【作者】 段彦丽；

【导师】 杨忠岐； 张永安；

【作者基本信息】 中国林业科学研究院 ， 森林保护， 2008， 博士

【摘要】 美国白蛾核型多角体病毒(Hyphantria cunea Nucleopolyhedrovirus,HcNPV)生物杀虫剂能有效控制寄主美国白蛾种群的数量,在生物防治中起到了重要的控害作用,具有重要的经济、生态、环保价值,应用前景十分广阔。本文应用生物测定、组织病理切片、电镜技术、分子生物学、免疫组化等方法,初步研究HcNPV与Bt混合致病机理及其病毒对寄主种群中的持续控制作用,力争从多个角度揭示HcNPV与寄主的互作机理。主要研究内容和结果如下:混合感染试验表明,当两病原浓度接近LC50时,美国白蛾幼虫可表现出病毒或细菌的症状,并依次出现细菌和病毒两个发病高峰期。用各病原浓度接近LC50混合感染,与各单剂单独作用相比,可提前发病高峰期12-24h,表明病原混合作用可加强各病的致病力;当Bt浓度为10、25mg/L时对HcNPV的毒力有增效作用;而Bt浓度为5mg/L时,对HcNPV的毒力表现出减效作用;而HcNPV浓度为1.6×105、1.6×106、1.6×107 PIBs/mL时,对Bt的毒力均有增效作用。混合感染增效可使半数致死时间(LT50)缩短0.5-2.1d。说明病原混合感染寄主对其病原的致病性有明显影响,浓度配比是混合侵染提高效力的关键因素。4龄初幼虫接种后按不同时间取样,制成石蜡切片,进行H.E染色。观察了HcNPV、Bt和HcNPV+Bt混合侵染寄主所引起的中肠上皮细胞、脂肪体细胞、表皮细胞、精巢的病变过程。观察发现,病毒和细菌混合感染,6-48h中肠上皮细胞顶端肿胀呈囊泡状,细胞质积聚在顶部;72h-144h表现病毒病症,与病毒单独感染相比,出现病毒症状的时间略早,病变的程度也较严重,组织细胞降解的速度也明显加快,说明混合感染加重了病理变化,加速了幼虫死亡。对寄主的超微结构观察表明,Bt感染24h,中肠上皮细胞、杯状细胞出现病理变化,48h细胞质出现空泡,72h病变加重,上皮细胞大多从基底膜脱落。混合感染幼虫,感染24h,中肠上皮细胞出现病理变化,96h出现病毒粒子复制,而脂肪体24h无明显病理变化,48h出现病理变化,120h出现病毒粒子复制,至144h-168h中肠、脂肪体细胞核内病毒粒子大量复制,出现包埋病毒粒子多角体,尤其中肠细胞核内充满了多角体,细胞核胀大,几乎占满了整个细胞。HcNPV感染精巢组织细胞核在144h出现病毒发生基质,168h有病毒核衣壳形成。上述结果表明,病毒侵染中肠细胞比脂肪体细胞早24h,中肠在中后期出现带囊膜病毒粒子的复制,在晚期有大量的多角体在细胞核内形成,而在多数鳞翅目昆虫中,这种情况比较少见。同时在精巢细胞发现病毒发生基质,这些变化在美国白蛾寄主中还未见报道。免疫组化观察HcNPV在寄主组织中的定位情况,结果表明其经口感染寄主的病理时相:感染后48h,在中肠检测到个别阳性信号;72-96小时,在气管、脂肪体、表皮同时检测到病毒抗原,中肠的阳性信号也在增多;感染后120小时,气管上皮细胞、脂肪体和真皮细胞的细胞核肿大,阳性信号增强;感染后144小时,在脂肪体、气管、真皮的上皮组织中全部检测到阳性信号。上述结果说明,中肠细胞阳性信号出现的早,但数量增加缓慢,其它组织阳性信号出现的晚,但病毒增殖速度快;HcNPV+Bt混合感染阳性信号出现的时间早于病毒单独感染,说明混合感染增强了病毒在寄主体内的复制。肌肉组织中始终未见阳性信号,表明肌肉未被侵染。需要指出的是,在感染144h后,精巢的精囊细胞内检测到病毒抗原的阳性信号,表明病毒侵染了精巢组织。对染病寄主的血淋巴蛋白浓度和SDS-PAGE分析表明,HcNPV使寄主的血淋巴蛋白在染病后连续6d中,除在第72h、96h突然升高,明显高于对照外,其余时间血淋巴蛋白含量浓度均低于对照;三种蛋白(普通蛋白、糖蛋白、脂蛋白)电泳图谱与对照相比变化不明显,普通蛋白在第72h、96h的血淋巴蛋白,在中分子量区域出现相对含量上的变化及迁移位置的变动。糖蛋白、脂蛋白图谱变化相似。上述结果说明,HcNPV对寄主的血淋巴代谢有抑制作用,可能是病毒感染破坏了脂肪体组织,损坏了合成代谢蛋白质的功能,导致了血淋巴蛋白浓度的变化。用HcNPV感染美国白蛾幼虫后,对其亲代、子一代、子二代的幼虫致死率、蛹重、产卵量、卵孵化率及染病成虫繁殖对子代影响进行了生物测定和观察研究,结果表明,与对照比较,各项检测指标经生物统计,均表现出差异显著。说明HcNPV在寄主种群中能够传代,对种群的数量起到了一定控制作用。应用PCR检测染病寄主当代、次代卵总DNA,发现HcNPV的扩增产物,证明HcNPV在分子水平上可以垂直传播给子代。HcNPV在寄主中连续传代7次后,对第0、3、5、7代进行了生物活性测定、电镜观察、病毒粒子蛋白SDS-PAGE电泳、基因组DNA限制性内切酶酶解分析等研究,生物活性测定结果说明,各代病毒毒力没有明显差异。第7代病毒与原始感染病毒电镜观察,在多角体和病毒粒子形态方面未发现明显变化。各代病毒的结构多肽基本一致,有25-26条多肽,只是第3、7代病毒出现了一条新增条带,分子量约为42.0 kD。病毒核酸经BGⅡ,EcoRI,PST,PVUⅡ4种酶解,各代NDA的酶切位点、片段大小基本完全一致。说明,HcNPV经传代7次后,病毒核酸遗传稳定,病毒粒子多肽的变化只是病毒与寄主互作的适应性改变,并未改变其遗传特性。对HcNPV的5种分离株进行了比较研究,结果发现,其多角体、病毒粒子在形态方面没有明显差异;生物活性比较,其中一株LC50、LT50明显优于其它毒株;结构多肽电泳、病毒DNA酶切图谱存在一定差异。更多还原

【Abstract】 Hyphantria cunea nucleopolyhedrovirus (HcNPV), an effective baculoviral insecticide to control population of the severe intrusive insect pest the Fall webworm, has been used on a large scale in China. The application of HcNPV will present very important value in economy, ecology and environment and will boost biological control in forestry insect pest management. Investigation of mechanism of its infection, transmission among its host and combination infectious mechanisms with Bt will contribute practical and theoretical references to the bio-insecticide application in control of the fall webworm.Bioassay experiments of the virus and Bt showed that the larvae of H. cunea exhibited either symptom of virus infection or that of Bt infection when the inoculating concentration approached their respective LC50 of the two pathogens, and the respective peaks of incidence of diseases also occurred under the same concentrations. Compared with inoculation of single pathogen, peak stage of combined pathogenesis was 12-24h earlier. The results indicated that combination of the two pathogens could enhance their respective pathogenesis. Enhancement of HcNPV virulence was observed when concentration of Bt was at 10 and 25 mg/L, while antagonism occurred at 5 mg/L. Virulence of Bt was enhanced when concentration of HcNPV was at 1.6×105、1.6×106、1.6×107 PIBs/mL, respectively. The LT50 was 0.5-2.1d shorter at HcNPV and Bt co-infection illustrated that application of the two pathogens could significantly effect their pathogenesis on the host insect. Allotment of the two pathogens concentrations was the key factor to enhance the efficiency of infection.The H. cunea larvae were sampled at different time after the two pathogens inoculation at early 4th instar and stained with H.E. after wax sectioned. Pathologies of the larval midgut epithelia, fat body cells and testis cells were observed microscopically. During 6 to 48 h co-infection of the virus and Bt, top of midgut epithelium showed swell and cystoid, where cytoplasm accumulated. Virus infectious symptom occurred during 72-144 h co-infection, which was earlier than that of virus single infection. The pathogenic changes and cellular dissociation also accelerated comparing to single infection, which led to rapid death of the larvae.Ultrastructural observation of the larvae infected with Bt showed that cytoplasm of the midgut epithelia and goblet cells appeared vacuoles 48 h after inoculation. At 72 h, pathological changes aggravated that most epithelia shed from the basal membrane. For the co-infected larvae, disease changes occurred in midgut epithelia at 24 h and virion amplified at 96 h, while no disease changes were observed in fat body at 24 h. Disease changes occurred in fat body at 48 h and virion amplified at 120 h. During 144 h to 168 h after co-infection, virion amplified actively in the nuclei of the epithelia and fat body cells. At the same time the nuclei swelled that were full of the viral polyhedra. In the nuclei of testis cells infected with HcNPV at 144 h virogenic stroma appeared. The results showed that infection of the virus occurred in midgut epithelia 24 h earlier than that in fat body cells.Location of HcNPV in the host tissue was studied by immunohistochemistry. Some positive signals were detected in midgut 48 h after infection. During infection of 72-96 h viral antigen was detected in the tissues of trachea, fat body and epidermis, while positive signal increased in midgut, At 120 h nuclei of tracheal epidermis, fat body cells and dermal cells swelled where positive signals increased. At 144 h positive signals were detected in the tissues mentioned above. The results indicated that positive signals appeared earliest in the midgut and increased slowly, while they appeared later in other tissues and proceeded faster. Occurrence of the signals in HcNPV and Bt co-infection were earlier than that in single infection of HcNPV indicated that co-infection enhanced amplification of the virus in the host. No signals were detected in muscle tissue showed no infection of the virus occurred in the tissue.Concentrations of the infected host hemolymph proteins were detected and SDS-PAGE was used to analyze. Concentrations of the infected hemolymph proteins were higher than those of no-infected controls except at 72 h and 96 h after infection. The results implied that infection of the virus probably suppressed synthesis of hemolymph protein via destroying fat body tissue. There were no significant differences in hemolymph protein patterns (including total proteins, glycoproteins and lipoproteins) between infected and no-infected host.Some impact indices, such as larval mortalities, pupal weight, oviposition, percentage of egg hatch of respective parental, first offspring, second offspring generations after infection, were studied by bioassay. Significant differences existed between treatments and controls. The results indicated that HcNPV could pass through different host’s generations and provided a sustainable control to the host population. Amplified PCR product of HcNPV polyh gene was detected in total DNA of the first and second generations of host’s eggs showed HcNPV could vertically pass through parent host to its offspring.There were no significant differences of the viral virulence after 7 passages through the host analyzing with the techniques of bioassay, ultrastructure, SDS-PAGE, genomic DNA restriction endonuclease analysis. However, a 42.0 kD band was observed in SDS-PAGE of virion proteins of the third and 7th viral generation. These results showed that genetic characteristics of the virus remained stable after 7 generation passages. The newly occurred 42.0 kD protein was probably the change of interaction between the virus and its host.This paper also compared biology of 5 HcNPV strains. One of a strain was more virulent than other 4 strains expressed in LC50 and LT50. There were some differences in viral proteins and restriction endonuclease patterns of the 5 viral strains.更多还原