【作者】 王晓红；

【导师】 黄大庄；

【作者基本信息】 河北农业大学 ， 森林保护， 2008， 硕士

【摘要】 本文以黄粉虫诱集法从土壤中采集并筛选到了对桑天牛（Apriona germari）幼虫具有较高毒性的球孢白僵菌（Beauveria bassiana）BbOO菌株,并研究了菌株的基本生物学特性、对桑天牛的致病性及其影响因子、白僵菌对桑天牛幼虫的入侵和致病过程、桑天牛幼虫染病后的病理学变化,旨在揭示白僵菌对桑天牛幼虫的致病机理及控制潜能,明确白僵菌与桑天牛幼虫的相互作用关系,为在桑天牛幼虫综合治理中有效利用白僵菌提供理论依据。结果如下:1本试验利用黄粉虫（Tenebrio molitor）诱集法从不同土壤中分离到球孢白僵菌共9株,分离率为45%,并对桑天牛幼虫进行了生物测定。结果表明不同菌株的致病力存在差异,其中以BbOO菌株的致病力最强,校正死亡率及感染率分别为100%和86.3%,致死中时LT₅₀为4.13 d,致死中浓度LCso为3.05x106 m L^-1。2营养条件及适宜的温、湿度是影响菌株生长及致病力高低的主要引子,该菌株营养生长的最适培养基为PPDA,最适温度和湿度分别为25℃和相对湿度100%。该菌产孢的最适培养基为察氏培养基。孢子萌发的最适温度为25℃,最适湿度为100%。BbOO菌株对桑天牛幼虫产生致病作用的最适温度为25℃,最适湿度为100%。3桑天牛幼虫感染球孢白僵菌BbOO后,虫体颜色及外部形态发生相应的变化。通过扫描及透射电镜的实验观察,桑天牛幼虫接菌后8h即可附着于幼虫体壁并紧密结合,接种后12h,孢子即可穿透体壁,之后进入血腔并大量繁殖,逐渐侵染体腔内的组织与器官,直至寄主死亡。在孢子侵入过程中伴随着保外酶对表皮及中肠的降解作用。侵染后期菌丝体在血液中大量增殖,而虫体的免疫系统对白僵菌只起暂时的阻碍和抑制作用,不能从根本上消除白僵菌的发展。4石蜡切片试验结果表明,白僵菌BbOO处理后24h,分生孢子即可以附着于桑天牛幼虫体表,48h即可以萌发并侵入幼虫体表,使内表皮分解;菌丝进入血腔后开始增殖并向侵入点附近的脂肪、肌肉、气管等组织入侵。随着菌丝在幼虫体内的增殖,各组织器官遭到不同程度的破坏。4d后感染幼虫死亡,5d后菌丝突破体表在虫体外形成菌丝层并有分生孢子产生。更多还原

【Abstract】 In this study,a high virulent isolate Beauveria bassiana Bb00 to Apriona germari larvae was screened from the isolates collected from the soil using the "Tenebrio molitor Bait Method".To this isolate,the basal biological characteristics,the pathogenicity against A.germari and factors affecting the pathogenicity,infection process and defense response of the host to the pathogen,the pathological changes of A.germari larvae infected by B.bassiana were studied.The purposes of this study were to establish the pathogenesis of B.bassiana to the A.germari and interaction between B.bassiana and A.germari,to evaluate the potential of the fungus as a biocontrol agent for A.germari,which can provide the basis for the application of the fungus in the integrated control of A.germari.The results showed that:1.The screening of the high virulent isolateNine isolates of B.bassiana were isolated from different soil using the "Tenebrio molitor Bait Method".The isolation rate was 45%.The pathogenicity of different strains to A.germari larvae was tested.The result showed that Bb00 was the most virulent one.The correct mortality was up to 100%and the infection rate was 86.3%.LTs0 was 4.13d,and LC₅₀on the A.germari larvae was determined to be 3.05×10⁶/mL.2.The study of biological characteristics of Bb00The culture medium,temperature and humidity were the most important dominant factors of the growth and pathogenicity to B.bassiana The most suitable media for the mycelia growth and conidia production of Bb00 were PPDA and Czapek,respectively.For the nutrient growth and the conidia germination,the optimal temperature and humidity were 25℃and 100%,respectively.At 25℃and RH 100%,the pathogenicity reached the highest.3.The study of infection process to the pathogenThere were some changes in color and morphology on the Apriona germari larvae infected with Beauveria bassiana Bb00.Penetration process of B.bassiana includes adherence,germination,appressoria and penetration.Through the scanning electron microscope we can observe most conidia can adhere to the cuticle after 8h.The conidia on the cuticle of A.germari larvae germinated 12h after inoculation.Most tissues and organs were infected and disintegrated with the heavy multiplication of the mycelium in body cavity until the larvae were dead.There are immune response in A.germari larvae body but the defense responses were just temporarily inhibition for the pathogen but could not clear them completely.4.The Symptom and Histopathological Study of Apriona germari Larvae Infected by Beauveria bassiana Bb00Paraffin sections showed the spores of B.bassian attached to the cuticle of A.germari larvae after 24h.After 48h,the conidium germinated and penetrated through the cuticle, the endocuticle were disintegrated.The mycelium multiplied and invaded the fat body, muscle,trachea tissue,etc nearby the point of penetration after the germ tubes into the body cavity.Most tissues and organs were infected and disintegrated with the multiplication of the mycelium,by 4d,infected larvae were dead.The hyphae emerged through the cuticle and formed hyphal layer on the surface of the dead insect 5d postinoculation and conidium emerged.更多还原