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Screening, Identification and Improvement of Pathogenicity of Nucleopolyhedroviruses against Several Noctuid Pests
|摘要:||為開發本土高致病力之病毒株以防治害蟲，自本省朴子、埔里及霧峰三地分別採得玉米穗蟲(Helicoverpa armigera)、甜菜夜蛾(Spodoptera exigua)及斜紋夜蛾(Spodoptera litura)等三種夜蛾科害蟲之核多角體病毒(nucleopolyhedrovirus)。各病毒多角體之形態、病毒粒子內所含有之核蛋白鞘數量、多角體之蛋白質圖譜及基因核酸酵素切位與大小均有明顯差異，可做為鑑定病毒株之依據。三種病毒之多角體蛋白質間具有極高之交互免疫反應(cross immune reaction)。各病毒僅對本身寄主害蟲具極高之致病力及專一性，對同科或同屬之害蟲致病率低，對小菜蛾(Plutella xylostella)均不具致病力，故應用性受到窄寄主域之限制。
探討廣寄主域之加州苜蓿夜蛾核多角體病毒(Autographa californica nucleopolyhedrovirus, AcMNPV)對本省九種鱗翅目害蟲之致病力。結果以擬尺蠖(Trichoplusia ni)、小菜蛾及甜菜夜蛾之罹病死亡率最高。菸芽夜蛾(Heliothis virescens)、斜紋夜蛾、亞洲玉米螟(Ostrinia furnacalis)與大蠟蛾(Galleria mellonella)、玉米穗蟲次之，未發現罹病之外米綴蛾(Corcyra cephalonica)。AcMNPV除造成寄主急性感染使幼蟲潰爛死亡之外，還會影響幼蟲的正常生長發育，使其脫皮時間延遲或失敗，化蛹率及羽化率明顯下降。AcMNPV之致死時間太長，可利用適當的起動子表現蠍神經毒以加速其致死時效，增強該病毒之防治效果。故將一段以色列黃蠍(Leiurus quinquestriatus hebraeus)之鎮定神經毒(LqhIT2)基因，分別插入早期起動子(Phcmv*-1)及非常晚期起動子(p10)之下游，構築成含蠍毒之AcMNPV重組病毒，分別命名為vAptcmIT2及vAp10IT2。前者較後者提早約18小時表現蠍神經毒，唯最終產量不及後者多。vAptcmIT2及vAp10IT2多角體在蟲體內之產量受到嚴重的影響，僅為野生型病毒之32及7%，但在細胞株內之產量影響較小。重組病毒可造成幼蟲神經麻痺停止取食，感染擬尺蠖、甜菜夜蛾及小菜蛾幼蟲時，其致死時間較野生型病毒顯著提早。在田間盆栽試驗，含蠍毒重組病毒處理組亦較螢光重組病毒及野生型病毒顯著減少甘藍菜葉片受害面積，而早期表現起動子表現神經毒亦較晚期表現者之殺蟲效果為佳。利用AcMNPV廣寄主域的特性，加上基因改造後可提高其殺蟲效力，確可增強病毒在害蟲防治上的效益。
Three nucleopolyhedroviruses (NPV) isolated from the larvae of Helicoverpa armigera, Spodoptera exigua, and S. litura with nucleopolyhedrosis in Taiwan for development of biopesticides. Distinct differences were found in their morphology, structures, polyhedral protein profiles, and restriction fragment length patterns and sizes of these viral genomic DNAs. There was a closely immunological relationship of polyhedrins between these viruses. They were highly pathogenic to their own host, and less pathogenic to hosts of other viruses, but not pathogenic to the larvae of Plutella xylostella. The application of these viruses was limited because of the narrow host spectrum. Pathogenicity and histopathology of Autographa californica nucleopolyhedrovirus (AcMNPV) to larvae of 9 lepidopteran species in Taiwan were investigated. Trichoplusia ni, P. xylostella, and S. exigua were highly susceptible to AcMNPV; Heliothis virescens, S. litura, Ostrinia furnacalis, and Galleria mellonella were found to be moderately susceptible; H. armigera was weakly susceptible; Corcyra cephalonica was not infected. In the infected larvae, target cells became swollen and finally lysed resulting from the formation of inclusion bodies. Furthermore, the infected larvae prolonged larval development, failed to molt or pupate, and finally died. The lethal time caused by AcMNPV was too long to gain a good control efficacy. The genetically improved AcMNPVs were thus constructed with an insect-selective toxin gene (LqhIT2) derived from venom of the Israel yellow scorpion, Leiurus quinquestriatus hebraeus, depressant neurotoxin expressed by the early phase promoter (Phcmv*-1) and the very late p10 promoter, named vAptcmIT2 and vAp10IT2. Toxin expression of vAptcmIT2 was ca. 18 h earlier than that of vAp10IT2. The yields of polyhedra of recombinant viruses were significantly reduced than those of wild type AcMNPV in larvae. These two recombinant viruses demonstrated a significant reduction in lethal time. The recombinant virus, vAptcmIT2, was improved in killing T. ni than vAp10IT2. In the field trials, larvae of S. exigua and T. ni infected with the recombinant virus containing scorpion toxin fell from the plant and stopped feeding, resulting in reduction in leaf area eaten compared to those larvae infected with wild type AcMNPV and vAp10G(L). The recombinant virus, vAp10G, which contains green fluorescent protein (GFP), was slightly less pathogenic to insect hosts than wild type AcMNPV. The larvae infected with vAp10G emitted strong green fluorescence irradiated by a long-wavelength UV light. Its FT50 (50% fluorescense-emitting time) was ca. 3 days earlier than LT50. The difference in persistence between wild type AcMNPV and GFP-containing recombinant virus was not significant. The percentage of original activity remaining of vAp10G in February was higher than that in April. Increase in viral concentration was positively correlated with the control efficacy, even though a negative effect on parasitism of the parasitic braconid wasps was found. It is suggested that this recombinant virus can be used as a biomarker to confirm its infection and persistence in the field.
|Appears in Collections:||昆蟲學系|
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