蚕豆萎蔫病毒2号北京玫瑰分离物的鉴定

doi:10.16420/j.issn.0513-353x.2023-0884

摘要/Abstract

摘要：

为明确引起北京怀柔玫瑰黄化、矮缩的病原，采用小RNA测序和反转录PCR对玫瑰病叶进行鉴定和分析。小RNA测序表明该病害与蚕豆萎蔫病毒2号（broad bean wilt virus 2，BBWV2）的侵染有关。根据深度测序结果，设计特异性引物对其全基因组进行克隆，序列分析表明该分离物基因组RNA1和RNA2分别为5 903和3 535 nt。基于RNA1和RNA2 CP基因序列的系统进化树分析表明，BBWV2-Rose RNA1与中国沈阳藜分离物BBWV2-CN：SY：Chenopodium album（MN786954）聚为一支，BBWV2-Rose RNA2与中国沈阳藜分离物BBWV2-CN：SY Chenopodium album（MN786955）聚为一支，亲缘关系最近。根据蚕豆病毒属（Fabavirus）的分类标准，该病毒是BBWV2的一个分离物。

关键词: 玫瑰, 深度测序, 蚕豆萎蔫病毒2号

Abstract:

In order to detect the pathogen of rose plants with yellow and stunt in Huairou，Beijing，small RNA deep sequencing（sRNA）and reverse transcription-polymerase chain reaction（RT-PCR）were conducted with leaf samples collected from the diseased plants. Broad bean wilt virus 2（BBWV2）was identified after sRNA deep sequencing followed by bioinformatic analyses. To further characterize the identified BBWV2（BBWV2-rose），the complete genome sequence was cloned with primer pairs designed according to the assembled contigs. Sequence analysis showed BBWV2-rose RNA1 and RNA2 were 5 903 and 3 535 nt，respectively. Phylogenetic tree analysis based on RNA1 and RNA2 CP gene sequences suggests that BBWV2-Rose RNA1 and isolate BBWV2-CN：SY：Chenopodium album（MN786954）clustered together，and BBWV2-Rose RNA2 and isolate BBWV2-CN：SY Chenopodium album（MN786955）clustered together，which was closely related. According to Fabavirus classification criteria，the virus should be an isolate of BBWV2.

Key words: rose, deep sequencing, broad bean wilt virus 2

梁宇卿, 孙春辉, 邓丛良, 史喜菊, 种焱, 李永强. 蚕豆萎蔫病毒2号北京玫瑰分离物的鉴定[J]. 园艺学报, 2024, 51(10): 2320-2328.

LIANG Yuqing, SUN Chunhui, DENG Congliang, SHI Xiju, ZHONG Yan, LI Yongqiang. Identification and Analysis of Complete Genomic Sequence of Broad Bean Wilt Virus 2 Beijing Rose Isolate[J]. Acta Horticulturae Sinica, 2024, 51(10): 2320-2328.

图/表 5

参考文献 40

[1]	Adams I, Fox A. 2016. Diagnosis of plant viruses using next-generation sequencing and metagenomic analysis. Current Research Topics in Plant Virology,323-335.
[2]	Adkins S, Baker C A. 2005. Tomato spotted wilt virus identified in desert rose in Florida. Plant Disease, 89 (5):526. doi: 10.1094/PD-89-0526C pmid: 30795435
[3]	Bolus S, Al Rwahnih M, Grinstead S C, Mollov D. 2021. Rose virus R,a cytorhabdovirus infecting rose. Archives of Virology, 166 (2):655-658.
[4]	Chen J H, Zhou Y C, Gu T X, Guo X, Zhuang X J, Zhang K. 2023. First report of broad bean wilt virus 2 on Mirabilis jalapa in China. Plant Disease, 107 (6):1957.
[5]	Chen X J, Xu X G, Li Y Z, Liu Y T. 2013. Development of a real-time fluorescent quantitative PCR assay for detection of Impatiens necrotic spot virus. Journal of Virological Methods, 189 (2):299-304.
[6]	Claros N A, Shires M, Mollov D, Hammond J, Jordan R, Ochoa-Corona F, Olson J, Ong K, Salamanca L R. 2022. Rose rosette disease:a diagnostic guide. Plant Health Progress, 23 (4):482-491.
[7]	Diaz-Lara A, Mollov D, Golino D, Rwahnih M A. 2020. Complete genome sequence of rose virus A,the first carlavirus identified in rose. Archives of Virology, 165 (1):241-244. doi: 10.1007/s00705-019-04460-1 pmid: 31701224
[8]	Elden K M E, Elawady M A M, Said M M, Elhallous E I, Eltarras A A. 2013. Improvement of serological diagnosis of viruses associated with Rose mosaic disease affecting egyptian-rose grown in Taif,kingdom of Saudi Arabia. Life Science Journal, 10 (4):1673-1679.
[9]	Ferrer R M, Ferriol I, Moreno P, Rubio L. 2011. Genetic variation and evolutionary analysis of Broad bean wilt virus 2. Archives of Virology,156:1445-1450.
[10]	Gang S, Zhang S R, Zhang S Y, Sun J, Yang C X. 2023. Broad bean wilt virus 2 in Commelina communis L. in China. Bangladesh Journal of Botany,52:569-574.
[11]	Gong Mingxia, Zhao Hu, Wang Meng, Wu Xing, Zhao Zengjing, He Zhi, Huang Jinmei, Meng Shengde, Wang Risheng. 2022. Identification of viruses infecting peppers in Guangxi by small RNA deep sequencing and RT-PCR. Acta Horticulturae Sinica, 49 (5):1060-1072. (in Chinse) doi: 10.16420/j.issn.0513-353x.2021-0673
	龚明霞, 赵虎, 王萌, 吴星, 赵曾菁, 何志, 黄金梅, 孟生德, 王日升. 2022. 广西辣椒病毒的sRNA深度测序和RT-PCR鉴定. 园艺学报, 49 (5):1060-1072. doi: 10.16420/j.issn.0513-353x.2021-0673
[12]	Hamilton A J, Baulcombe D C. 1999. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science, 286 (5441):950-952. doi: 10.1126/science.286.5441.950 pmid: 10542148
[13]	Han Shuai, Wu Jie, Zhang Heqing, Xi Yadong. 2022. Identification and sequence analysis of tomato spotted wilt orthotospovirus infecting lettuce in Sichuan. Acta Horticulturae Sinica, 49 (9):2007-2016. (in Chinse) doi: 10.16420/j.issn.0513-353x.2021-0592
	韩帅, 吴婕, 张河庆, 席亚东. 2022. 四川莴笋上番茄斑萎病毒的电镜观察与小RNA测序鉴定. 园艺学报, 49 (9):2007-2016. doi: 10.16420/j.issn.0513-353x.2021-0592
[14]	Kim H S, Byun H S, Choi Y J, Choi H Y, Seo J K, Choi H S, Lee B C, Kim M, Kwak H R. 2023. Characterization of broad bean wilt virus 2 isolated from Perilla frutescens in Korea. Korean Journal of Environmental Biology, 41 (1):1-13.
[15]	Kreuze J F, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R. 2009. Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs:a generic method for diagnosis,discovery and sequencing of viruses. Virology, 388 (1):1-7. doi: 10.1016/j.virol.2009.03.024 pmid: 19394993
[16]	Kuang Yunbo, Hou Ling, Fang Xiaoqin, Wang Jialu, Yang Yanjun, Ye Zuyun. 2018. Nested RT-PCR assay for detecting Broad bean wilt virus 2 and Turnip mosaic virus in Pseudostellaria heterophylla. Fujian Journal of Agricultural Sciences, 33 (3):288-292. (in Chinese)
	匡云波, 侯令, 方晓勤, 王佳璐, 杨燕君, 叶祖云. 2018. 太子参BBWV2和TuMV的巢式RT-PCR检测. 福建农业学报, 33 (3):288-292.
[17]	Kwak H R, Kim M K, Lee Y J, Seo J K, Choi H S. 2013a. Molecular characterization and variation of the Broad bean wilt virus 2 isolates based on analyses of complete genome sequences. The Plant Pathology Journal, 29 (4):397.
[18]	Kwak H R, Kim M K, Nam M, Kim J S, Choi H S. 2013b. Genetic compositions of Broad bean wilt virus 2 infecting red pepper in Korea. The Plant Pathology Journal, 29 (3):274.
[19]	Kwak H R, Lee Y J, Kim J, Kim M K, Kim J S, Choi H S, Seo J K. 2016. A determinant of disease symptom severity is located in RNA2 of broad bean wilt virus 2. Virus Research,21: 25-28.
[20]	Li Liang. 2020. Detection and identification of viruses on the Euonymu sbungeanus Maxin and Chenopodium album L. plants[M. D. Dissertation]. Shenyang University. (in Chinese)
	李梁. 2020. 卫矛与藜上病毒的检测与鉴定[硕士论文]. 沈阳:沈阳大学.
[21]	Li R, Gao S, Hernandez A G, Wechter W P, Fei Z, Ling K S. 2012. Deep sequencing of small RNAs in tomato for virus and viroid identification and strain differentiation. PLoS ONE, 7 (5):e37127.
[22]	Luo Hailin, Yuan Lei, Weng Hua, Yan Jiahui, Guo Qingyun, Wang Wenqing, Ma Xinming. 2023. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 pepper isolate in Qinghai Province. Acta Horticulturae Sinica, 50 (1):161-169. (in Chinese) doi: 10.16420/j.issn.0513-353x.2021-0756
	罗海林, 袁雷, 翁华, 闫佳会, 郭青云, 王文清, 马新明. 2023. 蚕豆萎蔫病毒2号青海辣椒分离物的鉴定与全基因组序列克隆. 园艺学报, 50 (1):161-169. doi: 10.16420/j.issn.0513-353x.2021-0756
[23]	Mollov D, Lockhart B, Zlesak D. 2009. Genome characterization and transmission of Rose yellow vein virus,a new caulimovirus occurring in garden rose. Phytopathology, 99 (6):S87-S87.
[24]	Mollov D, Lockhart B, Zlesak D. 2013. Complete nucleotide sequence of Rose yellow mosaic virus,a novel member of the family potyviridae. Archives of Virology, 158 (9):1917-1923.
[25]	Moury B, Cardin L, Onesto J P, Candresse T, Poupet A. 2001. Survey of Prunus necrotic ringspot virus in rose and its variability in rose and Prunus spp. Phytopathology, 91 (1):84-91. doi: 10.1094/PHYTO.2001.91.1.84 pmid: 18944282
[26]	Murphy F A, Fauquet C M, Bishop D H L, Ghabrial S A, Summers M D. 1995. Virus taxonomy:classification and nomenclature of viruses sixth report of the international committee on taxonomy of viruses. Archives of Virology(Supplementum),10:415-433.
[27]	Niu Yanbing, Shi Xiaoli, Zhang Ximei, Zhao Huiqi, Zhao Baojia. 2015. Molecular identification and sequence analysis of Broad bean wilt Virus 2 isolates from Atractylodes Macrocephala Koidz. Chinese Journal of Virology, 31 (1):58-64. (in Chinese) pmid: 25997332
	牛颜冰, 时晓丽, 张西梅, 赵慧琪, 赵保佳. 2015. 侵染白术的蚕豆萎蔫病毒2号的分子检测与序列分析. 病毒学报, 31 (1):58-64. pmid: 25997332
[28]	Qi Yijun, Zhou Xueping, Li Debao. 1999. Characterization of Broad bean wilt virus 2 isolate from Glycine max. Chinese Journal of Applied and Environmental Biology, 5 (1):70-73. (in Chinese)
	戚益军, 周雪平, 李德葆. 1999. 侵染大豆的蚕豆萎蔫病毒研究. 应用与环境生物学报, 5 (1):70-73.
[29]	Salem N M, Golino D A, Falk B W, Rowhani A. 2008a. Complete nucleotide sequences and genome characterization of a novel double-stranded RNA virus infecting Rosa multiflora. Archives of Virology, 153 (3):455-462.
[30]	Salem N M, Golino D A, Falk B W, Rowhani A. 2008b. Identification and partial characterization of a new luteovirus associated with rose spring dwarf disease. Plant Disease, 92 (4):508-512.
[31]	Seo J K, Kwak H R, Choi B, Han S J, Kim M K, Choi H S. 2017. Movement protein of broad bean wilt virus 2 serves as a determinant of symptom severity in pepper. Virus Research,242:141-145.
[32]	Sertkaya G. 2010. An investigation on Rose mosaic disease of rose in Hatay-Turkey. Julius-Kühn-Archiv,427:309.
[33]	Stubbs L L. 1947. A destructive vascular wilt virus disease of broad bean(Vicia faba L.) in Victoria. Journal of the Department of Agriculture,Victoria,46:323-332.
[34]	Thomas B J. 1980. The detection by serological methods of viruses infecting the rose. Annals of Applied Biology, 94 (1):91-101.
[35]	Vazquez-Iglesias I, Boonham N, Robinson R, Clover G, Fox A. 2023. A survey of rose viruses in England. Crop Protection,168:106231.
[36]	Wang Baoxia, Qi Yonghong, Xin Min, Cui Liyan, Wang Defu, Niu Yanbing. 2019. Identification and analysis of genomic sequence of Broad bean wilt virus 2 isolated from Isatidis Radix. Chinese Journal of Biochemistry and Molecular Biology, 35 (3):333-340. (in Chinese) doi: 10.13865/j.cnki.cjbmb.20193.03.14
	王宝霞, 齐永红, 辛敏, 崔丽艳, 王德富, 牛颜冰. 2019. 蚕豆萎蔫病毒2号板蓝根分离株全基因组序列测定与分析. 中国生物化学与分子生物学报, 35 (3):333-340. doi: 10.13865/j.cnki.cjbmb.20193.03.14
[37]	Yan Dankan, Zheng Hongying, Zhang Haishan, Shen Yan, Gu Jiangtao, Zhang Dongfang, Yan Fei. 2018. Identification and analysis of complete genomic sequence of Broad bean wilt virus 2 Anhui isolate. Journal of Plant Protection, 44 (1):67-73. (in Chinese)
	严丹侃, 郑红英, 张海珊, 沈艳, 顾江涛, 章东方, 燕飞. 2018. 蚕豆萎蔫病毒2号安徽分离物全基因组序列测定与分析. 植物保护, 44 (1):67-73.
[38]	Yardimci N, Culal H. 2009. Occurrence and incidence of Prunus necrotic ringspot virus,Arabis mosaic virus,and Apple mosaic virus on oil rose(Rosa damascena)in the Lakes region of Turkey. New Zealand Journal of Crop and Horticultural Science, 37 (2):95-98.
[39]	Zhou Xueping, Yu Yongjie, Liu Yong, Li Debao. 1995. Studies on two isolates of Broad bean wilt virus from Pisum sativum and Vicia faba. Journal of Zhejiang Agricultural University, 21 (3):221-226. (in Chinese)
	周雪平, 余永杰, 刘勇, 李德葆. 1995. 侵染豌豆和蚕豆的蚕豆萎蔫病毒研究. 浙江农业大学学报, 21 (3):221-226.
[40]	Zhou Xueping, Qi Yijun, Li Fan, Huang Xiaozao, Li Debao. 2001. Complete nucleotide sequence and possible genomic expression strategy of a Chinese isolate of Broad bean wilt virus 2. Acta Biochimica et Biophysica Sinica, 33 (1):46-52. (in Chinese)
	周雪平, 戚益军, 李凡, 黄晓璪, 李德葆. 2001. 蚕豆萎蔫病毒2中国分离物全基因组结构及可能的基因表达方式. 生物化学与生物物理学报, 33 (1):46-52.

病毒序列 Virus sequence	引物名称 Primer name	引物序列（5′-3′） Sequence	片段大小/bp Length
BBWV2	BBWV2-F	TCTCATTCAACGCCACAGGA	530
BBWV2	BBWV2-R	TGGCCAGCAAATTCCACATT	530
RNA1	BBWV2-1-F1	AAACAAACAGCTTTCGTTCCG	1 080
	BBWV2-1-R1	CTGAAATAGCTGCCATAGCAG	1 080
	BBWV2-1-F2	TTTGCAGCGTGGATGCC	1 075
	BBWV2-1-R2	CTTGTAGCCTGACCAAAAAGG	1 075
	BBWV2-1-F3	SGCAGATTGGTTATSWGAR	880
	BBWV2-1-R3	ACCGTGAGRCCCACWAR	880
	BBWV2-1-F4	MGRCGAAGRCACGCAGTRRT	1 083
	BBWV2-1-R4	AAGCACACTTGTGTYCTMACAT	1 083
	BBWV2-1-F5	CAAAGTGATRTKCCTAAYC	1 561
	BBWV2-1-R5	CCRTCAAAYCTGCTGTAA	1 561
	BBWV2-1-F6	CGSCTWGATCGTGGTCC	1 766
	BBWV2-1-R6	TTCCCTCACTACTGAAATTTACTC	1 766
RNA2	BBWV2-2-F1	CAAACAGCTTTCGTTCCGAA	927
	BBWV2-2-R1	AAGCCATCTGCCACCAATTC	927
	BBWV2-2-F2	CGCATA(G)GTC(T)TCTT(C)GTA(G)GCA(T)GA	900
	BBWV2-2-R2	GCCATGTCAGGTGCTGGA	900
	BBWV2-2-F3	TACAAT(C)TTG(A)CCTGGG(A)CCTAGC	1 119
	BBWV2-2-R3	CATGTTCCCAACCTTTTCAATGT	1 119
	BBWV2-2-F4	ATTT(C)TA(G)GATTTTACT(C)CTC(T)ATGAG	1 163
	BBWV2-2-R4	AGGTCATGGAACCCATTTTAATGG	1 163

病毒序列 Virus sequence	引物名称 Primer name	引物序列（5′-3′） Sequence	片段大小/bp Length
BBWV2	BBWV2-F	TCTCATTCAACGCCACAGGA	530
BBWV2	BBWV2-R	TGGCCAGCAAATTCCACATT	530
RNA1	BBWV2-1-F1	AAACAAACAGCTTTCGTTCCG	1 080
	BBWV2-1-R1	CTGAAATAGCTGCCATAGCAG	1 080
	BBWV2-1-F2	TTTGCAGCGTGGATGCC	1 075
	BBWV2-1-R2	CTTGTAGCCTGACCAAAAAGG	1 075
	BBWV2-1-F3	SGCAGATTGGTTATSWGAR	880
	BBWV2-1-R3	ACCGTGAGRCCCACWAR	880
	BBWV2-1-F4	MGRCGAAGRCACGCAGTRRT	1 083
	BBWV2-1-R4	AAGCACACTTGTGTYCTMACAT	1 083
	BBWV2-1-F5	CAAAGTGATRTKCCTAAYC	1 561
	BBWV2-1-R5	CCRTCAAAYCTGCTGTAA	1 561
	BBWV2-1-F6	CGSCTWGATCGTGGTCC	1 766
	BBWV2-1-R6	TTCCCTCACTACTGAAATTTACTC	1 766
RNA2	BBWV2-2-F1	CAAACAGCTTTCGTTCCGAA	927
	BBWV2-2-R1	AAGCCATCTGCCACCAATTC	927
	BBWV2-2-F2	CGCATA(G)GTC(T)TCTT(C)GTA(G)GCA(T)GA	900
	BBWV2-2-R2	GCCATGTCAGGTGCTGGA	900
	BBWV2-2-F3	TACAAT(C)TTG(A)CCTGGG(A)CCTAGC	1 119
	BBWV2-2-R3	CATGTTCCCAACCTTTTCAATGT	1 119
	BBWV2-2-F4	ATTT(C)TA(G)GATTTTACT(C)CTC(T)ATGAG	1 163
	BBWV2-2-R4	AGGTCATGGAACCCATTTTAATGG	1 163