番红花种球中虎眼万年青花叶病毒的分子鉴定与序列分析

doi:10.3864/j.issn.0578-1752.2017.20.019

摘要/Abstract

摘要： 【目的】对一批荷兰进境番红花种球（Crocus sativus）进行病毒鉴定，以防止危险性病毒传入危害。【方法】根据已报道的马铃薯Y病毒科（Potyviridae）通用引物(Sprimer和M4T)，采用RT-PCR方法对一批番红花种球样品进行检测，取其中一个RT-PCR产物进行克隆测序，利用GenBank上的基本局部比对搜索工具（Basic Local Alignment Search Tool，BLAST）和MEGA 6中的基于对数期望的多重序列比较（Multiple Sequence Comparison by Log-Expectation，MUSCLE）进行序列比对分析，并根据外壳蛋白基因（coat protein，CP）序列利用最大似然法（maximum likelihood method）进行系统发育分析。【结果】RT-PCR检测结果显示该批样品扩增到一条约1.7 kb的预期大小片段，说明该批番红花种球样品中存在Potyviridae科病毒。测序获得长度为1 666 bp的序列（命名为2677-NL），含有部分核内含体b基因（nuclear inclusion b，NIb）、完整的CP和3′端非编码区（3′-UTR），其核苷酸序列与虎眼万年青花叶病毒（Ornithogalum mosaic virus，OrMV）的SW3.3分离物具有最高序列一致性（99.6%），与OrMV参考基因组序列（Reference genomic sequences，RefSeq；NC_019409）的序列一致性为99.1%。该分离物的CP与6个OrMV印度分离物（JQ686722、JQ686720、JN692498、JN692497、JN692496、JF682235）的核苷酸和氨基酸序列一致性分别为71.5%—77.3%和65.7%—79.7%，与其他32个OrMV分离物的核苷酸和氨基酸序列一致性分别为77.9%—99.5%和82.9%—99.2%。基于CP进行的系统发育分析表明，OrMV可分为两个类群，而2677-NL与5个澳大利亚分离物（JQ807995、JQ807996、NC_019409、AF185964、AF185965）相聚成簇，表明其在系统发育关系上与OrMV澳大利亚分离物的亲缘关系最近。【结论】根据国际病毒分类委员会（ICTV）关于Potyviridae科病毒种类的分类标准，2677-NL为OrMV的一个分离物，证实该批番红花种球受到OrMV的侵染。这是世界上首次在番红花中发现OrMV的报道。

关键词: 虎眼万年青花叶病毒, 番红花, RT-PCR, 新寄主

Abstract: 【Objective】The objective of this study is to identify viruses in saffron (Crocus sativus) corms imported from the Netherlands and to prevent the entry of dangerous virus into China. 【Method】Using the Potyviridae universal primers (Sprimer and M4T), the saffron corms samples were detected by reverse transcription polymerase chain reaction (RT-PCR) assay. After cloning and sequencing of the RT-PCR products, the Basic Local Alignment Search Tool (BLAST) and Multiple Sequence Comparison by Log-Expectation (MUSCLE) methods were used for multiple sequence alignment. The maximum likelihood method was used to construct the phylogenetic tree according to the coat protein (CP) nucleotide sequences. 【Result】The about 1.7 kb RT-PCR products were amplified from the samples, indicating a member of Potyviridae virus was present in the samples of saffron corms. A sequence of 1 666 bp segments was obtained from a positive sample (designated 2677-NL) and it contained a portion of nuclear inclusion b gene (NIb), the complete CP and 3′ untranslated region (3′-UTR). This isolate shared the highest nucleotide sequence identity (99.6%) with the SW3.3 isolate of Ornithogalum mosaic virus (OrMV) and shared 99.1% nucleotide sequence identity with the Reference genomic sequences (RefSeq) of OrMV (GenBank accession number: NC_019409). Of those, the CP of the 2677-NL isolate shared 71.5%-77.3% and 65.7%-79.7% nucleotide and amino acid identities, respectively, with those of six OrMV isolates from India (GenBank accession number: JQ686722, JQ686720, JN692498, JN692497, JN692496 and JF682235) whereas 77.9%-99.5% and 82.9%-99.2%, respectively, with the other 32 OrMV isolates. Phylogenetic analysis based on the CP genes showed that the OrMV could be divided into two groups, in which 2677-NL was clustered together with five OrMV isolates from Australia (GenBank accession number: JQ807995, JQ807996, NC_019409, AF185964 and AF185965), suggesting these OrMV were highly homologous in the phylogenetic relationship. 【Conclusion】According to the species demarcation criteria of the family Potyviridae on the International Committee on Taxonomy (ICTV), the 2677-NL was an isolate of OrMV, and it showed that the saffron corms sample was infected by OrMV. this is the first report on OrMV infecting C. sativus in the world.

Key words: Ornithogalum mosaic virus, Crocus sativus, RT-PCR, new host

廖富荣，林武镇，陈细红，陈青，陈红运，黄蓬英，方志鹏，吴媛，沈建国，林石明. 番红花种球中虎眼万年青花叶病毒的分子鉴定与序列分析[J]. 中国农业科学, 2017, 50(20): 4046-4054.

LIAO FuRong, LIN WuZhen, CHEN XiHong, CHEN Qing, CHEN HongYun, HUANG PengYing, FANG ZhiPeng, WU Yuan, SHEN JianGuo, LIN ShiMing. Molecular identification and sequence analysis of Ornithogalum mosaic virus in saffron (Crocus sativus) corms[J]. Scientia Agricultura Sinica, 2017, 50(20): 4046-4054.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.20.019

https://www.chinaagrisci.com/CN/Y2017/V50/I20/4046

参考文献

[1] 兰卫, 朱金芳, 耿直. 新疆西红花的种植技术. 新疆医科大学学报, 2010, 33(8): 874-875.

Lan W, Zhu J F, Geng Z. Planting techniques of Xinjang saffron. Journal of Xinjiang Medical University, 2010, 33(8): 874-875. (in Chinese)

[2] Ahrazem O, Rubio-Moraga A, Castillo-López R, Mozos A T, Gómez-Gómez L. Crocus sativus pathogens and defence responses//Husaini A M. Saffron. Functional Plant Science and Biotechnology, 2010, 4(Special Issue 2): 81-90.

[3] Caiola M G, Faoro F. Latent virus infections in Crocus sativus and Crocus cartwrightianus. Phytopathologia Mediterranea, 2011, 50(2): 175-182.

[4] Miglino R, Jodlowska A, Van Schadewijk A R. First report of Narcissus mosaic virus infecting Crocus spp. cultivars in the Netherlands. Plant Disease, 2005, 89(3): 342.

[5] Corona F M O, Lebas B S M, Elliott D R, Tang J Z, Alexander B J R. New host records and new host family range for Turnip mosaic virus in New Zealand. Australasian Plant Disease Notes, 2007, 2(1): 127-130.

[6] Ehrig F, Kegler H, Fuchs E. Detection of a virus hitherto unknown in Crocus vernus hybrids. Archives of Phytopathology & Plant Protection, 1997, 30(5): 453-455.

[7] Samuitien? M, Navalinskien? M, Jackevi?ien? E. Arabis mosaic virus on ornamental plants. Biologija, 2008, 54(4): 264-268.

[8] Samuitien? M, Navalinskien? M. Occurrence of Cucumber mosaic cucumovirus on ornamental plants in Lithuania. Zemdirbyste- Agriculture, 2008, 95(3): 135-143.

[9] 谢礼, 吕明芳, 董峰丽, 饶君凤, 毛碧增, 洪健. 藏红花病毒病原的分子鉴定. 中草药, 2013, 44(8): 1033-1036.

Xie L, Lü M F, Dong F L, Rao J F, Mao B Z, Hong J. Molecular identification of viral pathogens in Crocus sativus. Chinese Traditional and Herbal Drugs, 2013, 44(8): 1033-1036. (in Chinese)

[10] 赵培洁, 许彩霞, 徐正, 高其昌. 由栽培番红花中检出鸢尾轻花叶病毒. 中药材, 1990, 13(5): 4-6.

Zhao P J, Xu C X, Xu Z, Gao Q C. Identification ofIris mild mosaic virus in the cultivated Crocus sativus. Chinese Traditional and Herbal Drugs, 1990, 13(5): 4-6. (in Chinese)

[11] 陈集双, 李德葆. 侵染番红花的芜菁花叶病毒. 植物病理学报, 1995, 25(4): 361-365.

Chen J S, Li D B. A Turnip mosaic virus infecting Crocus sativus. Acta Phytopathologica Sinica, 1995, 25(4): 361-365. (in Chinese)

[12] Burger J T, Brand R J, Rybicki E P. The molecular cloning and nucleotide sequencing of the 3′-terminal region of Ornithogalum mosaic virus. Journal of general virology, 1990, 71(11): 2527-2534.

[13] Adams M J, Antoniw J F, Fauquet C M. Molecular criteria for genus and species discrimination within the family Potyviridae. Archives of Virology, 2005, 150: 459-479.

[14] Burger J T, Von Wechmar M B. Purification and some properties of South African isolates of Ornithogalum mosaic virus. Phytopathology, 1989, 79(4): 385-391.

[15] 松本勤, 山本英樹, 高橋春實, 神田啓臣, 勝田茂満, 相馬玲子, 藤晋一, 井上正保. オーニソガラムモザイクウイルスによるオーニソガラムモザイク病の発生と宿主範囲を異にする分離株の存在. 日本植物病理学会報, 2007, 73(2): 102-105.

Matsumoto T, Yamamoto H, Takahashi H, Kanda H, Katsuta S, Soma R, Fuji S, Inoue M. The first report on the occurrence of Ornithogalum mosaic virus in Japan and the presence of isolates differing in pathogenicity to dicot plants. Japanese Journal of Phytopathology, 2007, 73(2): 102-105. (in Japanese)

[16] Wylie S J, Li H, Dixon K W, Richards H, Jones M G K. Exotic and indigenous viruses infect wild populations and captive collections of temperate terrestrial orchids (Diuris species) in Australia. Virus Research, 2013, 171: 22-32.

[17] Cho S Y, Lim S, Kim H, Yi S I, Moon J S. Complete genome sequence of Ornithogalum mosaic virus infecting Gladiolus spp. in South Korea. Genome Announcements, 2016, 4(4): e00816-16.

[18] Bouwen I, van der Vlugt R A A. Natural infection of Alstroemeria caryophyllea with Ornithogalum mosaic virus. Plant Disease, 2000, 84(2): 202.

[19] Grisoni M, Moles M, Farreyrol K, Rassaby L, Davis R, Pearson M. Identification of potyviruses infecting vanilla by direct sequencing of a short RT-PCR amplicon. Plant pathology, 2006, 55(4): 523-529.

[20] Zeidan M, Cohen J, Watad A, Gera A. Improved purification and molecular properties of Omithogalum mosaic virus in Israel. Annals of Applied Biology, 1998, 133(2): 167-176.

[21] Chandel V, Kulshrestha S, Hallan V, Zaidi A A. Natural infection of Ornithogalum mosaic virus on Iris from India. Plant Pathology, 2006, 55: 284.

[22] Kaur C, Raj S K, Snehi S K, Goel A K, Roy R K. Natural occurrence of Ornithogalum mosaic virus newly reported on gladiolus in India. New Disease Reports, 2011, 24: 2.

[23] Yoon H I, Ryu K H. Molecular identification and sequence analysis of coat protein gene of Ornithogalum mosaic virus isolated from Iris plant. The Plant Pathology Journal, 2002, 18(5): 251-258.

[24] Fuji S, Inoue M, Yamamoto H, Furuya H, Naito H, Matsumoto T. Nucleotide sequences of the coat protein gene of potyviruses infecting Ornithogalum thyrsoides. Archives of Virology, 2003, 148: 613-621.

[25] Wei T, Pearson M N, Cohen D. First report of Ornithogalum mosaic virus and Ornithogalum virus 2 in New Zealand. Plant Pathology, 2006, 55(6): 820.

[26] Gibbs A, Mackenzie A, Blanchfield A, Cross P, Wilson C R, Kitajima E, Nightingale M, Clements M. Viruses of orchids in Australia: their identification, biology and control. Australian Orchid Review, 2000, 65(3): 10-21.

[27] 陈金枝, 张幼莉, 张清安, 蔡惠婷. 于进口之天鹅绒发生严重嵌纹之病株中发现由天鹅绒嵌纹病毒及Pterostylis virus Y复合感染之分子生物学证据. 植物病理学会刊, 2000, 9(4): 184-185.

Chen J Z, Zhang Y L, Zhang Q A, Cai H T. Molecular evidences for the dual infection of Ornithogalum mosaic virus and Pterostylis virus Y in an imported Ornithogalum thyrsoides plant with severe mosaic symptom. Plant Pathology Bulletin, 2009, 9(4): 184-185. (in Chinese)

[28] 郑红英, 鲁宇文, 沈嘉乐, 林林, 陈炯, 陈剑平. 侵染水仙的虎眼万年青花叶病毒外壳蛋白的原核表达、抗血清制备及应用. 浙江农业学报, 2009, 21(3): 198-201.

Zheng H Y, Lu Y W, Shen J L, Lin L, Chen J, Chen J P. Prokaryotic expression of coat protein gene of Ornighogalum mosaic virus and its use in the antiserum preparation. Acta Agriculturae Zhejiangensis, 2009, 21(3): 198-201. (in Chinese)

[29] Burger J T, von Wechmar M B. Rapid diagnosis of Ornithogalum and Lachenalia viruses in propagation stock. Acta Horticulturae, 1988, 234: 31-38.

[30] Chen J, Chen J P, Langeveld S A, Derks A F L M, Adams M J. Molecular characterization of carla- and potyviruses from Narcissus in China. Journal of Phytopathology, 2003, 151(1): 26-29.

[31] James D, Varga A, Pallas V, Candresse T. Strategies for simultaneous detection of multiple plant viruses. Canadian Journal of Plant Pathology, 2006, 28(1): 16-29.

[32] 李鑫, 陈雅诗, 朱连, 张寅寅, 黑多尔, 廖富荣. 进境荷兰百合中车前草花叶病毒的分子鉴定及序列分析. 植物病理学报, 2017, 47(2): 197-202.

Li X, Chen Y S, Zhu L, Zhang Y Y, Hei D E, Liao F R. Molecular identification and sequence analysis of Plantago asiatica mosaic virus in Lily from Netherlands. Acta Phytopathologica Sinica, 2017, 47(2): 197-202. (in Chinese)

[33] 叶志红, 廖富荣, 郭木金, 方志鹏, 陈青, 陈红运, 林石明, 林毅. 同时检测豇豆花叶病毒属与蚕豆病毒属病毒的通用RT-PCR检测方法. 中国农业科学, 2015, 48(8): 1527-1537.

Ye Z H, Liao F R, Guo M J, Fang Z P, Chen Q, Chen H Y, Lin S M, Lin Y. A universal RT-PCR method for the simultaneous detection of the viruses in genera Comovirus and Fabavirus. Scientia Agricultura Sinica, 2015, 48(8): 1527-1537. (in Chinese)

[34] Chen J, Chen J, Adams M J. A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Archives of Virology, 2001, 146(4): 757-766.

[35] Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 2013, 30(12): 2725-2729.

[36] Adams M J, Zerbini F M, French R, Rabenstein F, Stenger D C, Valkonen J P T. Family Potyviridae//King A M, Adams M J, Carstens E B, Lefkowitz E J. Virus Taxonomy: Classification and Nomenclature of Viruses: Ninth Report of the International Committee on Taxonomy of Viruses. London, UK: Elsevier Academic Press, 2012: 1069-1089.

[37] Wylie S J, Adams M, Chalam C, Kreuze J, López-Moya J J, Ohshima K, Praveen S, Rabenstein F, Stenger D, Wang A, Zerbini F M, ICTV Report Consortium. ICTV virus taxonomy profile: Potyviridae. Journal of General Virology, 2017, 98(3): 352-354.