臺灣博碩士論文加值系統

近年來在台灣中南部發生嚴重之木瓜細菌病害，其病徵如木瓜黑腐病，罹病組織於切口處可見大量細菌湧出。本研究分別自屏東及彰化地區採回罹病之木瓜組織進行病原細菌分離純化，共獲得 36 個病原細菌菌株，經生理生化特性測試，其中 21 個菌株為 Erwinia sp.、10 個菌株為 Pseudomonas aeruginosa、5 個菌株為 Pectobacterium carotovorum subsp. carotovorum。Erwinia sp. 菌株之生理生化特性與 1980 年由木瓜所分離之 Pectobacterium cypripedii (原Erwinia cypripedii) 相似。利用 16S rDNA 序列分析顯示 Erwinia sp. 菌株與 Erwinia papayae 較為接近。利用 40 組隨機引子進行 RAPD (random amplified polymorphic DNA) 分析，結果顯示隨機引子 OPC5 可對 Erwinia sp. 增幅出具專一性之 DNA 片段 (約950 bp)。將此專一性的 DNA 片段選殖及進行核苷酸序列分析，並依其序列設計出對 Erwinia sp. 菌株具有專一性的引子對 C5-1R/C5-1L。以此引子對應用聚合酵素連鎖反應檢測 Erwinia sp. 菌株皆能增幅出 300 bp 之 DNA 專一性片段，而非標的細菌則無法增幅出此條帶，此引子對測試 Erwinia sp. 之 DNA 時，其靈敏度為 10 pg。此專一性引子對可應用於台灣木瓜黑腐病菌 Erwinia sp. 之鑑定與偵測之用。

A bacterial disease with symptoms as that of black rot on papaya, seriously occurred in central and southern Taiwan in recent years. Based on physiological, biochemical and pathogenicity tests, there were 36 strains of plant pathogenic bacteria isolated from diseased papayas from Pingtung and Changhua, including 21 strains of Erwinia sp., 5 strains of Pectobacterium carotovorum subsp. carotovorum and 10 strains of Pseudomonas aeruginosa. Physiological and biochemical assays revealed that the characteristics of the Erwinia sp. strains were similar to that of Pectobacterium cypripedii (Erwinia cypripedii) strain from papaya reported in 1980. Phylogenetic trees based on the sequences of 16S rDNA indicated that the Erwinia sp. strains isolated from papaya were more closely related to the Erwinia papayae than Pectobacterium cypripedii. Sixty different random primers were tested to amplify specific DNA fragments from strains of Erwinia sp. using random amplified polymorphic DNA (RAPD) analysis. A specific DNA fragment (about 950 bp in size) form Erwinia sp. strains was amplified by the random primer OPC 5. This specific DNA fragment was cloned and sequenced to design specific primer pair C5-1L/C5-1R. This primer pair could amplify a distinct band of 300 bp that was specific to strains of Erwinia sp. isolated from papaya by polymerase chain reaction (PCR). The minimum amount of DNA from Erwinia sp. that could be amplified by PCR was 10 pg. The results indicated that the primer pair C5-1L/C5-1R could be a useful tool for identification and detection of Erwinia sp. strains from black rot of papaya.

壹、前言-------------------------------------------------1
貳、材料與方法-------------------------------------------4
一、病原菌分離---------------------------------------4
二、供試菌株-----------------------------------------4
三、供試植物-----------------------------------------4
四、接種試驗-----------------------------------------5
五、病原菌的鑑定-------------------------------------6
(一)生理生化特性測定----------------------------6
(二) Biolog 鑑定分析----------------------------8
六、細菌全 DNA 的抽取與濃度測定----------------------8
(一)細菌全 DNA 之抽取---------------------------8
(二) DNA濃度及純度測定--------------------------9
七、16S rDNA序列之增幅-------------------------------9
八、DNA 片段之回收及純化----------------------------10
九、DNA 片段之選殖----------------------------------11
十、質體 DNA (plasmid DNA) 之抽取與選殖株篩選-------11
十一、16S rDNA核苷酸序列分析------------------------12
十二、以RAPD篩選 Erwinia sp. 專一性 DNA 片段--------13
十三、南方雜合法 (Southern hybridization)-----------14
(一) 轉漬 (transfer) 及聯結 (UV-crosslinking)--14
(二) 核酸探針的製備----------------------------14
(三) 核酸雜合反應(hybridization)及偵測反應
(detection)-------------------------------15
十四、選殖株重組質體DNA嵌入片段之核苷酸定序及專一性引子
對設計----------------------------------------16
十五、聚合酵素連鎖反應及引子對專一性與靈敏度之測定--16
(一) 聚合酵素連鎖反應之黏合溫度測試------------16
(二) 聚合酵素連鎖反應之引子對專一性測試--------16
(三) 靈敏度測試--------------------------------17
參、結果------------------------------------------------18
一、病原菌之分離------------------------------------18
二、病原性接種試驗----------------------------------18
三、病原菌之鑑定------------------------------------19
四、16S rDNA 核苷酸序列分析-------------------------20
五、Erwinia sp. 之 DNA 專一性片段之篩選-------------21
(一) RAPD 之引子之篩選-------------------------21
(二) 利用南方雜合法測試專一性DNA片段-----------21
六、Erwinia sp. 專一性 DNA 片段之選殖與分析---------22
七、Erwinia sp. 專一性引子對之設計------------------22
八、引子對 C5-1L/C5-1R 之黏合溫度、專一性與靈敏度測試22
(一) 引子對C5-1L/C5-1R之黏合溫度測試-----------22
(二) 引子對C5-1L/C5-1R之專一性測試-------------22
(三) 引子對C5-1L/C5-1R之靈敏度測試-------------23
肆、討論------------------------------------------------24
伍、引用文獻--------------------------------------------27
陸、中文摘要--------------------------------------------32
柒、英文摘要--------------------------------------------33
捌、圖表------------------------------------------------35
玖、附錄------------------------------------------------53

王德男。1995。番木瓜。p.109-116。台灣農家要覽農作篇(二)。農業委員會台灣農家要覽增修訂在版策劃委員會編。財團法人豐年社出版。台北。698 pp。
朱木貴。1995。Erwinia chrysanthemi 之遺傳差異性藍色素基因選殖及 PCR 偵測。國立中興大學植物病理學系博士論文。127pp。
呂昀陞。2003。鑑定及偵測茄科植物細菌性斑點病菌Xanthomonas vesicatoria之聚合酵素連鎖反應技術。國立中興大學植物病理學系碩士論文。63pp。
呂理燊、李啟彰、黃 昌。1980。Erwinia cypripedii 引起之木瓜黑腐病。植保會刊。22:377-384。
宋子承。2002。鑑定及偵測楊桃細菌性斑點病菌之聚合脢連鎖反應技術。國立中興大學植物病理學系碩士論文。61pp。
宋秉峰。1999。鑑定及偵測瓜類細菌性果斑病之聚合酵素連鎖反應技術。國立中興大學植物病理學系碩士論文。63pp。
徐世典、張東柱、張清安、蔡進來、蔡東纂編。2002。台灣植物病害名彙。修訂第四版。p50-52。中華民國植物病理學會。台中。386pp。
孫守恭。2001。木瓜病害。p.74-98。台灣果樹病害。第三版。世維出版社。台中。429 pp。
黃 昌。1995。木瓜病害。p.92-95。台灣農家要覽 農作篇(三)。農業委員會台灣農家要覽增修訂在版策劃委員會編。財團法人豐年社出版。台北。500 pp。
楊雯馨、呂昀陞、曾國欽、鄧文玲、徐世典。2006。台灣中南部木瓜之病原細菌特性分析。植病會刊15:302。
鄭安秀、吳雅方、郭源燿。2006。木瓜黑腐病防治研究。植病會刊15:301-302。
Audy, P., Breaat, C. E., Saindon, G., Huang, H. C., and Laroche, A. 1996. A rapid and sensitive PCR-based assay for concurrent detection of bacteria causing common and halo blights in bean seed. Phytopathology 86:361-366.
Beriam, L.O.S.; Almeida, I.M.G.; Destefano, S.A. L.; Grabert, E.; Grabert, E.,; Balani, D.M.; Ferria, M.; Rodrigues Neto, J. Pseudomonas syringae pv. tabaci in papaya seedlings. Summa Phytopathologica, v. 32. p. 21-26, 2006.
Berswell, S., Pah, A., Bellemann, P., Zeller, W., and Geider, K.1992. Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis. Appl. Environ Microbiol. 58:3522-3526.
Bradbury, J. F. 1986. Guide to Plant Pathogenic Bacteria. CBA International, U.K. 332pp.
Burr, T. J., and Schroth, M. N. 1977. Occurrence of soft-rot Erwinia spp. In soil and plant material. Phytopathology 67:1382-1387.
Cother, E. J., Darbyshire, B., and Brewer, J. 1976. Pseudomonas aeruginosa: cause of internal brown rot of onion. Phytopathology 66:828-834.
Drrasse A., Prious S., Kotoujansky A., Bertheau Y. 1994. PCR and restriction fragment-length-polymorphism of a pel gene as a tool to identify Erwinia carotovora in relation to potato disease. Appl. Environ. Microbiol. 60:1437-1443.
Dye, D. W. 1968. A taxonomic study of genus Erwinia. I. The “amylovora” group. N. Z. J. Sci. 11:590-607.
Elrod, R.P., and Braun, A. C. 1942. Pseudomonas aeruginosa: its role as a plant pathogen. J. Bacteriol. 44:633-644.
Gardan, L., Christen, R., Achouak, W., and Prior, P., 2004. Erwinia papayae sp. nov., a pathogen of papaya (Carica papaya) Int. J. Syst. Evol. Microbiol. 54:107-113.
Hartung, J. S., Daniel, J. F., and Pruvost, O. P. 1993. Detection of Xanthomonas campestris pv. citri by the polymerase chain reaction method. Appl. Environ. Microbiol. 59:1143-1148.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J. T., and Williams, S. T. 1994. Bergey’s Mannual of Determinative Bacteriology, 9th ed. p.717-730, Williams & Wilkins, Baltimore, Maryland, 964 pp.
King, E. O., Ward, M. D., and Raney, E. E. 1954. Two simple media for the demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44:301-307.
Kwon, S. W., Go, S. J., Kang, H. W., Ryu, J. C. and Jo, J. K. 1997. Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequence. Int. J. Syst. Bacteriol. 47:1061-1067.
Maki-Valkama, T., and Karjalainen, R. 1994. Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR. Ann. Appl. Biol. 125:301-309.
Mullis, K. B., and Faloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Medods in Enzymology. 155:335-350.
Nelson, M. N., and Alvarez A. M. 1980. Purple stain of Carica papaya. Plant Dis. 64:93-95.
Nishijima, K. A., Couey, H. M., and Alvarez, A. M. 1987. Internal yellowing, a bacterial disease of papaya fruits caused by Enterobacter cloacae. Plant Dis. 71:1029-1032.
Opina, N., Tavner, F., Hollway, G., Wang, J. F., Li, T. H.,Maghirang, R., Fegan, M., Hayward, A. C., Krishnapillai, V., Hong, W. F., Holloway, B. W., and Timmis , J. N. 1997. A novel method for development of species and strain-specific DNA probes and PCR primers for identifying Burkholderia solanacearum (Formerly Pseudomonas solanacearum). Asia Pacific Journal of Molecular Biology and Biotechnology Volume 5, No. 1, pp. 19-30.
Perombelon, M. C. M., and Kelman, A. 1980. Ecology of the soft rot erwinias. Annu. Rev. Phytopathol. 18:361-387.
Rahme, L. G., Stevens, E. J., Wolfort, S. F., Shao, J., Tompkins, R. G., and Ausubel, F. M. 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:1899-1902.
Prosen, D., Hatziloukas, E., Schaad, N. W., and Panopoulos, N. J, 1993, Specific detection of Pseudomonas syringae pv. phaseolicola DNA in bean seed by PCR-based amplification of a phaseolotoxin gene region. Phytopathology 83:965-970.
Sambrook, J., Maniatis, T. I. and Fritsch, E. F. 1989. Molecular cloning: a loboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
Schaad, N. W., Jones, J. B. and Chun, W. 2001. Laboratory guide for identification of plant pathogenic bacteria 3rd ed., APS Press. Minnesota, 373 pp.
Southern, E. M. 1975. Detection of specific sequences among DNA framents separated by gel electrophoresis. J. Mol. Biol. 98:503.
Toth, I. K., Avrova, A. O., and Hyman, L. J. 2001. Rapid Identification and Differentiation of the Soft Rot Erwinias by 16S-23S Intergenic Transcribed Spacer-PCR and Restriction Fragment Length Polymorphism Analyses. Appl. Environ. Microbiol. 67:4070-4076.
Trujillo, E. E., and Schroth, M. N. 1982. Two bacterial diseases of papaya trees caused by Erwinia species in the northern Mariana Islands. Plant Dis. 66:116-120.
Williams, J.G. K., Kubelik, A. R., Livak, K. J.m Rafalski, J. A., and Tingey, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:6531-6535.
Webb, R. R.1985. Epidemiology and control of bacterial canker of papaya caused by an Erwinia sp. On St. Croix, U.S. Virgin islands. Plant Dis. 69:305-309.
Weisburg, W. G., Barns, S. M., Pelletier, D., A., and Lane, D. J. 1991. 16S ribosomal DNA amplification for Phylogenetic study. J. Bacteriol 173:687-703.