,, 刘琴, 李传明, 祁建杭, 徐彬, 陆玉荣, 徐健,江苏里下河地区农业科学研究所/国家农业微生物扬州观测实验站,江苏扬州 225007The Persistent Infection and Detection of Cnaphalocrocis medinalis Granulovirus in Cnaphalocrocis medinalis
HAN GuangJie,, LIU Qin, LI ChuanMing, QI JianHang, XU Bin, LU YuRong, XU Jian,Jiangsu Lixiahe District Institute of Agricultural Sciences/National Agricultural Experimental Station for Agricultural Microbiology, Yangzhou, Yangzhou 225007, Jiangsu通讯作者:
责任编辑: 岳梅
收稿日期:2020-02-15接受日期:2020-03-16网络出版日期:2020-10-01
| 基金资助: |
Received:2020-02-15Accepted:2020-03-16Online:2020-10-01
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韩光杰,E-mail：
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韩光杰, 刘琴, 李传明, 祁建杭, 徐彬, 陆玉荣, 徐健. 稻纵卷叶螟颗粒体病毒的持续感染及检测[J]. 中国农业科学, 2020, 53(19): 3988-3995 doi:10.3864/j.issn.0578-1752.2020.19.012
HAN GuangJie, LIU Qin, LI ChuanMing, QI JianHang, XU Bin, LU YuRong, XU Jian.
0 引言
【研究意义】稻纵卷叶螟（Cnaphalocrocis medinalis）是水稻上重要的迁飞性害虫[1],大量化学农药的使用并未从根本上控制其暴发危害且造成了农药面源污染风险。稻纵卷叶螟颗粒体病毒（Cnaphalocrocis medinalis granulovirus,CnmeGV）是稻纵卷叶螟的专性病毒,对稻纵卷叶螟具有较强的致病力,田间应用可以显著降低害虫种群增长趋势指数,有效抑制稻纵卷叶螟种群增长[2]。杆状病毒的持续感染是一种非显症的、但在生物体内存在病毒粒子或遗传信息的潜伏性感染,其对控制害虫特别是迁飞性害虫的暴发危害具有重要的生态意义。但是,目前CnmeGV特异探针的构建工作尚未开展,且对该病毒持续感染的认识也不全面。因此,开发CnmeGV的巢式PCR探针,用于低剂量病毒感染的检测,对研究CnmeGV的持续感染具有重要的应用价值,同时明确病毒持续感染对害虫的控制作用,可为CnmeGV病毒杀虫剂的应用提供理论依据。【前人研究进展】杆状病毒作为生物控制因子的关键在于病毒在自然界中长期存活并持续感染（或称为covert infection）[3,4]。潜伏侵染是一种狭义的持续感染,是一种可转化的、非增殖性的感染,病毒基因组在宿主中保留,但通常并不产生感染性的病毒[5]。自然界存在的病毒可以通过接触感染的水平传播途径使敏感害虫持续感染,也可以通过潜伏侵染长期存在于宿主体内并通过繁殖垂直传播给子代[6]。潜伏侵染直接影响昆虫子代的生长发育和产卵繁殖,使幼虫发育历期延长、产卵量和卵孵化率显著下降,对害虫种群消长发挥重要的调控作用[7,8,9]。一些常规PCR检测探针的开发证实了棉铃虫（Helicoverpa armigera）核型多角体病毒（HaNPV）的经卵传播[10]和家蚕（Bombyx mori）核型多角体病毒（BmNPV）的跨代传播[11],从而提高了对于杆状病毒潜伏侵染的认识。CnmeGV是一种β属杆状病毒,主要感染稻纵卷叶螟幼虫脂肪体细胞,表现出颗粒体病毒Ⅱ型特征[12,13]。CnmeGV制剂对田间稻纵卷叶螟控制效果在70%以上,持效期达50 d,且可以保护蜘蛛等天敌[14],发挥了较好的生态效应,但病毒的感染特征还不太明确。【本研究切入点】常规PCR探针对于研究杆状病毒的潜伏侵染还稍显不足,巢式PCR具有更高灵敏度可以检测微量的病毒感染,利用该标记可以分析CnmeGV经蛹传播的潜伏侵染特征。【拟解决的关键问题】建立高灵敏度的CnmeGV病毒检测探针,利用探针分析CnmeGV在害虫种群中的持续感染,明确病毒持续感染特征及对稻纵卷叶螟发生的影响。1 材料与方法
1.1 稻纵卷叶螟饲养
稻纵卷叶螟幼虫采自扬州市槐泗稻田,在室温（25±1）℃,相对湿度70%条件下,使用玉米苗饲养[15]。幼虫饲养多代后,挑选健康,大小一致的2龄或4龄幼虫供试。1.2 CnmeGV的增殖与接种
以106 OB/mL的CnmeGV病毒口服感染2龄中期的稻纵卷叶螟幼虫,正常饲养至5龄（显症率达90%以上）后收集病虫尸体,研磨匀浆,过滤经差速离心（12 000 r/min离心10 min,3 000 r/min离心30 s）后,收集发白的颗粒体病毒,用血球计数板计数颗粒体病毒数量。以106 OB/mL（该剂量为实验室前期试验结果,未发表,死亡率约30%）的CnmeGV病毒作为亚致死浓度口服感染4龄稻纵卷叶螟幼虫80头/重复,共3个重复,正常饲养96 h后,收集部分幼虫后继续饲养,后续收集蛹和成虫,幼虫和蛹使用10%甲醛处理10 min。
1.3 蛹表面处理方法
使用健康的稻纵卷叶螟蛹作为CnmeGV病毒蛹表面处理的供试虫。以105 OB/mL的CnmeGV病毒液（添加0.1%羧甲基纤维素钠增加黏性）处理蛹,处理10 min后晾干。晾干的蛹使用10%的甲醛处理10、30 min、16 h及按照苏志坚等[16]描述的碱液-Dnase酶处理方法处理,提取所有样本总DNA。同样的方法适用幼虫表面处理。1.4 样品DNA的提取
昆虫及病毒DNA的提取：选取经体表处理的不同发育阶段的稻纵卷叶螟,经液氮研磨后,使用700 μL裂解液（0.1 mol·L-1 Na2CO3,0.1 mol·L-1 NaCl,0.008 mol·L-1 EDTA,pH 10.7）37℃温浴裂解10 min,添加7 μL蛋白酶K和1 μL Rnase酶55℃水浴30 min,之后使用酚氯仿抽提一次后等体积异丙醇沉淀,经12 000 r/min离心10 min后,75%乙醇洗涤一次,晾干后在沉淀中加入100 μL ddH2O溶解。病毒DNA的提取不使用液氮研磨,其余方法一致。土壤总DNA的提取：在2017年施用CnmeGV制剂的田块（东经119°23′,北纬32°29′）,2018年采用5点取样法选取不同位置的水稻表层土壤为样本,经室内风干后研磨,按照Solarbio公司土壤基因组DNA提取试剂盒（D2600-50T）方法,提取土壤总DNA。
1.5 病毒granulin巢式PCR引物的设计及检测
通过NCBI nr/nt数据库下载CnmeGV等8个颗粒体病毒granulin,利用GENEDOC软件多重比较分析,设定第1轮PCR上游引物位于该基因5′端200 bp以内,通过Primer 5.0软件设计巢式PCR外侧引物Cm-gran1和内侧引物Cm-gran2（表1）,建立检测探针。Table 1
表1
表1引物设计
Table 1
| 引物 Primer | 序列 Sequence (5′→3′) | 产物大小 Product size (bp) |
|---|---|---|
| Cm-gran1 | F: TGATGCCGAGTATGAACCG | 541 |
| R: TTCCTCAACCTCTCCCGTAG | ||
| Cm-gran2 | F: CGCATCACTCTGTTCAAGG | 422 |
| R: ACGAGAGGACGGTAGAAGTAG |
新窗口打开|下载CSV
PCR检测试剂为诺唯赞公司2×Taq Master Mix产品,PCR仪器为BIO-RAD公司T100。外侧引物Cm-gran1和内侧引物Cm-gran2使用相同的PCR反应程序,为95℃ 3 min;95℃ 30 s,55℃ 30 s,72℃ 30 s,30个循环;72℃ 5 min。PCR反应体系为20 μL,包括Taq Mix 10 μL,上下游引物各1 μL,模板1 μL,ddH2O 7 μL。每轮PCR检测需同时至少做8个无DNA模板的阴性对照。巢式PCR顺序为先使用外侧引物Cm-gran1进行第1轮PCR扩增,扩展产物稀释100倍后作为第2轮内侧引物Cm-gran2 PCR反应的模板。第2轮PCR产物使用1%的琼脂糖电泳进行检测。
1.6 调查及统计分析
田间稻纵卷叶螟幼虫调查采用5点取样法,每个点剥查20头幼虫,统计幼虫感病率。室内亚致死浓度病毒处理的稻纵卷叶螟待正常羽化后,统计羽化率。图形制作使用GraphPad Prism软件,数据处理使用SPSS 21.0。羽化率和田间幼虫感病率差异分析采用T-test法,不同虫态病毒检出率统计分析采用卡方测验。2 结果
2.1 CnmeGV granulin分子标记的建立
多重比较分析来自CnmeGV、AdorGV、ChocGV等8种颗粒体病毒的granlin,CnmeGV granulin与其他7个基因核苷酸一致性为72.3%—75.8%。除5′端200 bp以内,其他区域核苷酸较保守。因此,选择特异性较高的区段为分子标记位点,建立了巢式PCR探针（图1）,该标记可以有效扩出目标片段。以实验室常备材料（水稻、玉米、稻纵卷叶螟健康虫、斜纹夜蛾核型多角体病毒、小菜蛾颗粒体病毒、黏虫颗粒体病毒、茶尺蠖核型多角体病毒）的DNA为模板,利用该标记均未检测到目标条带（图略）。图1
新窗口打开|下载原图ZIP|生成PPT图1稻纵卷叶螟颗粒体病毒granulin分子标记
Fig. 1The molecular marker based on granulin of C. medinalis
CnmeGV: KP658210.1:1-750; AdorGV: AF547984.1:1-747; ChocGV: NC_008168.1:1-747; ClanGV: HQ116624.1:1-747; CrleGV: AY229987.1:1-747; EpapGV: JN408834.1:1-747; HearGV: EU255577.1:1-747; PiraGV: AY428513.1:1-744
2.2 巢式PCR标记的敏感性
将浓度为85 ng·μL-1的病毒DNA依次10倍稀释作为模板,检测分子标记的灵敏度,经4次重复检测发现病毒DNA的最低检出限为0.85 fg·μL-1,是常规PCR的1 000倍（图2）。不同处理方式处理蛹表后,检测结果发现稻纵卷叶螟健康蛹、10%甲醛处理10 min—16 h的病毒处理蛹均未检测到病毒,但甲醛处理16 h后的蛹不能正常羽化。参考苏志坚等[16]方法,使用碱液-Dnase处理30 min,并不能去除蛹表的病毒DNA（图3）。在增加24个个体的试验重复后,结果一致。图2
新窗口打开|下载原图ZIP|生成PPT图2granulin分子标记检测的灵敏度
1-11: 85, 8.5, 0.85 ng·μL-1, 85, 8.5, 0.85 pg·μL-1, 85, 8.5, 0.85 fg·μL-1, 85, 8.5 ag·μL-1
A：Cm-gran1探针常规PCR的检测灵敏度The sensitivity of probe based on Cm-gran1 of conventional PCR;B：巢式PCR探针的检测灵敏度The sensitivity of nested PCR probes
Fig. 2The sensitivity of granulin molecular marker
图3
新窗口打开|下载原图ZIP|生成PPT图3不同方式处理昆虫体表病毒检测
1—4：CnmeGV处理的稻纵卷叶螟蛹The pupa of C. medinalis treated with CnmeGV;5—8：稻纵卷叶螟健康蛹The healthy pupa of C. medinalis;9—12：10%甲醛处理稻纵卷叶螟蛹（病毒处理）10 min The infected pupa treated with 10% formaldehyde for 10 min;13—16：10%甲醛处理蛹
Fig. 3Detection of CnmeGV on the surface of pupa treated with different ways
2.3 CnmeGV经蛹传播及对蛹羽化的影响
使用亚致死浓度的病毒处理稻纵卷叶螟幼虫96 h后,挑选24头未显症幼虫经体表处理后检测病毒感染率。经巢式PCR检测发现,幼虫病毒感染率为100%,表明所有幼虫均有病毒定殖。待幼虫成功化蛹后,收集部分蛹进行体表处理后,提取蛹、蛹蜕和成虫DNA检测病毒。发现蛹的病毒检出率为87.5%,蛹蜕为83.3%,成虫为16.7%。经卡方检测,幼虫到化蛹阶段,病毒感染率未有显著变化（χ2=3.2,P=0.234）;而蛹羽化为成虫,病毒检出率显著降低（χ2=32.356,P=0）（表2）。Table 2
表2
表2稻纵卷叶螟不同虫态中稻纵卷叶螟颗粒体病毒检出率统计分析
Table 2
| 变态 Metamorphosis | 样本类型 Sample | 总样本数 Total number | 阳性样本数 Number of positives | 检出率 Positive rate (%) | 卡方值 χ2 value | P值 P value |
|---|---|---|---|---|---|---|
| 幼虫-蛹Larva-pupa | 幼虫Larva | 24 | 24 | 100 | 3.2 | 0.234 |
| 蛹Pupa | 24 | 21 | 87.5 | |||
| 蛹-成虫Pupa-adult | 蛹Pupa | 24 | 21 | 87.5 | 32.356 | 0 |
| 蛹蜕Pupal molt | 24 | 20 | 83.3 | |||
| 成虫Adult | 24 | 4 | 16.7 |
新窗口打开|下载CSV
进一步待蛹羽化,发现与对照群体93.4%的羽化率相比,病毒感染群体羽化率仅有30.8%（P<0.0001）（图4）。
图4
新窗口打开|下载原图ZIP|生成PPT图4稻纵卷叶螟感病群体和健康群体的羽化率
CK：稻纵卷叶螟健康群体Healthy population of C. medinalis;Treated：病毒处理群体Infected population of C. medinalis
Fig. 4The emergence rate of infected and healthy populations of C. medinalis
2.4 CnmeGV田间水平传播分析
2017年在扬州单一田块施用106 OB/mL病毒制剂后,当代稻纵卷叶螟幼虫感病率达70%[2]。次年,调查同一田块,发现稻纵卷叶螟幼虫的病毒显症率仍有4%,而附近田块未发现感病个体（图5）。考虑到病毒水平传播的可能性,检测了田间土样带毒水平。利用该分子标记检测24份土样,发现田间土壤带毒率为58%（图6）。图5
新窗口打开|下载原图ZIP|生成PPT图5稻纵卷叶螟田间种群感病后表征（A）和显症率（B）
CK：未使用病毒制剂田块Control field without the use of CnmeGV;Inf-Field：使用病毒制剂的田块,次年稻纵卷叶螟幼虫显症率The prevalence rate of larvae in the next year after using CnmeGV
Fig. 5Characterization (A) and prevalence rate (B) of C. medinalis in the field
图6
新窗口打开|下载原图ZIP|生成PPT图6病毒制剂处理后田间次年土壤带毒检测
Fig. 6The rate of soil carrying CnmeGV in the field at one year following CnmeGV treatment
3 讨论
潜伏侵染（狭义的持续感染）在杆状病毒[4,5]、植物病原细菌[17]、动物病毒[18]等中被广泛研究,虽然宿主未表现症状,但潜伏侵染严重影响了宿主种群的健康和病原物的后期流行规模。分子标记的开发对病原微生物潜伏侵染的研究具有重要作用,包括常规PCR探针[10,11]、定量PCR探针[18]、巢式PCR探针[19]等。对葡萄霜霉病菌（Plasmopara viticloa）的检测结果显示定量PCR探针灵敏度是常规PCR探针的100倍[17],对美人蕉黄斑驳病毒（canna yellow mottle virus）的检测结果显示巢式PCR探针灵敏度是常规PCR探针的1 000倍[19],二者横向比较,巢式PCR探针的灵敏度更高。因此,为了更好地检测病毒的潜伏侵染,笔者选择开发CnmeGV的巢式PCR探针。该探针检测CnmeGV的最低限为0.85 fg·μL-1,检测灵敏度是常规PCR探针的1 000倍。同时,为了保证检测的特异性,CnmeGV的巢式PCR探针是选取颗粒体病毒granulin变异度较大的区域作为候选区来设计的,且在稻纵卷叶螟的食物源（水稻、玉米）、其他多角体和颗粒体病毒中均未检测到目标片段。在昆虫饲养中,甲醛可以使蛋白质变性、DNA分子断裂,一般被用来蛹表消毒。本研究发现10%的甲醛处理10 min就能完全清除蛹表污染病毒,而参照苏志坚等[16]的碱液-DNase酶处理（其主要作用机理为利用碱液裂解病毒蛋白,使病毒DNA暴露,再使用DNase酶消解DNA）方法未能有效清除病毒,造成这种差异的原因可能是常规PCR灵敏度或者DNase酶活力不同。因此,在该探针检测的灵敏度范围内,10%甲醛可以被认为能够清除稻纵卷叶螟体表的CnmeGV。杆状病毒潜伏侵染普遍存在,KHURAD等以亚致死剂量的家蚕多角体病毒感染幼虫,存活成虫和子代幼虫体内都扩增到473 bp的病毒早期基因ie-1[20]。印度谷螟（Plodia interpunctella）感染颗粒体病毒后,存活的幼虫和蛹内检测到病毒晚期表达颗粒体蛋白基因（granulin）mRNA,成虫卵巢和睾丸中也检测到该基因的mRNA[21]。本研究使用亚致死浓度的CnmeGV感染稻纵卷叶螟4龄幼虫,同样在幼虫、蛹及成虫中检测到病毒granulin,但成虫的病毒感染率显著下降。进一步研究发现,蛹蜕的病毒检出率高达83.3%。昆虫的细胞凋亡或蜕皮是清除病毒和细菌感染的一个重要防御机制,死亡的细胞被新分化细胞代替,从而降低了感染水平[22]。因此可以推断,成虫在变态过程中,大部分病毒随蛹蜕一起被排出体外,从而降低了后代持续感染病毒的风险。但是笔者也注意到,在幼虫化蛹的变态中,病毒的感染率未有明显的下降,幼虫-蛹的变态在昆虫病毒清除中的作用目前并不清楚。有研究报道,黏虫成虫潜伏的核型多角体病毒多数集中于足、翅和触角[23]。这些结果是否间接表明昆虫成虫体表潜伏病毒的数量远大于体内,值得进一步去探究。由于鳞翅目昆虫成虫鳞片具有疏水特性,10%甲醛体表处理很难有效清除体表病毒,这也是本试验尚未解决的难题。
潜伏侵染是杆状病毒与宿主昆虫协同进化共存的一种方式,同时也是病毒选择和丰度控制昆虫种群密度的因素之一[24]。斜纹夜蛾（Spodoptera litura）成虫感染斜纹夜蛾核型多角体病毒后,平均寿命及单雌产卵量明显下降,对子代幼虫种群控制作用干扰指数为0.1807[25]。本研究发现潜伏感染的稻纵卷叶螟可以正常化蛹,但是成虫羽化率显著降低,未羽化的蛹呈现发黑状,显示CnmeGV对稻纵卷叶螟种群的控制效果显著。前期研究也发现,CnmeGV对稻纵卷叶螟种群具有明显的干扰控制作用,种群控制指数为0.31—0.32[2],显示了其作为生物杀虫剂的优势。田间调查发现,使用病毒制剂田块次年稻纵卷叶螟幼虫显症率仍达4%,而相邻田块未发现病毒感染幼虫,进一步研究发现该发病田块土壤带毒率为58%。CnmeGV于1979年首次在广东恩平县被分离[26],时隔30年后,该地区仍然存在因感染CnmeGV而死亡的稻纵卷叶螟幼虫[27]。因此,CnmeGV在土壤中存活,通过水平传播进行持续感染可能是诱发稻纵卷叶螟感病的主要原因。
无论是杆状病毒水平传播造成持续感染还是垂直传播形成的潜伏侵染,对害虫种群控制都具有重要的生态意义。稻纵卷叶螟是重要的迁飞性害虫,潜伏侵染的种群可以将病毒带到其他地方,并形成种群内的持续感染,从而有效控制种群暴发。一定条件下持续感染能被激发并在宿主体内复制增殖转化为增殖感染,引起害虫死亡并形成持续传播[4,28]。这些激发条件可能包括种群密度[6]、微毒化合物[29]、其他病毒[9,30]等。因此,稻纵卷叶螟持续感染种群病毒激发的条件将是后期研究重点。
4 结论
巢式PCR检测体系可以应用于CnmeGV的持续感染研究,其灵敏度是常规PCR的1 000倍。CnmeGV在宿主体内可以经蛹传播,并显著降低稻纵卷叶螟的羽化率。CnmeGV的持续感染对控制害虫种群暴发具有重要的生态意义。参考文献 原文顺序
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被引期刊影响因子
[本文引用: 1]
[本文引用: 1]
[本文引用: 3]
[本文引用: 3]
DOI:10.1016/j.jip.2005.03.005URLPMID:15893760 [本文引用: 1]
Transmission plays a central role in the ecology of baculoviruses and the population dynamics of their hosts. Here, we report on the horizontal and vertical transmission dynamics of wild-type Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV-WT) and a genetically modified variant (HaSNPV-AaIT) with enhanced speed of action through the expression of an insect-selective scorpion toxin (AaIT). In caged field plots, horizontal transmission of both HaSNPV variants was greatest when inoculated 3rd instar larvae were used as infectors, transmission was intermediate with 2nd instar infectors and lowest with 1st instar infectors. Transmission was greater at a higher density of infectors (1 per plant) than at a lower density (1 per 4 plants); however, the transmission coefficient (number of new infections per initial infector) was lower at the higher density of infectors than at the lower density. HaSNPV-AaIT exhibited a significantly lower rate of transmission than HaSNPV-WT in the field cages. This was also the case in open field experiments. In the laboratory, the vertical transmission of HaSNPV-AaIT from infected females to offspring of 16.7+/-2.1% was significantly lower than that of HaSNPV-WT (30.9+/-2.9%). Likewise, in the field, vertical transmission of HaSNPV-AaIT (8.4+/-1.1%) was significantly lower than that of HaSNPV-WT (12.6+/-2.0%). The results indicate that the recombinant virus will be transmitted at lower rates in H. armigera populations than the wild-type virus. This may potentially affect negatively its long-term efficacy as compared to wild-type virus, but contributing positively to its biosafety.
DOI:10.3389/fmicb.2017.01337URLPMID:28769903 [本文引用: 3]
Baculoviruses (Baculoviridae) are occluded DNA viruses that are lethal pathogens of the larval stages of some lepidopterans, mosquitoes, and sawflies (phytophagous Hymenoptera). These viruses have been developed as biological insecticides for control of insect pests and as expression vectors in biotechnological applications. Natural and laboratory populations frequently harbor covert infections by baculoviruses, often at a prevalence exceeding 50%. Covert infection can comprise either non-productive latency or sublethal infection involving low level production of virus progeny. Latency in cell culture systems involves the expression of a small subset of viral genes. In contrast, covert infection in lepidopterans is associated with differential infection of cell types, modulation of virus gene expression and avoidance of immune system clearance. The molecular basis for covert infection may reside in the regulation of host-virus interactions through the action of microRNAs (miRNA). Initial findings suggest that insect nudiviruses and vertebrate herpesviruses may provide useful analogous models for exploring the mechanisms of covert infection by baculoviruses. These pathogens adopt mixed-mode transmission strategies that depend on the relative fitness gains that accrue through vertical and horizontal transmission. This facilitates virus persistence when opportunities for horizontal transmission are limited and ensures virus dispersal in migratory host species. However, when host survival is threatened by environmental or physiological stressors, latent or persistent infections can be activated to produce lethal disease, followed by horizontal transmission. Covert infection has also been implicated in population level effects on host-pathogen dynamics due to the reduced reproductive capacity of infected females. We conclude that covert infections provide many opportunities to examine the complexity of insect-virus pathosystems at the organismal level and to explore the evolutionary and ecological relationships of these pathogens with major crop and forest pests.
[本文引用: 2]
[本文引用: 2]
DOI:10.1146/annurev.ecolsys.34.011802.132402URL [本文引用: 2]
URL [本文引用: 1]
室内试验表明,甜菜夜蛾核型多角体病毒对宿主种群有明显的控制作用.当饲毒浓度在1.14×108PIBs/mL以上时,能引起97%的甜菜夜蛾幼虫死亡;饲毒后1～8天内,致死中浓度LC50值在8.4718×105～2.6098×10 12PIBs/mL之间;在饲毒浓度范围内,致死中时间LT50在1.98～6.99天之间;感病幼虫每日病死时间分布可用时间-剂量-条件死亡率模型较好地拟合,累计病死时间分布可用Gompertz模型拟合.室内试验还表明,病毒对甜菜夜蛾种群有明显的亚致死作用.免于病死蛹重量减轻,羽化所得雌成虫产卵量下降,产卵期延长;从感病高龄幼虫发育的成虫能将病毒传播至子代,引起20%～48%的子代幼虫死亡,并使子代成虫产卵量下降.田间试验表明,病毒除具有明显的控制作用外,还有一定的后效作用.
URL [本文引用: 1]
室内试验表明,甜菜夜蛾核型多角体病毒对宿主种群有明显的控制作用.当饲毒浓度在1.14×108PIBs/mL以上时,能引起97%的甜菜夜蛾幼虫死亡;饲毒后1～8天内,致死中浓度LC50值在8.4718×105～2.6098×10 12PIBs/mL之间;在饲毒浓度范围内,致死中时间LT50在1.98～6.99天之间;感病幼虫每日病死时间分布可用时间-剂量-条件死亡率模型较好地拟合,累计病死时间分布可用Gompertz模型拟合.室内试验还表明,病毒对甜菜夜蛾种群有明显的亚致死作用.免于病死蛹重量减轻,羽化所得雌成虫产卵量下降,产卵期延长;从感病高龄幼虫发育的成虫能将病毒传播至子代,引起20%～48%的子代幼虫死亡,并使子代成虫产卵量下降.田间试验表明,病毒除具有明显的控制作用外,还有一定的后效作用.
URLPMID:23136793 [本文引用: 1]
It is demonstrated for the first time that viral infection can be formed in insects survived after infection of gypsy moth larvae by nucleopolyhedrovirus (NPV), and cause subsequent mortality of individuals during, at minimum, two generations (the period of observations). The vertical virus transmission is carried by both male and female insects survived after infection. It is shown by means of PCR that the level of virus carrying in gypsy moth embryos of generations F1 and F2 is higher than the number of insects killed by NPV.
DOI:10.3390/v11040337URL [本文引用: 2]
URL [本文引用: 2]
Primers were designed according to sequence of polyhedrin gene of HaNPV, polymerase chain reaction(PCR) technology was employed to detect Helicoverpa armigera nucleopolyhedrovirus DNA sequences from viral polyhedral inclusion bodies (PIB). The level of sensitivity by the technology was as low as 10 fg. PCR amplification of polyhedrin gene sequences demonstrated that the virus was present in eggs and pupaes whose parents infected with LC50 (5.26×104PIB). This method may provide a better understanding of HaNPV epizootiology as well as evidence of vertical transmission of the virus at molecular level.
URL [本文引用: 2]
Primers were designed according to sequence of polyhedrin gene of HaNPV, polymerase chain reaction(PCR) technology was employed to detect Helicoverpa armigera nucleopolyhedrovirus DNA sequences from viral polyhedral inclusion bodies (PIB). The level of sensitivity by the technology was as low as 10 fg. PCR amplification of polyhedrin gene sequences demonstrated that the virus was present in eggs and pupaes whose parents infected with LC50 (5.26×104PIB). This method may provide a better understanding of HaNPV epizootiology as well as evidence of vertical transmission of the virus at molecular level.
[D].
[本文引用: 2]
[D].
[本文引用: 2]
DOI:10.1371/journal.pone.0147882URLPMID:26848752 [本文引用: 1]
Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are homologous to other baculovirus genes including 37 baculovirus core genes. Thirty-two (32) ORFs are unique to CnmeGV with no homologues detected in the GeneBank and 53 tandem repeats (TRs) with sequence length from 25 to 551 nt intersperse throughout the genome of CnmeGV. Six (6) homologous regions (hrs) were identified interspersed throughout the genome. Hr2 contains 11 imperfect palindromes and a high content of AT sequence (about 73%). The unique ORF28 contains a coiled-coil region and a zinc finger-like domain of 4-50 residues specialized by two C2C2 zinc finger motifs that putatively bound two atoms of zinc. ORF21 encoding a chit-1 protein suggesting a horizontal gene transfer from alphabaculovirus. The putative protein presents two carbohydrate-binding module family 14 (CBM_14) domains rather than other homologues detected from betabaculovirus that only contains one chit-binding region. Gene synteny maps showed the colinearity of sequenced betabaculovirus. Phylogenetic analysis indicated that CnmeGV grouped in the betabaculovirus, with a close relation to AdorGV. The cladogram obtained in this work grouped the 17 complete GV genomes in one monophyletic clade. CnmeGV represents a new crambidae host-isolated virus species from the genus Betabaculovirus and is most closely relative of AdorGV. The analyses and information derived from this study will provide a better understanding of the pathological symptoms caused by this virus and its potential use as a microbial pesticide.
[本文引用: 1]
[本文引用: 1]
DOI:10.1016/S2095-3119(18)62097-0URL [本文引用: 1]
DOI:10.1016/S1672-6308(12)60047-XURL [本文引用: 1]
[本文引用: 3]
[本文引用: 3]
DOI:10.3864/j.issn.0578-1752.2019.09.005URL [本文引用: 2]
【目的】葡萄霜霉病是葡萄生产上重要的单年流行病害,研究旨在构建葡萄霜霉病菌（Plasmopara viticola）的实时荧光定量PCR（real-time PCR）检测体系,为葡萄霜霉病的早期诊断和预测预报提供依据。【方法】依据GenBank中葡萄霜霉病菌cox2基因序列设计1对特异性引物（F-cox-Pv/R-Pv）,建立并优化常规PCR和real-time PCR反应体系,利用葡萄霜霉病菌、葡萄炭疽病菌（Colletotrichum gloeosporioides）、葡萄白粉病菌（Uncinula necator）、葡萄灰霉病菌（Botrytis cinerea）、葡萄白腐病菌（Coniella diplodiella）、葡萄溃疡病菌（Botryosphaeria dothidea）、白菜黑斑病菌（Alternaria brassicae)、辣椒炭疽病菌（C. capsica）、甘草根腐病菌（Fusarium solani）、西葫芦白粉病菌（Sphaerotheca fuliginea）、番茄菌核病菌（Sclerotinia sclerotiorum）、马铃薯干腐病菌（F. equiseti）、西瓜枯萎病菌（F. oxysporum）、哈茨木霉（Trichoderma harzianum）等14种葡萄及其他作物病原菌和拮抗菌的菌丝DNA进行常规PCR和real-time PCR特异性检测,并对灵敏度和可重复性进行评价。运用已构建的real-time PCR体系对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行定量检测,利用SPSS 19.0软件分析接种时间与叶片内葡萄霜霉病菌潜伏侵染量的关系。【结果】研究设计的引物特异性高,常规PCR仅对葡萄霜霉病菌DNA有扩增条带,为139 bp;real-time PCR检测结果表明该对引物对葡萄霜霉病菌有唯一的产物吸收峰,对其他供试菌株均未检测到产物吸收峰。常规PCR检测的灵敏度为10 pg·μL -1,real-time PCR的灵敏度可达到0.1 pg·μL -1,是常规PCR检测灵敏度的100倍。以携带目的基因片段的重组质粒为标准品,构建real-time PCR循环阈值（Ct）与模板浓度的线性关系,标准曲线y=42.27-3.36x,相关系数R 2=0.997,扩增效率为98.50%,线性范围达7个数量级,在2.4×10 3—2.4×10 9 copies/μL呈现良好的线性关系。对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行real-time PCR检测,结果表明叶片内病原菌潜伏侵染量随接种时间的变化呈指数关系增长,y=6.34×10 4·e 0.084 x,相关系数R 2=0.936。该real-time PCR检测体系在接种6 h后就可以检测到葡萄霜霉病菌DNA,检测量为5.68×10 4 copies/μL病原菌DNA。 【结论】构建的葡萄霜霉病菌real-time PCR检测体系的灵敏度远高于常规PCR,且特异性强、重复性好,其Ct值与模板浓度呈较好的线性关系,扩增效率高,可用于定量检测葡萄霜霉病菌的潜伏侵染量。
DOI:10.3864/j.issn.0578-1752.2019.09.005URL [本文引用: 2]
【目的】葡萄霜霉病是葡萄生产上重要的单年流行病害,研究旨在构建葡萄霜霉病菌（Plasmopara viticola）的实时荧光定量PCR（real-time PCR）检测体系,为葡萄霜霉病的早期诊断和预测预报提供依据。【方法】依据GenBank中葡萄霜霉病菌cox2基因序列设计1对特异性引物（F-cox-Pv/R-Pv）,建立并优化常规PCR和real-time PCR反应体系,利用葡萄霜霉病菌、葡萄炭疽病菌（Colletotrichum gloeosporioides）、葡萄白粉病菌（Uncinula necator）、葡萄灰霉病菌（Botrytis cinerea）、葡萄白腐病菌（Coniella diplodiella）、葡萄溃疡病菌（Botryosphaeria dothidea）、白菜黑斑病菌（Alternaria brassicae)、辣椒炭疽病菌（C. capsica）、甘草根腐病菌（Fusarium solani）、西葫芦白粉病菌（Sphaerotheca fuliginea）、番茄菌核病菌（Sclerotinia sclerotiorum）、马铃薯干腐病菌（F. equiseti）、西瓜枯萎病菌（F. oxysporum）、哈茨木霉（Trichoderma harzianum）等14种葡萄及其他作物病原菌和拮抗菌的菌丝DNA进行常规PCR和real-time PCR特异性检测,并对灵敏度和可重复性进行评价。运用已构建的real-time PCR体系对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行定量检测,利用SPSS 19.0软件分析接种时间与叶片内葡萄霜霉病菌潜伏侵染量的关系。【结果】研究设计的引物特异性高,常规PCR仅对葡萄霜霉病菌DNA有扩增条带,为139 bp;real-time PCR检测结果表明该对引物对葡萄霜霉病菌有唯一的产物吸收峰,对其他供试菌株均未检测到产物吸收峰。常规PCR检测的灵敏度为10 pg·μL -1,real-time PCR的灵敏度可达到0.1 pg·μL -1,是常规PCR检测灵敏度的100倍。以携带目的基因片段的重组质粒为标准品,构建real-time PCR循环阈值（Ct）与模板浓度的线性关系,标准曲线y=42.27-3.36x,相关系数R 2=0.997,扩增效率为98.50%,线性范围达7个数量级,在2.4×10 3—2.4×10 9 copies/μL呈现良好的线性关系。对人工接种葡萄霜霉病菌的潜育期叶片内病原菌DNA进行real-time PCR检测,结果表明叶片内病原菌潜伏侵染量随接种时间的变化呈指数关系增长,y=6.34×10 4·e 0.084 x,相关系数R 2=0.936。该real-time PCR检测体系在接种6 h后就可以检测到葡萄霜霉病菌DNA,检测量为5.68×10 4 copies/μL病原菌DNA。 【结论】构建的葡萄霜霉病菌real-time PCR检测体系的灵敏度远高于常规PCR,且特异性强、重复性好,其Ct值与模板浓度呈较好的线性关系,扩增效率高,可用于定量检测葡萄霜霉病菌的潜伏侵染量。
[本文引用: 2]
[本文引用: 2]
[本文引用: 2]
[本文引用: 2]
DOI:10.1016/j.jip.2004.05.008URLPMID:15491594 [本文引用: 1]
Nucleopolyhedrovirus (NPV) was tested for vertical transmission in the silkworm, Bombyx mori. Fifth instar larvae were exposed to four different dosages of BmNPV (830, 1300, 1800, and 2000OBs/larva) and a dosage of about 2000OBs/larva was found suitable for obtaining infected adults. Histopathological studies revealed the infection in susceptible tissues and organs initially, and at later stages of infection cycles the spermatocytes and nurse cells in the young oocytes were infected in the larval rudiments of testis and ovary, respectively. The mating of infected females with uninfected males resulted in significant reduction in fecundity (P < 0.01) and hatching of eggs (P < 0.001) due to transovarial transmission of BmNPV. Mating tests of uninfected females and infected males also confirmed venereal transmission as there was a significant reduction in hatching of eggs (P < 0.01). Further, among the F1 hybrid offspring (infected female x uninfected male) that were infected transovarially, larval progeny died at first and second instar stages, whereas those infected venereally developed acute lethal infection late and died by the end of third and fourth instar stage. PCR amplification and sequencing of 473bp of immediate early-1 (ie-1) gene of BmNPV isolated from the viral-infected parent and the F1 offspring confirmed that the viral infection is vertically transmitted to the progeny.
DOI:10.1046/j.0962-1083.2001.01439.xURLPMID:11918789 [本文引用: 1]
Knowledge of the mechanisms of pathogen persistence in relation to fluctuations in host density is crucial to our understanding of disease dynamics. In the case of insect baculoviruses, which are typically transmitted horizontally via a lifestage that can persist outside the host, a key issue that remains to be elucidated is whether the virus can also be transmitted vertically as a sublethal infection. We show that RNA transcripts for the Plodia interpunctella GV granulin gene are present in a high proportion of P. interpunctella insects that survive virus challenge. Granulin is a late-expressed gene that is only transcribed after viral genome replication, its presence thus strongly indicates that viral genome replication has occurred. Almost all insects surviving the virus challenge tested positive for viral RNA in the larval and pupal stage. However, this proportion declined in the emerging adults. Granulin mRNA was also detected in both the ovaries and testes, which may represent a putative mechanism by which reduced fecundity in sublethally affected hosts might be manifested. RNA transcripts were also detected in 60-80% of second-generation larvae that were derived from mating surviving adults, but there was no difference between the sexes, with both males and females capable of transmitting a sublethal infection to their offspring. The data indicate that low-level persistent infection, with at least limited gene expression, can occur in P. interpunctella following survival of a granulovirus challenge. We believe that this is the first demonstration of a persistent, sublethal infection by a baculovirus to be initiated by a sublethal virus dose. We hypothesize that the 'latent' baculovirus infections frequently referred to in the literature may also be low level persistent, sublethal infections resulting from survival from initial baculovirus exposure.
DOI:10.1146/annurev-ento-112408-085450URLPMID:19775239 [本文引用: 1]
The insect midgut is an important site of entry for pathogens and insect control agents. This review focuses on recent information related to midgut epithelial growth, metamorphosis, and repair as a defense against pathogens. The roles of stem cell mitogens and differentiation factors are described. Included is a discussion of apoptosis and autophagy in the yellow body. Sloughing, also described, protects the midgut from virus infections and bacterial toxins through death and replacement of affected cells. The mechanisms by which the repair process reduces the effectiveness of pest control strategies are discussed. Primary tissue culture methods also are described, and their value in understanding the mechanisms by which biologically based insecticides work is discussed.
DOI:10.3390/insects6030746URLPMID:26463414 [本文引用: 1]
Many pathogens and parasites are present in host individuals and populations without any obvious signs of disease. This is particularly true for baculoviruses infecting lepidopteran hosts, where studies have shown that covert persistent viral infections are almost ubiquitous in many species. To date, the infection intensity of covert viruses has rarely been quantified. In this study, we investigated the dynamics of a covert baculovirus infection within the lepidopteran crop pest Spodoptera exempta. A real-time quantitative polymerase chain reaction (qPCR) procedure using a 5' nuclease hydrolysis (TaqMan) probe was developed for specific detection and quantification of Spodoptera exempta nucleopolyhedrovirus (SpexNPV). The qPCR assay indicated that covert baculovirus dynamics varied considerably over the course of the host life-cycle, with infection load peaking in early larval instars and being lowest in adults and final-instar larvae. Adult dissections indicated that, contrary to expectation, viral load aggregation was highest in the head, wings and legs, and lowest in the thorax and abdomen. The data presented here have broad implications relating to our understanding of transmission patterns of baculoviruses and the role of covert infections in host-pathogen dynamics.
DOI:10.1134/S1062359019030038URL [本文引用: 1]
URL [本文引用: 1]
Effects of SpltMNPV on survival, development, fecundity of Spodoptera litura adults and their progeny were investigated in this paper. Survival of adults and eggs deposited were reduced in adults exposed to diet added with SpltMNPV. Fecundity was 1530 eggs when the females were confined to diet without SpltMNPV, and 1213, 1340 and 1087 eggs when the males only, the females only and both the males and females were exposed to diets with SpltMNPV, respectively. Adult ingestion of SpltMNPV inhibited progeny population growth, IIPC being 0.2658, 0.3046 and 0.1807 when the males only, the females only and both the males and females ingested diet with SpltMNPV, respectively.
URL [本文引用: 1]
Effects of SpltMNPV on survival, development, fecundity of Spodoptera litura adults and their progeny were investigated in this paper. Survival of adults and eggs deposited were reduced in adults exposed to diet added with SpltMNPV. Fecundity was 1530 eggs when the females were confined to diet without SpltMNPV, and 1213, 1340 and 1087 eggs when the males only, the females only and both the males and females were exposed to diets with SpltMNPV, respectively. Adult ingestion of SpltMNPV inhibited progeny population growth, IIPC being 0.2658, 0.3046 and 0.1807 when the males only, the females only and both the males and females ingested diet with SpltMNPV, respectively.
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
[本文引用: 1]
DOI:10.1016/j.virol.2010.07.033URLPMID:20800868 [本文引用: 1]
MicroRNAs (miRNAs) have emerged as key players in host-pathogen interaction. Recently, many virus-encoded miRNAs have been identified from different mammalian species. However, the large family of invertebrate viruses of Baculoviridae, which infects diverse species of beneficial insects and agriculture pests, has hardly been investigated for elucidating the role of miRNAs in host-pathogen interaction. In the study reported here, we have identified four Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNAs using a combination of in silico and experimental methods. Unlike other reported viral miRNAs, the BmNPV-encoded miRNAs identified in the present study were found to be evolutionarily conserved among many closely related baculoviruses. Besides, we have computationally predicted 8 viral and 64 cellular targets of these virus-encoded miRNAs and the putative functions of these targets suggest a key role of viral miRNAs in insect-pathogen interactions by modulating several viral replication genes as well as those involved in host immune defense machinery.
DOI:10.1111/jen.2017.141.issue-5URL [本文引用: 1]
DOI:10.1099/vir.0.028027-0URL [本文引用: 1]
