具杀松材线虫活性的苏云金芽胞杆菌筛选及其毒力测定  

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

作　　者：李恩杰 李娜[3] 王青华[2] 张永安[1,2] 王玉珠[2] 曲良建[2] LI Enjie;LI Na;WANG Qinghua;ZHANG Yongan;WANG Yuzhu;QU Liangjian(Experimental Center of Forestry in North China,Chinese Academy of Forestry,Beijing 102300,China;Key Laboratory of Forest Protection,State Forestry Administration/Research Institute of Forest Ecology,Environment and Protection,Chinese Academy of Forestry,Beijing 100091,China;College of Forestry,Inner Mongolia Agricultural University,Huhehot 010019,China)

机构地区：[1]中国林业科学研究院华北林业实验中心,北京102300 [2]中国林业科学研究院森林生态环境与保护研究所/国家林业局森林保护重点实验室,北京100091 [3]内蒙古农业大学林学院,呼和浩特010019

出　　处：《中国生物防治学报》2018年第4期539-545,共7页Chinese Journal of Biological Control

基　　金：国家重点研发计划(2016YFC1201200);公益性行业(林业)科研专项(201504302)

摘　　要：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 r DNA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p H 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 r DNA 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。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 16 S r DNA 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 p H 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 16 S r DNA 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.

关 键 词：苏云金芽胞杆菌 发酵液 松材线虫 毒力 

分 类 号：S718.8[农业科学—林学] S476.1