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National Chung Hsing University Institutional Repository - NCHUIR > 農業暨自然資源學院 > 昆蟲學系 > 依資料類型分類 > 碩博士論文 >  綠殭菌(Nomuraearileyi)毒素對斜紋夜蛾(Spodopteralitura)幼蟲之影響

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/104049

標題: 綠殭菌(Nomuraearileyi)毒素對斜紋夜蛾(Spodopteralitura)幼蟲之影響
Effect of the toxin produced by the entomopathogenic fungus, Nomuraea rileyi, on larvae of Spodoptera litura
作者: 施淑蓮
Contributors: 侯豐男
Feng-nan Hou
國立中興大學
關鍵字: Nomuraea rileyi;Spodoptera litura;toxin;protein;after
蟲生真菌綠殭菌;斜紋夜蛾;毒素;蛋白質
日期: 2005
Issue Date: 2012-09-04 13:27:41 (UTC+8)
Publisher: 昆蟲學系
摘要: 蟲生真菌綠殭菌(Nomuraea rileyi)雖可感染30多種鱗翅目蟲類,但在真菌毒素的產生之研究方面幾無報告,不如黑殭菌(Metarhizium anisopliae)及白殭菌(Beauveria bassiana)皆已純化出毒素 ; 故本研究主要是針對綠殭菌在致病過程中,是否產生真菌毒素及其對斜紋夜蛾(Spodoptera litura)幼蟲之毒性進行研究。將實驗室中的17株綠殭菌菌株(isolate)以PDB+Y液體培養基培養12天,經過濾後將濾液注射五齡斜紋夜蛾幼蟲,結果顯示SH1和TC菌株在第10天時會對90%以上的幼蟲造成明顯不取食、脫皮不全和生長遲緩等影響,PZ1菌株約58%,而其他菌株之影響約在25%以下。將17株綠殭菌之培養液注射五齡幼蟲後,連續測量體重7天,結果顯示注射SH1和TC菌株培養液之幼蟲重量會明顯逐漸下降,最後造成死亡;由以上結果顯示此二菌株可產生真菌毒素,並具有劑量依賴( dose-dependent )之特性。另分別將SH1菌株培養液經高溫、蛋白酶(proteinase K) 及乙醇處理後,皆能降低SH1菌株對斜紋夜蛾幼蟲的毒性,因此推測此培養液內之真菌毒素應為蛋白質類。以濃縮10倍培養液進行SDS-PAGE分析,顯示SH1菌株較其他菌株在50k Da左右產生一獨特之蛋白質帶,推測此蛋白質可能為SH1菌株造成斜紋夜蛾幼蟲致死之主要成分。另將SH1菌株培養液處理Sf-21細胞株,結果顯示SH1培養液在1/600、1/800和1/1000稀釋下,能抑制Sf-21細胞株的生長。亦取SH1培養液之相同劑量注射其他鱗翅目昆蟲,發現SH1培養液對四齡黑角舞蛾(Lymantria xylina)幼蟲具最高之毒性影響,可達96%亡死率,而對四齡玉米穗蛾(Helicoverpa armigera)及大蜡蛾(Galleria mellonella)幼蟲分別有76%及86%之死亡率。
The entomopathgenic fungus, Nomuraea rileyi, is pathogenic to over 30 species of Lepidoptera. Investigations on the toxin produced by N. rileyi were little, and not as many as those by Metarhizium anisopliae or Beauveria bassiana. The purpose of this study was to find out whether N. rileyi produces mycotoxin and causes toxicity to Spodoptera litura larvae during pathogenic process. Seventeen isolates of N. rileyi were cultivated with PDB+Y medium for 12 days in the laboratory. Then, the culture filtrates were injected into haemocoel of S. litura 5th instar larvae. The results showed that the toxin produced by isolates SH1 and TC caused 90% or more deterioration to larval growth of S. litura on tenth day, resulting in feeding reduction, incomplete ecdysis and hypoplasia. However, the isolate PZ1 caused 58%, while other isolates caused only 25% or lower. Treatment of S. litura larvae with the filtrated cultures of 17 isolates for 7 days showed that only isolates SH1 and TC were able to significantly decrease the larval weight, causing death finally, indicating that both isolates may produce mycotoxin. The SH1 toxin exhibited a dose-dependent character. The toxic activity on S. litura decreased as treated SH1 culture filtrates with heat, proteinase K or ethanol. This indicated that the toxin molecule in SH1 culture filtrate was protein in nature. A 50k Da protein band was obtained from SH1 culture filtrate using SDS-PAGE. This protein was suspicious to be the active ingredient against S. litura larvae. The cell number of Sf-21 cell lines was reduced after treating with isolate SH1 culture filtrate in vitro at dilutions of 1/600、1/800 and 1/1000. In addition, injection of 3 lepidopteran 4th instar larvae with SH1 culture filtrate resulted in 96% larval mortality in Lymantria xylina, 76% in Helicoverpa armigera and 86% in Galleria mellonella.
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