基于宏病毒组测序技术的苹果病毒病鉴定与分析

doi:10.16420/j.issn.0513-353x.2021-0450

摘要/Abstract

摘要：

采用宏病毒组测序技术（Virome Sequencing Technology）对河南地区染病苹果树进行病毒检测与分析,从建库测序的苹果叶片中共检测出13种病毒,其中有5种苹果病毒:苹果茎沟病毒（apple stem grooving virus,ASGV）、苹果茎痘病毒（apple stem pitting virus,ASPV）、苹果褪绿叶斑病毒（apple chlorotic leaf spot virus,ACLSV）、苹果坏死花叶病毒（apple necrotic mosaic virus,ApNMV）和苹果绿皱缩相关病毒（apple green crinkle associated virus,AGCaV）。对病毒检测结果中的5种苹果病毒进行RT-PCR验证,扩增后均获得目的条带,与宏病毒组测序检测结果一致,表明宏病毒组测序检测结果精准可靠。根据测序结果设计特异性引物对ASPV全长进行扩增,得到1条长度为9 246 nt的ASPV-HN分离物基因组,与NCBI数据库中已报道的19个ASPV分离物的基因组核苷酸序列一致性为75.48% ~ 79.85%;宏病毒组测序拼接所得的两条长度均为6 475 nt的ASGV基因组全长基因ASGV-HN1和ASGV-HN2,与NCBI数据库中已报道的18个ASGV分离物的基因组核苷酸序列一致性为79.98% ~ 98.08%。对53份河南不同产区的苹果叶片样品进行苹果花叶病毒（apple mosaic virus,ApMV）和ApNMV的检测,结果显示花叶病症样品中均检测出ApNMV,未检测到ApMV,表明ApNMV可能是引起河南地区苹果花叶病的主要病原。

关键词: 苹果, 病毒病, 宏病毒组测序技术, RT-PCR, 检测, 苹果花叶病

Abstract:

Virome Sequencing Technology was used for virus detection and analysis of virus-infected apple trees in Henan Province,a total of 13 viruses were detected from apple leaves in the sequenced database,including five apple viruses:apple stem grooving virus（ASGV）,apple stem pitting virus（ASPV）,apple chlorotic leaf spot virus（ACLSV）,apple necrotic mosaic virus（ApNMV）and apple green crinkle associated virus（AGCaV）. RT-PCR was used to verify the detection results of five apple viruses,and objective bands were obtained after amplification,it was consistent with virome sequencing results,indicating that the virome sequencing detection results were accurate and reliable. Based on the sequencing results,specific primers were designed to amplify the full length of ASPV,a length of 9 246 nt ASPV-HN isolate genome was obtained and shared sequence identities 75.48% to 79.85% with complete genome of 19 ASPV isolates published in NCBI database;the two assembly sequences of ASGV genomes ASGV-HN1 and ASGV-HN2 based on virome sequencing results were both 6 475 nt in length and shared sequence identities 79.98% to 98.08% with complete genome of 18 ASGV isolates published in NCBI database. Detection of apple mosaic virus（ApMV）and ApNMVwere carried out in 53 apple leaf samples collected from different apple producing areas in Henan Province. ApNMV was detected in all samples with mosaic disease,while ApMV was not detected in 53 samples. It indicated that ApNMV may be the main pathogen which can cause apple mosaic disease in Henan Province.

Key words: apple, virus disease, Virome Sequencing Technology, RT-PCR, detection, apple mosaic disease

中图分类号:

S661.1

夏炎, 黄松, 武雪莉, 刘一琪, 王苗苗, 宋春晖, 白团辉, 宋尚伟, 庞宏光, 焦健, 郑先波. 基于宏病毒组测序技术的苹果病毒病鉴定与分析[J]. 园艺学报, 2022, 49(7): 1415-1428.

XIA Yan, HUANG Song, WU Xueli, LIU Yiqi, WANG Miaomiao, SONG Chunhui, BAI Tuanhui, SONG Shangwei, PANG Hongguang, JIAO Jian, ZHENG Xianbo. Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology[J]. Acta Horticulturae Sinica, 2022, 49(7): 1415-1428.

图/表 15

参考文献 30

[1]	Barba, Czosnek, Hadidi. 2014. Historical perspective,development and applications of next-generation sequencing in plant virology. Viruses-Basel, 6 (1):106-136. doi: 10.3390/v6010106 URL
[2]	Buchfink B, Xie C, Huson D H. 2015. Fast and sensitive protein alignment using DIAMOND. Nature Method, 12 (1):59-60. doi: 10.1038/nmeth.3176 URL
[3]	Campbell A I. 2015. The effect of some latent virus infections on the growth and cropping of apples. Journal of Horticultural Science, 38 (1):15-19. doi: 10.1080/00221589.1963.11514054 URL
[4]	Candresse T, Marais A, Faure C, Gentit P. 2013. Association of little cherry virus 1 (LChV1) with the Shirofugen stunt disease and characterization of the genome of a divergent LChV1 isolate. Phytopathology, 103 (3):293-298. doi: 10.1094/PHYTO-10-12-0275-R pmid: 23402630
[5]	Coetzee B, Freeborough M J, Maree H J, Celton J M, Rees D J G, Burger J T. 2010. Deep sequencing analysis of viruses infecting grapevines:Virome of a Vineyard. Virology, 400 (2):157-163. doi: 10.1016/j.virol.2010.01.023 URL
[6]	Fan Sheng-tao, Gao Yu-wei, Xia Xian-zhu. 2014. Amplication of viral metagenomics in medical field. Chinese Journal of Biologicals, 27 (2):285-288. (in Chinese)
	范胜涛, 高玉伟, 夏咸柱. 2014. 病毒宏基因组学在医学领域的应用. 中国生物制品学杂志, 27 (2):285-288.
[7]	Giampetruzzi A, Roumi V, Roberto R, Malossin U, Yoshikawa N, Notte P L, Terlizzi F, Credi R, Saldarelli P. 2012. A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris. Virus Research, 163 (1):262-268. doi: 10.1016/j.virusres.2011.10.010 pmid: 22036729
[8]	Grabherr M G, Haas B J, Yassour M, Levin J Z, Thompson D A, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q D, Chen Z H, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren B W, Nusbaum C, Lindblad T K, Friedman N, Regev A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnology, 29 (7):644-652. doi: 10.1038/nbt.1883 pmid: 21572440
[9]	Grimova L, Winkowska L, Zíka L, Pavel P. 2016. Distribution of viruses in old commercial and abandoned orchards and wild apple trees. Journal of Plant Pathology, 98 (3):549-554.
[10]	Huang Ai-jun, Wang Ying, Ding Min, Lu Zhan-jun, Yi Long. 2019. Establishment and application of multiplex PCR rapid detection of four citrus viruses. Acta Horticulturae Sinica, 46 (8):1616-1622. (in Chinese)
	黄爱军, 王莹, 丁敏, 卢占军, 易龙. 2019. 柑橘4种病毒多重PCR检测技术的建立及应用. 园艺学报, 46 (8):1616-1622.
[11]	Hye-Kyung S, Kyu-Hyon H, Chang-Ki S, Su-Wan S, Dong-Giun K, Yong-Mun C, Young-Jea C, Dea-Hyun K, Hyeong-Jin Jee, Suk-Chan L. 2006. The pear black necrotic leaf spot disease virus transmitted by Talaromyces flavus displays pathogenicity similar to Apple stem grooving virus strains. Plant Pathology Journal, 22 (3):255-259.
[12]	Komorowska B, Malinowski T, Michalczuk L. 2010. Evaluation of several RT-PCR primer pairs for the detection of Apple stem pitting virus. Journal of Virological Methods, 168 (1):242-247. doi: 10.1016/j.jviromet.2010.04.024 URL
[13]	Martelli G P, Jelkmann W. 1998. Foveavirus,a new plant virus genus. Archives of Virology, 143 (6):1245-1249. pmid: 9687881
[14]	Martin D, Rybicki E. 2000. RDP:detection of recombination amongst aligned sequences. Bioinformatics, 16 (6):562-563. pmid: 10980155
[15]	Masotti A, Preckel T. 2006. Analysis of small RNAs with the Agilent 2100 Bioanalyzer. Nature Method, 3 (8):658-658.
[16]	Noda H, Yamagishi N, Xing F, Xie J P, Li S F, Zhou T, Ito T, Yoshikawa N. 2017. Apple necrotic mosaic virus,a novel ilarvirus from mosaic diseased apple trees in Japan and China. Journal of General Plant Pathology, 83 (2):1-8. doi: 10.1007/s10327-016-0691-6 URL
[17]	Plese N, Hoxha E, Milicic D. 1975. Pathological anatomy of trees affected with Apple stem grooving virus. Phytopathology, 82 (4):315-325.
[18]	Rasmussen T S, Mentzel C M J, Kot W, Castro-Mejia J L, Zuffa S, Swann J R, Hansen L H, Vogensen S K, Hansen A K, Nielsen D S. 2020. Faecal virome transplantation decreases symptoms of type 2 diabetes and obesity in a murine model. Gut,69 (12):gutjnl-2019-320005.
[19]	Rowhani A, Maningas M A, Lile L S, Daubert S D, Golino D A. 1995. Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions. Phytopathology, 85 (3):347-352. doi: 10.1094/Phyto-85-347 URL
[20]	Sheikh M A, Bhat K M, Mir J I, Mir M A, Raja W H. 2017. Phenotypic and molecular screening for disease resistanceof apple cultivars and selections against apple scab(Venturia inaequalis). International Journal of Chemical Studies, 5 (4):1107-1111.
[21]	Sono T, Sastry K S. 2015. Plant virus and viroid diseases in the tropics,Volume 1:Introduction of plant viruses and sub-viral agents,classification,assessment of loss,transmission,and diagnosis. Virus Disease, 26 (4):324.
[22]	Tao Yuan, Wu Xing-quan. 2017. Research progress on methods for detection of plant virus. Molecular Plant Breeding, 15 (7):2901-2906. (in Chinese)
	陶源, 吴兴泉. 2017. 植物病毒检测方法的研究进展. 分子植物育种, 15 (7):2901-2906.
[23]	Tiziano C, Folwell R J, Wandschneider P, Eastwell K C, Howell W E. 2003. Economic implications of a virus prevention program in deciduous tree fruits in the U.S. Crop Protection, 22 (10):1149-1156. doi: 10.1016/S0261-2194(03)00156-X URL
[24]	Wang L P, Hong N, Matic S, Mytra A, Song Y S, Wang G P. 2011. Pome fruit viruses at the Canadian clonal Genebank and molecular characterization of Apple chlorotic leaf spot virus isolates. Scientia Horticulturae, 130 (3):665-671. doi: 10.1016/j.scienta.2011.08.001 URL
[25]	Wang Ya-nan, Hu Tong-le, Liu Shu-xiang, Zhao Xu-sheng, Yang Jun-yu, Cao Ke-qiang. 2010. Research on apple virus in China. Journal of Anhui Agricultural Sciences, 38 (15):7933-7936. (in Chinese)
	王亚楠, 胡同乐, 刘淑香, 赵绪生, 杨军玉, 曹克强. 2010. 我国苹果病毒病的研究现状. 安徽农业科学, 38 (15):7933-7936.
[26]	Wei Ning-sheng. 1959. Preliminary report of apple mosaic disease test. Journal of Northwest A & F University(Natural Science Edition),(3):41-74,125-134. (in Chinese)
	魏宁生. 1959. 苹果花叶病试验初报. 西北农林科技大学学报(自然科学版),(3):41-74,125-134.
[27]	Xing Fei. 2018. Identification,detection,techniques and pathogenicity of viral pathogen associated with mosaic disease of apple tress in China[Ph. D. Dissertation]. Beijing:China Agricultural University. (in Chinese)
	邢飞. 2018. 我国苹果花叶病病原的鉴定、检测体系建立及其致病性分析究[博士论文]. 北京: 中国农业大学.
[28]	Yoon J Y, Joa J H, Choi K S, Do K S, Lim H C, Chung B N. 2014. Genetic diversity of a natural population of Apple stem pitting virus isolated from apple in Korea. The Plant Pathology Journal, 30 (2):195-199. doi: 10.5423/PPJ.NT.02.2014.0015 URL
[29]	Yoshikawa N, Sasamoto K, Sakurada M, Takahashi T, Yanase H. 1996. Apple stem grooving and citrus tatter leaf capilloviruses obtained from a single shoot of Japanese pear(Pyrus serotina). Jpn J Phytopathol, 62 (2):119-124. doi: 10.3186/jjphytopath.62.119 URL
[30]	Yozwiak N L, Skewes-Cox P, Stenglein M D, Balmaseda A, Derisi J L. 2012. Virus identification in unknown tropical febrile illness cases using deep sequencing. PLoS Neglected Tropical Diseases, 6 (2):e1485. doi: 10.1371/journal.pntd.0001485 URL

引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增大小/bp Amplification length	退火温度/℃ Annealing temperature	来源 Source
ASGV-F	ATGAGTTTGGAAGACGTGCTTC	745	58	Hye-Kyung et al.,2006
ASGV-R	CTAACCCTCCAGTTCCAAGTTA	745	58	Hye-Kyung et al.,2006
ASPF1CP	GGGTGTACTTTGAGGCAGTATT	235	56	Komorowska et al.,2010
ASPR3CP	GAGCGGATGCGGTACATCTGTAT	235	56	Komorowska et al.,2010
ACLSV-F	CCATCTTCGCGAACATAGC	660	51	本研究合成 Synthesized in this study
ACLSV-R	GTAGTAAAATATTTAAAAGTCTAC	660	51	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
AGCaV-F	TCGGAAAGACTGTCCCGTTG	647	58	本研究合成 Synthesized in this study
AGCaV-R	ACTCTTGCCCCCGATTCTTG	647	58	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
ApMV-F1	CAACATGGTCTGCAAGTACT	640	50	Noda et al.,2017
ApMV-R1	GGAGCTAATCGCTCCATCAT	640	50	Noda et al.,2017

引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增大小/bp Amplification length	退火温度/℃ Annealing temperature	来源 Source
ASGV-F	ATGAGTTTGGAAGACGTGCTTC	745	58	Hye-Kyung et al.,2006
ASGV-R	CTAACCCTCCAGTTCCAAGTTA	745	58	Hye-Kyung et al.,2006
ASPF1CP	GGGTGTACTTTGAGGCAGTATT	235	56	Komorowska et al.,2010
ASPR3CP	GAGCGGATGCGGTACATCTGTAT	235	56	Komorowska et al.,2010
ACLSV-F	CCATCTTCGCGAACATAGC	660	51	本研究合成 Synthesized in this study
ACLSV-R	GTAGTAAAATATTTAAAAGTCTAC	660	51	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
AGCaV-F	TCGGAAAGACTGTCCCGTTG	647	58	本研究合成 Synthesized in this study
AGCaV-R	ACTCTTGCCCCCGATTCTTG	647	58	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
ApMV-F1	CAACATGGTCTGCAAGTACT	640	50	Noda et al.,2017
ApMV-R1	GGAGCTAATCGCTCCATCAT	640	50	Noda et al.,2017

引物名称 Primer name	用途 Amplification	引物序列（5′-3′） Primer sequence	扩增位置 Amplification position
5'RACE外引物 Outer Primer	5′端扩增 5′ RACE	TGTTCCTCAGAAGTGAATGA	NC003462（101 ~ 121）
5'RACE内引物 Inner primer	5′端扩增 5′ RACE	TCCATGCATCCAGGAATGAG	NC003462（553 ~ 571）
ASPV-1F	ASPV	GGATACGCAAACAAACTCTGAA	NC003462（1 ~ 22）
ASPV-1R	ASPV	CATGCADGGYTTRAARTGRTTRTC	NC003462（3627 ~ 3650）
ASPV-2F	ASPV	AATCAYTTYAARCCHTGCATGCC	NC003462（3627 ~ 3650）
ASPV-2R	ASPV	CAAAYTTBGTGCAVAGYTGRGACT	NC003462（5725 ~ 5748）
ASPV3-F	ASPV	AGTCYCARCTBTGCACVAARTTTG	NC003462（5725 ~ 5748）
ASPV-3R	ASPV	AATATTAAACAGATACGCCCTGACC	NC003462（7862 ~ 7886）
ASPV-4F	ASPV	GGTCAGGGCGTATCTGTTTAATATT	NC003462（7862 ~ 7886）
ASPV-4R	ASPV	GAAAATCTAGTTAAAACAAAAATAAG	NC003462（9307 ~ 9332）
3'RACE外引物 Outer Primer	3′端扩增 3′ RACE	AGCATGTCTGGAACCTCATGCT	NC003462（8896 ~ 8917）
3'RACE内引物 Inner primer	3′端扩增 3′ RACE	GACTTCTTCTTTGGAGTTGAG	NC003462（8988 ~ 9008）

引物名称 Primer name	用途 Amplification	引物序列（5′-3′） Primer sequence	扩增位置 Amplification position
5'RACE外引物 Outer Primer	5′端扩增 5′ RACE	TGTTCCTCAGAAGTGAATGA	NC003462（101 ~ 121）
5'RACE内引物 Inner primer	5′端扩增 5′ RACE	TCCATGCATCCAGGAATGAG	NC003462（553 ~ 571）
ASPV-1F	ASPV	GGATACGCAAACAAACTCTGAA	NC003462（1 ~ 22）
ASPV-1R	ASPV	CATGCADGGYTTRAARTGRTTRTC	NC003462（3627 ~ 3650）
ASPV-2F	ASPV	AATCAYTTYAARCCHTGCATGCC	NC003462（3627 ~ 3650）
ASPV-2R	ASPV	CAAAYTTBGTGCAVAGYTGRGACT	NC003462（5725 ~ 5748）
ASPV3-F	ASPV	AGTCYCARCTBTGCACVAARTTTG	NC003462（5725 ~ 5748）
ASPV-3R	ASPV	AATATTAAACAGATACGCCCTGACC	NC003462（7862 ~ 7886）
ASPV-4F	ASPV	GGTCAGGGCGTATCTGTTTAATATT	NC003462（7862 ~ 7886）
ASPV-4R	ASPV	GAAAATCTAGTTAAAACAAAAATAAG	NC003462（9307 ~ 9332）
3'RACE外引物 Outer Primer	3′端扩增 3′ RACE	AGCATGTCTGGAACCTCATGCT	NC003462（8896 ~ 8917）
3'RACE内引物 Inner primer	3′端扩增 3′ RACE	GACTTCTTCTTTGGAGTTGAG	NC003462（8988 ~ 9008）

分类 Classify	病毒名称 Virurs name	相关Contig数量 Quantity of related contig
植物病毒 Plant virus	苹果茎沟病毒Apple stem grooving virus,ASGV	25
	苹果茎痘病毒Apple stem pitting virus,ASPV	71
	苹果褪绿叶斑病毒Apple chlorotic leaf spot virus,ACLSV	8
	苹果坏死花叶病毒Apple necrotic mosaic virus,ApNMV	7
	苹果绿皱缩相关病毒Apple green crinkle associated virus,AGCaV	6
	柑橘叶斑病毒Citrus leaf blotch virus,CLBV	1
真菌病毒 Mycovirus	树孢链格菌丝裂病毒1 Alternaria arborescens mitovirus 1	1
	球孢白僵菌分体病毒2 Beauveria bassiana partitivirus 2	1
	葡萄座腔菌镰刀病毒1 Botryosphaeria dothidea fusarivirus 1	5
	葡萄座腔菌分体病毒1 Botryosphaeria dothidea partitivirus 1	2
	葡萄座腔菌病毒1 Botryosphaeria dothidea virus 1	2
	白粉病菌相关病毒11 Erysiphe necator associated ourmia-like virus 11	1
	葡萄霜霉病菌相关病毒Plasmopara viticola lesion associated ourmia-like virus	8