100%;并发现部分菌株发酵液能使线虫虫体发生渗漏或消解。发酵条件优化后能使发酵液杀线虫效果提高4倍。4株高活性菌株产生的杀线虫物质均对蛋白酶稳定、耐热不耐酸碱且长时间保藏活性不下降。经过鉴定DP1和CCM7是枯草芽孢杆菌(Bacillus subtilis),BCM2和SZ5是蜡样芽孢杆菌(Bacillus cereus)。【结论】经济作物体内存在一定数量的能产生杀线虫活性物质的内生细菌,其中一些细菌产生的杀线虫物质具有较强的稳定性。认为杀线虫活性的植物内生细菌具有很大的生防潜力。
【Objective】 Plant parasitic nematodes are important pathogens that endanger plants. In order to screen endophytic bacteria biocontrol bacteria which can be colonized stably in plants and have high nematicidal activity against plant parasitic nematodes. 【Method】 The pine wood nematode was used as the target and screened by direct contact method. The orthogonal test was used to optimize the fermentation conditions for the highly active strains. The stability of the nematicidal activity in the fermentation broth was determined and the strains were identified. 【Result】 Thirteen strains of endophytic bacteria with higher nematicidal activity against Bursaphelenchus xylophilus were screened from 6 kinds of plants. The fermentation supernatant of these strains had a nematicidal rate of 100 %. Among them, the four strains of BCM2, SZ5, CCM7 and DP1 showed high nematicidal activity. The nematicidal rates of all three strains of BC1, SZ5, CCM7 and DP1 were higher than those of the control. The rate of nematodes was 95%, and the DP1 and SZ5 strains reached 100% Some strains of fermentation broth can nematode parasites leakage or digestion. Optimized fermentation conditions make the fermentation liquid nematicidal effect increased by 4 times. The nematicidal substances produced by 4 highly active strains were all stable to protease, resistant to heat and acid and alkali, and had no decrease in preservation activity for a long time. DP1 and CCM7 were identified as Bacillus subtilis, BCM2 and SZ5 as Bacillus cereus. 【Conclusion】 There is a certain amount of endophytic bacteria producing nematicidal active substances in economic crops, some of which produce nematicidal substances with strong stability. Endophytic bacteria that are considered nematicidal have great potential for biocontrol.