【作者】 王刚；

【导师】 董建臻； 冯书亮；

【作者基本信息】 河北农业大学 ， 农业昆虫与害虫防治， 2010， 硕士

【摘要】 球孢白僵菌(Beauveria bassiana (Balsamo)Vuillemin)是目前国内外应用最广泛的昆虫病原真菌之一,能够侵染15个目149个科的700多种昆虫,并可在田间形成昆虫流行病,防治效果持久。HFW-05菌株是河北省农科院植保所杀虫微生物实验室分离筛选出的,对小菜峨、粉虱具有很高的杀虫活性,在前期的工作基础上,本研究扩大了该菌株的杀虫谱,明确了HFW-05菌株对烟粉虱和棉铃虫的不同侵染方式,通过合适的高温胁迫处理方法,在一定程度上提高了HFW-05菌株对较高温度的耐受力,并得到了耐热性状较稳定的新菌株。通过对烟粉虱、小菜蛾、棉铃虫、东亚飞蝗和蛴螬类(华北大黑鳃金龟、暗黑鳃金龟和铜绿丽金龟)进行室内生物测定,其中烟粉虱采用浸渍法,小菜蛾采用喷雾法,棉铃虫采用浸渍和饲喂两种方法,东亚飞蝗采用背板点滴法,蛴螬采用拌土法,表明HFW-05菌株对以上害虫均有毒力,除棉铃虫外,其余供试昆虫均可由表皮侵染,其中对烟粉虱和小菜蛾的效果最好,LC50分别为4.20×105孢子/mL和1.59×106孢子/mL,且田间小区试验防效均可达80%;HFW-05菌株对其他几种昆虫的作用时间较长,以浓度为107孢子/mL的孢悬液接种,9d时东亚飞蝗2龄蝗蝻的死亡率为62.67%;蛴螬类害虫中,对14d铜绿异丽金龟、12d华北大黑鳃金龟和9d暗黑鳃金龟幼虫的校正死亡率分别为60%、46%和40.74%。对于棉铃虫的胃毒作用较明显,6d后的校正死亡率为72%,且对试虫的体重和取食量有显著的抑制作用,但通过表皮侵染效果不佳。组织学研究表明,该菌株对烟粉虱主要从体壁接触侵染,菌丝优先从腹部侵入,也最早从腹部穿出体表;但未观察到从体壁侵入棉铃虫的现象,而通过胃毒侵染在消化道内发现分生孢子,并萌发生长至体内充满菌丝时导致试虫死亡。已报道的白僵菌对棉铃虫的侵染方式多为体壁侵染,这一现象进一步说明白僵菌对寄主侵入方式因昆虫的种类、虫态、环境条件等不同而有差别。为了提高HFW-05菌株对较高温度环境的适应性,采用高温胁迫的方法对HFW-05菌株进行了定向驯化。选用48℃下分别水浴处理30、40、50、60、70、80、90、100、110、120min,以孢子残存率、产孢量、生长速率、萌发中时、对小菜蛾的毒力以及产生的胞外蛋白酶和几丁质酶的活性为指标,将胁迫后一代中耐热性较好的处理以相同的处理方式连续胁迫至第8代,通过比较各测定指标,结果表明在48℃下处理60和110min并连续胁迫至第8代时菌种的耐热性最好,萌发率、产孢量及在较高温度环境下对小菜蛾的毒力均显著高于对照菌株。将筛选出的耐热菌株和对照菌株同时在30℃下继代培养,5个世代后30℃下对照菌株的萌发率仅为15%,而耐热菌株的萌发率均达50%;对照菌株对小菜蛾致死率极低,耐热菌株在30℃和模拟田间环境的条件下对小菜蛾校正死亡率均在50%以上,32℃下也有30%以上的校正死亡率。本研究表明,合适的热处理方式可以在较大程度上提高白僵菌的耐热性,为解决白僵菌在田间高温环境下应用具有局限性的问题提供了科学依据。更多还原

【Abstract】 Beauveria bassiana (Balsamo) Vuillemin is one of the entomopathogenic fungi which applied the most twidely presently at home and abroad, to pest control effeetively.I tcould infect more than 700 Species insects of 149 Families in 15 Orders, and caused the epidemicdis of pest, lasted control. A more virulent strain, HFW-05, to nymph of B. tabaci and P. xylostella was obtained by Laboratory of Microbial insecticide Institute of Plant Protection, Hebei Agricultural and Forestry Science. In this paper, the expansion of insecticidal spectrum of the HFW-05 strain, and infection of Bemisia tabaci and Plutella xylostella would be studied. The thermotolerance of the strain could be improved by high temperature stress, and got a new more stable strain.The HFW-05 strain has virulence against B. tabaci, P. xylostella, Helicoverpa armigera, Locusta migratoria manilensis, Holotrichia oblira, Holotrichia parallela and Anomala corpulenta was showed by the bioassay. All of those pest could be infected through the skin, except Helicoverpa armigera. The formulation brought on high mortality of two tested Pest in the lab and field. HFW-05 strain was toxic to the 3rd larvae of Bemisia tabaci with an LC50 of 4.20×105conidia/mL, to the 2rd of Plutella xylostella with an LC50 of 1.59×106 conidia/mL, and the mortality could be 80% in the field. The action time to the other pests would be longer .The mortality of HFW-05 with 107 conidia/mL to locusta migratoria manilensis after 9 days and to Holotrichia oblira, Holotrichia parallela and Anomala corpulenta after 12 days could be 62.67%, 60%, 46% and 40.74%. Stomach infection had be found on Helicoverpa armigera, Weight and food consumption bad be inhibited obviously.The paraffin slice of and Helicoverpa armigera infected by HFW-05 showed that the conidia infected Bemisia tabaci through the skin, hypha first penetrated the abdomen,and firstly pierced surface from the abdomen . Skin infection was not found on Helicoverpa armigera, conidias were found in digestive tract,germination and growth was showed ,and tissues was full of hypha when the pest was dead. Skin infection on Helicoverpa armigera by B. bassiana had been reported mostly. Further explanation of the phenomenon is the infection to the host by Beauveria bassiana is different for differernt insect species, instars and environmental conditions.HFW-05 strain was domesticated directionaly with high temperature stress for improving the thermotolerance. Conidia suspension were exposed to a thermal stress of water bath under 48°C for 30, 40, 50, 60, 70, 80, 90, 100, 110, 120min, then mensurated the residue viabilities of conidia, rate of growth, sporulation, hydrophobicity virulence, extracellular protease and chitinase. The treatment which have high heat resistance after thermal stress were teratde with the same approach to the 8th generation.The results showed that thermotolerance of the strain which were exposed to a thermal stress of under 48°C water bath for 60 and 110 min to the 8th generation have been obviously improved.The heat-resistant and control strains were subcultured at the same time at 30℃. After 5 generation, the germination rate of the control strain was 15%,but the heat-resistant strains, were 50%, the Virulence against Plutella xylostella by the control was too little ,but the heat-resistant strains, were more than 50% at 30℃and more than 30% at 32℃. This study shows that the appropriate heat treatment can greatly improve the heat resistance of Beauveria bassiana,it provided a scientific basis for solving the problem that the high-temperature environment limitations Beauveria bassiana faced when applicated in the field.更多还原