Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31014
標題: 梨葉緣焦枯病菌之PCR鑑定與偵測技術及其與國外其他寄主植物來源之 Xylella fastidiosa 菌株之親緣關係
A PCR technique for identification and detection of pear leaf scorch bacterium and the phylogenetic relationship between strains of the bacterium and strains of Xylella fastidiosa from other hosts
作者: 蘇秋竹
Su, Chiou-Chu
關鍵字: 梨葉緣焦枯病;pear leaf scorch;聚合酵素連鎖反應;偵測;基因體指紋圖譜技術;16S rRNA 基因序列;16S-23S rRNA基因區間序列;親緣關係;Xylella fastidiosa;polymerase chain reaction;detection;genomic fingerprinting;16S rRNA gene sequence;16S-23S rRNA intergenic spacer region sequence;phylogenetic relationship
出版社: 植物病理學系所
引用: 第一章、 緒言 1. 林嘉興、林信山、張榕生、傅阿炳. 1979. 橫山梨高接溫帶梨試驗研究初步報 告. 台灣農業15:29-39. 2. 林嘉興、廖萬正、林信山、林長仁. 1991. 梨栽培之回顧與展望. P. 379-396. 台灣果樹之生產及研究發展研討會專刊. 510pp. 3. Banks D., Albibi R., Chen, J., Lamikanra O., Jarret R. L., and Smith B. J. 1999. Specific detection of Xylella fastidiosa Pierce’s disease strains. Curr. Microbiol. 39:85-88. 4. Brlansky, R. H., Lee, R. F., Timmer, L. W., Purcifull, D. E., and Raju, B. C. 1982. Immunofluorescent detection of xylem-limmited bacteria in situ. Phytopathology 71: 1444-1448. 5. Chagas, C. M., Rossetti, V., and Beretta, J. G. 1992. Electron microscopy studies of a xylem-limited bacterium in sweet orange affected with citrus variegated chlorosis disease in Brazil. J. Phytopathol. 134:306-312. 6. Chang, C. J., Garnier, M., Zreik, L., Rossetti, V., and Bove, J. M. 1993. Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain of Xylella fastidiosa. Curr. Microbiol. 27:137-142. 7. Chen, J., Hartung, J. S., Chang, C. J., and Vidaver, A. K. 2002. An evolutionary perspective of Pierce''s disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases. Curr. Microbiol. 45:423-428. 8. Davis, M. J., Purcell, A. H., and Thomson. S. V. 1978. Pierce’s disease of grapevines: isolation of the causal bacterium. Science 199:75-77 9. Davis, M. J., Purcell, A. H., and Thomson, S. V. 1980. Isolation medium for the Pierce’s disease bacterium. Phytopathology 70:425-429. 10. de Lima, J. E. O., Miranda, V. S., Hartung, J. S., Brlansky, R. H., Coutinho, A., Roberto, S. R., and Carlos, E. F. 1998. Coffee leaf scorch bacterium: Axenic culture, pathogenicity, and comparison with Xylella fastidiosa of citrus. Plant Dis. 82:94-97. 11. Goheen, A. C., Nyland, G., and Lowe, S. K. 1973. Association of a rickettsia-like organism with Pierce’s of grapevines and alfalfa dwarf and heat therapy of the disease in grapevines. Phytopathology 63:341-345. 12. Hearon, S. S., Sherald, J. L., and Kostka, S. J. 1980. Association of xylem-limited bacteria with elm, sycamore and oak leaf scorch. Can. J. Bot. 58:1986-1996. 13. Hewitt, W. B., Frazier, N. W., and Houston, B. R. 1942. Transmission of Pierce’s disease of grapevines with leaf hopper. (Abstr.) Phytopathology 32:8. 14. Hewitt, W. B., and Houston B. R. 1941. Association of Pierce’s disease of grapevine and alfalfa dwarfin California. Plant Dis. Rep. 25:475-476. 15. Hewitt, W. B., Houston, B. R., Frazier, N. W., and Freitag, J. H., 1946. Pierce’s disease of grapevine. Calif. Agric. Exp. Stn. Circ. 353:1-32. 16. Hillis, M. D. and Dixon, M. T. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Quart. Rev. Biol. 66:411-453. 17. Hopkins, D. L. 1989. Xylella fastidiosa : xylem-limited bacterial pathogen of plants. Annu. Rev. Phytopathol. 27:271-290. 18. Hopkins, D. L., and Adlerz, W. C. 1988. Natural hosts of Xylella fastidiosa in Florida. Plant Dis. 72:492-431. 19. Hopkins, D. L., and Mollenhauer, J. A. 1971. Suppression of Pierce’s disease symptoms by tetracycline antibiotics. Plant Dis. Rep. 55:610-612. 20. Hopkins, D. L., and Mollenhauer, H. H. 1973. Rickettsia-like bacterium associated with Pierce’s disease of grapes. Science 179:298-300. 21. Hopkins, D. L., and Purcell, A. H. 2002. Xylella fastidiosa : cause of Pierce''s disease of grapevine and other emergent diseases. Plant Dis. 86: 1056-1066. 22. Jesús, G.-M., Ignacio, B., Jesús, J. R.-S., and Francisco, R.-V. 2001. RISSC: a novel database for ribosomal 16S-23S RNA genes spacer regions. Nucleic Acids Res. 29:178-180. 23. Kostka, S. J., Tattar, T. A., Sherald, J. L., and Hurtt, S. S. 1986. Mulberry leaf scorch, a new disease caused by a fastidious xylem-inhabiting bacterium. Plant Dis. 70:690-693. 24. Leu, H. H., Leu, L. S., and Lin, C. P. 1998. Development and application of monoclonal antibodies against the causal bacterium of pear leaf scorch, Xylella fastidiosa. J. Phytopathol. 146: 31-37. 25. Leu, L. S., and Su, C. C. 1990. Preliminary report on pear leaf scorch induced by xylem-limited bacteria in Taiwan. (Abstr.) Plant Prot. Bull. 32:329. 26. Leu, L. S. and Su, C. C. 1993. Isolation, cultivation, and pathogenicity of Xylella fastidiosa, the causal bacterium of pear leaf scorch disease in Taiwan. Plant Dis. 77:642-646. 27. Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Michell, T., Andrew, P., Prudhomme, M., Alloing, G., Hakenbeck, R., Morrison, A. D., Boulnois, J. G., and Claverys, J. 1992. A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 20:3479-3483. 28. Mehta, A., and Rosato, Y. B. 2001. Phylogenetic relationships of Xylella fastidiosa strains from different hosts, based on 16S rDNA and 16-23S intergenic spacer sequences. Int. J. Syst. Evol. Microbial. 51:311-318. 29. Minsavage, G. V., Thompson C. M., Hopkins, D. L., Leite, R. M. V. B. C., and Stall, R. E. 1994. Development of polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84:456-461. 30. Mircetich, S. M., Lowe, S. K., Moller, W. J., and Nyland, G. 1976. Etiology of almond leaf scorch and transmission of the causal agent. Phytopathology 84:456-461. 31. Mullis, K. B., and Falloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol. 155:335-350. 32. Nomé, S. F., Raju, B. C., Goheen, A. C., Nyland, G., and Docampo, D. 1980. Enzyme-linked immunosorbent assay for Pierce’s disease bacteria in plant tissues. Phytopathology 70:746-749. 33. Pooler, M. R., and Hartung, J. S. 1995. Specific PCR detection and identification of Xylella fastidiosa strains causing citrus variegated chlorosis. Curr. Microbiol. 31:377-381. 34. Pooler, M. R., Myung, A. S., Bentz, J., Sherald J., and Hartung J. S. 1997. Detection of Xylella fastidiosa in potential insect vector by immunomagnetic separation and nested polymerase chain reaction. Letters Appl. Microbiol. 25:123-126. 35. Purcell, A. H., and Hopkins, D. L. 1996. Fastidious xylem-limited bacterial plant pathogens. Annu. Rev. Phytopathol. 34:131-151. 36. Qin, X., Miranda, V. S., Machado, M. A., Lemos, E. G. M., and Hartung, L. S. 2001. An evaluation of the genetic diversity of Xylella fastidiosa isolated from diseased citrus and coffee in São Paulo, Brazil. Phytopathology 91:599-605. 37. Raju, B. C., Wells, J. M., Nyland, G., Brlansky, R. H., and Lowe, S. K. 1982. Plum leaf scald: isolation, culture, and pathogenicity of the causal agent. Phytopathology 72:1460-1466. 38. Sharples, G. J., and Lloyd, R. G. 1990. A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes. Nucleic Acids Res. 18:6503-6508. 39. Schaad, N. W., Postnikova, E., Lacy, G., Fatmi, M., and Chang, C. J. 2004. Xylella fastidiosa subspecies: X. fastidiosa subsp. piercei, subsp. nov., X. fastidiosa subsp. multiplex subsp. nov., and X. fastidiosa subsp. pauca subsp. nov. Syst. Appl. Microbiol. 21:290-300. 40. Sheraid, J. L., and Lei, J. D. 1991. Evaluation of a rapid ELISA test kit for detection of Xylella fastidiosa in landscape. Plant Dis. 75:200-203. 41. Su, C. C. and Leu, L. S. 1995. Distribution of pear leaf scorch and monthly isolation of its causal organism, Xylella fastidiosa from infected trees. Plant Pathol. Bull. 4:30-33. 42. Su, C. C., Yang, W. J., Hsu, S. T., and Tzeng, K. C. 2002. Development of polymerase chain reaction technique for detection xylem-limited bacterial pathogen of pear leaf scorch. (Abstr.) Plant Prot. Bull. 44: 368-369. 43. Wells, J. M., Raju, B. C., and Nyland, G. 1983. Isolation, culture, and pathogenicity of the bacterium causing phony disease of peach. Phytopathology 73:859-862. 44. Wells, J. M., Raju, B. C., Hung, H. Y., Weisburg, W. G., Mandelco-Paul, L., and Brenner, D. J. 1987. Xylella fastidiosa gen. nov. sp. nov.: Gram-negative xylem-limited, fastidious plant bacteria related to Xanthomaonas spp. Int. J. syst. Bacteriol. 36:136-143. 45. 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 Acids Res. 18:6531-6535. 46. Yonce, C. E. and Chang, C. J. 1987. Detection of xylem-limited bacteria from sharpshooter leafhoppers and their feeding hosts in peach environs monitored by culture isolations and ELISA techniques. Environ. Entomol. 16:68-71. 第二章、應用聚合酵素連鎖反應偵測台灣梨葉緣焦枯 病菌 1. 林嘉興、林信山、張榕生、傅阿炳. 1979. 橫山梨高接溫帶梨試驗研究初步報 告. 台灣農業15:29-39. 2. 林嘉興、廖萬正、林信山、林長仁. 1991. 梨栽培之回顧與展望. P. 379-396. 台灣果樹之生產及研究發展研討會專刊. 510pp. 3. 蘇秋竹、楊文仁、徐世典、曾國欽. 2002. 梨葉緣焦枯病聚合酵素連鎖反應偵 測技術之研究. 植保會刊44(4):368-369. (摘要) 4. Albibi, R., Chen, J., Lamikanra, O., Bank, R. L., Jarret R. L., and Smith, B. J. 1998. RAPD fingerprinting Xylella fastidiosa Pierce’s disease strains isolated from a vineyard in North Florida. FEMS Microbiol.Letters 165:347-352. 5. Banks D., Albibi R., Chen, J., Lamikanra O., Jarret R. L., and Smith B. J. 1999. Specific detection of Xylella fastidiosa Pierce’s disease strains. Curr. Microbiol. 39:85-88. 6. Benson, D. R., Stephens, D. W., Clawson, M. L., and Silvester, W. B. 1996. Amplification of 16S rDNA genes from Frankia strains in root nodules of Ceanothus griseus, Coriaria arborea, Coriaria plumose, Discaria toumatou, and Purshia tridentate. Appl. Environ. Microbiol. 62:2904-2909. 7. Brlansky, R. H., Lee, R. F., Timmer, L. W., Purcifull, D. E., and Raju, B. C. 1982. Immunofluorescent detection of xylem-limmited bacteria in situ. Phytopathology 71: 1444-1448. 8. Chagas, C. M., Rossetti, V., and Beretta, J. G. 1992. Electron microscopy studies of a xylem-limited bacterium in sweet orange affected with citrus variegated chlorosis disease in Brazil. J. Phytopathol. 134:306-312. 9. Chang, C. J., Garnier, M., Zreik, L., Rossetti, V., and Bove, J. M. 1993. Culture and serological detection of the xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain of Xylella fastidiosa. Curr. Microbiol. 27:137-142. 10. Davis, M. J., Purcell, A. H., and Thomson. S. V. 1978. Pierce’s disease of grapevines: isolation of the causal bacterium. Science 199:75-77 11. Davis, M. J., Purcell, A. H., and Thomson, S. V. 1980. Isolation medium for the Pierce’s disease bacterium. Phytopathology 70:425-429. 12. de Lima, J. E. O., Miranda, V. S., Hartung, J. S., Brlansky, R. H., Coutinho, A., Roberto, S. R., and Carlos, E. F. 1998. Coffee leaf scorch bacterium: Axenic culture, pathogenicity, and comparison with Xylella fastidiosa of citrus. Plant Dis. 82:94-97. 13. Goheen, A. C., Nyland, G., and Lowe, S. K. 1973. Association of a rickettsia-like organism with Pierce’s of grapevines and alfalfa dwarf and heat therapy of the disease in grapevines. Phytopathology 63:341-345. 14. Hearon, S. S., Sherald, J. L., and Kostka, S. J. 1980. Association of xylem-limited bacteria with elm, sycamore and oak leaf scorch. Can. J. Bot. 58:1986-1996. 15. Hopkins, D. L., and Purcell, A. H. 2002. Xylella fastidiosa : cause of Pierce''s disease of grapevine and other emergent diseases. Plant Dis. 86: 1056-1066. 16. Kostka, S. J., Tattar, T. A., Sherald, J. L., and Hurtt, S. S. 1986. Mulberry leaf scorch, a new disease caused by a fastidious xylem-inhabiting bacterium. Plant Dis. 70:690-693. 17. Leu, H. H., Leu, L. S., and Lin, C. P. 1998. Development and application of monoclonal antibodies against the causal bacterium of pear leaf scorch, Xylella fastidiosa. J. Phytopathol. 146: 31-37. 18. Leu, L. S., and Su, C. C. 1990. Preliminary report on pear leaf scorch induced by xylem-limited bacteria in Taiwan. (Abstr.) Plant Prot. Bull. 32:329. 19. Leu, L. S. and Su, C. C. 1993. Isolation, cultivation, and pathogenicity of Xylella fastidiosa, the causal bacterium of pear leaf scorch disease in Taiwan. Plant Dis. 77:642-646. 20. Minsavage, G. V., Thompson C. M., Hopkins, D. L., Leite, R. M. V. B. C., and Stall, R. E. 1994. Development of polymerase chain reaction protocol for detection of Xylella fastidiosa in plant tissue. Phytopathology 84:456-461. 21. Mircetich, S. M., Lowe, S. K., Moller, W. J., and Nyland, G. 1976. Etiology of almond leaf scorch and transmission of the causal agent. Phytopathology 84:456-461. 22. Mullis, K. B., and Falloona, F. A. 1987. Specific synthesis of DNA in vitro via a polymerase-catalysed chain reaction. Methods Enzymol. 155:335-350. 23. Nomé, S. F., Raju, B. C., Goheen, A. C., Nyland, G., and Docampo, D. 1980. Enzyme-linked immunosorbent assay for Pierce’s disease bacteria in plant tissues. Phytopathology 70:746-749. 24. Oliveira, A. C., Vallim, M. A., Semighini, C. 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Plum leaf scald: isolation, culture, and pathogenicity of the causal agent. Phytopathology 72:1460-1466. 30. Rowhani, A., Chay, C., Golino, D. A., and Falk, B. W. 1993. Development of a polymerase chain reaction technique for the detection of grapevine fan leaf virus in grapevine tissue. Phytopathology 83:749-753. 31. Sambrook, J., Maniatis, T. I., and Frisch, E. F. 1989. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 32. Schaad, N. W., Cheng, S. S., Tamski, S., Hatziloukas, E., and Panopoulos, N. J. 1995. Identification of Pseudomonas syringae pv. phaseolicola by DNA hybridization probe. Phytopathology 79:903-907. 33. Sheraid, J. L., and Lei, J. D. 1991. Evaluation of a rapid ELISA test kit for detection of Xylella fastidiosa in landscape. Plant Dis. 75:200-203. 34. Southern, E. M. 1975. Detection of specific sequences among DNA fragment separated by gel electrophoresis. J. Mol. Biol. 98:503-517. 35. Su, C. C. and Leu, L. S. 1995. Distribution of pear leaf scorch and monthly isolation of its causal organism, Xylella fastidiosa from infected trees. Plant Pathol. Bull. 4:30-33. 36. Warren, Charles R., Adams, M. A., and Chen, Zuliang. 2000. Is photosynthesis related to concentration of nitrogen and rubisco in leaves of Australian native plants? Austral. J. Plant Physiol. 27:407-416. 37. Wells, J. M., Raju, B. C., and Nyland, G. 1983. Isolation, culture, and pathogenicity of the bacterium causing phony disease of peach. Phytopathology 73:859-862. 38. 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 Acids Res. 18:6531-6535. 39. Yonce, C. E. and Chang, C. J. 1987. Detection of xylem-limited bacteria from sharpshooter leafhoppers and their feeding hosts in peach environs monitored by culture isolations and ELISA techniques. Environ. Entomol. 16:68-71. 第三章、應用基因體指紋圖譜技術鑑別台灣梨葉緣焦 枯病菌與國外其他植物來源 Xylella fastidiosa 之差 異 1. 蘇秋竹、楊文仁、徐世典、曾國欽. 2002. 梨葉緣焦枯病菌聚合酵素連鎖反應偵測技術之研究. 植保會刊44(4):368-369. (摘要) 2. Albibi, R., Chen, J., Lamikanra, O., Banks, D., Jarret, R.L., and Smith, B. J. 1998. RAPD fingerprinting Xylella fastidiosa Pierce’s disease strains isolated from vineyard in North Florida. FEMS Microbiol. Letters 165:347-352. 3. Chang, C. J., Garnier, M., Zreik, L., Rossett, I. V., and Bove, J. M. 1993. Culture and serological detection of xylem-limited bacterium causing citrus variegated chlorosis and its identification as a strain of Xylella fastidiosa. Curr. Microbiol. 27:137-142. 4. Chen, J., Banks, D., Jarret, R. L., Chang, C. J., and Smith, B. J. 2000. Use of 16S rDNA sequences as signature characters to identify Xylella fastidiosa. Curr. Microbiol. 40:29-33. 5. Chen, J., Chang, C. J., Jarret, R. L., and Gawel, L. 1992. Genetic variation among Xylella fastidiosa strains. Phytopathology 82:973-977. 6. Chen, J., Hartung, J. S., Chang, C. J., and Vidaver, A. K. 2002. An evolutionary perspective of Pierce''s disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases. Curr. Microbiol. 45:423-428. 7. Chen, J., Jarret, R. L., Qin, X., Hartung, J. S., Banks, D., Chang, C. J., and Hopkins, D. L. 2000. 16S rDNA sequence analysis of Xylella fastidiosa strains. System. Appl. Microbiol. 23:349-354. 8. Davis, M. J., Purcell, A. H., and Thomson, S. V. 1978. Pierce''s disease of grapevines: isolation of the causal bacterium. Science 199:75-77. 9. Davis, M. J., Purcell, A. H., and Thomson, S. V. 1980. Isolation medium for the Pierce’s disease bacterium. Phytopathology 70:425-429. 10. de Lima, J. E. O., Miranda, V. S., Hartung, J. S., Brlansky, R. H., Coutinho, A., Roberto, S. R., and Carlos, E. F. 1998. Coffee leaf scorch bacterium: Axenic culture, pathogenicity, and comparison with Xylella fastidiosa of citrus. Plant Dis. 82:94-97. 11. Goheen, A. C., Nyland, G., and Lowe, S. K. 1973. 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Xylella fastidiosa : Cause of Pierce''s disease of grapevine and other emergent diseases. Plant Dis. 86:1056-1066. 18. Kamper, S. M., French, W. J., and Dekloet, S. R. 1985. Genetic relationships of some fastidious xylem-limited bacteria. Int. J. Syst. Bacteriol. 35:185-188. 19. Kim, W., Hong, Y., Yoo, J., Lee, W., Choi, C., and Chung, S. 2001. Genetic relationship of Bacillus anthracis and closely related species BOX-PCR genomic fingerprinting. FEMS Microbiol. Letters. 207:21-27. 20. Koeuth, T., Versalovic, J., and Lupski, J. R. 1995. Differential subsequence conservation of interspersed repetitive Streptococcus pneumoniae BOX elements in diverse bacteria. Genome Res. 5:408-418. 21. Kostka, S. J., Tattar, T. A., Sherald, J. L., and Hurtt, S. S. 1986. Mulberry leaf scorch, a new disease caused by a fastidious xylem-inhabiting bacterium. Plant Dis. 70:690-693. 22. Leu, H. H., Leu, L. S., and Lin, C. P. 1998. 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Bull. 4:30-33. 33. Su, C. C., Yang, W. J., Hsu, S. T., and Tzeng, K. C. 2002. Development of polymerase chain reaction technique for detection xylem-limited bacterial pathogen of pear leaf scorch. (Abstr.) Plant Prot. Bull. 44: 368-369. 34. Su, C. C., Yang, W. J., Hsu, S. T., and Tzeng, K. C. 2003. Genetic relationships of pear leaf scorch bacteria and strains of Xylella fastidiosa from other hosts. (Abstr.) Plant Prot. Bull. 45: 391-392. 35. Vandamme, P., Pot, B., Gillis, M., de Vos, P., Kersters, K., and Swings, J. 1996. Polyphasic taxonomy, a consensus approach to bacterial systematics. Microbiol. Rev. 60:407-438. 36. Wells, J. M., Raju, B. C., and Nyland, G. 1983. Isolation, culture and pathogenicity of the bacterium causing phony disease of peach. Phytopathology 73:859-862. 37. Wells, J. M., Raju, B. C., Hung, H. Y., Weisburg, W. G., Mandelco-Paul, L., and Brenner, D. J. 1987. Xylella fastidiosa gen, nov., sp. nov.: Gram-negative xylem-limited, fastidious plant bacteria related to Xanthomonas spp. Int. J. Syst. Bacteriol. 36:136-143.
摘要: 
梨葉緣焦枯病 (pear leaf scorch) 為台灣特有之病害,普遍發生於横山梨(Pyrus pyrifolia) 栽植區,為梨產業之重要限制因子。梨葉緣焦枯病菌之培養性狀及具波浪狀細胞壁 (ripple cell wall) 的特性與侷限導管細菌 (xylem-limited bacteria) Xylella fastidiosa 相同,但梨葉緣焦枯病菌與其他寄主植物來源之 X. fastidiosa 菌株間無血清相關性,且以 X. fastidiosa 廣效性引子對 272-int-1/272-int-2 及 RST31/RST33 對梨葉緣焦枯病菌菌株進行 polymerase chain reaction (PCR) 反應時,亦無任何基因產物。為研發快速鑑定及偵測梨葉緣焦枯病菌之 PCR 技術,本研究利用隨機引子 (OPA11) 進行 random amplified polymorphic DNA (RAPD) 分析,成功獲得對梨葉緣焦枯病菌獨有之約1400 bp 大小核酸片段,進一步將此核酸片段轉殖及序列解序,確認此核酸片段大小為1412 bp,從核酸序列中設計得到一組專一性引子對 PLS-F/PLS-R 可增幅出416 bp大小之專一性基因產物。此一引子對可直接進行梨葉緣焦枯病田間罹病梨樹組織之 PCR 檢測,具有潛力可應用於田間中間寄主植物及蟲媒內病原菌偵測。另本研究利用 random amplified polymorphic DNA-PCR (RAPD-PCR)、enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR) 及 BOX-PCR 等三種基因體指紋圖譜技術分析由國內7個產區收集之梨葉緣焦枯病菌30個菌株及國外7種其他寄主植物 X. fastidiosa 34個菌株之 DNA。由此三種指紋圖譜技術分析所得之親緣樹狀圖皆可將8種寄主植物之 X. fastidiosa 菌株區分為4個菌群,分別為:梨菌群,桑椹及葡萄菌群,胡桃、桃、李及無花果菌群,及夾竹桃菌群。利用此等三種指紋圖譜技術均可有效鑑別梨葉緣焦枯病菌,並顯示其為 X. fastidiosa 菌群中唯一之獨立菌系。本研究進一步針對台灣梨葉緣焦枯病菌4個菌株及國外9種其他寄主植物之 X. fastidiosa 15個菌株進行 16S rRNA 基因及 16S-23S rRNA 基因區間序列之分析,以瞭解台灣與國外菌株之親緣關係。選殖之 16S rRNA 基因序列片段大小為1537~1540 bp;另選殖之16S-23S rRNA 基因區間序列片段大小為510~540 bp,序列比較之結果顯示,梨葉緣焦枯病菌之 16S rRNA 基因及 16S-23S rRNA 基因區間序列基因圖譜 (gene map) 中可發現獨有之基因序列,可作為其鑑定之重要依據。利用鄰聚法 (neighbor joining) 進行系統親緣性分析 (phylogenetic analysis),由 16S rRNA基因序列及 16S-23S rRNA 基因區間序列所構建的親緣演化樹 (phylogenetic tree) 皆可明顯區分為2個菌群,一為梨葉緣焦枯病菌菌群,另一為國外其他寄主植物之 X. fastidiosa 菌群,而後者之菌群又可再細分為4個次菌群,分別為夾竹桃菌群、葡萄及桑椹菌群、咖啡及柑橘菌群及胡桃樹、李樹、桃樹及無花果菌群。由以上結果顯示,梨葉緣焦枯病菌為 Xylella 屬內僅侷限於台灣獨立的菌群與其他寄主植物之 X. fastidiosa 菌株間之關係較疏遠,因此,可能屬於 Xylella fastidiosa 亞種之分類地位,甚或可能為一新種,但其他相關證據仍待研討。

Pear leaf scorch (PLS) disease, the only Xylella fastidiosa-induced disease reported from Taiwan, is commonly found in areas where the variety Hengshan (Pyrus pyrifolia) is grown. The disease is an important limiting factor for the pear industry. Strains of PLS bacterium shares similarities to strains of X. fastidiosa from other host origins in the requirement of complex medium and the rippled cell wall in cell structure. However, PLS strains are not serologically related to strains of X. fastidiosa from other hosts. Likewise, no DNA fragment is amplified from PLS strains with two general primer sets of RST31/RST33 and 272-int/272-int-2 used to detect strains of X. fastidiosa from other hosts. In this study, we developed a polymerase chain reaction (PCR) technique specifically to identify and detect PLS bacterium. The random amplified polymorphic DNA technique was employed to screen a specific DNA fragment of PLS strains. A fragment of about 1400 bp in size amplified with a random primer OPA11 was identified as specific for PLS strains and was further cloned, sequenced, and found to be 1412bp in length. A set of specific primer PLS-F/PLS-R amplified a 416 bp fragment was designed from the sequence data for PLS strains. The PLS bacterium could be detected by using the specific primer set in PLS diseased leaf tissues collected from fields. This PCR technique could be a useful tool for rapid detection of PLS bacterium in insect vectors and alternative hosts in Taiwan. Another study was undertaken to compare the genomic fingerprints between strains of PLS bacterium and strains of X. fastidiosa from other hosts using techniques of random amplified polymorphic DNA-PCR (RAPD-PCR)、enterobacterial repetitive intergenic consensus sequence-PCR (ERIC-PCR) and BOX-PCR. Thirty strains of PLS bacterium collected from seven locations in Taiwan and 34 strains of other hosts including oleander, pecan, plum, peach, mulberry, grape, citrus, coffee and sycamore obtained from foreign countries were compared. The phylogenetic trees constructed showed that all strains of PLS bacterium were separate from strians of X. fastidiosa from other hosts, and the latter strains were clustered into three groups: one consisted of oleander strains, another consisted of grape and mulberry strains, and the third comprised pecan, peach, plum and sycamore strains. The results of this study revealed that the three fingerprinting techniques could be useful tools for differentiation of pear leaf scorch strains from other strains of X. fastidiosa. Further study was to compare four strains of PLS bacterium with 15 strains of X. fastidiosa originally isolated from oleander, pecan, plum, peach, mulberry, grapes, citrus, coffee and sycamore using sequence analyses of 16S rRNA gene and 16S-23S rRNA gene spacer region. The cloned 16S rRNA gene fragment sizes of 1537-1540 bp and cloned 16S-23S rRNA intergenic spacer region fragment sizes of 510-540 bp were sequenced. Specific gene sequences for PLS strains could be found in the 16S rRNA gene and 16S-23S rRNA intergenic spacer region sequences of PLS strains. The phylogenetic trees constructed based on 16S rRNA gene sequences and 16S-23S rRNA intergenic spacer region sequences using neighbor joining method revealed that strains of PLS bacterium were separate from strains of X. fastidiosa from other hosts. Strains of the other host group could be divided into four subgroups: strains from oleander, strains from grape and mulberry, strains from citrus and coffee, and strains from pecan, peach, plum and sycamore. The results of this study indicate that PLS strains were not closely related to strains of X. fastidiosa from other hosts and could belong to a subspecies of X. fastidiosa or even a new species of the genus Xylella, but more taxonomic data are needed before the reclassification can be considered.
URI: http://hdl.handle.net/11455/31014
其他識別: U0005-0502200808282600
Appears in Collections:植物病理學系

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