Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30756
標題: Pathogenic mechanism of Nomuraea rileyi SH1 cultured fluid against Galleria mellonella and development of a molecular technique for its diagnosis
綠殭菌SH1分離株培養液對大蠟蛾之致病機制暨其分子診斷技術之開發
作者: 曾羽凱
Tseng, Yu-Kai
關鍵字: http://etds.lib.nchu.edu.tw/etdservice/view_metadata?etdun=U0005-1208201017262400;綠殭菌;培養液;大蠟蛾;細胞性免疫;專一性引子
出版社: 昆蟲學系所
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摘要: 
In this study, it was found that high larval mortalities of the greater wax moth, Galleria mellonella, occurred when treated with the cultured fluid of Nomuraea rileyi SH1 isolate. The insecticidal activity of the fungal cultured fluid initiated from the 7th day after culturing, resulting in ca. 24% larval mortality and reached 100% at the 11th day. In addition, the number of days causing 100% larval mortality decreased as elevated the fluid concentration, indicating a dose-dependent activity. When the cultured fluids were pretreated with proteinase K and heating at 100℃ to deprive the insecticidal activity, SF21 cell line showed growth inhibition and apoptosis at 18 h after treatment with N. rileyi cultured fluid, but it remained normal if treated with those of Beauveria bassiana or Metarhizium anisopliae. This indicates that toxic components inducing apoptosis are present only in N. rileyi. Furthermore, phagocytic rates of hemocytes decreased over 80% in G. mellonella larvae treated with N. rileyi cultured fluid, and the number of nodules containing conidia decreased 74.5% in both in vitro and in vivo tests. The inhibition of phagocytosis by this cultured fluid was also found in G. mellonella larvae against conidia of B. bassiana and M. anisopliae, and Escherichia coli, indicating its non-specificity of phagocytic inhibition to microbial invaders. The cultured fluid pretreated with proteinase K was deprived of the insecticidal activity and its impairment on cellular immunity of G. mallonella hemocytes, revealing that its active components seem to be proteinaceous substances. Therefore, N. rileyi SH1 isolate could secrete toxic proteinaceous components to cause mortality and impairment of cellular immunity in G. mellonella larvae, and apoptosis of SF21 cells. For developing a molecular diagnosis of N. rileyi, a 1.4 kb of DNA fragment specific to N. rileyi was amplified using a random primer, OPF18 (AACCCGGGTT). The 289 bp of DNA fragment was amplified using a pair of specific primers, NS1/NS2, designed from it. The predicted size of DNA fragment was able to be amplified from 17 domestic N. rileyi isolates and other two from the United States using NS1/NS2 primers pair, but was not from B. bassiana and M. anisopliae. In sensitivity and interference assays, a predicted amplicon could be detected using as low as 0.1 ng DNA template and was not disturbed with Spodoptera litura DNA. A 289 bp amplicon was detected from living and mummified S. litura larvae infected with N. rileyi isolates but was not with B. bassiana and M. anisopliae using this specific primers pair. This result indicates that diagnosis of insects infected with N. rileyi can be correctly carried out one day after infection using NS1/NS2 primers pair. Thus, the NS1/NS2 primers pair could extensively be utilized to precisely diagnose field samples infected with N. rileyi.

在綠殭菌 (Nomuraea rileyi) 培養液的殺蟲效果測試發現SH1分離株對大蠟蛾幼蟲 (Galleria mellonella) 造成高死亡率,而且在第七天的培養液開始有毒殺作用,引起24%死亡率,在第十一日達100%死亡率。另隨著培養液濃度的提升,毒殺幼蟲達100%死亡率的日數隨之遞減,顯示有劑量依變的特性。當經蛋白酶 (proteinase K) 及100℃熱處理過的綠殭菌培養液,則失去對大蠟蛾幼蟲的高死亡率。又以綠殭菌SH1培養液處理SF21細胞株,細胞生長受到明顯抑制並可在18小時開始產生細胞凋亡 (apoptosis) ,但黑殭菌 (Metarhizium anisopliae) 與白殭菌 (Beauveria bassiana) 的培養液則無此現象,顯示綠殭菌培養液的有毒成分與其他蟲生真菌所分泌者不同。此外,在in vitro及in vivo試驗發現綠殭菌SH1培養液,可以抑制大蠟蛾幼蟲血球80%以上之吞噬率與降低74.5%含有孢子之瘤結率;同時,也抑制血球對黑殭菌、白殭菌分生孢子與大腸菌的吞噬作用,顯示該細胞性免疫所受之抑制對微生物不具專一性。當經蛋白酶處理過的綠殭菌培養液,則喪失對大蠟蛾幼蟲的毒殺及細胞性免疫之抑制,顯示有毒物質具蛋白質成分。因此,綠殭菌SH1可分泌有毒的蛋白質成分,一方面造成大蠟蛾幼蟲的高死亡率,也可直接抑制大蠟蛾幼蟲之細胞性免疫;另一方面,也會引起細胞凋亡。在綠殭菌分子診斷技術的開發上,將OPF18 (AACCCGGGTT) 隨機引子增幅的綠殭菌專一性1.4 kb 之DNA片段,從中設計一對sequence characterized amplified region (SCAR) 的專一性引子對NS1/NS2,以此可對綠殭菌genomic DNA增幅約289 bp大小的片段。取十七種國內收集的綠殭菌分離株及二種美國的綠殭菌品系,均可利用NS1/NS2增幅出預期的DNA片段,但對白殭菌及黑殭菌均無任何增幅片段。另進行靈敏度與干擾測試,顯示只要0.1 ng genomic DNA即可被增幅出預期片段,而且不受斜紋夜蛾 (Spodoptera litura) DNA的干擾。進一步將受綠殭菌感染的斜紋夜蛾之活蟲或殭蟲,也發現可被此專一性引子對增幅289 bp的片段,但受白殭菌與黑殭菌感染的殭蟲,並無任何的增幅片段,顯示NS1/NS2引子對可以準確診斷出受綠殭菌感染的蟲體,而且在感染後的第一天即可被正確的診斷,因此將可廣泛應用於田間標本的診斷。
URI: http://hdl.handle.net/11455/30756
其他識別: U0005-1208201017262400
Appears in Collections:昆蟲學系

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