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題名 | 含蘇力菌毒素基因cry1Aa1之轉形葉表生菌Erwinia herbicola的殺蟲效力與拮抗植物病原菌之分析=Insecticidal Efficacy and Phytopathogenic Antagonism of the Epiphytic Erwinia herbicola Strains Transformed with the Bacillus thuringiensis cry1Aa1 Gene |
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作者 | 林志輝; 曾經洲; 高穗生; 陳良築; Lin, Chi-hui; Tzeng, Ching-chou; Kao, Suey-sheng; Chen, Liang-jwu; |
期刊 | 中華農學會報 |
出版日期 | 20030200 |
卷期 | 4:1 2003.02[民92.02] |
頁次 | 頁17-29 |
分類號 | 433.4 |
語文 | chi |
關鍵詞 | 葉表生菌; 蘇力菌殺蟲晶體蛋白; 小菜蛾; 拮抗作用; 黑腐病; 軟腐病; Epiphytic erwinia herbicola; Bacillus thuringiensis insecticidal crystal protein; Diamondback moth; Antagonism; Black rot; Soft rot; |
中文摘要 | 葉表生菌Erwinia herbicola常見於蔬菜及果樹等植物葉面,與大腸桿菌Escherichia coli同屬於腸道科細菌,適合作為蘇力菌基因轉形的新目標。經由大腸桿菌的質體pUN4,已經成功地將蘇力菌毒素蛋白基因cry1Aal轉形至E. herbicola菌體內。由其表現之CrylAal毒素蛋白對小菜蛾具有顯著之殺蟲效果。利用葉表生菌能在葉面形成菌落之特性,將轉形菌株之一的Eh4直接噴灑於甘監葉面,測試活菌對小菜蛾之殺蟲效力,並以未轉形菌株作為試驗對照組。菌體在葉面上維持1、4、11、14、17天後,以圓形鋼管切下15c□之葉圓片餵食小菜蛾,結果顯示在試驗處理48小時後,小菜蛾取食含Eh4的葉片所造成之死亡率可達36.7~60%,處理72小時後,死亡率則在40~83.3%之間,相當於使用含Cry1Aal之E. herbiicola菌體總蛋白質劑量0.312~0.625mg/mgl在處理72小時後所造成之死亡率。而未轉形之表生菌不會造成小菜蛾死亡。由死亡率與蟲體取食葉片的狀況觀察,轉形菌株的殺蟲效果會隨著時間遞減。此外,在植物病原菌拮抗能力分析方面,在PDA培養基上塗抹胡瓜細菌性斑點病之Pseudomonas syringae、十字花科蔬菜黑腐病之Xanthomonas campestris pv. Campestris及軟腐病之Erwinia carotovora subsp. Carotovora細菌草皮,測試E. herbicola排斥其他細菌生長之情形。結果顯示E. herbicola對P. syringae生長的抑制效果較差,但對X. campestris 與E. carotovora生長的抑制效果顯著。本試驗結果首度顯示應用轉形蘇力菌毒素基因之殺蟲效力,結合葉表生菌的葉面附生特性與特有之微生物拮抗作用,可使轉形之葉表生菌成為新一代的生物性農藥。 |
英文摘要 | The epiphytic Erwinia herbicola (also known as Pantoea agglomerans) is commonly found on the foliar surface of vegetables and fruits. Falling in the same family of the Escherichia coli (E. coli), the E. herbicola presents common accessibility for genetic transformation as the E. coli system. The E. herbicola has been successfully transformed with a Bacillus thuringiensis toxin gene cry1Aal presented in plasmid pUN4. Protein extracts isolated from transformed E. herbicola strains showed significant insecticidal activity against diamondback moth (DBM), Plutella xylostella. Based on the characteristic of E. herbicola, one of the transformed e. herbicola strains, Eh4, was sprayed on cabbage leaves for colonization to test its insecticidal efficacy and persistency against the DBM. The cabbage leaf discs with an area of 15c□ cut from the growing cabbage, 1, 4, 11, 14 and 17 days respectively after spraying, and were used for feeding assays. The mortality rates of DBM were 36.7~60% and 40~83.3% after feeding for 48 and 72 hours, respectively. The mortality caused by living bacteria after 72 hours was equivalent to that caused by 0.312~0.625 mg/ml of the extracted proteins. Non-transformed E. herbicola strain Eh1 presented no insecticidal properties. From the mortality and leaf consumption assays, a decreased of insecticidal efficacy with a concomitant increase of time interval after spraying e. herbicola was observed. The antagonistic effects of E. herbicola on the phytopathogens Pseudomonas syringe, Xanthomonas campestris pv. Campestris and Erwinia carotovora subsp. catrotovora were performed on PDA media by spotting E. herbicola on the top of the phytopathogenic bacterial lawns. The results showed that there was a significant suppression against X. campestris pv. Campestris and E. carotovora subsp. carotovora subsp. carotovora after hours of incubation; however, only a lesser degree of suppression against P. syringae was observed as weak inhibition zones appeared only after 24 hours of incubation. This report is the first example demonstrating the combinations of the insecticidal activity (by transforming B. thuringiensis endotoxin gene), and the characteristics of antagonism and foliar colonization in the same epiphytic bacteria. Accordingly, these genetic-engineered epiphytic bacteria could present effective measures in controlling both insects and pathogens as well as a novel bio=pesticide in routine practices. |
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