具杀松材线虫活性的苏云金芽胞杆菌筛选及其毒力测定

松材线虫是松树萎蔫病的主要病原，为了筛选到具有较高杀线虫活性的苏云金芽胞杆菌Bacillus thuringiensis菌株，采用直接触杀法对苏云金芽胞杆菌发酵液进行杀线虫活性以及稳定性的检测，并对筛选出的高活性菌株通过扩增16S rDNA片段进行菌株的鉴定。结果表明，4个菌株中具有最高活性的菌株D发酵液处理松材线虫8 h的死亡率超过70%，24 h其死亡率达到100%；在温度为-20、25、40、60、80和121℃条件下处理的菌株D发酵液，其对松材线虫的毒力都在85%以上；在pH 5.0~10.0时，菌株D发酵液对松材线虫的毒力都在85%~94%，并无显著的差异；将菌株D发酵液进行蛋白酶K处理后，其对松材线虫的毒力仍在98%以上；在4℃环境下保存50 d，菌株D发酵液的毒力没有明显下降。通过16S rDNA 1.5 kb片段的序列分析，初步得出菌株D可能是苏云金芽胞杆菌KNU-07。可见，菌株D发酵液具有耐热、耐储藏和对蛋白酶K稳定的特性，是一株具有开发潜力和应用前景的高毒力菌株。

Screening of Bacillus thuringiensis Strains against the Pine Wood Nematode and Determination of Its Toxicity

The pine wood nematode (PWN, Bursaphelenchus xylophilus) is the main pathogen causing pine wilt disease. Direct contact killing method was used to determine the nematocidal activity and stability of Bacillus thuringiensis fermentation broth to screen highly toxic B. thuringiensis strain. The screened strain was identified with 16S rDNA fragment. The results showed that the nematocidal activity of strain D fermentation broth was the highest among four strains. The mortality of PWN treated by strain D fermentation broth was over 70% at 8 h and reached 100% at 24 h. The toxicities of strain D fermentation broth against PWN were more than 85% when the strain was treated to temperatures of -20, 25, 40, 60, 80 and 121℃. At pH of 5.0-10.0, toxicities of strain D fermentation broth against PWN ranged between 85%-94%, which showed no significant difference. When treated with protease K, the strain D fermentation liquid still showed a toxicity above 98%. When the strain D fermentation liquid was stored at 4℃ for 50 d, no obvious decrease in its toxicity was observed. The sequence analysis of the 16S rDNA 1.5 kb fragment showed that that the strain D may be B. thuringiensis KNU-07. It can be concluded that the strain D fermentation broth is heat resistant, shelf stable and not degradable by protease K, and thus is a highly virulent strain with potential for development and application.