Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (13): 2256-2267.doi: 10.3864/j.issn.0578-1752.2019.13.006

• PLANT PROTECTION • Previous Articles     Next Articles

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

TANG YaFei,PEI Fan,LI ZhengGang,SHE XiaoMan,YU Lin,LAN GuoBing,DENG MingGuang,HE ZiFu()   

  1. Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640
  • Received:2019-03-15 Accepted:2019-04-29 Online:2019-07-01 Published:2019-07-11
  • Contact: ZiFu HE E-mail:hezf@gdppri.com

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

Fig. 1

Symptoms of pepper plants infected by virus in the field"

Table 1

Family and genus of the viruses analyzed by small RNA deep sequencing"

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

Table 2

The viruses derived from small RNA deep sequencing of pepper samples collected from Guangdong"

病毒
Virus
拼接得到与病毒有同源性的contings(条)Numbers of assembled contings
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 - -

Table 3

Comparison of results between small RNA deep sequencing and RT-PCR"

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
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 + + + + + + - - - + - + - +

Table 4

Primers used to detect 14 viruses in this study"

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
TMGMV TMGMV1-F CCTCCCTGGTAATAGTACTATAC 407
TMGMV1-R CCAACTGTCCAATAGTACTTCT
ChiRSV ChiRSV2-F GAGTTCCACAGAGTAATGATCT 500
ChiRSV2-R GACTAACTGACTCTCACTCTTAC
BBWV-2 BBWV-2-1-F GAGTTCAGTTACAGAAATTGGG 674
BBWV-2-1-R CATATGGGTTCCTTGAAATGAC
BPEV BPEV1-F AGGAGATGTGATTTCAGTAGAC 558
BPEV1-R TCTAATATTCTCTCCCCTCCTAG
PeVYV-1 PeVYV1-1-F CTAGTCTACCTTAAGATCTCAGC 678
PeVYV1-1-R GTACTACCTTCTACCTATTTCGG
PVMV PVMV2-F GGTTTGGTGTATAGAAAATGGG 758
PVMV2-R CAGACTCTATCAGGTGGTATAAC
PeVYV-6 PeVYV6-1-F GAAAAGAAAGTGGTGGAAGTAG 794
PeVYV6-1-R GGCTACCAATTGATCTACTAGAG
CaCV CaCV1-F CAGCTCTTAAATCAGTACTAGGG 472
CaCV1-R GTGTTTCCTGCATTAGTTCTAG
PCV1 PCV1-F CAACACATATTCAGCATACTCC 608
PCV1-R GGGCGTATCTCTGATAATGATAG
PMMoV PMMoV2-F GCAGATCCCCTACGGTTCAA 761
PMMoV2-R CGCTATTCATCATCGCCAAAGT
PVY PVY2-F GAAGCGCGAGGAGAGAGAAA 728
PVY2-R TTATCGCTGCAACTCTGCCA
CMV CMV-F ATGGACAAATCTGRATCWMCC 764
CMV-R CTGGATGGACAACCCGTTC
TMV TMV-F CGGTCAGTGCCGAACAAGAA 600
TMV-R ATTTAAGTGGASGGAAAAVCACT

Table 5

The viruses in 125 samples detected by RT-PCR "

病毒
Virus
检出率Detection rate (%)
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

Table 6

Incidence of single or mixed infection of 14 viruses in samples collected from each region (%)"

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
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