【作者】 王子坚；

【导师】 罗建勋； 曾巧英； 殷宏；

【作者基本信息】 甘肃农业大学 ， 预防兽医学， 2010， 硕士

【摘要】 蜱隶属于蜱螨目、蜱总科,寄生于动物体表,广泛分布于我国各省区,是严重危害畜牧业生产的一类外寄生虫,不仅可通过叮咬吸血导致动物生产性能的下降,还可以传播动物和人的多种传染病（包括病毒病和细菌病）和寄生虫病（如:泰勒虫病和巴贝斯虫病）,给畜牧业造成严重经济损失。目前对蜱的防制方式主要以化学药物防治为主,但随着蜱耐药性的产生以及人们对环境污染和药物残留等公共卫生问题的关注,该方法的应用在一定程度上受到限制,人们越来越将目光转移到免疫预防和生物防治等绿色控制方法上来。致病性病原真菌作为一种很有潜力的生物控制因子,已在农业害虫生物防治领域得到广泛地应用,而在兽医线虫和外寄生虫病防治的研究才刚刚起步。本文以对蜱具有高致病力的菌株为研究对象,研究其毒力因子——胞外蛋白酶和几丁质酶与致病力之间的相关性;对发酵条件进行优化,摸索绿僵菌的产酶最佳条件,制备粗酶制剂;将其与微生物农药Bt进行配伍,观察协同作用效果。主要研究结果如下:1﹑观察了10株绿僵菌对微小牛蜱饥饿幼虫的致病性作用,通过生物测定,得知Ma002菌株对微小牛蜱饥饿幼虫致病力最高,致死率达到82.1%。半数致死时间最短的是Ma002的LT₅₀为4.83 d;所以将Ma002作为所选菌株。测定了10株绿僵菌的蛋白酶和几丁质酶的活性,用生物统计学的方法分析了其与菌株对蜱的毒力之间的关系,相关系数r₁=0.8183,r₂=0.6828。结果表明,绿僵菌对微小牛蜱饥饿幼虫的毒力与其胞外酶之间呈现正向相关,并发现几丁质酶与菌株毒力之间的相关性要明显高于蛋白酶（r1>r2）,检验结果极显著。2﹑应用微生物发酵的方法研究了Ma002产几丁质酶的培养特性。Ma002产几丁质酶的最佳组分为:胶体几丁质为碳源,蛋白胨为氮源,0.05% MgSO₄·7H₂O,0.001%ZnSO₄·7H₂O。优化的培养条件:装基量12%、24 h的种子液、起始pH值7.5、发酵温度26℃、搅拌速度180 r/min、产酶时程96 h。通过条件优化,产酶菌株Ma002酶活力从1.67 U/mL提高到2.63 U/mL,酶活力提高57%。3﹑观察了几丁质酶作为一种生物添加剂与Bt农药配伍的增效作用。结果显示,随着几丁质酶溶液浓度的提高,混合药液的杀虫效果也随之提高。当在1.6×10² mg/kg浓度的Bt溶液中添加浓度为2 U/mL的酶时,杀虫效果最强,累计死亡率从52%提高到85.8%,表明几丁质酶可以作为一种增效剂,提高微生物农药的作用。更多还原

【Abstract】 Ticks are ectoparasites classified in the class Acari and parasitize on the surface of animals, which are widely spread in all over of China,constricting the development of animal industry. The parasites not only cause degrading of animal production by bloodsucking, but also transmit various pathogens, including viruses, bacteria and parasites such as Theileria and Babesia. Ticks and tick borne diseases usually cause heavy economic loss in husbandry industry. Currently, tick control is predominantly by chemical therapy. However, due to the issues concerning resistance, environmental pollution and public sanitation, the wide application of this method has been limited. Therefore, more and more scientists shed their eyes on immune prophylaxis and biocontrol.As potential factors for biocontrol,entomopathogenic fungi have been widely applied in the control of insect pests in agriculture. However, its use has just started in laboratory in control of nematodes and ectoparasites.This study is taking pathogenic strains as target, focuses on their virulence factors----extracellular protease and chitinase, and their correlation to pathogenicity. Then the individual effects of the extracellular protease and chitinase was evaluated by induction of enzyme yields using selective cultural media. The optimized conditions for chitinase production were determined and crude enzyme was prepared. And the synergy effects in compatible with the microbial pesticide Bt was studied,which provides the basis of development for new biological agents. The major achievements are as following:1. Through study on the pathogenecity of 10 isolates of Metarhizium against unfed larvae of B. microplus and bioassay, Ma002 strain was assessed to be the highest in pathogenecity with lethality of 82.1% and the shortest LT50 of 4.83d.The fermentation of 10 Metarhizium isolates protease and chitinase, the virulence of the strain with different yields of these enzymes was assessed by biometrics.The coefficient correlation of r1,r2 and r0.05 were 0.8183,0.6828,respectively.The results showed that there was a significant correlation, in which the virulence of the strain with high yield of chitinase is significantly higher than that of the protease （r1> r₂）.2. The characteristic of Ma002 for producing chitinase was studied by fermentation. The method showed optimal fermentation conditions was that colloid chitin was used as carbon source and peptone as nitrogen source; the inoculating periods was 24 h.The optimized cultural conditions were in flask with 12% volume medium of pH7.5-8.0 at 26℃, culturing for 96 h on 180 r/min shaker. Under these conditions, the chitinase activity increased 57% comparised to the original basal medium from 1.67 U/mL to 2.63 U/mL.3. The effection of chitinase as biological additive was observed.By combination with Bt pesticides under a certain percentage, its synergistic effect was observed. With the increase of the concentration of chitinase, the insecticidal effect also increased. Adding a final concentration of 2 U/mLchitinase to 1.6×102 mg/kg Bt solution the cumulative mortality from 52% to 85.8% and the insecticidal effect could increase by 62.2%, indicating that chitinase could be a synergist to enhance microbial pesticides effect of significantly.更多还原