跳到主要內容

臺灣博碩士論文加值系統

(3.237.38.244) 您好!臺灣時間:2021/07/24 17:11
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:李亞芸
研究生(外文):Ya-Yun Li
論文名稱:台灣七種柑橘類病毒快速偵測方法之建立及應用
論文名稱(外文):Development and application of a fast detection for seven citrus viroids in Taiwan
指導教授:沈湯龍
口試委員:洪挺軒詹富智陳煜焜
口試日期:2015-07-17
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物病理與微生物學研究所
學門:農業科學學門
學類:植物保護學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:41
中文關鍵詞:柑橘鱗砧類病毒啤酒花矮化類病毒柑橘曲葉類病毒柑橘矮化類病毒柑橘樹皮裂紋類病毒柑橘類病毒第五型柑橘類病毒第六型反轉錄聚合酶連鎖反應
外文關鍵詞:Citrus exocortis viroid (CEVd)Hop stunt viroid (HSVd)Citrus bent leaf viroid (CBLVd)Citrus dwarf viroid (CDVd)Citrus bark cracking viroid (CBCVd)Citrus viroid V and VI (CVd-V and VI)reverse transcription polymerase chain reaction (RT-PCR)
相關次數:
  • 被引用被引用:0
  • 點閱點閱:114
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
類病毒為不具有蛋白質外鞘且無蛋白編碼之單股環狀RNA分子 (non-coding single-stranded circular RNA),大小為249至401個核酸,並藉由分子間氫鍵形成桿狀或具分枝之二級結構。儘管如此,類病毒為具有感染力並可造成多種植物病害的病原菌。柑橘是重要的經濟果樹作物之一,其受類病毒的危害甚廣;曾在臺灣發生而被報導的柑橘類病毒有柑橘鱗砧類病毒 (Citrus exocortis viroid)以及啤酒花矮化類病毒 (Hop stunt viroid)。國外尚有柑橘曲葉類病毒 (Citrus bent leaf viroid, CBLVd)、柑橘矮化類病毒 (Citrus dwarf viroid, CDVd)、柑橘樹皮裂紋類病毒 (Citrus bark cracking viroid, CBCVd)、柑橘類病毒第五型 (Citrus viroid V, CVd-V)及柑橘類病毒第六型 (Citrus viroid VI, CVd-VI)等柑橘類病毒病害之報導。反轉錄聚合酶連鎖反應 (RT-PCR)為目前普遍運用於柑橘類病毒之偵測技術,而國內外田間病害報導常發現作物受柑橘類病毒複合感染,又現行偵測技術中針對複合感染的柑橘植株,僅能分別進行檢測,費時且繁瑣,故本論文研究目標為改良柑橘類病毒偵測技術,簡化現行檢測流程並運用於台灣田間柑橘類病毒之偵測調查。本研究目前已完成台灣各地的類病毒檢測,包括新竹、彰化、斗六、嘉義、屏東、宜蘭、花蓮及台東等地,偵測結果發現台灣除了柑橘鱗砧類病毒 (CEVd)以及啤酒花矮化類病毒 (HSVd)外,可能存在另外三種柑橘類病毒,柑橘曲葉類病毒 (Citrus bent leaf viroid, CBLVd)、柑橘矮化類病毒 (Citrus dwarf viroid, CDVd)、柑橘樹皮裂紋類病毒 (Citrus bark cracking viroid, CBCVd)。進一步利用或得到的田間類病毒序列進行類緣分析顯示,台灣島內的CEVd可分為兩群,且該兩群與已知之分類群並無顯著相似性; 相反的,其他于台灣存在的其他柑橘類病毒則分散性的分佈于世界各地之分類型內,顯現這些柑橘類病毒可能為近年來由世界各地傳至台灣島內。

Viroids consist of solely non-coding ssRNA (249-401 bp) and frequently form secondary structure by intramolecular interactions. They are known as the smallest plant pathogens and infect a wide variety of crops. In spite of its small and simple property, plant viroids cause a great deal of agricultural loss. Citrus is one of the most important crops, of which numerous cultivars are susceptible to several viroids. In Taiwan, two plant viroids have been reported, namely Citrus exocortis viroid (CEVd) and Hop stunt viroid (HSVd). Nevertheless, Citrus bent leaf viroid (CBLVd), Citrus dwarf viroid (CDVd), Citrus bark cracking viroid (CBCVd), Citrus viroid V and VI (CVd-V and VI) are also reported globally to be able to infect citrus crops worldwide. The molecular biology technique reverse transcription polymerase chain reaction (RT-PCR) is commonly employed to detect the citrus viroid. Complex infections can generally be found in citrus crops. Due to the fact that a single plant may be infected by multiple viroids simultaneously, the present detection method for citrus viroids is time-consuming and laborious. Thus, the purpose of this study aims to improve the detection technique for multiple citrus viroids in a time-saving and cost-effective manner, thereby facilitating the field survey and inspection of citrus viroids. The study concludes a survey of citrus viroids over various locations in Taiwan, including Hsinchu, Changhwa, Douliu, Chiayi, Pingtung, Yilan, Hualien and Taitun. Our result suggests that rather than CEVd and HSVd, additional three viroids, CBLVd, CDVd and CBCVd, might also exist in the citrus fields of Taiwan. In addition, via phylogenetic analyses, we suspected that CEVd exists in Taiwan for a long time to form two specific clades, whereas other 3 detectable citrus viroids might be imported in recent years from all over of the world.

口試委員會審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
Contents vi
Introduction - 1 -
Viroids - 1 -
Perspectives on citrus viroids - 1 -
The pathogenicity of citrus viroids - 2 -
The phylogenetic analysis for viroids - 3 -
Diagnosis of citrus viroids - 3 -
Research aim and rationale - 4 -
Materials and methods - 5 -
Plant materials and viroid source - 5 -
in vitro transcription - 5 -
One step RT-PCR for simultaneous detection assay - 6 -
Sampling method in field survey - 6 -
Total RNA extraction - 7 -
Two-step RT-PCR - 7 -
Phylogenetic analyses - 8 -
Results - 9 -
1. Comparison of detection by one-step RT-PCR and two-step RT-PCR - 9 -
2. RT-PCR detection of Taiwanese field citrus populations - 9 -
3. Phylogenetic analysis for citrus viroids - 11 -
Discussion - 12 -
Reference - 17 -
Table 1. Citrus cultivar and number collected for field surveys in Taiwan - 23 -
Table 2. Incidences of citrus viroid infection in citrus fields.a - 24 -
Table 3. Number of citrus plant infected with citrus viroid: results of citrus sample detectiona . - 25 -
Figure 1. One-step RT-PCR for simultaneous detection assay - 26 -
Figure 2. Multiple infection ratio in field surveys - 27 -
Figure 3. HSVd detection result, a. Douliu, b. Hsinchu, c. PingtungA , d. Chiayi (one-step RT-PCR); e. Douliu ,Hsinchu, PingtungA, Chiayi (Two-step RT-PCR ). - 28 -
Figure 4. CDVd detection result, a. Douliu, b. Hsinchu, c. PingtungA, d. Chiayi (one-step RT-PCR); e. Douliu ,Hsinchu, PingtungA, Chiayi (Two-step RT-PCR ). - 29 -
Figure 5. Symptoms of viroid infection on the filed citrus. a. Citrus (Douliu-No.08 ) was infected with CEVd and HSVd expressing symptom of typical exocortis. b. Another citrus plant (Hsinchu-No. 12) infected by CEVd and HSVd showing no symptoms. - 30 -
Figure 6. The phylogenetic tree based on the alignment of the obtained CEVd cDNA sequences amplified from Taiwan’s citrus fields and other geographic isolates derived from NCBI. - 31 -
Figure 7. The phylogenetic tree based on the alignment of the obtained HSVd cDNA sequences amplified from Taiwan’s citrus fields and other geographic isolates derived from NCBI. - 32 -
Figure 8. The phylogenetic tree based on the alignment of the obtained CDVd cDNA sequences amplified from Taiwan’s citrus fields and other geographic isolates derived from NCBI. - 33 -
Appendix - 34 -
Table Appendix 1. Sequences of primer employed in this study. - 34 -
Table Appendix 2. Incidences of citrus viroid infection: results of single sample detectiona . - 35 -
Figure Appendix 1. One step RT-PCR for citrus viroids - 40 -
Figure Appendix 2. The field survey in Taiwan. - 41 -


Ashulin, L., Lachman, O., Hadas, R. and Bar-Joseph, M., 1991. Nucleotide sequence of a new viroid species, citrus bent leaf viroid (CBLVd) isolated from grapefruit in Israel. Nucleic Acids Research 19, 4767.
Bernad, L. and Duran-Vila, N., 2006. A novel RT-PCR approach for detection and characterization of citrus viroids. Molecular Cell Probes 20, 105-13.
Daros, J.-A. and Flores, R., 2004. Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proceedings of the National Academy of Sciences of the United States of America 101, 6792-6797.
Diener, T.O., 1971. Potato spindle tuber virus: a plant virus with properties of a free nucleic acid. 3. Subcellular location of PSTV-RNA and the question of whether virions exist in extracts or in situ. Virology 43, 75-89.
Diener, T.O., 1976. Towards an understanding of viroid nature and replication. Annales de Microbiologie 127a, 7-17.
Diener, T.O., 1995. Origin and evolution of viroids and viroid-like satellite RNAs. Virus Genes 11, 119-31.
Diener, T.O., 2003. Discovering viroids--a personal perspective. Nature Review Microbiology 1, 75-80.
Diener, T.O. and Raymer, W.B., 1967. Potato spindle tuber virus: a plant virus with properties of a free nucleic acid. Science (New York, N.Y.) 158, 378-81.
Ding, B., 2009. The biology of viroid-host interactions. Annual Review Phytopathology 47, 105-31.
Ding, B. and Itaya, A., 2007. Viroid: a useful model for studying the basic principles of infection and RNA biology. Molecular Plant Microbe Interaction 20, 7-20.
Ding, B. and Wang, Y., 2009. Viroids: uniquely simple and tractable models to elucidate regulation of cell-to-cell trafficking of RNA. DNA Cell Biology 28, 51-6.
Duran-Vila, N., Flores, R. and Semancik, J.S., 1986. Characterization of viroid-like RNAs associated with the citrus exocortis syndrome. Virology 150, 75-84.
Elena, S.F., Dopazo, J., de la Pena, M., Flores, R., Diener, T.O. and Moya, A., 2001. Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment. Journal of Molecular Evolution 53, 155-9.
Flores, R., Hernandez, C., Martinez de Alba, A.E., Daros, J.A. and Di Serio, F., 2005. Viroids and viroid-host interactions. Annual Review Phytopathology 43, 117-39.
Fonseca, M.E., Marcellino, L.H. and Gander, E., 1996. A rapid and sensitive dot-blot hybridization assay for the detection of citrus exocortis viroid in Citrus medica with digoxigenin-labelled RNA probes. Journal of Virological Methods 57, 203-7.
Gago, S., Elena, S.F., Flores, R. and Sanjuan, R., 2009. Extremely high mutation rate of a hammerhead viroid. Science (New York, N.Y.) 323, 1308.
Hajeri, S., Ramadugu, C., Manjunath, K., Ng, J., Lee, R. and Vidalakis, G., 2011. In vivo generated Citrus exocortis viroid progeny variants display a range of phenotypes with altered levels of replication, systemic accumulation and pathogenicity. Virology 417, 400-9.
Hammond, R.W., 1992. Analysis of the Virulence Modulating Region of Potato Spindle Tuber Viroid Pstvd by Site-Directed Mutagenesis. Virology 187, 654-662.
Hsu, Y.-H., Chen, W. and Owens, R.A., 1995. Nucleotide sequence of a hop stunt viroid variant isolated from citrus growing in Taiwan. Virus Genes 9, 193-195.
Itaya, A., Folimonov, A., Matsuda, Y., Nelson, R.S. and Ding, B., 2001. Potato spindle tuber viroid as inducer of RNA silencing in infected tomato. Mol Plant Microbe Interact 14, 1332-1334.
Ito, T., Ieki, H. and Ozaki, K., 2001. Characterization of a new citrus viroid species tentatively termed Citrus viroid OS. Archives of Virology 146, 975-82.
Ito, T., Ieki, H. and Ozaki, K., 2002a. Simultaneous detection of six citrus viroids and Apple stem grooving virus from citrus plants by multiplex reverse transcription polymerase chain reaction. Journal of Virological Methods 106, 235-9.
Ito, T., Ieki, H., Ozaki, K., Iwanami, T., Nakahara, K., Hataya, T., Ito, T., Isaka, M. and Kano, T., 2002b. Multiple citrus viroids in citrus from Japan and their ability to produce exocortis-like symptoms in citron. Phytopathology 92, 542-547.
Jiang, D., Peng, S., Wu, Z., Cheng, Z. and Li, S., 2009. Genetic diversity and phylogenetic analysis of Australian grapevine viroid (AGVd) isolated from different grapevines in China. Virus Genes 38, 178-83.
Keese, P. and Symons, R.H., 1985. Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proceedings of the National Academy of Sciences of the United States of America 82, 4582-6.
Lin, C.Y., Wu, M.L., Shen, T.L., Yeh, H.H. and Hung, T.H., 2015. Multiplex detection, distribution, and genetic diversity of Hop stunt viroid and Citrus exocortis viroid infecting citrus in Taiwan. Virology Journal 12, 11.
Ling, P., 1972. A report on exocortis in Taiwan. In : Price, W. C. (ed) Proc. 5th Conf. Intern. Organ. Citrus Virol. pp. 102-104. Fla. Press, Gainseville, Florida.
Martin, R., Arenas, C., Daros, J.A., Covarrubias, A., Reyes, J.L. and Chua, N.H., 2007. Characterization of small RNAs derived from Citrus exocortis viroid (CEVd) in infected tomato plants. Virology 367, 135-46.
Matsushita, Y., Usugi, T. and Tsuda, S., 2010. Development of a multiplex RT-PCR detection and identification system for Potato spindle tuber viroid and Tomato chlorotic dwarf viroid. European Journal of Plant Pathology 128, 165-170.
Murcia, N., Bernad, L., Duran-Vila, N. and Serra, P., 2011. Two nucleotide positions in the Citrus exocortis viroid RNA associated with symptom expression in Etrog citron but not in experimental herbaceous hosts. Molecular Plant Pathology 12, 203-8.
Nakahara, K., Hataya, T. and Uyeda, I., 1999. A simple, rapid method of nucleic acid extraction without tissue homogenization for detecting viroids by hybridization and RT-PCR. Journal of Virological Methods 77, 47-58.
Ohno, T., Takamatsu, N., Meshi, T. and Okada, Y., 1983. Hop stunt viroid: molecular cloning and nucleotide sequence of the complete cDNA copy. Nucleic Acids Research 11, 6185-97.
Owens, R.A., 2007. Potato spindle tuber viroid: the simplicity paradox resolved? Molecular Plant Pathology 8, 549-60.
Owens, R.A. and Hammond, R.W., 2009. Viroid pathogenicity: one process, many faces. Viruses 1, 298-316.
Pelissier, T., Thalmeir, S., Kempe, D., Sanger, H.-L. and Wassenegger, M., 1999. Heavy de novo methylation at symmetrical and non-symmetrical sites is a hallmark of RNA-directed DNA methylation. Nucleic Acids Research 27, 1625-1634.
Puchta, H., Ramm, K., Luckinger, R., Hadas, R., Bar-Joseph, M. and Snger, H.L., 1991. Primary and secondary structure of citrus viroid IV (CVd IV), a new chimeric viroid present in dwarfed grapefruit in Israel. Nucleic Acids Research 19, 6640.
Qi, Y. and Ding, B., 2003. Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA. The Plant Cell 15, 1360-74.
Rakowski, A.G., Szychowski, J.A., Avena, Z.S. and Semancik, J.S., 1994. Nucleotide sequence and structural features of the group III citrus viroids. Journal of General Virology 75, 3581-4.
Reanwarakorn, K. and Semancik, J.S., 1999. Correlation of hop stunt viroid variants to cachexia and xyloporosis diseases of citrus. Phytopathology 89, 568-74.
Sano, T., Candresse, T., Hammond, R.W., Diener, T.O. and Owens, R.A., 1992. Identification of multiple structural domains regulating viroid pathogenicity. Proceedings of the National Academy of Sciences of the United States of America 89, 10104-8.
Sano, T., Mimura, R. and Ohshima, K., 2001. Phylogenetic analysis of hop and grapevine isolates of hop stunt viroid supports a grapevine origin for hop stunt disease. Virus Genes 22, 53-9.
Schumacher, J., Randles, J.W. and Riesner, D., 1983. A two-dimensional electrophoretic technique for the detection of circular viroids and virusoids. Analytical Biochemistry 135, 288-95.
Semancik, J.S., Szychowski, J.A., Rakowski, A.G. and Symons, R.H., 1993. Isolates of citrus exocortis viroid recovered by host and tissue selection. Journal of General Virology 74 ( Pt 11), 2427-36.
Semancik, J.S. and Weathers, L.G., 1972. Exocortis disease: evidence for a new species of "infectious" low molecular weight RNA in plants. Nature New Biology 237, 242-4.
Semancik, J.S.a.W., L. G., 1968. Exocortis virus of citrus: association of infectivity with nucleic acid preparation. Virology 36, 326-328.
Serra, P., Barbosa, C.J., Daros, J.A., Flores, R. and Duran-Vila, N., 2008a. Citrus viroid V: molecular characterization and synergistic interactions with other members of the genus Apscaviroid. Virology 370, 102-12.
Serra, P., Gago, S. and Duran-Vila, N., 2008b. A single nucleotide change in Hop stunt viroid modulates citrus cachexia symptoms. Virus Research 138, 130-4.
Singh, R.P., Dilworth, A.D., Ao, X., Singh, M. and Misra, S., 2010. Molecular and biological characterization of a severe isolate of Tomato chlorotic dwarf viroid containing a novel terminal right (T-R) domain sequence. European Journal of Plant Pathology 127, 63-72.
Skoric, D., Conerly, M., Szychowski, J.A. and Semancik, J.S., 2001. CEVd-induced symptom modification as a response to a host-specific temperature-sensitive reaction. Virology 280, 115-23.
Vernière, C., Perrier, X., Dubois, C., Dubois, A., Botella, L., Chabrier, C., Bové, J. M., and Duran Vila, N., 2006. Interactions between citrus viroids affect symptom expression and field performance of clementine trees grafted on trifoliate orange. Phytopathology 96, 356-368.
Visvader, J.E. and Symons, R.H., 1985. Eleven new sequence variants of citrus exocortis viroid and the correlation of sequence with pathogenicity. Nucleic Acids Research 13, 2907-20.
Wang, X., Zhou, C., Tang, K., Zhou, Y. and Li, Z., 2009. A rapid one-step multiplex RT-PCR assay for the simultaneous detection of five citrus viroids in China. European Journal of Plant Pathology 124, 175-180.
Wassenegger, M., Heimes, S., Riedel, L. and Saenger, H.L., 1994. RNA-directed de novo methylation of genomic sequences in plants. Cell 76, 567-576.


QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top