Comparative activity of Choristoneura fumiferana nucleopolyhedrovirus propagated in different hosts

The biological activity of the Ireland strain of Choristoneura fumiferana (Clem) nucleopolyhedrovirus (CfMNPV) propagated in different hosts was determined to provide the basis upon which genetically modified CfMNPV, or other naturally occurring isolates, should be compared. Occlusion bodies (OB) derived from CF-203 cells were significantly larger and more pathogenic than those propagated in vivo when tested against the fifth larval instar of C fumiferana (Clem) and C occidentalis Freeman. The dose-responses (LD50 and LD95, expressed as occlusion bodies per larva) of C fumiferana larvae to in vitro-propagated OBs were 274 and 5785, respectively. The values of LD50 and LD95 to C occidentalis larvae were 19 and 118, respectively. There were no significant differences in pathogenicity or size when OBs propagated in C fumiferana larvae were tested against either insect species, nor were there significant differences for OBs propagated in C occidentalis larvae. The LD50 and LD95 of in vivo-produced OBs to C fumiferana were 925 and 61988, respectively. The LD50 and LD95 to C occidentalis were 50 and 453, respectively. OBs propagated in vitro had a mean volume of 13.13 microm3, whereas those propagated in vivo ranged from 0.84 to 1.41 microm3. The median survival time-responses (ST50) of fifth-instar C fumiferana or C occidentalis larvae to OBs propagated in vivo were not significantly different from those propagated in vitro at the dosage levels tested. Values of ST50 of C fumiferana larvae to in vitro- and in vivo-produced OBs at dosages causing less than 50% mortality rangedfrom 9.6 to 9.8 days post-inoculation (dpi), whereas a LD95 dose resulted in ST50 values ranging from 7.3 to 7.7 days. ST50 values of C occidentalis larvae at dosages causing less than 50% mortality ranged from 9.8 to 10.2 dpi, whereas a LD95 dose resulted in ST50 values ranging from 9.5 to 9.8 dpi. The median feeding cessation time-response (FT50) of fifth-instar C fumiferana larvae to OBs propagated in vitro (5.7 days) was not significantly different from the FT50 of those propagated in vivo in either insect species (5.3 and 5.7 days) at the dosage level tested (LD95). No significant differences in FT50 values were observed between OBs propagated in either larval host. The FT50 of C occidentalis larvae to OBs propagated in vitro (7.7 days) was not significantly different from that to those propagated in vivo in C occidentalis larvae (7.6days), but somewhat different (7.2 days) from that to those propagated in C fumiferana larvae. Results indicate that CfMNPV can be propagated in vivo in either C fumiferana or C occidentalis larvae (or sequentially through both) without alteration in infectivity, although the use of the CF-203 cell line yields the most biologically active OBs.     

蓖麻蚕核型多角体病毒p6.9基因的克隆及序列分析

基于丰富蓖麻蚕核型多角体病毒(Philosamia cynthia ricininucleopolyhedrovirus,PcrNPV)分子信息的目的,对PcrNPV DNA部分片段进行了测序分析,获得1个DNA结合蛋白基因p6.9的完整序列。该基因的读码框由240个核苷酸组成,编码79个氨基酸,分子质量约为9.8 kD,转录起始位点ATG侧翼序列符合Kozak规则,其上游34bp处具有晚期基因转录的保守起始序列taag,表明该基因为晚期表达基因。将PcrNPV与12种昆虫核型多角体病毒P6.9蛋白氨基酸序列作同源性比较的结果显示:PcrNPV P6.9蛋白氨基酸序列与家蚕(Bombyx mori)NPV P6.9的同源性为59%,与柞蚕(Antheraea pernyi)NPV的同源性为100%,表明PcrNPV与ApNPV的亲缘关系很近。     

家蚕核型多角体病毒LAMP可视化检测技术的建立

[目的]建立针对家蚕核型多角体病毒(BmNPV)的环介导等温扩增(LAMP)可视化检测技术,为生产现场进行家蚕血液型脓病早期诊断提供技术支持.[方法]以BmNPV多角体蛋白基因po如为扩增靶标,分别设计5组内/外引物和5条环引物,根据扩增效率筛选最佳引物组合;优化LAMP反应条件,并进行LAMP检测的特异性、敏感性及临床样本检测试验;使用羟基萘酚蓝(HNB)染色对结果进行比色观察,对简化样品处理的条件进行摸索.[结果]使用环引物后LAMP对BmNPV基因组DNA的最低检测浓度为7fg/μL,检测灵敏度得到有效提高,且反应时间缩短.建立的LAMP检测方法对靶标DNA扩增具有特异性,对样本的检出率高于常规PCR,其最低检测限是常规PCR的100倍.在反应前加入HNB,结果易判断且能减少交叉污染.感染家蚕血淋巴进行100℃煮沸处理后即可直接用于LAMP反应,既简化了操作步骤,又降低了检测成本.[结论]针对BmNPV建立的LAMP可视化检测技术具有灵敏、快捷、可靠的特点,适合用于生产现场的BmNPV感染早期诊断.     

桦褐孔菌不同菌株培养特性及人工栽培比较

以来自不同国家及地区的21个桦褐孔菌菌株为试材,比较菌丝长势、长速、干重及多糖产量,并分别对每个国家的1个菌株进行人工栽培比较．结果表明,不同国家的桦褐孔菌菌株菌丝培养特性及菌核形成特性明显不同,其中,中国菌株JL01菌丝长势最强,长速最快,达3．62mm／d;中国、芬兰和朝鲜菌株均产生菌核,但中国菌株的菌核干重、生物学效率及多糖产量显著高于其它2个菌株,生物学效率达27％．     

家蚕核型多角体病毒ORF 75基因的生物信息学分析

通过对家蚕核型多角体病毒(Bombyx mori,nucleopolyhedrovirus:BmNPV)ORF 75基因核苷酸序列进行生物信息学分析,BmNPVORF 75基因位于病毒基因组70 485 bp和71 264 bp之间,编码259个氨基酸残基的多肽,预测分子相对质量为30.8 kDa,等电点为7.67,分子式为C1409H2157N351O390S19;蛋白在大肠杆菌内的半衰期大于10 h;酸性氨基酸(Asp Glu)占10.5%,碱性氨基酸(Arg Lys)占10.8%;蛋白的不稳定指数为42.67,是不稳定蛋白.利用Prosite数据库扫描,查到4类9个蛋白修饰位点.通过Blast搜索氨基酸同源序列发现,BmNPVORF 75同源蛋白存在于所有测序的鳞翅目昆虫杆状病毒中,不存在于其他非鳞翅目昆虫杆状病毒中.同源序列比对结果,BmNPVORF75蛋白的氨基酸序列与苜蓿银纹夜蛾核型多角体病毒(Autographa californica MNPV)ORF92之间的同源性达到100%,应为相同基因.依照BmNPVORF75同源基因序列相似性,对13种昆虫杆状病毒进行进化树构建分析,其中,BmNPVORF75与薄荷灰夜蛾多粒包埋型核型多角体病毒(Rachiplusia ouMN-PV)ORF89基因进化距离很近,同源性较高.     

云南蚕区家蚕品种资源对家蚕核型多角体病毒的抗性评价分析

[目的]全面掌握云南蚕区家蚕品种资源的抗家蚕核型多角体病毒(BmNPV)能力,为挖掘具有特色的家蚕抗病材料及培育适应当地的抗病品种提供参考.[方法]采用三龄起蚕经口添食相同浓度病毒、逐日调查存活率的方法,对200个家蚕品种(品系)进行BmNPV抗性评价和筛选;并通过接种后每日存活数、各发育时期存活率等指标分析云南蚕区家蚕品种资源对BmNPV的抗性水平、发病死亡规律、不同抗性品种的发病死亡时期及同一品种不同品系间抗性差异.[结果]云南蚕区家蚕品种资源中抗BmNPV的品种有731、795、854B、872A、966B、B黄肉色茧、P50、山河B、选二甲白和竹印,感病品种包括955、242A、242B、963B、DW3、锦秀A、锦秀B、日、野乙和云夏A油.各抗病品种和感病品种首次出现死亡差异的时间截点分别是接种后96和48 h,二者相差48 h;部分抗病品种在秋季对BmNPV的抗性水平低于春季;同一家蚕品种A系和B系的抗病性差异不显著(P>0.05).[结论]云南蚕区家蚕品种资源中大多数品种的抗BmNPV能力属于中等水平,其中P50为BmNPV抗性最强品种.对未知品种进行抗BmNPV测定时,可选择接种后168 h作为抗性评价的时间截点.     

EoNPV对茶尺蠖两近缘种的毒力差异

茶尺蠖核型多角体病毒(Ectropis obliqua nucleopolyhedrosis virus,EoNPV)是一种重要的病原微生物,为进一步明确该病毒对茶尺蠖两近缘种的毒力差异,采用叶盘法对3龄茶尺蠖和灰茶尺蠖幼虫进行了EoNPV的毒力测定。研究显示,EoNPV对灰茶尺蠖的半致死剂量LD₅₀(median lethal dose)为茶尺蠖的28.9倍,EoNPV对灰茶尺蠖的致死中时间LT₅₀(median lethal time)大于茶尺蠖。结果表明,EoNPV对茶尺蠖两近缘种间的毒力存在差异,EoNPV对茶尺蠖具有更高的致病力,研究结果对EoNPV田间防治茶尺蠖两近缘种具有现实指导意义。     

灰茶尺蛾性信息素的初步研究

灰茶尺蛾是我国茶园中重要的食叶害虫。本研究对求偶高峰期的灰茶尺蛾雌虫,采用正己烷浸提腹部末端方法提取性信息素,利用气相色谱-触角电位联用仪(GC-EAD)筛选性信息素提取物中的活性成分,运用气相色谱-质谱联用仪(GC-MS)对活性成分进行鉴定,最后将人工合成的性信息素进行田间诱捕试验。GC-EAD结果显示,灰茶尺蛾性信息素提取物中有两种成分可引起雄虫触角的电位反应。GC-MS分析结果确定两种活性成分分别为顺3,顺6,顺9-十八碳三烯(Z3,Z6,Z9-18:H)和顺3,顺9-6,7-环氧十八碳二烯(Z3,epo6,Z9-18:H)。田间引诱试验表明,不同比例性信息素在田间显示不同的引诱效果,当Z3,Z6,Z9-18:H和Z3,epo6,Z9-18:H以4∶6的比例配比,总剂量为1 mg时,田间引诱效果最佳。该研究结果为灰茶尺蛾性信息素的应用研究提供了理论基础。     

两个EoNPV毒株对茶尺蠖和灰茶尺蠖的毒力差异

通过确定不同茶尺蠖核型多角体病毒(Ectropis obliqua nucleopolyhedrovirus,EoNPV)毒株对茶尺蠖(Ectropis obliqua)和灰茶尺蠖(Ectropis grisescens)毒力水平的差异,为有效提高茶尺蠖病毒的防效提供理论基础.采用浸渍法,测定EoNPV浙江毒株(EoN...     

茶尺蠖核型多角体病毒与农药混用试验

本文测定了茶尺蠖核型多角体病毒（EoNPV）与几种杀虫剂混用的杀虫效果。生物测定结果表明,苏云金杆菌（Bt）与EoNPV混用时,表现有一定的颉颃作用,而且Bt浓度越高,往往颉颃作用越强。敌灭灵、喹硫磷与EoNPV混用,虽然有协同作用,但无增效作用。