仙人掌X病毒與紅龍果X病毒於紅龍果植株之感染和體內分布

Infection and distribution of Cactus virus X and Pitaya virus X in pitaya plants

10.6342/NTU201800282

林沛延

仙人掌X病毒 ； 紅龍果X病毒 ； 紅龍果 ； 感染 ； 分布 ； Cactus virus X ； Pitaya virus X ； pitaya plants ； infection ； distribution

臺灣大學植物病理與微生物學研究所學位論文

2017年

碩士

張雅君

繁體中文

紅龍果 (Pitaya)多指屬於仙人掌科 (Cactaceae)、三角柱屬 (Hylocereus)的紅皮白肉紅龍果 (H. undatus)，以及有許多雜交品系的紅皮紅肉品種 (Hylocereus spp.)。台灣已知感染紅龍果的病毒皆為Potexvirus屬：仙人掌X病毒 (Cactus virus X, CVX)、紅龍果X病毒 (Pitaya virus X, PiVX)與蟹爪蘭X病毒 (Zygocactus virus X, ZyVX)，且田間調查發現，幾乎全台皆有這些病毒分佈。本研究主要目的為建立CVX與PiVX病毒接種系統以及研究病毒在植株體內移動與分佈情形。由於目前沒有將紅龍果病毒接種至紅龍果植株的有效方法，因此希望能夠建立一套高成功率之接種系統，以利後續實驗進行。前人文獻指出仙人掌科植株對於受傷反應會快速形成隔離組織，可能使病毒接種不易成功；因此我們選擇白肉紅龍果實生苗為材料，應用金剛砂接種子葉，分析病毒的感染情形。當以ELISA進行檢測時，子葉單獨接種CVX 9日後可獲得80%受CVX感染的葉狀枝；而單獨接種PiVX時，於11日後，有40%葉狀枝受PiVX感染。藉由子葉接種系統觀察CVX在白肉紅龍果實生苗的植物體內感染與移動的變化情形，發現在接種後3日到12日為病毒進行長距離移動的關鍵期。在CVX與PiVX病毒濃度相同時，不論是單獨或複合接種，從MNC RT-PCR結果發現CVX在紅龍果實生苗中的移動能力比PiVX好。在複合接種下，PiVX的感染率較單獨接種佳，暗示PiVX在移動上有可能受到CVX的幫助。在病毒總濃度相同下，以MNC RT-PCR檢測單獨或複合接種之結果顯示，3日後於接種的子葉可100%測得病毒感染；而從第9天的結果發現，PiVX的感染情形在單獨接種之下較佳，推測PiVX對PiVX在移動上的協助大於CVX對PiVX的協助。依據組織轉印免疫分析法之結果，感染CVX或是PiVX的植株，無論在莖頂端或根部皆可測得病毒訊號。以簡單組織固定法配合免疫組織化學染色法，目前可在罹病株的不含莖頂分生組織的周圍細胞測得較明顯的紅龍果病毒訊號，顯示CVX與PiVX可系統性感染紅龍果，但可能無法入侵分生組織。

Pitayas usually indicate the fruit crops belonging to the genus Hylocereus (Cactaceae) and their fruits can be grouped into red skin white flesh (H. undatus) and red skin red flesh (Hylocereus spp.). Cactus virus X (CVX), Pitaya virus X (PiVX) and Zygocactus virus X (ZyVX) are known pitaya viruses in Taiwan and they all belong to the genus Potexvirus. Field surveys showed that these viruses exist in most areas in Taiwan. The purposes of this study are to build up pitaya virus inoculation system and to investigate the movement and distribution of the viruses. Because there is no efficient inoculation method for pitaya viruses, we want to establish an inoculation system with high successful rate for further studies. Based on previous studies, cactus plants quickly form barrier tissues after they are wounded. This property may interfere with the success of virus inoculation. Therefore, pitaya (H. undatus) seedlings were chosen as plant materials, the cotyledons were mechanically inoculated with the help of carborundum, and then viral infection was investigated. By ELISA test, 80% of CVX-infected phyllocaldes were obtained after 9 days of CVX single inoculation but only 40% of PiVX-infected phyllocaldes were acquired after 11 days of PiVX single inoculation. Using cotyledon inoculation method to study CVX infection and movement inside the pitaya seedlings, the result indicated that the period between 3 to 12 days post inoculation is the key stage of CVX long-distance movement. When inoculated with the same concentration of CVX and PiVX, CVX showed better movement ability than PiVX in single- and mixed-inoculation according to MNC RT-PCR. PiVX infection rate was higher in mixed-inoculation than in single-inoculation. The result suggested that CVX may help PiVX move inside the plant. When single- or mix-inoculated with the same total virus concentration, the results of MNC RT-PCR revealed cotyledons were 100% infected after 3 days; PiVX showed higher infection rate in single inoculation after 9 days. Therefore, it is proposed the assistance in PiVX movement by PiVX might be better than by CVX. Based on tissue blot immunoassay, viral signals could be detected in the shoot tips and roots of CVX- or PiVX-infected pitaya seedlings. By using simple fixation combined with immunohistochemistry, we observed obvious virus signals around the shoot meristem of virus-infected pitaya seedling but not in the central of shoot meristem. These results demonstrated that CVX and PiVX can systemically infect the pitaya seedling but maybe they cannot invade the shoot meristem.

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   連結：
2. 呂有其。2007。仙人掌病毒X新分離株之特性分析與感染性選殖株之構築。國立台灣大學植物病理與微生物學研究所 碩士論文。
   連結：
3. 李勇賜。2010。紅龍果X病毒之特性分析、感染性選植株構築與抗血清製備。國立台灣大學植物病理與微生物學研究所 碩士論文。
   連結：
4. 余建美。2016。臺灣紅龍果產業發展現況。臺中區農業改良場特刊131: 1-12。
   連結：
5. 邱禮弘。2009。紅龍果有機栽培技術。有機農業產業發展研討會專輯 96: 133-139。
   連結：
6. 廖吉彥、張清安、顏昌瑞、陳昱初、鄧汀欽。2003。感染紅龍果之仙人掌病毒X之鑑定與分佈調查。植物病理學會刊12: 225-234。
   連結：
7. 張佑瑋。兩種仙人掌X病毒感染性選殖株之研究與紅龍果原生質體系統之建立。國立台灣大學植物病理與微生物學研究所 碩士論文。
   連結：
8. 張林仁。2000。將仙人掌作為栽培作物。台中區農業改良場八十九年度試驗研究暨推廣學術研討會報告摘要特刊 49: 3-4。(摘要)
   連結：
9. 張雅玲。2016。紅龍果品種特性及栽培管理。苗栗區農業專訊 74: 9-10。
   連結：
10. 黃士晃。2013。優質紅龍果種苗繁殖生產技術。臺南區農業專訊 85: 1-5。
    連結：
11. 楊玉婷和劉依蓁。2014。臺灣紅龍果生產技術改進研討會採訪紀實。植物種苗生技 37: 89-92。
    連結：
12. 蔡志濃、林筑蘋、安寶貞、鄧汀欽、廖吉彥、倪惠芳、楊宏仁。2013。紅龍果的重要病害及其防治(下)。農業試驗所技術服務 96: 4-7。
    連結：
13. 劉命如、洪建龍、劉瑞芬。2004。引起紅龍果斑駁病徵之Cactus virus X的鑑定與免疫檢測。植物病理學會刊13: 27-34。
    連結：
14. Almeida, O. J. G., Paoli, A. A. S., Souza, L. A., and Cota-Sánchez, J.H. 2013. Seedling morphology and development in the epiphytic cactus Epiphyllum phyllanthus (L.) Haw. (Cactaceae: Hylocereeae). J. Torrey Bot. Soc. 140: 196-214.
    連結：
15. Asano, S., Hirayama, Y., and Matsushita, Y. 2017. Distribution of Tomato spotted wilt virus in dahlia plants. Lett. Appl. Microbiol. 64: 297-303.
    連結：
16. Boke, N. H. 1980. Developmental morphology and anatomy in Cactaceae. BioScience 30: 605-610.
    連結：
17. Chang, K. H., Chang, L. T., Huang, Y. W., Lai, Y. C., Lee, C. W., Liao, J. T., Lin, N. S., Hsu, Y. H., and Hu, C. C. 2017. Transmission of Bamboo mosaic virus in Bamboos mediated by insect in the order Diptera. Front. Microbiol. 8: 870. doi: 10.3389/fmicb.2017.00870.
    連結：
18. Hsu, HT. 2009. Development of enzyme linked, tissue blot and dot blot immunoassays for plant virus detection. Methods Mol. Biol. 508: 15-25.
    連結：
19. Hull, R. 2009. Mechanical inoculation of plant viruses. Curr. Protoc. Microbiol. 16B.6.1-16B.6.4.
    連結：
20. Ju, J., Bai, H., Zheng, Y., Zhao, T., Fang, R., and Jiang, L. 2012. A multi-structural and multi-functional integrated fog collection system in cactus. Nat. Commu. 3: 1247.
    連結：
21. Just, K., Arif, U., Luik, A., and Kvarnheden, A. 2016. Monitoring infection of tomato fruit by Tomato yellow leaf curl virus. J. Plant Pathol. doi:10.1111/ppa.12596.
    連結：
22. Khaimov, A., and Mizrahi, Y. 2012. Effects of day-length, radiation, flower thinning and growth regulators on flowering of the vine cacti Hylocereus undatus and Selenicereus megalanthus. J. Hort. Sci. Biotech. 81: 465-470.
    連結：
23. Kliot, A., and Ghanim, M. 2016. Fluorescent in situ hybridization for the localization of viruses, bacteria, and other microorganism in insect and plant tissues. Methods 98: 74-81.
    連結：
24. Kogovšek, P., Kladnik, A., Mlakar, J., Tušek Žnidarič, M., Dermastia, M., Ravnikar, M., and Pompe-Novak, M. 2011. Distribution of Potato virus Y in potato plant organs, tissues, and cells. Phytopathology 101: 1292-1300.
    連結：
25. Le Bellec, F., Vaillant, F., and Eric, I. 2006. Pitahaya (Hylocereus spp.): a new fruit crop, a market with a future. Fruits 61: 237-250.
    連結：
26. Liou, M. R., Hung, C. L., and Liou, R. F. 2001. First report of Cactus virus X on Hylocereus undatus (Cactaceae) in Taiwan. Plant disease 85: 229.
    連結：
27. Martelli, G. P., Adams, M. J., Kreuze, J. F., and Dolja, V. V. 2007. Family Flexiviridae: A case study in virion and genome plasticity. Annu. Rev. Phytopathol. 45: 73-100.
    連結：
28. Martin, S. 2003. A review of Hylocereus Production in the United States. J. PACD 5: 98-105.
    連結：
29. Mizrahi, Y. 2014. Vine-cacti pitayas-The new crops of the world. Rev. Bras. Frutic. 36: 124-138.
    連結：
30. Mochizuki, T., and Ohki, S. T. 2015. Detection of plant virus in meristem by immunochemistry and in situ hybridization. Methods Mol. Biol. 1236: 275-287.
    連結：
31. Peng, C., Yu, N. T., Luo, W. Z., Fan, H. Y., He, F., Li, X. H., and Zhang, Z. L. 2016. Molecular identification of Cactus virus X infecting Hylocereus polyrhizus (Cactaceae) in Hainan island, China. Plant Disease 100: 1965.
    連結：
32. Santovito, E., Mascia, T., Siddiqui, S. A., Minutillo, S. A., Valkonen, J. P. T., and Gallitelli, D. 2014. Infection cycle of Artichoke Italian latent virus in tobacco plants: Meristem invasion and recovery from disease symptoms. PLoS One 9:e99446. 10.1371/journal.pone.0099446.
    連結：
33. Sauer, M., and Friml, J. 2010. Immunolocalization of proteins in plants. Methods Mol. Biol. 655: 253-263.
    連結：
34. Shargil, D., Zemach, H., Belausov, E., Lachman, O., and Kamenetsky, R. 2015. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues. J. Virol. Methods 223: 55-60.
    連結：
35. Soukup, A., and Tylová, E. 2014. Essential methods of plant sample preparation for light microscopy. Methods Mol. Biol. 1080: 1-23.
    連結：
36. Valencia-Botín, A. J., Kokubu, H.,and Ortiz-Hernández , Y. D. 2013. A brief overview on pitahaya (Hylocereus spp.) diseases. Australasian Plant Pathol. 42: 437-440.
    連結：
37. 毛青樺。2008。蟹爪蘭X病毒與紅龍果X病毒之分子特性與偵測。國立台灣大學植物病理與微生物學研究所 碩士論文。
    連結：
38. 呂有其。2007。仙人掌病毒X新分離株之特性分析與感染性選殖株之構築。國立台灣大學植物病理與微生物學研究所 碩士論文。
    連結：
39. 李勇賜。2010。紅龍果X病毒之特性分析、感染性選植株構築與抗血清製備。國立台灣大學植物病理與微生物學研究所 碩士論文。
    連結：
40. 余建美。2016。臺灣紅龍果產業發展現況。臺中區農業改良場特刊131: 1-12。
    連結：
41. 邱禮弘。2009。紅龍果有機栽培技術。有機農業產業發展研討會專輯 96: 133-139。
    連結：
42. 廖吉彥、張清安、顏昌瑞、陳昱初、鄧汀欽。2003。感染紅龍果之仙人掌病毒X之鑑定與分佈調查。植物病理學會刊12: 225-234。
    連結：
43. 張佑瑋。兩種仙人掌X病毒感染性選殖株之研究與紅龍果原生質體系統之建立。國立台灣大學植物病理與微生物學研究所 碩士論文。
    連結：
44. 張林仁。2000。將仙人掌作為栽培作物。台中區農業改良場八十九年度試驗研究暨推廣學術研討會報告摘要特刊 49: 3-4。(摘要)
    連結：
45. 張雅玲。2016。紅龍果品種特性及栽培管理。苗栗區農業專訊 74: 9-10。
    連結：
46. 黃士晃。2013。優質紅龍果種苗繁殖生產技術。臺南區農業專訊 85: 1-5。
    連結：
47. 楊玉婷和劉依蓁。2014。臺灣紅龍果生產技術改進研討會採訪紀實。植物種苗生技 37: 89-92。
    連結：
48. 蔡志濃、林筑蘋、安寶貞、鄧汀欽、廖吉彥、倪惠芳、楊宏仁。2013。紅龍果的重要病害及其防治(下)。農業試驗所技術服務 96: 4-7。
    連結：
49. 劉命如、洪建龍、劉瑞芬。2004。引起紅龍果斑駁病徵之Cactus virus X的鑑定與免疫檢測。植物病理學會刊13: 27-34。
    連結：
50. Almeida, O. J. G., Paoli, A. A. S., Souza, L. A., and Cota-Sánchez, J.H. 2013. Seedling morphology and development in the epiphytic cactus Epiphyllum phyllanthus (L.) Haw. (Cactaceae: Hylocereeae). J. Torrey Bot. Soc. 140: 196-214.
    連結：
51. Asano, S., Hirayama, Y., and Matsushita, Y. 2017. Distribution of Tomato spotted wilt virus in dahlia plants. Lett. Appl. Microbiol. 64: 297-303.
    連結：
52. Boke, N. H. 1980. Developmental morphology and anatomy in Cactaceae. BioScience 30: 605-610.
    連結：
53. Chang, K. H., Chang, L. T., Huang, Y. W., Lai, Y. C., Lee, C. W., Liao, J. T., Lin, N. S., Hsu, Y. H., and Hu, C. C. 2017. Transmission of Bamboo mosaic virus in Bamboos mediated by insect in the order Diptera. Front. Microbiol. 8: 870. doi: 10.3389/fmicb.2017.00870.
    連結：
54. Hsu, HT. 2009. Development of enzyme linked, tissue blot and dot blot immunoassays for plant virus detection. Methods Mol. Biol. 508: 15-25.
    連結：
55. Hull, R. 2009. Mechanical inoculation of plant viruses. Curr. Protoc. Microbiol. 16B.6.1-16B.6.4.
    連結：
56. Ju, J., Bai, H., Zheng, Y., Zhao, T., Fang, R., and Jiang, L. 2012. A multi-structural and multi-functional integrated fog collection system in cactus. Nat. Commu. 3: 1247.
    連結：
57. Just, K., Arif, U., Luik, A., and Kvarnheden, A. 2016. Monitoring infection of tomato fruit by Tomato yellow leaf curl virus. J. Plant Pathol. doi:10.1111/ppa.12596.
    連結：
58. Khaimov, A., and Mizrahi, Y. 2012. Effects of day-length, radiation, flower thinning and growth regulators on flowering of the vine cacti Hylocereus undatus and Selenicereus megalanthus. J. Hort. Sci. Biotech. 81: 465-470.
    連結：
59. Kliot, A., and Ghanim, M. 2016. Fluorescent in situ hybridization for the localization of viruses, bacteria, and other microorganism in insect and plant tissues. Methods 98: 74-81.
    連結：
60. Kogovšek, P., Kladnik, A., Mlakar, J., Tušek Žnidarič, M., Dermastia, M., Ravnikar, M., and Pompe-Novak, M. 2011. Distribution of Potato virus Y in potato plant organs, tissues, and cells. Phytopathology 101: 1292-1300.
    連結：
61. Le Bellec, F., Vaillant, F., and Eric, I. 2006. Pitahaya (Hylocereus spp.): a new fruit crop, a market with a future. Fruits 61: 237-250.
    連結：
62. Liou, M. R., Hung, C. L., and Liou, R. F. 2001. First report of Cactus virus X on Hylocereus undatus (Cactaceae) in Taiwan. Plant disease 85: 229.
    連結：
63. Martelli, G. P., Adams, M. J., Kreuze, J. F., and Dolja, V. V. 2007. Family Flexiviridae: A case study in virion and genome plasticity. Annu. Rev. Phytopathol. 45: 73-100.
    連結：
64. Martin, S. 2003. A review of Hylocereus Production in the United States. J. PACD 5: 98-105.
    連結：
65. Mizrahi, Y. 2014. Vine-cacti pitayas-The new crops of the world. Rev. Bras. Frutic. 36: 124-138.
    連結：
66. Mochizuki, T., and Ohki, S. T. 2015. Detection of plant virus in meristem by immunochemistry and in situ hybridization. Methods Mol. Biol. 1236: 275-287.
    連結：
67. Peng, C., Yu, N. T., Luo, W. Z., Fan, H. Y., He, F., Li, X. H., and Zhang, Z. L. 2016. Molecular identification of Cactus virus X infecting Hylocereus polyrhizus (Cactaceae) in Hainan island, China. Plant Disease 100: 1965.
    連結：
68. Santovito, E., Mascia, T., Siddiqui, S. A., Minutillo, S. A., Valkonen, J. P. T., and Gallitelli, D. 2014. Infection cycle of Artichoke Italian latent virus in tobacco plants: Meristem invasion and recovery from disease symptoms. PLoS One 9:e99446. 10.1371/journal.pone.0099446.
    連結：
69. Sauer, M., and Friml, J. 2010. Immunolocalization of proteins in plants. Methods Mol. Biol. 655: 253-263.
    連結：
70. Shargil, D., Zemach, H., Belausov, E., Lachman, O., and Kamenetsky, R. 2015. Development of a fluorescent in situ hybridization (FISH) technique for visualizing CGMMV in plant tissues. J. Virol. Methods 223: 55-60.
    連結：
71. Soukup, A., and Tylová, E. 2014. Essential methods of plant sample preparation for light microscopy. Methods Mol. Biol. 1080: 1-23.
    連結：
72. Valencia-Botín, A. J., Kokubu, H.,and Ortiz-Hernández , Y. D. 2013. A brief overview on pitahaya (Hylocereus spp.) diseases. Australasian Plant Pathol. 42: 437-440.
    連結：
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