基于小RNA深度测序技术鉴定侵染广东辣椒的病毒种类

doi:10.3864/j.issn.0578-1752.2019.13.006

摘要/Abstract

摘要：

【目的】病毒病是广东省辣椒生产上主要病害之一,田间病株率一般为5%—30%,严重时可达100%。本研究旨在探明危害广东辣椒的病毒种类,为辣椒病毒病的防控提供理论依据。【方法】2013—2016年,从广东省广州、佛山、惠州、江门、梅州、湛江、茂名和韶关8市辣椒主要种植区采集疑似病毒病辣椒样品125份,分别提取每份辣椒病样总RNA,对从茂名、梅州、韶关3市采集的病样按地点和症状混合成7份混合病样进行小RNA深度测序分析,根据小RNA深度测序分析结果,对每种病毒分别根据小RNA深度测序拼接的基因片段序列和GenBank数据库中与该拼接序列同源性最高的病毒基因组序列保守区设计2对特异性引物,以小RNA深度测序的病样RNA为模板进行RT-PCR扩增,根据扩增效果对引物进行筛选,进一步应用筛选出的引物,对采集于广东省的125份辣椒病样分别进行RT-PCR检测,根据检测结果明确危害广东辣椒的病毒种类。【结果】从采集于广东省8市的辣椒主要种植区的125份病样中检测到14种病毒,按照检出率从高到低依次为辣椒轻斑驳病毒（Pepper mild mottle virus,PMMoV）(44.0%）、甜椒内源RNA病毒（Bell pepper endornavirus,BPEV）（32.8%）、烟草轻型绿花叶病毒（Tobacco mild green mosaic virus,TMGMV）（31.2%）、辣椒脉斑驳病毒（Chilli veinal mottle virus,ChiVMV）（29.6%）、辣椒黄脉病毒1（Pepper vein yellow virus 1,PeVYV-1）（26.4%）、甜椒斑驳病毒（Pepper veinal mottle virus,PVMV）（25.6%）、黄瓜花叶病毒（Cucumber mosaic virus,CMV）（18.4%）、辣椒环斑病毒（Chilli ringspot virus,ChiRSV）（16.8%）、辣椒黄脉病毒6（Pepper vein yellow virus 6,PeVYV-6）（16.8%）、马铃薯 Y 病毒（Potato virus Y,PVY）（15.2%）、辣椒褪绿病毒（Capsicum chlorosis virus,CaCV）（14.4%）、蚕豆萎蔫病毒2号（Broad bean wilt virus 2,BBWV-2）（9.6%）、辣椒隐症病毒1（Pepper cryptic virus 1,PCV1）（8.8%）、烟草花叶病毒（Tobacco mosaic virus,TMV）（4.0%）。其中,PMMoV、BPEV、TMGMV、ChiVMV、PeVYV-1和PVMV 6种病毒的检出率在25%以上,但PMMoV、ChiVMV和PVMV分布广泛,PMMoV除了茂名外,ChiVMV除了韶关外,其他7市辣椒产区均有分布;PVMV广泛分布于8市辣椒产区,根据检出率和分布范围,得出PMMoV、ChiVMV和PVMV是危害广东辣椒的优势病毒。同时发现,广东辣椒上多种病毒复合侵染现象普遍,在本研究125份病样中病毒复合侵染率达到88.0%,其中2种、3种、4种、5种、6种、7种和8种病毒复合侵染检出率分别为28.0%、25.6%、12.0%、9.6%、6.4%、1.6%和2.4%,由此可知,2种和3种病毒复合侵染是主要侵染形式。【结论】危害广东辣椒的病毒有14种,其中PMMoV、ChiVMV和PVMV为优势病毒,且复合侵染普遍,2种和3种病毒复合侵染是主要侵染形式。

关键词: 辣椒病毒病, 种类鉴定, 小RNA深度测序, RT-PCR, 广东省

Abstract:

【Objective】 The viral disease is one of the major diseases of pepper production in Guangdong Province. The disease incidence is generally 5%-30% in the field, which can up to 100% in serious field. The objective of this study is to identify virus species infecting pepper in Guangdong Province, and to provide a theoretical basis for the prevention and control of pepper virus disease. 【Method】 From 2013 to 2016, a total of 125 susceptible pepper plant samples were collected from 8 major pepper growing areas of Guangzhou, Foshan, Huizhou, Jiangmen, Meizhou, Zhanjiang, Maoming and Shaoguan in Guangdong Province. Total RNA was extracted respectively from the leaves of 125 pepper samples. The 7 mixed samples collected from Maoming, Meizhou and Shaoguan were analyzed by small RNA deep sequencing. According to the results of small RNA deep sequencing analysis, two pairs of specific primers of each virus were designed to RT-PCR. The first pair of specific primers was designed according to the sequences of spliced gene fragments from small RNA deep sequencing. The second pair of specific primers was designed according to the conserved region sequences of viral genome in the GenBank database with the highest homology to sequences of spliced gene fragments from small RNA deep sequencing. Using disease samples RNA involved in small RNA deep sequencing as a template, the RT-PCR amplification was carried out with the two pairs of primers. Based on RT-PCR amplification results, the better pair of specific primers of each virus was selected. Furthermore, all 125 samples collected from Guangdong Province were subjected to detect viruses with the better pair of specific primers by RT-PCR. 【Result】 Fourteen viruses were identified in 125 samples collected from major pepper growing areas in 8 cities of Guangdong Province. According to the order of detection rate from high to low, they were Pepper mild mottle virus (PMMoV) (44.0%), Bell pepper endornavirus (BPEV) (32.8%), Tobacco mild green mosaic virus (TMGMV) (31.2%), Chilli veinal mottle virus (ChiVMV) (29.6%), Pepper vein yellow virus 1 (PeVYV-1) (26.4%), Pepper veinal mottle virus (PVMV) (25.6%), Cucumber mosaic virus (CMV) (18.4%), Chilli ringspot virus (ChiRSV) (16.8%), Pepper vein yellow virus 6 (PeVYV-6) (16.8%), Potato virus Y (PVY) (15.2%), Capsicum chlorosis virus (CaCV) (14.4%), Broad bean wilt virus 2 (BBWV-2) (9.6%), Pepper cryptic virus 1 (PCV1) (8.8%), Tobacco mosaic virus (TMV) (4.0%). The detection rates of PMMoV, BPEV, TMGMV, ChiVMV, PeVYV-1 and PVMV were over 25%. PMMoV, ChiVMV and PVMV were widely distributed in Guangdong Province. PMMoV (except Maoming), ChiVMV (except Shaoguan) were detected in pepper producing areas of other 7 cities. PVMV was detected in pepper producing areas of 8 cities. According to detection rate and distribution range, it was concluded that PMMoV, ChiVMV and PVMV were the dominant viruses infecting pepper in Guangdong Province. The mixed infection phenomenon was common on peppers in Guangdong Province. The detection rate of mixed infection was up to 88.0% in 125 samples. Among them, the mixed detection rates of 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds, 7 kinds and 8 kinds of viruses were 28.0%, 25.6%, 12.0%, 9.6%, 6.4%, 1.6%, 2.4%, respectively. So, 2 kinds and 3 kinds of viruses mixed infection were the main infection forms on peppers in Guangdong Province. 【Conclusion】 There are 14 kinds of viruses endangering pepper plants in Guangdong Province, among which PMMoV, ChiVMV and PVMV are the dominant viruses. The phenomenon of mixed infection is common. The main infection forms on peppers are 2 kinds and 3 kinds of viruses mixed infection in Guangdong Province.

Key words: pepper virus diseases, virus identification, small RNA deep sequencing, RT-PCR, Guangdong Province

汤亚飞,裴凡,李正刚,佘小漫,于琳,蓝国兵,邓铭光,何自福. 基于小RNA深度测序技术鉴定侵染广东辣椒的病毒种类[J]. 中国农业科学, 2019, 52(13): 2256-2267.

TANG YaFei,PEI Fan,LI ZhengGang,SHE XiaoMan,YU Lin,LAN GuoBing,DENG MingGuang,HE ZiFu. Identification of Viruses Infecting Peppers in Guangdong by Small RNA Deep Sequencing[J]. Scientia Agricultura Sinica, 2019, 52(13): 2256-2267.

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病毒Virus	科Family	属Genus
Broad bean wilt virus 2 (BBWV-2)	Secoviridae	Fabavirus
Bell pepper endornavirus (BPEV)	Endornaviridae	Alphaendornavirus
Cucumber mosaic virus (CMV)	Bromoviridae	Cucumovirus
Pepper vein yellows virus 1 (PeVYV-1)	Luteoviridae	Polerovirus
Pepper vein yellows virus 6 (PeVYV-6)	Luteoviridae	Polerovirus
Chilli veinal mottle virus (ChiVMV)	Potyviridae	Potyvirus
Pepper veinal mottle virus (PVMV)	Potyviridae	Potyvirus
Chilli ringspot virus (ChiRSV)	Potyviridae	Potyvirus
Potato virus Y (PVY)	Potyviridae	Potyvirus
Capsicum chlorosis virus (CaCV)	Tospoviridae	Orthotospovirus
Tobacco mild green mosaic virus (TMGMV)	Virgaviridae	Tobamovirus
Pepper mild mottle virus (PMMoV)	Virgaviridae	Tobamovirus
Pepper cryptic virus 1 (PCV1)	Partitiviridae	Deltapartitivirus

病毒Virus	引物Primer	序列Sequence (5′-3′)	目的片段Product (bp)
ChiVMV	ChiVMV1-F	CGAGTCTCATAAGTAATTGCAC	752
	ChiVMV1-R	AAGACATTTACTGATACCCTCC	752
TMGMV	TMGMV1-F	CCTCCCTGGTAATAGTACTATAC	407
	TMGMV1-R	CCAACTGTCCAATAGTACTTCT	407
ChiRSV	ChiRSV2-F	GAGTTCCACAGAGTAATGATCT	500
	ChiRSV2-R	GACTAACTGACTCTCACTCTTAC	500
BBWV-2	BBWV-2-1-F	GAGTTCAGTTACAGAAATTGGG	674
	BBWV-2-1-R	CATATGGGTTCCTTGAAATGAC	674
BPEV	BPEV1-F	AGGAGATGTGATTTCAGTAGAC	558
	BPEV1-R	TCTAATATTCTCTCCCCTCCTAG	558
PeVYV-1	PeVYV1-1-F	CTAGTCTACCTTAAGATCTCAGC	678
	PeVYV1-1-R	GTACTACCTTCTACCTATTTCGG	678
PVMV	PVMV2-F	GGTTTGGTGTATAGAAAATGGG	758
	PVMV2-R	CAGACTCTATCAGGTGGTATAAC	758
PeVYV-6	PeVYV6-1-F	GAAAAGAAAGTGGTGGAAGTAG	794
	PeVYV6-1-R	GGCTACCAATTGATCTACTAGAG	794
CaCV	CaCV1-F	CAGCTCTTAAATCAGTACTAGGG	472
	CaCV1-R	GTGTTTCCTGCATTAGTTCTAG	472
PCV1	PCV1-F	CAACACATATTCAGCATACTCC	608
	PCV1-R	GGGCGTATCTCTGATAATGATAG	608
PMMoV	PMMoV2-F	GCAGATCCCCTACGGTTCAA	761
	PMMoV2-R	CGCTATTCATCATCGCCAAAGT	761
PVY	PVY2-F	GAAGCGCGAGGAGAGAGAAA	728
	PVY2-R	TTATCGCTGCAACTCTGCCA	728
CMV	CMV-F	ATGGACAAATCTGRATCWMCC	764
	CMV-R	CTGGATGGACAACCCGTTC	764
TMV	TMV-F	CGGTCAGTGCCGAACAAGAA	600
	TMV-R	ATTTAAGTGGASGGAAAAVCACT	600

病毒 Virus	检出率Detection rate (%)
病毒 Virus	湛江 Zhanjiang	茂名 Maoming	江门 Jiangmen	佛山 Foshan	广州 Guangzhou	惠州 Huizhou	韶关 Shaoguan	梅州 Meizhou	合计 Total
PMMoV	73.5	0	66.7	100.0	31.8	55.6	12.5	100.0	44.0
BPEV	32.4	59.3	0	0	40.9	0	6.3	40.0	32.8
TMGMV	44.1	29.6	0	0	0	44.4	75.0	0	31.2
ChiVMV	17.6	22.2	66.7	50.0	54.5	44.4	0	50.0	29.6
PeVYV-1	5.9	44.4	0	0	40.9	0	37.5	40.0	26.4
PVMV	2.9	7.4	33.3	25.0	45.5	77.8	12.5	70.0	25.6
CMV	0	48.1	66.7	0	0	22.2	37.5	0	18.4
ChiRSV	8.8	33.3	0	0	4.1	0	0	0	16.8
PeVYV-6	2.9	29.6	0	0	27.3	0	25.0	20.0	16.8
PVY	8.8	7.4	33.3	0	22.7	66.7	6.3	10.0	15.2
CaCV	8.8	11.1	0	75.0	0	0	31.2	40.0	14.4
BBWV-2	0	40.7	0	25.0	0	0	0	0	9.6
PCV1	0	3.7	33.3	0	4.5	0	50.0	0	8.8
TMV	0	0	0	0	0	0	31.3	0	4.0

地点Region	1	2	3	4	5	6	7	8
湛江Zhanjiang	23.5	41.2	20.6	8.8	0	0	0	0
茂名Maoming	22.2	25.9	3.7	0	3.7	22.2	7.4	11.1
江门Jiangmen	0	33.3	66.7	0	0	0	0	0
佛山Foshan	0	25.0	75.0	0	0	0	0	0
广州Guangzhou	0	36.4	36.4	9.1	18.2	0	0	0
惠州Huizhou	11.1	22.2	0	33.3	33.3	0	0	0
韶关Shaoguan	0	6.3	43.8	31.3	12.5	6.3	0	0
梅州Meizhou	0	10.0	40.0	20.0	20.0	10.0	0	0
合计Total	12.0	28.0	25.6	12.0	9.6	6.4	1.6	2.4