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

Abstract

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

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

Identification of Viruses Infecting Peppers in Guangdong by Small RNA Deep Sequencing

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病毒 Virus	拼接得到与病毒有同源性的contings（条）Numbers of assembled contings
病毒 Virus	茂名-1 Maoming-1	茂名-2 Maogming-2	茂名-3 Maoming-3	茂名-4 Maoming-4	梅州 Meizhou	韶关-1 Shaoguan-1	韶关-2 Shaoguan-2
ChiRSV	5	9	10	-	-	-	-
BBWV-2	7	2	5	-	-	-	-
BPEV	12	11	5	-	21	-	-
CMV	5	6	16	5	-	-	-
PeVYV-1	6	5	2	-	-	-	-
ChiVMV	9	6	19	-	-	-	-
PVMV	18	7	-	-	5	-	-
PeVYV-6	2	3	2	-	-	-	-
CaCV	-	-	15	-	16	23	80
TMGMV	-	-	-	2	-	-	-
PCV1	-	-	-	2	-	3	-
PMMoV	-	-	-	-	3	-	2
PVY	-	-	-	-	3	-	-

病毒 Virus	茂名-1 Maoming-1		茂名-2 Maoming-2		茂名-3 Maoming-3		茂名-4 Maoming-4		梅州 Meizhou		韶关-1 Shaoguan-1		韶关-2 Shaoguan-2
病毒 Virus	sRNA	RT-PCR	sRNA	RT-PCR	sRNA	RT-PCR	sRNA	RT-PCR	sRNA	RT-PCR	sRNA	RT-PCR	sRNA	RT-PCR
ChiVMV	+	+	+	+	+	+	-	-	-	+	-	-	-	-
TMGMV	-	-	-	-	+	+	+	+	-	-	-	+	-	-
BBWV-2	+	+	+	+	+	+	-	-	-	-	-	-	-	-
PCV1	+	+	-	-	-	-	+	+	-	-	+	+	+	+
CaCV	-	-	-	-	+	+	-	-	+	+	+	+	+	+
ChiRSV	+	+	+	+	+	+	-	-	-	-	-	-	-	-
PVMV	+	+	-	-	-	-	-	-	+	+	-	+	-	+
PMMoV	-	-	-	-	-	-	-	-	+	+	+	+	-	+
PVY	-	+	-	-	-	-	-	-	+	+	-	+	-	-
TMV	-	-	-	-	-	-	-	-	-	-	-	+	-	+
CMV	+	+	+	+	+	+	+	+	-	-	-	+	-	+
PeVYV-6	+	+	+	+	+	+	-	-	-	+	-	-	-	+
BPEV	+	+	+	+	+	+	-	-	+	+	-	+	-	-
PeVYV-1	+	+	+	+	+	+	-	-	-	+	-	+	-	+

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