武汉地区油蟠桃病毒种类的小RNA测序鉴定

doi:10.16420/j.issn.0513-353x.2024-0086

摘要/Abstract

摘要：

为明确侵染湖北武汉地区油蟠桃的病毒种类，采用小RNA测序技术对采集的5份病害样品进行了鉴定分析。结果表明样品中含有桃相关黄症病毒属病毒（peach-associated luteovirus，PaLV）、桃病毒D（peach virus D，PeVD）和桃潜隐花叶类病毒（peach latent mosaic viroid，PLMVd）。采用RT-PCR技术对测序结果进行了验证，获得的PaLV、PeVD和PLMVd基因序列（GenBank登录号：OP244073、OP244074和OP244075）与GenBank中相应的PaLV分离物T01-2（MK361479）、PeVD分离物T02-1（MK361476）和PLMVd分离物HUEI-Ob166/1-1（MW046337）的核苷酸序列一致性为92.15% ~ 98.59%。进一步利用PaLV、PeVD、PLMVd及其他6种桃病毒特异性引物对109份油蟠桃样品进行RT-PCR检测，仅检测到PLMVd、PaLV和PeVD，检出率依次为33.94%、29.36%和5.50%。PLMVd可能为侵染油蟠桃的优势类病毒。病毒和类病毒复合侵染率为22.93%，高于单一的侵染率，表明复合侵染可能是油蟠桃上病毒、类病毒侵染的主要形式。

关键词: 油蟠桃, 病毒, 类病毒, 小RNA测序, RT-PCR

Abstract:

To determine the viruses infecting flat nectarine in Wuhan，Hubei，five shoot samples with the virus-like symptoms were collected and analyzed by small RNA sequencing. The results showed that peach associated luteovirus（PaLV），peach virus D（PeVD），and peach latent mosaic viroid（PLMVd）were detected. RT-PCR with specific primers was conducted to confirm the result. Partial sequences of PaLV，PeVD and PLMVd were obtained by RT-PCR，sequenced and deposited in GenBank. Sequences of PaLV，PeVD and PLMVd（GenBank accession No. OP244073，OP244074 and OP244075）were 92.15%-98.59% identical with the corresponding nucleotide sequences of PaLV isolate T01-2（MK361479），PeVD isolate T02-1（MK361476）and PLMVd isolate HUEI-Ob166/1-1（MW046337） in GenBank. One hundred and nine flat nectarine samples were collected and detected by RT-PCR with primers specific for PaLV，PeVD，PLMVd and six other peach viruses. Only PLMVd，PaLV and PeVD were detected in these samples，with a detection rate of 33.94%，29.36% and 5.50%，respectively. The highest incidence of PLMVd indicated that it was the dominant viroid infecting flat nectarine in Wuhan. The mixed infection of viruses and viroid was 22.93%，higher than that of a single virus or viroid infection，indicating that mixed infection of virus and viroid was common in flat nectarine.

Key words: flat nectarine, virus, viroid, small RNA sequencing, RT-PCR

刘勇, 王富荣, 王会良, 艾小艳, 朱炜, 张杨, 甘志猛, 龚林忠, 何华平. 武汉地区油蟠桃病毒种类的小RNA测序鉴定[J]. 园艺学报, 2025, 52(3): 727-736.

LIU Yong, WANG Furong, WANG Huiliang, AI Xiaoyan, ZHU Wei, ZHANG Yang, GAN Zhimeng, GONG Lingzhong, HE Huaping. Identification and Analysis of Viruses Infecting Flat Nectarine in Wuhan by Small RNA Sequencing[J]. Acta Horticulturae Sinica, 2025, 52(3): 727-736.

图/表 7

表1 RT-PCR检测使用的引物

Table 1 Primers used for RT-PCR

病毒名 Name of virus	引物名 Primer name	引物序列（5ʹ-3ʹ） Primer sequence	长度/bp Length	参考文献 Reference
PaLV	PaLV-981	F：ATCCAGCATGATTCGTTCGT；R：CGTGACAGAGCCCATCTAAAA	981	Xu et al.，2019
PeVD	PeVD-695	F：CCGACTCTGTCATTGACTACCC；R：GAGGACTCGTGCGGACTTTA	695	Xu et al.，2019
PLMVd	Primer	1：ATCACACCCTCCTCGGAACCAA；2：CCAGGTAACGCCGTAGAAACTG	337	Xu et al.，2006
PNRSV	PNRSV	L：TGGTCCCACTCAGGGCTCAACAAAG；R：ACGCGCAAAAGTGTCGAAATAAA	455	Zong et al.，2017
PDV	PDV	L：CGAAGTCTATTTCCGAGTGG；R：CCACTGGCTTGTTTCGCTGT	304	Zong et al.，2017
PBNSPaV	det	F：TACCGAAGAGGGTTTGGATG；R：AGTCGCACCACCAGTCTTCT	400	Rwahnih et al.，2007
CGRMV	CGRMV	F：TGCGGGAAATCAACTCTTGTC；R：TGTGCCACCAAACACCTTAC	363	卢美光等，2015
NSPaV	NSPaV	F1：CCTGTACCGCAGAAGAAAGC；R1：ATGAGTGTGCAGGGTGATGA	474	Lu et al.，2017
PPV	PPV	F3：GGAATGTGGGTGATGATGG；RR：CTCTTCTTGTGTTCCGACGTTTC	345	Singh et al.，2019

图1 田间染病桃树植株症状 A：枝叶生长缓慢；B：叶片扭曲；C：叶片褪绿黄化（右）；D：枝条上没有果实（右）

Fig. 1 Symptoms of infected peach tree in the field A：The branches and leaves grow slowly；B：Leaves distorted；C：Leaves chlorosis（right）；D：No fruit on the branch（right）

图2 基于核苷酸序列的PaLV系统进化树各分支上的数字为bootstrap检验的可信度。距离标尺的数值代表序列替代率。★本研究获得的分离物。下同

Fig. 2 Phylogenetic analysis of PaLV based on nucleotide sequences Values indicate the bootstrap confidence values for each branch. The value of the distance scale represents the sequence substitution rate. ★The isolates obtained in this study. The same below

图3 基于核苷酸序列的PeVD系统进化树

Fig. 3 Phylogenetic analysis of PeVD based on nucleotide sequences

图4 基于核苷酸序列的PLMVd系统进化树

Fig. 4 Phylogenetic analysis of PLMVd based on nucleotide sequences

图5 部分样品中PLMVd（A）、PaLV（B）和PeVD（C）的RT-PCR检测结果 M：DNA marker；N：阴性对照；1 ~ 22：检测样品

Fig. 5 RT-PCR detection results of PLMVd（A）、PaLV（B）and PeVD（C）in part samples M：DNA marker；N：Negative control；1-22：Test samples

表2 109份武汉油蟠桃样品中病毒检测结果

Table 2 Detection results of the viruses in 109 flat nectarines from Wuhan

病毒侵染类型 Types of virus infection	阳性样品数Positive samples
病毒侵染类型 Types of virus infection	总数Total	有症状Symptoms	无症状Symptomless
PLMVd	12	9（叶褪绿黄化Leaf chlorosis and yellowing）	3
PaLV	7	2（叶轻微褪绿黄化Leaf mild chlorosis and yellowing）	5
PeVD	0
PLMVd + PaLV	19	17（叶褪绿黄化Leaf chlorosis and yellowing）	2
PLMVd + PaLV + PeVD	6	6（叶褪绿黄化皱缩，不结果Leafchlorosis and yellowing，wrinkle，acarpous	0
总计Total	44

参考文献 30

[1]	Bu Fangdi, Chen Junguang, Liu Zhen, Xiang Benchun, Shen Mian, Cui Baiming, Zheng Yinying. 2021. Nectarine stem pitting-associated virus and Asian prunus virus found in Prunus persica of Xinjiang. Acta Horticulturae Sinica, 48 (1):49-59. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-0215 URL
	卜方迪, 陈俊光, 刘贞, 向本春, 申冕, 崔百明, 郑银英. 2021. 新疆蟠桃中发现油桃茎痘相关病毒和亚洲李属病毒. 园艺学报, 48 (1):49-59. doi: 10.16420/j.issn.0513-353x.2020-0215 URL
[2]	Chan P, Lowe T M. 2008. GtRNAdb:a database of transfer RNA genes detected in genomic sequence. Nucleic Acids Research, 37 (suppl 1):93-97.
[3]	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
[4]	Fan Hongyan. 2011. Incident of viroids from peach and gene diversity analysis of PLMVd[M. D. Dissertation]. Changchun: Jilin Agricultural University. (in Chinese)
	范宏艳. 2011. 桃树类病毒发生率调查及基因多样性分析[硕士论文]. 长春: 吉林农业大学.
[5]	Feng Jia, Li Gang, Zhu Xiaoping, Song Hongri, Chen Wenyu, Peng Futian, Li Shifang, Liu Huixiang. 2017. Molecular detection and identification of viruses and viroids infecting Feicheng peach. Plant Protection, 43 (2):146-151. (in Chinese)
	冯佳, 李刚, 竺晓平, 宋红日, 陈文玉, 彭福田, 李世访, 刘会香. 2017. 侵染肥城桃的病毒和类病毒的分子检测与鉴定. 植物保护, 43 (2):146-151.
[6]	Gao Rui, Liang Yingbo, Li Shifang, Mahmut Mijit, Lu Meiguang. 2016. Preparation and application of cRNA poly 2 probe for the detection of cherry green ring mottle virus and peach tatent mosaic viroid. Journal of Plant Protection, 43 (6):1055-1056. (in Chinese)
	高蕊, 梁颖博, 李世访, 麦合木提江 · 米吉提, 卢美光. 2016. 同时检测樱桃绿环斑驳病毒和桃潜隐花叶类病毒的二聚cRNA探针制备和应用. 植物保护学报, 43 (6):1055-1056.
[7]	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
[8]	Griffiths-Jones S, Bateman A, Marshall M, Khanna A, Eddy S R. 2003. Rfam:an RNA family database. Nucleic Acids Research, 31 (1):439-441.
[9]	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 Chinese) doi: 10.16420/j.issn.0513-353x.2021-0592 URL
	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 URL
[10]	He Y, Cai L, Zhou L L, Yang Z K, Hong N, Wang G P, Li S F, Xu W X. 2018. Author correction:deep sequencing reveals the first fabavirus infecting peach. Scientific Reports, 8 (1):5268. doi: 10.1038/s41598-018-23058-2 pmid: 29568034
[11]	Herranz M C, Niehl A, Rosales M, Fiore N, Zamorano A, Granell A, Pallas V. 2013. A remarkable synergistic effect at the transcriptomic level in peach fruits doubly infected by Prunus necrotic ringspot virus and peach latent mosaic viroid. Virology Journal, 10 (164):1-15.
[12]	Igori D, Lim S, Baek D, Kim S Y, Seo E, Cho I S, Cho G S, Lim H S, Moon J S. 2017. Complete nucleotide sequence and genome organization of peach virus D,a putative new member of the genus Marafivirus. Archives of Virology, 162:1769-1772.
[13]	Jin Xiaoxiang, Yao Junxi, Yin Yueyan, Li Tingting, Guo Miao, Zhong Jing, He Shouxing, Chen Yue, He Jiawei, Yan Bo, Ding Ming. 2024. Molecular characterization of the odontoglossum ringspot virus and cymbidium chlorotic mosaic virus mix-infecting Cymbidium hybridum in Yunnan. Acta Horticulturae Sinica, 51 (8):1844-1852. (in Chinese)
	金晓香, 姚俊汐, 尹跃艳, 李婷婷, 郭淼, 钟静, 和寿星, 陈越, 和家卫, 鄢波, 丁铭. 2024. 云南大花蕙兰复合感染的ORSV和CyCMV病毒基因组序列特征分析. 园艺学报, 51 (8):1844-1852.
[14]	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.
[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]	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. (in Chinese) 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.
[17]	Lu M G, Zhang C, Zhang Z X, Wang C A, Li S F. 2017. Nectarine stem-pitting-associated virus detected in peach trees in China. Plant Disease, 101 (3):513. doi: 10.1094/PDIS-09-16-1256-PDN
[18]	Lu Meiguang, Wu Bing, Gao Rui, Zhang Zhixiang, Xiao Hong, Chen Ranran, Li Shifang. 2015. Preliminary investigation on cherry virus diseases and detection of the pathogen in some regions of China. Plant Protection, 41 (1):98-103. (in Chinese)
	卢美光, 吴冰, 高蕊, 张志想, 肖红, 陈冉冉, 李世访. 2015. 我国部分地区樱桃病毒病害初步调查和病原检测. 植物保护, 41 (1):98-103.
[19]	Pruesse E, Quast C, Knittel K, Fuchs B M, Ludwig W, Peplies J, Glockner 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
[20]	Rwahnih M A, Uyemoto J K, Falk B W, Rowhani A. 2007. Molecular characterization and detection of plum bark necrosis stem pitting-associated virus. Archives of Virology, 152 (12):2197-2206. pmid: 17885796
[21]	Singh K, Neubauerová T, Kundu J K. 2019. Quantitative analysis of the interaction of heterologous viruses with plum pox virus in C 5 HoneySweet transgenic plums. Journal of Integrative Agriculture, 18 (10):2302-2310.
[22]	Su Z W, Sun M, Cai Z X, Guo S L, Li J Y, Zhao B T, Shen Z J, Ma R J, Yan J, Yu M L. 2023. Identification and expression analysis of chlorogenic acid biosynthesis key gene PpHCT in peach. Horticultural Plant Journal, 9 (4):670-680.
[23]	Wang L P, He Y, Kang Y P, Hong N, Farooq A N U, Wang G P, Xu W X. 2013. Virulence determination and molecular features of peach latent mosaic viroid isolates derived from phenotypically different peach leaves:a nucleotide polymorphism in 11 contributes to symptom alteration. Virus Research, 177:171-178.
[24]	Wang Lirong. 2021. Current situation and development suggestions of peach industry in China. China Fruits,(10):1-5. (in Chinese)
	王力荣. 2021. 我国桃产业现状与发展建议. 中国果树,(10):1-5.
[25]	Xu W X, Hong N, Zhang J K, Wang G P. 2006. Improving the sensitivity of single-strand conformation polymorphism(SSCP)to study the variability of PLMVd. Journal of Virological Methods, 135 (2):276-280.
[26]	Xu Y X, Yang L J, Zhou J, Yang Y, Li S F. 2019. Multiplex RT-PCR to simultaneously detect three viruses that infect peach. Letters in Applied Microbiology, 69:318-324. doi: 10.1111/lam.13220 pmid: 31529706
[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]	Zhou J, Zhang Z, Lu M G, Xiao H, Li S F. 2018. First report of peach-associated luteovirus from flat peach and nectarine in China. Plant Disease, 102 (12):2669.
[29]	Zhou Jun. 2020. Study on the virome of peach and its wild relative species[Ph. D. Dissertation]. Beijing: Chinese Academy of Agricultural Sciences. (in Chinese)
	周俊. 2020. 桃及其野生近缘种中病毒组研究[博士论文]. 北京: 中国农业科学院.
[30]	Zong X J, Liu Q Z, Wang W W, Wei H R, Wang J W. 2017. Detection of prunus necrotic ringspot virus and prune dwarf virus in sweet cherry (Prunus avium)and Chinese cherry(Prunus pseudocerasus)in Shandong Province,China. Acta Horticulturae, 1161:469-474.