核型多角体病毒基因组学研究进展

昆虫病毒是专性侵染节肢动物的一类微生物,包括核型多角体病毒、颗粒体病毒、质型多角体病毒、痘病毒、虹彩病毒等很多类群。核型多角体病毒属于杆状病毒科(Rhabdoviridae)核型多角体病毒属(Nucleopolyhedrovirus),是昆虫病毒中最大的类群。它能够专一性地侵染并杀死一种或者几种农林害虫,并且对害虫天敌、环境、人畜无害,是一种值得推广的绿色生物农药。本文总结了获得全基因组序列的核型多角体病毒,综述了核型多角体病毒的基因组主要功能基因(RNA转录相关基因、DNA复制相关基因、结构相关基因)及其他基因,并对核型多角体病毒今后的发展方向进行了展望。     

利用Red/ET系统结合rpsL反向筛选敲除AcMNPV lef-10基因

【目的】利用Red/ET系统结合rpsL反向筛选建立苜蓿银纹夜蛾核型多角体病毒(AcMNPV)晚期表达因子lef-10点突变载体,为进一步研究AcMNPVlef-10的功能奠定基础。【方法】利用Red/ET系统结合rpsL反向筛选BAC修饰系统对AcMNPV lef-10进行点突变,由于lef-10和必需基因vp1054有重叠,所以在敲除lef-10时只能通过引进点突变使lef-10失活,避免影响vp1054的正常表达。首先,构建rpsL-AMP筛选盒,然后在野生型AcBacmid中引进点突变(方法是通过2次Red/ET重组:第1次重组在野生型AcBacmid lef-10中引进rpsL-AMP抗性筛选标记,形成一次重组Bacmid,第2次重组是在一次重组Bacmid中引进含点突变的lef-10片段),再利用链霉素反向筛选去除rpsL-AMP抗性筛选标记,同时引入相应的点突变。将从lef-10基因点突变重组菌中提取得到的AcBacmid与质粒pTriEx1.1和pTriEx-innateP-lef10分别共转染草地贪夜蛾Sf9细胞,同时用野生型AcBacmid与质粒pTriEx1.1共转染Sf9细胞作为对照,显微镜下观察病毒的复制情况,确定lef-10基因是否为AcMNPV的必需基因。【结果】通过对重组Bacmid的PCR鉴定和点突变序列的测定,证明AcMNPVlef-10基因已经发生点突变;通过共转染试验发现,随着转染时间的延长,lef-10补回型病毒和野生型病毒一样,均能在Sf9细胞中复制产生具有感染性的子代病毒粒子BV,从而对邻近细胞造成二次感染,使得细胞死亡,且lef-10补回型重组病毒产生感染性病毒粒子的能力与野生型基本一致;而由于lef-10缺失型重组病毒不能产生有感染性的病毒粒子,所以在被lef-10缺失型重组病毒转染的细胞中,细胞数目不会随着转染时间的增加而发生明显改变。【结论】利用Red/ET重组系统结合rpsL反向筛选系统可以在Ac MNPVlef-10基因中引入点突变,且lef-10是杆状病毒生活周期中的一个必需基因,其缺失会影响杆状病毒的复制。     

茶尺蠖NPV对茶尺蠖幼虫取食及生长发育的影响

喂饲茶尺蠖核型多角体病毒(EoNPV)后,茶尺蠖幼虫的总食量极显著下降。1、2龄初饲毒,减少90.99～99.39％;3、4、5龄初饲毒,减少47.24～54.18％。总食量减少的主要原因是取食速率的显著减缓。1龄初饲毒,总取食期极显著缩短;2、5龄初饲毒,与对照无显著差异;3、4龄初饲毒,则总取食期极显著延长。各龄初饲毒后,该龄和下几龄的历期一般都极显著延长。饲毒后,幼虫体重明显减轻,能化蛹的蛹重也显著下降,但蛹历期和成虫寿命不受影响,上述结果表明,以EoNPV防治茶尺蠖幼虫宜在2龄初或其以前应用。在3龄初及其以后饲毒,高温条件下虽然幼虫死亡率不高,但总食量明显下降,仍有一定的效果。     

Application technology of the sex pheromone of the tea geometrid Ectropis grisescens (Lepidoptera: Geometridae)

The sex pheromone of Ectropis grisescens, one of the primary defoliator insects of tea plantations, has been identified, but its trapping parameters have not been optimized for field application. In this study, we investigated the effects of pheromone dose, trap height and trap type on the effectiveness of trapping E. grisescens in the field. Our results show that the optimal pheromone dose is 800 μg. The bucket-funnel trap hung 40 cm below the tea canopy had the highest trapping efficiency. This study establishes optimized parameters for the pheromone trapping of E. grisescens in tea plantations.     

灰茶尺蠖性信息素田间应用技术研究

灰茶尺蠖是我国茶树重要的食叶害虫,严重影响茶叶的质量和产量。为了给灰茶尺蠖性信息素的田间应用提供指导,本文对其性信息素缓释载体类型、诱捕器类型、诱捕器高度以及使用间距等参数进行了研究。结果显示,灰茶尺蠖性信息素以异戊二烯橡胶塞为缓释载体对灰茶尺蠖雄虫具有最好的引诱效果,效果优于硅胶塞和PVC毛细管;5种测试诱捕器中,船型诱捕器最适合引诱灰茶尺蠖;当诱捕器置于茶树上部25βcm时,引诱效果最好;诱捕器保持15βm的间距时,诱捕效率最高。本研究结果可为灰茶尺蠖性信息素应用于雄虫诱杀提供科学依据。     

茶尺蠖幼虫取食提高茶树儿茶素代谢响应强度

研究了茶尺蠖幼虫为害茶树叶片对儿茶素合成途径的影响。采用3龄茶尺蠖幼虫取食茶树新梢芽下第二叶,测定了儿茶素合成相关基因的表达水平和儿茶素含量。研究结果表明,与对照相比,茶尺蠖幼虫为害后3、6、12βh显著诱导了CsANR基因的相对表达水平,且在为害后3βh和12βh达到了极显著差异。CsLAR基因在茶尺蠖为害后6βh和12βh,与对照具有显著差异。茶尺蠖幼虫为害后24βh显著诱导了没食子酸、没食子儿茶素、表儿茶素、表没食子儿茶素没食子酸酯和表儿茶素没食子酸酯含量的升高,而茶尺蠖为害后48βh仅没食子酸和没食子儿茶素的含量显著高于对照;儿茶素、表没食子儿茶素和没食子儿茶素没食子酸酯在茶尺蠖为害后24βh和48βh均没有被显著诱导。上述结果表明茶尺蠖幼虫为害提高了茶树儿茶素合成途径的代谢强度和儿茶素类化合物的积累。     

Field evaluation of Lymantria obfuscata multiple nucleopolyhedrovirus for the management of Indian gypsy moth in Jammu & Kashmir,India

The Indian gypsy moth (Lymantria obfuscata) is a pest of national significance, which attacks a wide range of forest and fruit crops in India. The outbreaks of this pest over the past few years spurred interest towards sustainable biointensive approaches for quality fruit production of apple and walnut for agricultural export in Jammu & Kashmir (J & K). The exploratory survey's were conducted for the isolation of native strains of L. obfuscata multiple nucleopolyhedrovirus (LyobMNPV) and field evaluation was conducted to determine the most promising strain as a potential biological control agent of this pest. The virus was recovered from different locations of J&K with its natural incidence varying from 0.77 to 7.43 percent. This is the first report from the Indian sub-continent and worldwide of the occurrence of NPVs in natural larval populations of L. obfuscata. The in vivo mass production was optimised by inoculating per os late 3rd instar L. obfuscata larvae with an inoculum dose of 1.44 × 10⁵ OBs/larva and followed by a 10 day incubation period. To prevent the disintegration of larval cuticle and microbial contamination, moribund larvae were harvested with an average yield of 1.42 × 10⁸ OBs/larva. The field application of LyobMNPV against the host populations on apple and willow with the pre-standardised dosage of 2.5 × 10¹² OBs/ha reduced the larval population density by 25–63%. In the year following application, the larval population density in the treated blocks was 19–28% lower as compared to controls on both apple and willow. This suggests that the virus has considerable potential as a biocontrol agent and the timely application with LyobMNPV on non-fruit trees such as willow which are the preferred hosts for egg laying may help to initiate viral epizootics to keep population outbreaks under check.     

茶尺蠖发育历期的温度效应

观察研究结果表明,茶尺蠖(Ectropis Obliqua Prout)各虫态发育历期随着温度的上升而缩短,发育速率则随着温度的升高而加快,并探明了各虫态历期与期内均温之间的曲线关系模型。同时,用加权法和回归直线法分别计算了各虫态的发育起点温度和有效积温常数,结果表明两种方法计算的数据间差异不显著,表明两方法均可采用。验证结果表明,利用各虫态的发育起点和有效积温,推算茶尺蠖的发生代数及各虫态的发生期,与实际发生情况基本吻合。