Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification

doi:10.16420/j.issn.0513-353x.2024-0192

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (2): 503-512.doi: 10.16420/j.issn.0513-353x.2024-0192

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Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification

FAN Ronghui, WU Jianshe, FENG Zinan, ZHONG Shengyuan, ZHONG Huaiqin^*()

Institute of Crop Sciences（Fujian Provincial Germplasm Resources Center），Fujian Academy of Agricultural Science，Fujian Engineering Research Center for Characteristic Floriculture，Fuzhou 350013，China

Received:2024-10-29 Revised:2024-12-13 Online:2025-02-25 Published:2025-02-23
Contact: ZHONG Huaiqin

Abstract

Abstract:

This study collected 56 suspected virus samples of Phalaenopsis from Fuzhou，Xiamen，Zhangzhou，and Quanzhou in Fujian Province，which were divided into three types of mixed samplesbased on infection symptoms：chlorosis，yellowing brown spots，and yellowing wrinkles. Small RNA sequencing technology and RT-PCR were used for detection，and a total of 8 viruses were found. According to the detection rate，they were Cymbidium mosaic virus（CyMV）（85.71%），Odontoglossum ringspot virus （ORSV）（35.71%），white clover mosaic virus（WCMV）（32.14%），yam virus X（YaVX）（21.43%），Narcissus mosaic virus（NaMV）（10.71%），pepino mosaic virus（PeMV）（8.93%），potato virus X（PoVX）（7.14%），and Schlumbergera virus X（ScVX）（7.14%）from high to low. Among them， detection rates of CyMV，ORSV and WCMV are over 30%，and most of them are compound infections，accounting for 75.51% of the total infections. Using small RNA sequencing sequences as templates，specific primers were designed and a multiplex RT-PCR system was established for simultaneous detection of five viruses. The primer concentrations for ORSV，CyMV，WCMV，YaVX，and PoVX were 0.30，0.06，0.20，0.50 and 0.40 μmol · L^-1，respectively. At an annealing temperature of 56 ℃，target bands with fragment sizes of 1 156，908，561，292 and 162 bp，respectively，can be simultaneously amplified with good specificity. The sensitivity test results show that these 5 viruses can be detected from infected plant tissues equivalent to or greater than 0.0001 mg.

Key words: Phalaenopsis, small RNA deep sequencing, multiple RT-PCR, virus

FAN Ronghui, WU Jianshe, FENG Zinan, ZHONG Shengyuan, ZHONG Huaiqin. Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification[J]. Acta Horticulturae Sinica, 2025, 52(2): 503-512.

Figures/Tables 13

References 30

[1]	Bateman A, Marshall M, Khanna A, Griffiths-Jones S, Eddy S R. 2003. Rfam:an RNA family database. Nucleic Acids Research, 31 (1):439-441.
[2]	Chan P P, Lowe T M. 2009. GtRNAdb:a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Research, 37 (suppl 1):D93-D97.
[3]	Chai Ali, Chen Lida, Cao Jinqiang, Kang Huajun, Shi Yanxia, Xie Xuewen, Li Baoju. 2019. Identification of viruses causing eggplant purple mottle flower disease by siRNA high-throughput sequencing and RT-PCR detection. Acta Horticulturae Sinica, 46 (3):508-518. (in Chinese) doi: 10.16420/j.issn.0513-353x.2018-0097
	柴阿丽, 陈利达, 曹金强, 康华军, 石延霞, 谢学文, 李宝聚. 2019. 利用 siRNA 高通量测序和RT-PCR技术鉴定引起茄子斑驳紫花病的病毒种类. 园艺学报, 46 (3):508-518. doi: 10.16420/j.issn.0513-353x.2018-0097
[4]	Chen Dao, Zhang Jie, Wu Zujian, Ding Xinlun. 2021. Genome sequence of strawberry pallidosis-associated virus isolated in China. Acta Horticulturae Sinica, 48 (1):146-152. (in Chinese) doi: 10.16420/j.issn.0513-353x.2019-0989 URL
	陈道, 张洁, 吴祖建, 丁新伦. 2021. 草莓白化相关病毒中国分离物全基因组分析. 园艺学报, 48 (1):146-152. doi: 10.16420/j.issn.0513-353x.2019-0989 URL
[5]	Chen W H, Chen H H. 2017. Orchid Biotechnology III:Phalaenopsis Viruses and Their Detection Techniques. 10.1142/10022:165-188.
[6]	Chen Yahan, Ma Qiang, Sun Pingping, Zhang Lei, Li Zhengnan. 2020. Identification of viruses causing apricot decline and leaf chlorosis disease by siRNA high-throughput sequencing and RT-PCR detection. Acta Horticulturae Sinica, 47 (4):725-733. (in Chinese) doi: 10.16420/j.issn.0513-353x.2019-0433 URL
	陈雅寒, 马强, 孙平平, 张磊, 李正男. 2020. 杏衰退萎黄病病毒的siRNA高通量测序和RT-PCR鉴定. 园艺学报, 47 (4):725-733. doi: 10.16420/j.issn.0513-353x.2019-0433 URL
[7]	Fan Ronghui, Huang Minling, Zhong Huaiqin, Wu Jianshe, Luo Yuanhua. 2015. Multiple RT-PCR for simultaneous detection of three viruses in Oncidium. Fujian Journal of Agricultural Sciences, 30 (7):697-700. (in Chinese)
	樊荣辉, 黄敏玲, 钟淮钦, 吴建设, 罗远华. 2015. 文心兰3种主要病毒多重RT-PCR检测体系的建立. 福建农业学报, 30 (7):697-700.
[8]	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 Chinese) doi: 10.16420/j.issn.0513-353x.2021-0673 URL
	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 URL
[9]	Hu J S, Ferreira S. 1993. Detection of Cymbidium mosaic virus, Odontoglossum ringspot virus,Tomato spotted wilt virus,and Potyvirues infecting orchids in Hawaii. Plant Disease, 77 (5):464-467.
[10]	Inouye N. 1983. Host range and properties of a strain of Odontoglossum ringspot virus in Japan. Nogaku Kenkyu,60:53-67.
[11]	Inouye N. 2008. Viruses of orchids,symptoms,diagnosis,spread,and control. Blue Bird,the Netherlands:176.
[12]	Jurka J, Kapitonov V V, Pavlicek A, Klonowski P, Kohany O, Walichiewicz J. 2005. Repbase update,a database of eukaryotic repetitive elements. Cytogenetic and Genome Research, 110 (1-4):462-467.
[13]	Kado C I, Jensen D D. 1964. Cymbidium mosaic virus in Phalaenopsis. Phytopathology,54:947-974.
[14]	Khentry Y, Paradornuwat A, Tantiwiwat S, Phansiri S, Thaveechai N. 2006. Incidence of Cymbidium mosaic virus and Odontoglossum ringspot virus in Dendrobium spp.in Thailand. Crop Protection, 25 (9):926-932.
[15]	Langmead B, Trapnell C, Pop M, Salzberg S L. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology,10:R25.
[16]	Lawson R H, Hsu H T. 1995. Orchid∥Loebenstein et al.(eds.),virus and virus-like diseases of bulb and flower crops. John Wiley & Sons,West Sussex:409-420.
[17]	Lesemann D E. 1977. Long,filamentous virus-like particles associated with vein necrosis of Dendrobium phalaenopsis. Phytopathologische Zeitschrift,89:330-339.
[18]	Lesemann D, Begtrup J. 1971. Electron microscopic demonstration of a bacilliform virus in Phalaenopsis. Phytopathologische Zei tschrift,71:257-269.
[19]	Liang Yuqing, Sun Chunhui, Deng Congliang, Shi Xiju, Zhong Yan, Li Yongqiang. 2024. Identification and analysis of complete genomic sequence of broad bean wilt virus 2 Beijing rose isolate. Acta Horticulturae Sinica, 51 (10):2320-2328. doi: 10.16420/j.issn.0513-353x.2023-0884 URL
	梁宇卿, 孙春辉, 邓丛良, 史喜菊, 种焱, 李永强. 2024. 蚕豆萎蔫病毒2号北京玫瑰分离物的鉴定. 园艺学报, 51 (10):2320-2328
[20]	Liu Yong, Li Fan, Li Yueyue, Zhang Songbai, Gao Xiwu, Xie Yan, Yan Fei, Zhang Ansheng, Dai Liangying, Cheng Zhaobang, Ding Ming, Niu Yanbing, Wang Shengji, Che Haiyan, Jiang Tong, Shi Xiaobin, He Zifu, Wu Yunfeng, Zhang Deyong, Qing Ling, Yan Wanrong, Yang Xuehui, Tang Yafei, Zheng Hongying, Tang Qianjun, Zhang Songbai, Zhang Dongfang, Cai Li, Tao Xiaorong. 2019. Identification,distribution and occurrence of viruses in the main vegetables of China. Scientia Agricultura Sinica, 52 (2):239-261. (in Chinese) doi: 10.3864/j.issn.0578-1752.2019.02.005
	刘勇, 李凡, 李月月, 张松柏, 高希武, 谢艳, 燕飞, 张安盛, 戴良英, 程兆榜, 丁铭, 牛颜冰, 王升吉, 车海彦, 江彤, 史晓斌, 何自福, 吴云锋, 张德咏, 青玲, 严婉荣, 杨学辉, 汤亚飞, 郑红英, 唐前君, 章松柏, 章东方, 蔡丽, 陶小荣. 2019. 侵染我国主要蔬菜作物的病毒种类、分布与发生趋势. 中国农业科学, 52 (2):239-261. doi: 10.3864/j.issn.0578-1752.2019.02.005
[21]	Martínez R T, Severo de Almeida M M, Rodriguez R, Silva de Oliveira A, Melo F L, Resende R O. 2018. Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing. Virology Journal, 15 (1):24. doi: 10.1186/s12985-018-0931-9 pmid: 29373979
[22]	Pruesse E, Quast C, Knittel K, Fuchs B M, Ludwig W, Peplies J, Glöckner F O. 2007. SILVA:a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Research, 35 (21):7188-7196. doi: 10.1093/nar/gkm864 pmid: 17947321
[23]	Sun Aiqing, Wang Lihua, Zhang Yiping, Yang Xiumei, Xu Feng, Su Yan. 2024. Simultaneous detection of Cymbidium mosaic virus and Odontoglossum ringspot virus by TaqMan duplex real-time fluorescence quantitative PCR. Plant Protection, 50 (2):219-227. (in Chinese)
	孙爱青, 王丽花, 张艺萍, 杨秀梅, 许凤, 苏艳. 2024. TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒. 植物保护, 50 (2):219-227.
[24]	Xie Linna, Su Mengyun, Zhu Mingming, Gong Hongtao, Zhu Limei, Xu Min, Zhang Bo, Gan Liming, Luo Fengxia. 2017. ELISA detection and symptom expression of two viruses in different varieties of Phalaenopsis. Jiangsu Agricultural Science, 45 (3):80-83. (in Chinese)
	谢林娜, 苏梦芸, 朱明明, 贡宏涛, 朱丽梅, 徐敏, 张波, 甘黎明, 罗凤霞. 2017. 不同品种蝴蝶兰 2 种病毒的 ELISA 检测及其症状表现. 江苏农业科学, 45 (3):80-83.
[25]	Yoon J Y, Chung B N, Choi S K. 2011. High sequence conservation among Odotoglossum ringspot virus isolates from orchids. Virus Genes, 42 (2):261-267.
[26]	Zhang Liying, Wang Sen, Chen Dongliang, Yu Xin, Jiang Baolong, Cheng Yuhan, Li Yanhui, Huang Conglin. 2023. Establishment of a multiplex RT-PCR system for detection of five viruses/ viroids in Chrysanthemum × Morifolium. Chinese Journal of Virology, 39 (4):1045-1052. (in Chinese)
	张利英, 王森, 陈东亮, 喻锌, 姜宝龙, 程钰涵, 李彦慧, 黄丛林. 2023. 菊花5种病毒及类病毒多重RT-PCR检测方法的建立. 病毒学报, 39 (4):1045-1052.
[27]	Zerbino D R, Birney E. 2008. Velvet:algorithms for de novo short read assembly using de Bruijn graphs. Genome Research, 18 (5):821-829. doi: 10.1101/gr.074492.107 pmid: 18349386
[28]	Zheng Y X, Chen C C, Yang C J, Yeh S D, Jan F J. 2008. Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids. European Journal of Plant Pathology,120:199-209.
[29]	Zheng Y X, Chen C C, Jan F J. 2011. Identification and characterization of three new Phalaenopsis Orchid-infection viruses. Acta Hortic,901:127-132.
[30]	Zhu Lijuan, Bai Yani, Su Lanyi, Wang Chi, He Shiyun, Han Yanhong. 2023. Establishment of a rapid one-step multiplex RT-PCR assay for simultaneous detection of four viruses infecting jasmine. Acta Phytopathologica Sinica, 54 (4):819-828. (in Chinese)
	朱丽娟, 白雅妮, 苏兰怡, 王驰, 何诗芸, 韩艳红. 2023. 4种茉莉病毒一步法多重RT-PCR检测技术的建立. 植物病理学报, 54 (4):819-828.

病毒Virus	数量 Contigs count
病毒Virus	褪绿 Chlorosis	黄化褐斑 Yellowing brown spots	黄化皱缩 Yellowing wrinkles
建兰花叶病毒Cymbidium mosaic virus（CyMV）	44	13	25
水仙花叶病毒Narcissus mosaic virus（NaMV）	0	1	0
齿兰环斑病毒Odontoglossum ringspot virus（ORSV）	0	12	0
凤果花叶病毒pepino mosaic virus（PeMV）	0	1	0
马铃薯X病毒potato virus（PoVX）	0	1	0
仙人指X病毒Schlumbergera virus X（ScVX）	0	1	0
白三叶草花叶病毒white clover mosaic virus（WCMV）	0	3	2
淮山药X病毒yam virus X（YaVX）	0	3	1

病毒Virus	数量 Contigs count
病毒Virus	褪绿 Chlorosis	黄化褐斑 Yellowing brown spots	黄化皱缩 Yellowing wrinkles
建兰花叶病毒Cymbidium mosaic virus（CyMV）	44	13	25
水仙花叶病毒Narcissus mosaic virus（NaMV）	0	1	0
齿兰环斑病毒Odontoglossum ringspot virus（ORSV）	0	12	0
凤果花叶病毒pepino mosaic virus（PeMV）	0	1	0
马铃薯X病毒potato virus（PoVX）	0	1	0
仙人指X病毒Schlumbergera virus X（ScVX）	0	1	0
白三叶草花叶病毒white clover mosaic virus（WCMV）	0	3	2
淮山药X病毒yam virus X（YaVX）	0	3	1

样本Sample	原始数据Raw_reads	待分析数据Clean_reads	GC/%	Q30/%	重叠群N50 Contigs_N50	重叠群数量Contigs_Num
褪绿Chlorosis	13 558 557	10 586 409	50.25	97.20	74	82
黄化褐斑Yellowing brown spots	19 031 729	15 892 227	49.80	97.77	795	37
黄化皱缩 Yellowing wrinkles	13 710 865	10 084 402	50.63	97.74	455	44

样本Sample	原始数据Raw_reads	待分析数据Clean_reads	GC/%	Q30/%	重叠群N50 Contigs_N50	重叠群数量Contigs_Num
褪绿Chlorosis	13 558 557	10 586 409	50.25	97.20	74	82
黄化褐斑Yellowing brown spots	19 031 729	15 892 227	49.80	97.77	795	37
黄化皱缩 Yellowing wrinkles	13 710 865	10 084 402	50.63	97.74	455	44

病毒Virus	检出率/% Detection rate
病毒Virus	福州 Fuzhou	泉州 Quanzhou	厦门 Xiamen	漳州 Zhangzhou	合计Total
CyMV	86.67	84.62	78.57	92.86	85.71
NaMV	13.30	7.70	0	21.43	10.71
ORSV	40.00	23.08	35.71	42.86	35.71
PeMV	13.33	0	7.14	7.14	7.14
PoVX	0	15.38	14.29	7.14	8.93
ScVX	0	7.70	21.43	7.14	8.93
WCMV	40.00	23.08	28.57	35.71	32.14
YaVX	20.00	23.08	28.57	14.29	21.43

Detection and Identification of Virus Infecting Phalaenopsis Based on Small RNA Deep Sequencing Technology and Multiple RT-PCR Identification

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引物 Primer	引物序列（5ʹ-3ʹ）Primer sequence	目的片段/bp Product
CyMV	F：AGTCTCAACCTGACGTGGAG；R：GGGGAAGCGGTGAATATTA	908
YaVX	F：GGCAGAATCTATTGGAAAAA；R：CCATGAATGACTAAGGGAAG	292
NaMV	F：TGTCACTCACATTCCCCCTG；R：TCCTGAACGTTTGCCTCAAC	234
ORSV	F：TATGACAAATCCCAGAACGA；R：GCAAATAGAAACAAACTCCA	1 156
PeMV	F：ATCTTAGCAACCAGCGTTAT；R：GTCCCAAGCAACATAGGC	397
PoVX	F：GAGGGTCCCACTTTTGATGC；R：CAAGCGGTTGGTGATTTTAT	162
ScVX	F：CAATCACCGGACTTTGACCC；R：AATAGCTCCCAGTTTTTCCG	84
WCMV	F：GTTGTGCTCCCTGTTGAA；R：GTCCCAAATGGATAAGGATAG	561

组合Composition	CyMV	WCMV	YaVX	ORSV	PoVX
1	0.10	0.10	0.10	0.10	0.10
2	0.02	0.40	0.40	0.20	0.20
3	0.02	0.20	0.30	0.10	0.10
4	0.10	0.40	0.40	0.20	0.40
5	0.06	0.20	0.50	0.30	0.40

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