海南西番莲主要病毒种类的分子检测与鉴定

doi:10.16420/j.issn.0513-353x.2021-0418

摘要/Abstract

摘要：

2020年5—7月,在海南三亚市和五指山市采集表现花叶、皱缩、畸形等疑似病毒病的西番莲叶片样品176份,通过PCR和RT-PCR检测发现：侵染西番莲的病毒主要为黄瓜花叶病毒（cucumber mosaic virus,CMV）、南瓜蚜传黄化病毒（cucurbit aphid-borne yellows virus,CABYV）、东亚西番莲病毒（East Asian Passiflora virus,EAPV）、夜来香花叶病毒（Telosma mosaic virus,TeMV）、西番莲木质化病毒（passion fruit woodiness virus,PWV）、西番莲斑驳病毒（Passiflora mottle virus,PaMV）和番茄黄化曲叶病毒（tomato yellow leaf curl virus,TYLCV）。TeMV的检出率最高,为52.27%;其次为PaMV,43.75%;CMV和EAPV均为28.98%;PWV为22.73%;TYLCV为9.09%;CABYV为4.55%。TeMV、PaMV、CMV、EAPV和PWV可能是三亚和五指山西番莲的主要病毒,样品病毒复合侵染率为78.91%,存在32种复合侵染类型,以2 ~ 4种病毒复合侵染为主。3个TYLCV西番莲分离物的基因组全长均为2 781 bp,含6个ORF。序列相似性和系统进化分析结果表明：3个分离物均属于TYLCV-Israel株系,序列相似性为98.85% ~ 99.14%,与海南已报道的4个其他作物上的分离物聚在同一进化分支上。这是TYLCV侵染西番莲的首次报道。

关键词: 西番莲, 病毒病, 病原检测, 番茄黄化曲叶病毒

Abstract:

A total of 176 Passiflora edulis leaf samples showing mosaic,crinkle and deformation were collected from Sanya and Wuzhishan of Hainan Province from May to July 2020. Seven viruses were detected by PCR and RT-PCR including cucumber mosaic virus （ CMV ）,cucurbit aphid-borne yellows virus （ CABYV ）,East Asian Passiflora virus （ EAPV ）,Telosma mosaic virus （ TeMV ）,passion fruit woodiness virus （ PWV ）,Passiflora mottle virus （ PaMV ） and tomato yellow leaf curl virus （ TYLCV ）. The result showed that TeMV was the most prevalent virus with infection rate of 52.27%,followed by PaMV（43.75%）,CMV and EAPV （ 28.98% ）,PWV （ 22.73% ）,TYLCV （ 9.09% ） and CABYV （ 4.55% ）. TeMV,PaMV,CMV,EAPV and PWV may be the main viruses infecting P. edulis in Sanya and Wuzhishan. 78.91% of samples were detected co-infection with a total of 32 kinds of co-infection types,in which co-infection of two to four viruses commonly occurs. Three complete genome sequences of TYLCV from P. edulis were determined to be 2 781 bp and encoded six open reading frames （ ORFs ）. Both the identity and phylogenetic analysis based on the complete genome showed that all the three TYLCV isolates belonged to TYLCV Israel strain with nt identity ranging from 98.85% to 99.14%,and clustered in the same branch with the four TYLCV isolates from other crops in Hainan. To our knowledge,this is the first report of TYLCV in P. edulis.

Key words: Passiflora edulis, virus disease, pathogen detection, tomato yellow leaf curl virus

中图分类号:

S668.9

陈礼浪, 杨天章, 蔡儒平, 林小漫, 邓南康, 车海彦, 林雅婷, 孔祥义. 海南西番莲主要病毒种类的分子检测与鉴定[J]. 园艺学报, 2022, 49(8): 1785-1794.

CHEN Lilang, YANG Tianzhang, CAI Ruping, LIN Xiaoman, DENG Nankang, CHE Haiyan, LIN Yating, KONG Xiangyi. Molecular Detection and Identification of Viruses from Passiflora edulis in Hainan[J]. Acta Horticulturae Sinica, 2022, 49(8): 1785-1794.

图/表 6

表1 西番莲病毒检测所用引物和反应条件

Table 1 Primers and reation condition used for virus detection in Passiflora edulis

引物名称 Primer name	引物序列（5ʹ-3ʹ） Sequence	退火温度/℃ Annealing temperature	预期产物大小/bp Expected size	参考文献 Reference
CMV-CP fwd	TGGTCGTCCAACTATTAACCAC	53	~ 320	Kim et al.,2014
CMV-CP rev	TACTGATAAACCAGTACCGGTGA	53	~ 320	Kim et al.,2014
CABYV-F	CAAAGGACAATCTCACGGGCAGTTCC	58	~ 570	本研究This study
CABYV-R	GTTGCRTCGTGCCATTCCATCCCGTT	58
PfGSV RNA1 C13F	ATTYATGCGTTTCACGGTTA	50	~ 320	Ramos-González et al.,2020
PfGSV RNA1 C13R	CGAATGCCTCTGACAYAACT	50	~ 320	Ramos-González et al.,2020
EAPV-AO fwd	AGGATGCTAGCATGGATGAGCTAC	58	~ 900	本研究This study
EAPV-AO rev	ATGTTCTGATTGACGTCCCGTGCAG	58	~ 900	本研究This study
TeMV-CPf	TCAAGTAAGGTGGATGATGTT	49	~ 816	谢丽雪等,2017
TeMV-CPr	CTGCACAGAGCCAACCCCAA	49	~ 816	谢丽雪等,2017
PWV-F1	GGTKTGGTGCATHGAYAAYGGAAC	55	~ 400	本研究This study
PWV-R1	ACRTCVCKTGCAGTRTGCCTTTCAGT	55	~ 400	本研究This study
PaMV-F	TCACGTCTTGGACAGTTCCACAATCGC	55	~ 300	本研究This study
PaMV-R	CGACTTTAGTGACCTTGTACTGTCT	55	~ 300	本研究This study
PA	TAATATTACCKGWKGVCCSC	56	~ 580	谢艳等,2002
PB	TGGACYTTRCAWGGBCCGCACA	56	~ 580	谢艳等,2002
TYLCV-F	GTTGAGAAAACATTTGTGAATCCCC	54	~ 500	本研究This study
TYLCV-R	GGCAAAGCAACACAAAGTACGGGA	54	~ 500	本研究This study
PLDV-F	CCSATGGACTTTGGCCAGGTGTTCA	55	~ 680	本研究This study
PLDV-R	CAGGCAGAGAATGGCGTSTATATC	55	~ 680	本研究This study

图1 西番莲病毒病田间症状 A：花叶、皱缩和明脉;B：黄斑和皱缩;C：花叶、斑驳、小叶、畸形和缢缩;D：花叶、黄化、皱缩、疱斑和缢缩;E：曲叶和轻微皱缩;F：果实瘤状凸起。

Fig. 1 Field symptoms of Passiflora edulis caused by virus A：Mosaic,crinkling and vein clearing;B：Yellow spot and crinkling;C：Mosaic,mottle,little leaf,deformity and constriction;D：Mosaic,yellowing,crinkling,blister spot and constriction;E：Leaf curl and slight crinkling;F：Protuberance in fruit.

图2 西番莲样品中CMV、EAPV、TeMV、PWV、PaMV、CABYV和TYLCV的PCR扩增结果

Fig. 2 PCR amplification of CMV,EAPV,TeMV,PWV,PaMV,CABYV和TYLCV in Passiflora edulis M：DL2000 DNA marker;1：CMV,~ 320 bp;2：CABYV,~ 570 bp;3：EAPV,~ 900 bp;4：TeMV,~ 816 bp：5：PWV,~ 400 bp：6：PaMV,~ 300 bp：7：TYLCV,~ 500 bp.

表2 海南西番莲叶片样品病毒病检测结果

Table 2 Detection results of the major viruses in diseased Passiflora edulis in Hainan

样品来源 Sample origin	样品数 Sample number	感染病毒的样品数Number of virus-infected sample
样品来源 Sample origin	样品数 Sample number	CMV	CABYV	PfGSV	EAPV	TeMV	PWV	PaMV	TYLCV	PLDV
三亚市崖州区 Yazhou District,Sanya	10	3	0	0	0	0	5	2	2	0
三亚市吉阳区 Jiyang District,Sanya	40	6	1	0	22	36	10	37	10	0
五指山市南圣镇Nansheng Town,Wuzhishan	103	20	1	0	6	44	22	18	3	0
五指山市毛道乡Maodao Township,Wuzhishan	23	22	6	0	23	12	3	20	1	0
总数 Total	176	51	8	0	51	92	40	77	16	0

图3 西番莲中病毒单一侵染（5种）和复合侵染（32种类型）及相应的检出样品数量

Fig. 3 Virus single infection（5 types）and compound infection（32 types）in Passiflora edulis and the corresponding number of detected samples

图4 基于TYLCV完整核酸序列的系统进化树 ▲本研究中分离到的 TYLCV序列;△在海南已分离到的TYLCV序列。

Fig. 4 The phylogenetic tree based on complete nucleotide sequence of TYLCV ▲ Isolate of TYLCV sequenced in this study;△ Isolate of TYLCV sequenced from Hainan.

参考文献 30

[1]	Bai Xiao-juan, Li Ya-dong, Wang Guo-hua, Yin Qing-zhen, Geng Bao-jin, Dong Wen-qi, Zhou Chun-jiang, Wu Zhi-ming. 2015. Molecular identification and sequence analysis of tomato yellow leaf curl virus(TYLCV)Isolate from Shijiazhuang. Acta Horticulturae Sinica, 42 (1):167-173. (in Chinese)
	白晓娟, 李亚栋, 王国华, 尹庆珍, 耿保进, 董文琦, 周春江, 吴志明. 2015. 番茄黄化曲叶病毒石家庄分离物的分子鉴定及序列分析. 园艺学报, 42 (1):167-173.
[2]	Brown J K, Zerbini F M, Navas-Castillo J, Moriones E, Ramos-Sobrinho R, Silva J C F, Fiallo-Olivé E, Briddon R W, Hernández-Zepeda C, Idris A, Malathi V G, Martin D P, Rivera-Bustamante R, Ueda S, Varsani A. 2015. Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Archives of Virology, 160:1593-1619. doi: 10.1007/s00705-015-2398-y URL
[3]	Chen L J, Sun D L, Lu Y L, An Y X, 2020. First report of ramie mosaic virus on passion fruit in Guangdong,southern China. Journal of Plant Pathology, 102:1305. doi: 10.1007/s42161-020-00590-7 URL
[4]	Chen L J, Zhang X X, Lu Y L, An Y X. 2021. First report of turnip mosaic virus on yellow passion fruit in China. Journal of Plant Pathology, 103 (5):401. doi: 10.1007/s42161-020-00734-9 URL
[5]	Ding Ying-na, Li Shi-fang, Zhang Sheng, Cui Yuan-yu, Yang Huan, He Wei, Sun Xiao-jun. 2013. Molecular detection and analysis of the complete nucleotide sequences of TYLCV isolates from Xinjiang. Plant Protection, 39 (3):51-55. (in Chinese)
	丁英娜, 李世访, 张升, 崔元玗, 杨华, 何伟, 孙晓军. 2013. 新疆番茄黄化曲叶病毒检测及其DNA-A基因组序列分析. 植物保护, 39 (3):51-55.
[6]	Du Kai-tong, Wang Shao-jie, Yong Rong-jing, Zuo Lin-yu, Deng Cong-liang, Fan Zai-feng, Zhou Tao, Zhou Qi. 2016. Molecular identification and full-length sequence analysis of tomato yellow leaf curl virus on sweet pepper in China. Acta Phytopathologica Sinica, 46 (3):425-428. (in Chinese)
	杜开通, 王少杰, 雍容婧, 左琳彧, 邓丛良, 范在丰, 周涛, 周琦. 2016. 侵染甜椒的番茄黄化曲叶病毒的分子鉴定和全序列分析. 植物病理学报, 46 (3):425-428.
[7]	Huang Ai-jun, Wang Ying, Hu Xiao-lin. 2019. Research progress of passiflora virus disease. South China Fruits, 48 (3):141-146. (in Chinese)
	黄爱军, 王莹, 胡笑林. 2019. 西番莲病毒病研究进展. 中国南方果树, 48 (3):141-146.
[8]	Ji Ying-hua, Zhang Hui, Zhao Wen-hao, Wu Shu-hua, Hu Jie, Cheng Zhao-bang, Zhao Tong-min, Yu Wen-gui, Zhu Ye-qin, Zhou Yi-jun. 2016. Sequence analysis of tomato yellow leaf curl virus isolated from Phaseolus vulgaris. Acta Phytopathologica Sinica, 46 (5):591-597. (in Chinese)
	季英华, 张晖, 赵文浩, 吴淑华, 胡婕, 程兆榜, 赵统敏, 余文贵, 朱叶芹, 周益军. 2016. 江苏菜豆上分离的番茄黄化曲叶病毒基因组序列分析. 植物病理学报, 46 (5):591-597.
[9]	Kim M K, Jeong R D, Kwak H R, Lee S H, Kim J S, Kim K H, Cha B, Choi H S. 2014. First report of Cucumber mosaic virus isolated from wild Vigna angularis var. nipponensis in Korea. The Plant Pathology Journal, 30 (2):200-207. doi: 10.5423/PPJ.NT.01.2013.0012 URL
[10]	Liu Zhi-xin, Pan Jun-song, Wu Hao, Zheng Xue-qin. 1995. Molecular identification and specific detection of telosma mosaic virus infecting passion fruit. Chinese Journal of Tropical Crops, 16 (S1):49-53. (in Chinese)
	刘志昕, 潘俊松, 吴豪, 郑学勤. 1995. 侵染西番莲的CMV分离物的研究. 热带作物学报, 16 (S1):49-53.
[11]	Mabvakure B, Martin D P, Kraberger S, Cloete L, Brunschot S V, Geering A D W, Thomas J E, Bananej K, Lett J M, Lefeuvre P, Varsani A, Harkins G W. 2016. Ongoing geographical spread of Tomato yellow leaf curl virus. Virology, 498:257-264. doi: S0042-6822(16)30250-1 pmid: 27619929
[12]	Prasad A, Sharma N, Hari-Gowthem G, Muthamilarasan M, Prasad M. 2020. Tomato yellow leaf curl virus:impact,challenges,and management. Trends in Plant Science, 25 (9):897-911. doi: 10.1016/j.tplants.2020.03.015 URL
[13]	Ramos-González P L, Santos G F D, Chabi-Jesus C, Harakava R, Kitajima E W, Freitas-Astúa J. 2020. Passion fruit green spot virus genome harbors a new orphan ORF and highlights the flexibility of the 5′-end of the RNA 2 segment across cileviruses. Frontiers in Microbiology, 11:206. doi: 10.3389/fmicb.2020.00206 pmid: 32117189
[14]	Song Ruo-nan, Hou Yong-xiang, Cui Meng-di, Feng-yan, Han Qun-xin, Rao Xue-qin. 2020. Detection of virus infecting passion fruit in Guangdong. Journal of Zhongkai University of Agriculture and Engineering, 33 (1):11-15,20. (in Chinese)
	宋若楠, 侯永翔, 崔梦迪, 冯岩, 韩群鑫, 饶雪琴. 2020. 广东百香果病毒的检测. 仲恺农业工程学院学报, 33 (1):1-15,20.
[15]	Tang Y F, He Z F, Zhou G H. 2020. Passiflora edulis is a new host of cotton leaf curl Multan virus-betasatellite complex in China. Canadian Journal of Plant Pathology, 42 (1):493-498. doi: 10.1080/07060661.2019.1706640 URL
[16]	Tang Ya-fei, Zhou Yang, Zhang Li, Li Zheng-gang, She Xiao-man, Yu Lin, Lan Guo-bing, Deng Ming-guang, He Zi-fu. 2020. Tomato yellow leaf curl virus infecting tomato in Hainan and its molecular characteristics. Journal of Southern Agriculture, 51 (8):1970-1976. (in Chinese)
	汤亚飞, 周洋, 张丽, 李正刚, 佘小漫, 于琳, 蓝国兵, 邓铭光, 何自福. 2020. 番茄黄化曲叶病毒侵染危害海南番茄及其分子特征. 南方农业学报, 51 (8):1970-1976.
[17]	Wu J B, Dai F M, Zhou X P. 2006. First report of tomato yellow leaf curl virus in China. Plant Disease, 90 (10):1359.
[18]	Xie Hui-ting, Li Zhan-biao, Cui Li-xian, Qin Bi-xia, Liu Li-hui, Lin Shan-yu, Chen Jin-qing, Cai Jian-he. 2020. Identification of pathogens and analysis of the dominant viruses of passionfruit virus disease in some areas of Guangxi. Acta Phytopathologica Sinica, 50 (4):387-393. (in Chinese)
	谢慧婷, 李战彪, 崔丽贤, 秦碧霞, 刘丽辉, 林珊宇, 陈锦清, 蔡健和. 2020. 广西局地西番莲病毒病的病原鉴定及优势病毒分析. 植物病理学报, 50 (4):387-393.
[19]	Xie L X, Gao F L, Zheng S, Zhang X Y, Zhang L J, Li T. 2019. Molecular characterization of a new potyvirus infecting passion fruit. Archives of Virology, 164:1903-1906. doi: 10.1007/s00705-019-04251-8 URL
[20]	Xie Lixue, Zhang Lijie, Zhang Xiaoyan, Zheng Shan, Li Tao. 2018. First report of East Asian passiflora virus infecting Passiflora edulisin in Fujian,China. Acta Horticulturae Sinica, 45 (8):1587-1594. (in Chinese)
	谢丽雪, 张立杰, 张小艳, 郑姗, 李韬. 2018. 福建西番莲上首次检出东亚西番莲病毒. 园艺学报, 45 (8):1587-1594.
[21]	Xie Li-xue, Zhang Xiao-yan, Zheng Shan, Zhang Li-jie, Li Tao. 2017. Molecular identification and specific detection of Telosma mosaic virus infecting passion fruit. Scientia Agricultura Sinica, 50 (24):4725-4734. (in Chinese)
	谢丽雪, 张小艳, 郑姗, 张立杰, 李韬. 2017. 侵染西番莲的夜来香花叶病毒的分子鉴定及特异性检测. 中国农业科学, 50 (24):4725-4734.
[22]	Xie Li-xue, Zhang Xiao-yan, Zheng shan, Zhang Li-jie, Li Tao. 2019. Molecular identification of papaya leaf curl Guandong virus infecting passion fruit in Fujian Province. Southeast Horticulture, 7 (5):19-22. (in Chinese)
	谢丽雪, 张小艳, 郑姗, 张立杰, 李韬. 2019. 福建西番莲上广东番木瓜曲叶病毒的分子鉴定. 东南园艺, 7 (5):19-22.
[23]	Xie Yan, Zhang Zhong-kai, Li Zheng-he, Ding Ming, Zhou Xue-ping. 2002. Rapid detection of whitefly-transmitted geminiviruses by TAS-ELISA and PCR. Acta Phytopathologica Sinica, 32 (2):182-186. (in Chinese)
	谢艳, 张仲凯, 李正和, 丁铭, 周雪平. 2002. 粉虱传双生病毒的TAS-ELISA及PCR快速检测. 植物病理学报, 32 (2):182-186.
[24]	Xu Ping-dong, Ke Chong. 1990. The occurrence and etiological identification of virus diseases of passionfruit in Fujian Province. Journal of Fujian Academy of Agricultural Sciences, 5 (2):47-55. (in Chinese)
	徐平东, 柯冲. 1990. 福建省西番莲病毒病的发生及其病原鉴定. 福建省农科院学报, 5 (2):47-55.
[25]	Yan Jia-wen, Yuan Qi-feng, Xie Pu, Wang Li-juan, Ma Yu-hua, Wang Zheng-yuan. 2019. Identification of viruses infecting Passiflora edulisin Guizhou province by small RNA sequencing. Chinese Journal of Tropical Crops, 40 (8):1577-1584. (in Chinese)
	严佳文, 袁启凤, 解璞, 王立娟, 马玉华, 王正媛. 2019. 利用小RNA测序技术检测贵州西番莲病毒. 热带作物学报, 40 (8):1577-1584.
[26]	Yang K, Yan H X, Song L, Jin P F, Miao W G, Cui H G. 2018. Analysis of the complete genome sequence of a potyvirus from passion fruit suggests its taxonomic classification as a member of a new species. Archives of Virology, 163:2583-2586. doi: 10.1007/s00705-018-3885-8 pmid: 29789942
[27]	Yi Zi-lin, Xu Wei-qing. 2018. Present situation and prospect of introduction and cultivation of passion fruit in Hainan. Chinese Journal of Tropical Agriculture, 38 (7):25-28. (in Chinese)
	易籽林, 徐卫清. 2018. 百香果在海南引种栽培现状与展望. 热带农业科学, 38 (7):25-28.
[28]	Zhang Ai-hong, Yang Fei, Pan Yang, Sun Xiang-rui, Zhang Shang-qing, Miao Hong-qin, Di Dian-ping, Sun Qian. 2019. Identification and distribution of mixed infections of tomato yellow leaf curl virus and tomato chlorosis virus in Hebei Province,China. Acta Phytopathologica Sinica, 49 (2): 271-275. (in Chinese)
	张爱红, 杨菲, 潘阳, 孙祥瑞, 张尚卿, 苗洪芹, 邸垫平, 孙茜. 2019. 河北省番茄黄化曲叶病毒和番茄褪绿病毒复合侵染及分布. 植物病理学报, 49 (2):271-275.
[29]	Zhang Jie, Lin Wen-wu, Wan Bai-jie, Yu Xiao-fang, Wu Zu-jian. 2016. Molecular characterization of Tomato yellow leaf curl virus in Fuzhou. Journal of Fujian Agriculture and Forestry University(Natural Science Edition), 45 (5):501-504. (in Chinese)
	张洁, 林文武, 宛柏杰, 余小芳, 吴祖建. 2016. 福州番茄黄化曲叶病毒的全基因组结构特征. 福建农林大学学报(自然科学版), 45 (5):501-504.
[30]	Zhang S, Huang A J, Zhou X, Li Z H, Dietzgen R G, Zhou C Y, Cao M J. 2021. Natural defect of a plant Rhabdovirus glycoprotein gene:a case study of virus-plant coevolution. Phytopathology, 111 (1):227-236. doi: 10.1094/PHYTO-05-20-0191-FI URL