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  • 學位論文

線蟲捕捉菌Arthrobotrys musiformis之幾丁質分解酵素蛋白生化特性分析; 小花蔓澤蘭Mikania micrantha之本土真菌性病原生物防治潛力評估

Purification and characterization of chitinase from nematode-trapping fungi Arthrobotrys musiformis; Fungal pathogens of invaded Mikania micrantha biocontrol appraisal

指導教授 : 曾顯雄

摘要


線蟲捕捉菌Arthrobotrys musiformis之幾丁質分解酵素蛋白生化特性分析 幾丁質(chitin)是由N-acetyl-β-D-glucosamine(GlcNAc)單體,經β-1,4鍵結所聚合而成的一種直鏈狀多醣類聚合物,廣泛存在許多生物體中,包括甲殼類動物、真菌、線蟲及昆蟲。某些線蟲內部寄生菌已知在侵染線蟲過程中時,需分泌幾丁質分解酵素以突破線蟲卵壁幾丁質層之障礙。本研究的主要目的在於探討線蟲捕捉菌Arthrobtrys musiformis是否產生幾丁質分解酵素及其於侵染線蟲寄主時所賦有之功能。 Arthrobtrys musiformis培養於可誘導幾丁脢培養基,其幾丁脢及總蛋白含量於第四天到達高峰,以減壓濃縮,等電點分離,陰離子樹脂管柱,疏水性蛋白質管柱層析,純化並檢測各部分純化分割液之活性。獲知Arthrobtrys musiformis之幾丁脢偏酸性等電點為pH 3.4-pH 3.8,可能為trimer,由分子量大小約為29kDa,33kDa以及 38kDa之單體蛋白質所組成,擬由此純化之幾丁脢解序N端胺基酸序列進行相關基因選殖,因量偏低,並未獲得結果。 小花蔓澤蘭Mikania micrantha之本土真菌性病原生物防治潛力評估 於民國九十二年五月至九十三年六月期間在南投縣鹿谷鄉、竹山鎮、水里鎮、魚池鄉、埔里鎮、國姓鄉,台中縣太平市。彰化縣彰化市、埔心鄉。嘉義市,嘉義縣中埔鄉、竹崎鎮、梅山鄉,台南縣龍崎鄉、關廟鄉、新化鎮、玉井鄉,高雄縣杉林鄉、三民鄉、旗山鎮,屏東縣霧臺鄉、內埔鄉、泰武鄉,花蓮市,花蓮縣秀林鄉、吉安鄉、壽豐鄉、豐濱鄉,台東縣台東市等地調查、分離、鑑定可侵染小花蔓澤蘭之莖、葉、花部之本土性寄生性真菌天敵。由病班所分離之菌株共297株,初步鑑定至少涵蓋13屬真菌:Acremonium sp. Alternaria sp. Colletotrichum sp. Curcularia sp. Cyllindrocladium sp. Fusarium sp. Leptosphaeria sp. Macrophomina sp. Nigrospora sp. Pestalotiopsis sp. Phoma sp. Phomopsis sp. Phyllostica sp.。在此主要以Colletotrichum sp.侵染小花蔓澤蘭造成的葉、莖部炭疽病班最為常見;Phomposis sp.可引起小花蔓澤蘭葉部及莖部壞疽病班及葉脈及莖部褐化;Phoma sp.及Macrophomina sp. 感染葉片造成嚴重的黃化、水浸狀壞疽病班,常可導致野外小花蔓澤蘭區塊性風土病。此外,多種真菌性病原天敵也可同時感染小花蔓澤蘭,而加劇其罹病程度。經由菌絲圓盤接種、胞子懸浮液接種測試分離之真菌對小花蔓澤蘭之侵染病原性強弱,有9屬34種真菌可造成小花蔓澤蘭葉片黃化至壞疽病徵。但以真菌培養濾液噴灑小花蔓澤蘭葉片並無任何明顯之病徵呈現。

並列摘要


Purification and characterization of chitinase from nematode-trapping fungi Arthrobotrys musiformis Chitin, an essential component of exoskelton, cuticular layer, or cell wall of crustacean, insect, nematode or fungi, is a linear polymer of amino sugar N-acetyl glucosamine, linked by β-1,4-glycosidic linkage. Some reported nematode egg endoparasites with the aid of chitinase are able to penetrate the nematode egg shell. In a comparative study, we attempt to investigate the chitinase and encoding gene of Arthrobotry musiformis , a nematode trapping fungi. The chitinase in conducive culture medium was partially purified by anionic, hydrophobic and gel filtration high performance chromatography. The chitinase pI is around pH 3.4- pH 3.8, composed by three monomer, 29kDa, 33kDa and 38kDa in end purification step its activity, was proved both in gel or by spectrophotometry. Due to the low quantity of chitinase in the last purified fractionation, try to decode the N-terminal amino acid residue sequence fail, which further hinders the possibility for primer design to clone the encoding gene. Fungal pathogens of invaded Mikania micrantha biocontrol appraisal Investigation of the domestic fungal enemies against the invaded Mikania micrantha were carried out from May 2003 to June 2004 in more than 20 counties islandwide. Of totally 297 fungal strains have been isolated, 13 genera represent Acremonium , Alternaria , Colletotrichum , Curcularia , Cyllindrocladium , Fusarium , Leptosphaeria , Macrophomina , Nigrospora , Pestalotiopsis . Phoma . Phomopsis . Phyllostica genera were recovered. Of them, several Colletotrichum spices which invaded the leaf and stem of M. micrantha resulted in leaf spot and canker were mostly often encountered, and less than by Phomopsis, Phoma and Macrophomina. Some of these fungal pathogens can simultaneously infected M. micrantha and cause endemic. Inoculation of the invaded weed with the spore suspension in greenhouse, some Colletotrichum, Phomopsis, Phoma or Macrophomina spices can cause spot, necrosis, canker or blight of varied degree severity in leaves or stems. But the spraying culture filtrates cannot cause any symptoms, which preliminarily exclude the involvement of toxin in disease development.

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