臺灣博碩士論文加值系統

茭白細菌性基腐病為南投縣埔里魚池地區茭白近年來發生之新病害，罹病之植株莖基部呈現腐敗現象，並向上蔓延，造成罹病植株心葉黃化枯萎死亡。以光學顯微鏡鏡檢，可見大量細菌自罹病組織湧出。自罹病組織分離之13個病原細菌菌株，經接種試驗、生理生化特性測試，確認茭白細菌性基腐病係由 Enterobacter cloacae 引起。此菌為革蘭氏陰性細菌，具周生鞭毛，有游動性，為兼性嫌氣細菌，在PDA培養基上形成黏稠流體狀之白色菌落，此菌在罹病茭白植株殘體上可存活一年以上。以E. cloacae SM1全細胞製備抗血清，以瓊脂雙向擴散反應測試分離自茭白之E. cloacae菌株，顯示各菌株與抗血清所形成之反應帶完全融合，然與供試之其他細菌則無任何反應帶形成，以酵素連結抗體免疫反應（ELISA）測試結果顯示，此抗血清可偵測到茭白細菌性基腐病E. cloacae之最低菌量為1×105 cfu/ml，而對其他非標的細菌則呈負反應。利用60組隨機引子進行RAPD（random amplified polymorphic DNA）分析，結果顯示以隨機引子OPC12 可增幅出對茭白細菌性基腐病E. cloacae具專一性之DNA片段（約900bp）。將此專一性之DNA片段選殖及進行核甘酸序列分析，並依其序列設計出對茭白細菌性基腐病菌E. cloacae具專一性之引子對Fc12-1 / Rc12-2。以引子對Fc12-1 / Rc12-2對茭白細菌性基腐病菌E. cloacae菌株之DNA或經NaOH處理之細胞懸浮液進行聚合酵素連鎖反應測試，皆能增幅出706bp之DNA專一性片段。除E. cloacae ATCC13047標準菌株外，以引子對Fc12-1 / Rc12-2對供試之其他細菌皆無法增幅出任何DNA條帶，以此組引子對測試茭白細菌性基腐病菌E. cloacae之DNA時其靈敏度為10pg，而用於測試E. cloacae時其靈敏度為1.21×102 cfu。此外，非標的細菌並不會影響引子對Fc12-1 / Rc12-2對E. cloacae之偵測。應用PCR偵測技術以引子對Fc12-1 / Rc12-2可成功地偵測在茭白罹病組織中之E. cloacae。由上述各項結果顯示本研究所製備之血清及專一性引子對可應用於茭白細菌性基腐病菌E. cloacae之鑑定與偵測。

Zizania latifolia is an important crop in Nantou. During recent years, a new bacterial disease named bacterial basal rot of Zizania latifolia severely affected this crop in Puli and Yuichih areas of Nantou. It caused great losses to the growers. The disease caused internal decay from the basal part of the plants and the disease could progress upward to the leaf. The newly emerged leaves of the infected plants usually showed chlorotic symptoms. The infected plants could die at later stage. Bacterial streaming from the cut edge of the infected tissues was observed with light microscope. Thirteen strains of bacteria were isolated. These bacteria were gram-negative, motile with peritrichous flagella, and facultative anaerobic. All strains formed slimy mucoid and white colonies on potato dextrose agar. Based on the physiological, biochemical and pathogenicity tests, the causal agent of the bacterial basal rot of Zizania latifolia was identified as Enterobacter cloacae. Cells of E. cloacae from Zizania latifolia could survive in infected Zizania latifolia tissues over one year. The antiserum against cells of E. cloacae SM1 was prepared. In Ouchterlony double diffusion assays, cells of 13 strains of E. cloacae from Zizania latifolia formed complete identical band with antiserum against E. cloacae SM1, whereas there was no any band formed with other bacteria tested. The antiserum did not react with other tested bacteria either in indirect-ELISA reaction. The lowest concentration of E. cloacae from Zizania latifolia detected in indirect-ELISA reaction was 1×105 cfu/ml. Sixty different random primers were tested to amplify specific DNA fragments for strains of E. cloacae from Zizania latifolia by using random amplified polymorphic DNA (RAPD) analysis. A specific DNA fragment (about 900bp in size) for the strains of E. cloacae from Zizania latifolia was amplified by the primer OPC12. The specific DNA fragment was cloned and sequenced to design primer pair Fc12-1 / Rc12-2. This primer pair could amplify a distinct band of 706bp that was specific to strains of E. cloacae isolated from Zizania latifolia by polymerase chain reaction (PCR). With the exception of E. cloacae ATCC13047 type strain, there was no any DNA fragment amplified with primer pair Fc12-1 / Rc12-2 from any other tested strains of saprophytic bacteria from Zizania latifolia, E. cloacae from clinical specimens and phytopathogenic bacteria. The minimum amount of DNA from E. cloacae that could be amplified by PCR was 10pg. Sensitivity of PCR for the detection of E. cloacae with primer pair Fc12-1 / Rc12-2 was 1.21×102 cfu. Nontarget bacteria isolated from Zizania latifolia did not affect the efficiency of specific amplification of E. cloacae in PCR assay with primer pair Fc12-1 / Rc12-2. PCR technique with primer pair Fc12-1 / Rc12-2 could successfully detect E. cloacae in the diseased tissues of Zizania latifolia inoculated with E. cloacae. The results indicated that the antiserum and this primer pair Fc12-1 / Rc12-2 could be useful tools for rapid identification and detection of E. cloacae in tissues of Zizania latifolia infected with E. cloacae.

壹、 前言------------------------------------------------1
貳、材料與方法-----------------------------------------------4一、 病原菌之分離----------------------------------------4
二、 供試菌株--------------------------------------------4
三、 供試植物--------------------------------------------5
四、 接種源之製備----------------------------------------5
五、 病原性測試------------------------------------------5
（一） 茭白植株接種----------------------------------------5
（二） 茭白筍體接種----------------------------------------5
六、 病原菌鑑定------------------------------------------6
七、 E. cloacae病原菌感染途徑試驗------------------------7
八、 E. cloacae在茭白罹病植株殘體存活測試----------------7
九、 細菌全DNA的抽取與濃度測定---------------------------7
（一） 細菌全DNA的抽取-------------------------------------8（二） DNA濃度及純度測定-----------------------------------8
十、 E. cloacae專一性DNA片段之篩選-----------------------9（一） RAPD增幅反應----------------------------------------10（二） 引子的篩選------------------------------------------10十一、 E. cloacae專一性DNA片段之回收及純化-----------------10十二、 南方雜合法 ( Southern hybridization )---------------10（一） 轉漬 ( transfer ) 及聯結 ( UV-crosslinking )--------11（二） 核酸探針之製備--------------------------------------11（三） 核酸雜合反應 ( hybridization ) 及偵測反應 ( detection )--------------------------------------------------12
十三、 E. cloacae專一性DNA片段之選殖-----------------------13十四、 小量質體DNA（plasmid DNA）的製備及選殖株篩選--------14
十五、 選殖株重組質體DNA嵌入片段之核甘酸定序與其特性分析及專一性引子對之設計---------------------------------------------14
十六、 聚合酵素連鎖反應及引子對專一性與靈敏度之測定--------14
（一） 聚合酵素連鎖反應之引子黏合溫度測試及專一性測定------14
（二） 靈敏度測試------------------------------------------15
十七、 應用PCR技術偵測茭白罹病組織中之E. cloacae ----------15
十八、 茭白細菌性基腐病E. cloacae抗血清之製備--------------16
（一） 全細胞抗血清之製備----------------------------------16
（二） 免疫球蛋白G之純化-----------------------------------17
十九、 雙向擴散反應----------------------------------------17
二十、 酵素連結抗體免疫反應--------------------------------18
二十一、 免疫螢光染色----------------------------------------18
貳、 結果------------------------------------------------20
一、 茭白細菌性基腐病之田間病徵--------------------------20
二、 病原菌之分離----------------------------------------20
三、 病原性測試------------------------------------------20
（一） 茭白植株接種----------------------------------------20
（二） 茭白筍體接種----------------------------------------21
四、 病原菌鑑定------------------------------------------21
五、 病原菌感染途徑測試----------------------------------21
六、 病原菌之存活測試------------------------------------22
七、 E. cloacae專一性DNA片段之篩選-----------------------22
八、 E. cloacae專一性DNA片段之選殖-----------------------23
九、 選殖株重組質體DNA嵌入片段之序列分析-----------------23
十、 E. cloacae專一性引子對之設計------------------------24
十一、 聚合酵素連鎖反應之引子黏合溫度及引子對 EcFc12-1 / EcRc12-2 EcFD6-1 / EcRD6-2之專一性與靈敏度-------------------24
（一） 聚合酵素連鎖反應之引子黏合溫度及專一性--------------24
（二） 引子對之靈敏度測試----------------------------------25
十二、 非標的細菌對PCR偵測E. cloacae之影響-----------------26
十三、 應用PCR技術偵測茭白罹病組織中之E. cloacae ----------26
十四、 茭白細菌性基腐病E. cloacae抗血清之力價--------------26
十五、 雙向擴散反應（Ouchterlony double diffusion）--------27
十六、 酵素連結抗體免疫反應--------------------------------27
十七、 免疫螢光染色----------------------------------------28
肆、討論-----------------------------------------------------29
伍、參考文獻-------------------------------------------------33
陸、中文摘要-------------------------------------------------39
柒、英文摘要-------------------------------------------------40
捌、圖表-----------------------------------------------------42
玖、附錄-----------------------------------------------------67

1. 朱木貴。1995。Erwinia chrysanthmi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研究所畢業博士論文。
2. 朱軒宇。1999。Burkholderia caryophylli偵測計數之發展與應用。國立中興大學植物病理學研究所第二十九屆畢業碩士論文。8-16頁。
3. 李正應。1993。稀有蔬菜栽培技術。科學技術文獻出版社。北京。317頁。
4. 呂厚興。1994。梨葉緣焦枯病病原菌Xylella fastidiosa單元抗體之製備與應用。國立台灣大學植物病蟲害研究所畢業碩士論文。
5. 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研究所第二十九屆畢業碩士論文。5-16頁。
6. 林天枝、洪澨堂、莊杉行。1999。茭白筍。台中區農業專訊第26期，p:9-15。
7. 林金樹、廖君達。2000。茭白筍重要病蟲害發生調查。台中區農業改良場八十九年度試驗研究暨推廣論文發表會論文摘要。台中區農業改良場特刊第48號，台中區農業改良場編，p.30。
8. 洪聖峰、陳舜英。2000。茭白作物之演化、利用與育種方向。農政與農情，99：93-97。
9. 黃冬青。1997。Burkholderia caryophylli之特性及其血清偵測。國立中興大學植物病理學研究所第二十七屆畢業碩士論文。12頁。
10. 黃晉興、安寶貞、柯文雄。2001。茭白基腐病病害生態之初步探討，植物病理學會90年年會摘要。植物病理學會刊，4：202。
11. 廖君達。2000。茭白筍新病害，中部地區近來重要疫病蟲害發生與管理對策。台中區農業技術專刊第158期，台中區農業改良場編，p.10。
12. 廖君達、林金樹。2001。茭白筍基腐病發生與管理。台中區農業改良場九十年度試驗研究暨推廣論文發表會論文摘要，台中區農業改良場特刊第52號 台中區農業改良場編，p.11。
13. 廖君達、陳慶忠、陳榮五。2002。埔里地區茭白筍基腐病發生及防治現況。農業世界，216：56-59。
14. 廖君達、林金樹、陳慶忠。2002。臺灣茭白筍病蟲害種類及發生消長調查，台中區農業改良場研究彙報，79：(Accepted，2002/6出版)。
15. 蔡雲鵬 編 。 1991 。台灣植物病害名彙。3版。中華植物保護學會暨中華民國植物病理學會。台中。604頁。
16. 鄧文玲。1996。菜豆細菌性斑點病菌致病基因群中兩個外膜蛋白（HrcJ與HrcC）的特性分析。國立中興大學農業生物科技學研究所第一屆畢業碩士論文。
17. Audy, P., Braat, 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.
18. Audy, P., Laroche, A., Sainadon, G., Huang, H. C., and Gilbertson, R. L. 1994. Detection of the bean common blight bacteria, Xanthomonas campestris pv. phaseoli and X. c. var. fuscans, using the polymerase chain reaction. Phytopathology 84:1185-1192.
19. Benedict, A. A., Alzarez, A. M., Berestecky, J., Imanaka, W., Mizumoto, C. Y., Pollard, L. W., Mew, T. W., and Gonzalez, C. F. 1989. Pathovar-specific monoclonal antibodies for Xanthomonas campestris pv. oryzae and for Xanthomonas campestris pv. oryzicola. Phytopathology 79: 322-328.
20. Bishop, A. L., and Davis, R. M. 1990. Internal decay of onions caused by Enterobacter cloacae. Plant Dis. 74: 692-694.
21. Clark, M. F., and Adams, A. N. 1977. Characteristics of the microplate method of enzyme-linked immunosorbent assay for the detection of plant virus. J. Gen. Virol. 34: 475-483.
22. Fahy, P. C., and Persley G. J. 1983. Plant Bacterial Diseases: A Diagnostic Guide. Academic Press. Australia.
23. Hampton, R., Ball, E., and DeBoer, S. 1990. Serological Methods for Detection and Identification of Viral and Bacterial Plant Pathogens. APS Press. The American Phytopathological Society, St. Paul, Minnesota.
24. Jan, F. J., and Yeh, S. D. 1995. Purification, in situ localization, and comparative serological properties of passionfruit woodiness virus-encoded amorphous inclusion protein and two other virus proteins. Phytopathology 85: 64-71.
25. John, G. H., Noel, R. K., Peter, H. A. S., James, T. S., and Stanley T. W. 1994. Bergey’s manual of determinative bacteriology. 9th ed., Williams ＆Wilkins Press p: 178-179, 205.
26. Lin, C. P., and Chen, T. A. 1986. Comparison of monoclonal antibodies and polyclonal antibodies in detection of the aster yellows mycoplasma like organism. Phytopathology 76: 45-50.
27. Loper, J. E., Ishimaru, C. A., Carnegie, S. R., and Vanavichit, A. 1993. Cloning and characterization of aerobactin biosynthesis genes of the biological control agent Enterobacter cloacae. Appl. Environ. Microbiol. 59: 4189-4197.
28. Lorito, M., Di Pietro, A., Hayes, C. K., Woo, S. L., and Harman, G. E. 1993.Antifungal, synergistic interaction between chitinolytic enzymes from Trichoderma harianum and Enterobacter cloacae. Phytopathology 83: 721-728.
29. Mäki-Valkama, T., and Karjalainen, R. 1994. Differentiation of Erwinia carotovora subsp. atroseptica and carotovora by RAPD-PCR. Ann. Appl. Biol. 125:301-309.
30. Malin, E. M., Roth, D. A., and Belden, E. L. 1983. Indirect immunofluorescent staining and identification of Xanthomonas campestris pv. phaseoli in naturally infected bean seed. Plant Dis. 67: 645-647.
31. Matheson, V. G., Munakata-Marr, J., Hopkins, G. D., Mccarty, P. L., Tiedje, J. M., and Forney, L. J. 1997. A novel means to develop strain-specific DNA probes for detecting bacteria in the environment. Appl. Environ. Microbiol. 63: 2863-2869.
32. Mazarei, M., and Kerr, A. 1990. Distinguishing pathovars of Pseudomonas syringae on peas: nutritional, pathogenicity and serological tests. Plant Pathol. 39: 278-285.
33. Mullis, K. B., and Faloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods in Enzymol. 155: 335-350.
34. Narayanasamy, P. 1997. Plant Pathogen Detection and Disease Diagnosis. Marcel Dekker, Inc, New York.
35. Nelson, E. B., Chao, W.L., Norton, J. M., Nash, G. T. and Harman, G. E. 1986. Attachment of Enterobacter cloacae to hyphae of Pythium ultimum: possible role in the biological control of Pythium pre-emergence damping-off. Phytopathology 76: 327-335.
36. Nelson, E. B., and Maloney, A. P., 1992. Molecular approaches for understanding biological control mechanisms in bacteria: studies of the interaction of Pythium ultimum. Can. J. Plant Pathol. 14: 106-114.
37. 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: 1029-1034.
38. Puig, M., Jofre, J., Lucena, F., Allard, A., Wadell, G., and Girones, R. 1994. Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Appl. Environ. Microbiol. 60: 2963-2970.
39. Purcifull, D. E., and Hiebert, E. 1979. Serological distinction of watermelon mosaic virus isolates. Phytopathology 69: 112-116.
40. Saettler, A. W., Schaad, N. W., and Roth, D. A. 1989. Detection of Bacteria in Seed and Other Planting Material.. APS Press. The American Phytopathology Society, St. Paul, Minnesota.
41. Sambrook, J., Maniatis, T. I., and Fritsch, E. F. 1989. Molecular Cloning : a Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y.
42. Schaad, N. W. 1978. Use of direct and indirect immunofluorescence tests for identification of Xanthomonas campestris. Phytopathology 68: 249-252.
43. Schaad, N. W., Cheong, S. S., Tamaki, S., Hatziloukas, E., and Panopoulos, N. J. 1995. A combined biological and enzymatic amplification (BIO-PCR) technique to detect Pseudomonas syringae pv. phaseolicola in bean seed extracts. Phytopathology 85: 243-248.
44. Schaad, N. W., Jones, J. B., and Chun W. 2001. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed., APS Press. Minnesota.
45. Schisler, D.A., and Slininger, P. J. 1994. Selection and performance of bacterial strains for biologically controlling Fusarium dry rot of potatoes incited by Gibberella pulicaris. Plant Dis. 78:251-255
46. Schwartz, H. F. and Otto, K. 2000. Disease notes: First report of a bulb decay of onion by Enterobacter cloacae in Colorado. Plant Dis. 84:808.
47. Southern, E. M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503. (Abstr.).
48. Takahashi, Y., Takahashi, K., Sato M., Watanabe K., and Kawano T. 1997. Bacterial leaf rot of Odontioda orchids caused by Enterobacter cloacae. Ann. Phytopathol. Soc. Jpn. 63: 164-169.
49. Wang, H., Qi, M., and Cutler, A. J. 1993. A simple method of preparing plant samples for PCR. Nucleic Acids Res. 21:4153-4154.
50. Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingey, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Res. 18: 6531-6535.
51. Yeh, S. D., and Gonsalves, D. 1984. Purification and immunological analyses of cylindrical-inclusion protein induced by papaya ringspot virus and watermelon mosaic virus 1. Phytopathology 74:1273-1278.