【作者】 丁秀琼；

【导师】 刘世贵；

【作者基本信息】 四川大学 ， 微生物学， 2007， 硕士

【摘要】 蝗虫是一种世界性的害虫，是农作物和其他植物的害虫，对农业生产构成严重的威胁。蝗虫的防治已成为一项非常紧迫的任务。化学杀虫剂在大量而长期的使用过程中暴露出了许多缺点：污染环境，害虫产生抗药性，破坏生态平衡等。作为一种防治农林害虫的新技术手段，微生物农药在世界范围内受到广泛重视。蝗虫微孢子虫（Nosema Iocustae）是我国研究生物防治方法控制蝗害的第一个成功例子。利用昆虫病源真菌包括白僵菌（Beauveria bassiana）、绿僵菌（Metarhizium anisopliae）、和黄绿僵菌（Maxtrhizium flavoviride）等开发的真菌灭蝗剂对蝗虫有较好的防治效果。除此之外，由昆虫病源细菌类产碱假单孢菌（pseudomonas pseudoalcaligenes），蜡状芽孢杆菌（bacillus cereus）等制成的杀蝗剂也有一定的防治效果。但至目前，蝗害未能从根本上得到控制。开发新的微生物资源，研制成更加有效的杀蝗剂仍旧是微生物农药发展的一个重要方向。本文从自然病死全身发黑蝗虫虫尸中成功分离到一株蝗虫病原菌，命名为hb；用分离菌株纯培养物感染健康蝗虫，回复感染原宿主后的病死虫病症与以前相同，并从回复感染致死的虫尸中重新分离到了相同的病原菌，其致病性为科赫定律所证实；分别用液体石蜡法、甘油速冻保藏法和液氮法保藏分离菌株，供后续实验使用。为了对该菌株灭蝗效果作定量检测，本文作了室内毒力测定试验：用菌株hb发酵液口服感染健康蝗虫，记录死亡情况，观察病死虫病症，并收集具有典型死亡特征的虫体。对结果进行统计分析，采用统计分析软件SPSS（V11.0），以校正死亡率值法求毒力回归方程，得到毒力回归方程Y=1.199X-9.745，致死中浓度为LC₅₀=1.33×10⁸cfu／mL，相关系数R=0.9158。通过喂食感染家蚕测定了该菌对鳞翅目昆虫的杀虫活性。结果表明，该菌对鳞翅目的昆虫没有毒杀作用，可以初步将该目昆虫列在该菌杀虫谱之外。为了初步探讨所分离蝗虫病原菌杀虫作用机理，本文利用蛋白酶K水解消化蛋白质作用，在发酵菌液上清中加入蛋白酶K使其中酶失活，再喷雾到玉米苗上喂食感染蝗虫，观察并记录死亡情况。试验结果发现，蛋白酶K对菌株杀虫效果有明显的抑制作用。据此，可以初步确定杀虫物质是该菌产生的一种胞外蛋白。为了确定该菌在分类学上的地位，本文对强毒株hb进行了分子生物学鉴定，采用细菌16SrDNA通用引物，用PCR技术扩增出目的片段细菌的保守序列16SrDNA，将目的片段连接到pTA2-T载体上，转化大肠杆菌DH5a，提取质粒并通过酶切确认序列正确后送公司测序。对所测序列进行16SrDNA序列分析和DNA同源性分析。结合形态特征和生理生化特征，将分离菌株hb鉴定为产碱杆菌（Alcaligenes sp.）。更多还原

【Abstract】 Locust is a kind of worldwide pest to crops and other plants. It can make large lose and grave treatment to agriculture.Chemical pesticide causes many problems while killing pest:environmental pollution,chemical resistance, breakdown ecological balance,etal.So,pest biocontrol acqure high reconstruction in the world because its merits. Nosema locustae is the first succefful pesticide using to control locusts in china. Beauveria bassiana, Metarhizium anisopliae,and Maxtrhizium flavoviride are insects’ pathogenic fungus. They all have high toxicity to locusts. Pseudomonas pseudoalcaligenes and Bacillus cereus are insects’ pathogenic bacteria They also have some toxicity to locusts. But the truth is ,until now, locust is still out of control. Finding out new pathogenic bacteria and developing high toxicity pesticide is still our top responsibility.A pathogen hb was isolated from naturally dead locust, denominate hb.The pathogenicity of hb was testified by law of KOCH. Using follow method to preserve this strain: glycerol quick freezing method and petroline slant tube method.Inorder to conform the function of this strain in biocontrol, we determinated the toxicity of the strain hb to the locust. It was assayed by the way of oral infection. The results showed that the strain hb had a strong toxicity to locust.The linear regression relationship between the logarithm（x） of hb concentration and the probability（y） of corrected locusts morality is:Y=1.1993X-9.745 and the median lethal concentration is:LC₅₀=1.33×10⁸cfu/mL, the correlation coefficient R=0.9158.Toxicity of hb to silkworm was also assayed by the way of oral infection. The results showed that the strain hb had no toxicity to silkworm.It is to say, Lepidoptera insects is out of the spectrum of this isolated strain.Inorder to identify the insect disinfestation material,we add proteinase K into fermentation broth of the isolated strain hb after centrifuging.Then spraying the broth onto corn seedling and feed it to locusts.The result showed that proteinase K inhibits the toxicity of strain hb obviously.So,we can initially preliminary determin the insect disinfestation material as a kind of extracellular protein produced by strain hb.The strain hb was identificated by molecular biology method 16SrDNA. Using GP primers of bacterium to PCR the conserved sequence of the strain and then link the conserved sequence and pTA2-T vector, transformate Escherichia coli DH5a.. Selecte the positive colony. Extract plasmid after 24h culture.Then the plasmid was sent to determining sequence after confirming by enzyme cutting. Based on the results of physiological and biochemical fertures, 16SrDNA sequence and DNA homology analysis, the strain hb was identified asAlcaligenes sp.更多还原