Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31062
標題: 探討線蟲對殺線蟲劑的抗藥性
Discussion of the nematicide resistances in nematodes
作者: 林昱岑
Lin, Yuh-Tsen
關鍵字: 植物寄生性線蟲;free-living nematodes;抗藥性;乙醯膽鹼酯酶突變;plant-parasitic nematodes;nematicide-resistance;acetylcholinesterase;mutation
出版社: 植物病理學系所
引用: 伍、 參考文獻 林新強、蔡東纂。2004。田間殺線蟲劑抗藥性及人工誘導抗性線蟲族群之研究。 國立中興大學植物病理研究所第三十四屆碩士論文。54頁。 黃盈彥、蔡東纂、陳珮臻。2005。台灣地區松材線蟲之分布及鑑定分類。國立中興大學植物病理研究所第三十五屆碩士論文。64頁。 游培琪、蔡東纂。2004。山蘇花葉芽線蟲在台灣之發生。植物病理學會刊 13:35-44。 馮海東。1987。利用田間網龍研究抗藥性小菜峨之防治經濟。植保會刊 29:175-184。 廖龍盛。2004。實用農藥。修訂版。127-141頁。 陳嘉芬。2003。基礎遺傳學。第一版。藝軒出版社。33-43及177-205頁。 劉宗榮、康熙州、郭明良、翁祖輝。2003。基礎毒理學。再版。藝軒出版社。 33-43頁。 臺灣省政府農林廳。1999。植物保護手冊。209-218頁。 蔡東纂。1999。植物寄生性線蟲病害之化學防治。植物病理學會刊 8:41-50。 Agrios G. N. 2005. Plant diseases caused by nematode. Pages 826-872 in Plant pathology. Elsevier Inc. Burlington MA USA, 922 pp. Arpagaus M., Fedon Y., Cousin X., Chatonnet A., Berges J., Fournier D. and Toutant J. 1994. cDNA sequence, gene structure, and in vitro expression of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. J. Biol. Chem. (13) 269: 9957-9965. Batterby S., Le Patourel G. N. J., and Wright D. J. 1977. 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摘要: 
觀察具抗藥性的葉芽線蟲(Aphelenchoides besseyi)及腐生性線蟲(Rhabditis spp.)族群其抗藥性維持的能力;由於兩者生活史的差異,使大多數的腐生線蟲(Rhabditis spp.)族群失去其抗藥性,而葉芽線蟲A. besseyi則僅有AE10及AE100兩族群失去抗藥性。以有機磷類藥劑的普伏松(ethoprop)、芬滅松(phenamiphos),及氨基甲酸鹽類的加保伏(carbofuran)與歐殺滅(oxamyl)四種藥劑處理目前已被完整解序的Caenorhabditis elegans,計算其半數致死濃度(LC50),並以低濃度藥劑人工誘導及高濃度藥劑的篩選方式,建立兩種腐生線蟲(C. elegans及Rhabditis spp.)、葉芽線蟲(A. besseyi)及兩種松材線蟲(Bursaphelenchus xylophilus 及未知種 Bursaphelenchus sp.)的抗藥性族群。C. elegans的半數致死濃度以對歐殺滅的803 ppm最高,對普伏松的260.8 ppm最低。在誘導及篩選抗藥性族群方面,則是發現腐生線蟲C. elegans及葉芽線蟲對於有機磷類藥劑感受性較氨基甲酸鹽類為高;供試5種線蟲中的三種植物寄生性線蟲於藥劑的感受性較另兩種腐生性線蟲高;其中同屬的兩種松材線蟲對藥劑的感受性有高達450 ppm的差異;造成這些差異的原因可能是藥劑可在線蟲體內累積或經由代謝過程消耗、線蟲個體對藥劑的感受性差異及生殖方式的不同。待抗藥性線蟲族群建立之後,利用分子生物學的技術找尋藥劑目標分子--乙醯膽鹼酯酶(acetylcholinesterase,AChE)基因上的差異。使用C. elegans ace-1的引子對增幅10個C. elegans抗藥性族群(含未以藥劑處理者)可增幅到8個族群,片段大小約為2.0 kb;ace-2則僅有1個族群被增幅出預期片段;ace-3增幅到4個族群;ace-4引子對則沒有增幅到任何族群的條帶;表示這些具不同抗藥能力的C. elegans族群在Class A到Class D的四種AChE表現量上有所差異。進一步將上述的7個 ace-1片段進行解序,僅有CO1000族群的ace-1核酸序列產生改變;其它抗藥性族群未如預期於AChE序列上產生突變,可能是由於實驗的核酸來源單位為族群、AChE的表現量不同,甚至可能是其它有關解毒的基因變異才產生抗藥性。另外,也以C. elegans 4種ace的引子對增幅其它4種不同取食型態之線蟲, ace-1及ace-4引子對皆沒有增幅出任何片段,而ace-2及 ace-3引子對增幅4種不同線蟲,皆增幅出大小不一的數種片段,進一步挑選其中四個片段相同大小的進行解序,並與C. elegans ace-2比對,相似度僅24.7~24.1 %,但AC500B-1.1及AO500B-1.1與C. elegans小片段的X染色體基因相似。此外以腐生線蟲C. elegans、C. briggsae及根瘤線蟲Meloidogyne incognita、M. javanica的ace-1相似區間設計引子對,增幅本試驗的5種未施藥線蟲,成功在4種線蟲上增幅到cDNA片段,挑選其中2種線蟲的5條序列進行解序及比對,結果與C. elegans ace-1相似僅為23.6~27.6 %,但其中JCKmA-1.2及JCKmA-0.6分別與黃果蠅Drosophila melanogaster及黑猩猩Pan troglodytes性染色體X上的小片段序列相似。

Nine of the 11 nematicide-resistant Rhabditis sp. populations, but only two out of 12 Aphelenchoides besseyi populations, lost their resistance after 16 months in the absence of nematicides. Calculated LC50 of Caenorhabditis elegans with four nematicides, and selected nematicide- resistant populations. The free-living nematodes, Rhabditis sp. and three plant-parasitic nematodes, A. besseyi, Bursaphelenchus sp. and B. xylophilus were treated with low concentrations of the 4 nematicides to induce the resistant nematodes and to treated with high concentration of nematicide to select the nematodes. The LC50 of C. elegans with ethoprop, phenamiphos, carbofuran or oxamyl was 260.8 ppm, 511.6 ppm, 783.7 ppm and 803 ppm respectively. Seventy eight induced or selected nematode populations with different levels of nematicide resistance were obtained. C. elegans and A. besseyi tested were more sensitive to organophosphate nematicides than carbamate nematicides. Our results also showed that free-living nematodes were more resistant to all four nematicides than plant-parasitic nematodes tested. Moreover, B. xylophilus was more resistant to the tested nematicides than Bursaphelenchus sp.
Primer sets of C. elegans were applied to characterization the acetylcholinesterases from nematodes that have different nematicide resistances. Eight populations from ten C. elegans nematicide-resistance populations resulted a 2.0 kb cDNA region C. elegans ace-1 primers. But only one population from ace-2 primers, four populations from ace-3 primers and no population could be amplified by ace-4 primers. The results indicated the expression levels of these 4 genes are not equal. Only CO1000 population has mutations in the ace-1 cDNA sequence.
As to other four nematodes, no cDNA regions were amplified by C. elegans ace-1 and ace-4 primers, but there were various cDNA regions resulted by using ace-2 and ace-3 primers. Four ace-2 cDNA fragment from Rhabditis sp. and A. besseyi were compared to C. elegans ace-2, and only had 24.7~24.1 % similarity. When blasted in the NCBI, the sequences of AC500B-1.1 and AO500B-1.1 had somewhat similarity to C. elegans X chromosome.
Furthermore, a new set of the ace-1 primers was designed from the similar fragments of C. elegans, C. briggsae, M.incognita and M. javanica ace-1 gene to amplify five untreated nematodes. Different cDNA regions from four nematodes; C. elegans, Rhabditis sp., Bursaphelenchus sp. and B. xylophilus except A. besseyi were amplified and five sequences from 2 nematode species were blasted with C. elegans ace-1 and NCBI database. The results showed the 5 sequences had only 23.6~27.6 % similarity to C. elegans ace-1, among them, JCKmA-1.2 and JCKmA-0.6 were somewhat similar to a fragment of Drosophila melanogaster and Chimpanzee(Pan troglodytes) X chromosome.
URI: http://hdl.handle.net/11455/31062
其他識別: U0005-2408200719320000
Appears in Collections:植物病理學系

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