Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (14): 2752-2761.doi: 10.3864/j.issn.0578-1752.2022.14.006

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Characteristics and Pathogenicity Analysis of Youcai Mosaic Virus Guangdong Isolate Infecting Radish

LI ZhengGang(),TANG YaFei,SHE XiaoMan,YU Lin,LAN GuoBing,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:2022-01-04 Accepted:2022-02-28 Online:2022-07-16 Published:2022-07-26
  • Contact: ZiFu HE E-mail:lzaagg@cau.edu.cn;hezf@gdppri.com

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Abstract:

【Objective】Viral disease is one of the major factors threatening the production safety and radish farming industry. The objective of this study is to identify the viruses that infect radish in Guangdong Province, and to analyze the molecular characteristics and pathogenicity of youcai mosaic virus (YoMV), so as to provide theoretical basis for the prevention of viral disease in radish. 【Method】Three symptomatic and one asymptomatic samples were collected from a radish-planting base in Guangdong and were subjected to total RNA extraction. Small RNA sequencing and assembly (sRSA) was used to identify the virus species, followed by RT-PCR verification with specific primer pairs designed according to the sRSA results. To construct the infectious cDNA clones of YoMV-GD isolate, two primer pairs were designed, and the PCR products were introduced into pCB301 vector by seamless cloning and assembly. Then the full-length genomic sequences of YoMV-GD and other YoMV isolates were used to construct phylogenetic tree by MEGA7 software. Agrobacterium containing pCB301-YoMV-GD was infiltrated into Nicotiana benthamiana and Nicotiana tabacum to verify the infectivity of YoMV-GD infectious cDNA clones. Subsequently, the pathogenicity of YoMV-GD was tested in radish and Chinese flowering cabbage by agroinfiltration. 【Result】sRSA result showed that the viruses that infecting radish in Guangdong include Raphanus sativus cryptic virus 3 (RsCV3), Raphanus sativus chrysovirus 1 (RasCV1), turnip mosaic virus (TuMV), and YoMV. Sanger sequencing reveals that YoMV-GD isolate contains 6 304 nt and encodes four proteins, replication-associated protein, replicase, movement protein, and coat protein. YoMV-GD shares the highest similarity (99.18%) with YoMV British isolate (GenBank accession number: AY318866). Phylogenetic analysis shows that YoMV-GD has a closer relationship with isolates from British, Germany, Shanghai, and Chongqing. In addition, the YoMV-GD infectious cDNA clones could successfully infect N. benthamiana and N. tabacum, causing crinkle, mosaic, and necrosis symptoms in the upper leaves. Moreover, though YoMV-GD isolate could systemically infect radish and Chinese flowering cabbage, the symptoms were very weak. 【Conclusion】Viruses infecting radish in Guangdong include RsCV3, RasCV1, TuMV, and YoMV, and mixed infection is the main type. There was no clear regional difference among YoMV isolates. YoMV-GD is highly pathogenic in tobacco, but weak in radish and Chinese flowering cabbage.

Key words: viral disease in radish, youcai mosaic virus (YoMV), molecular characteristics, infectious clone, pathogenicity

Table 1

Primers used in this study"

引物名称
Primer name		 引物序列
Primer sequence		 对应位置
Localization (nt)		 产物长度
Product length (bp)		 退火温度
Annealing temperature (℃)
RsCV3-F ACAGGATTCATCTCGGATCTT 140-160 1042 55
RsCV3-R ACTAGTGAATCGTCTCCTAATGT 1182-1160
RasCV1-F AATGTGATTGTCACACATGAACTAG 498-522 1030 55
RasCV1-R CTAATAGCCCCATCCTTCAGG 1528-1508
YoMV-F AATATCAGAGAGCGGCATTCTAT 550-572 1104 55
YoMV-R CTGTCGGTAAAAGTGCAATATAACT 1654-1630
TuMV-F GTCGCCGTTCTGTTGGTCGTTTTGA 1509-1533 1371 55
TuMV-R TGCCTGCGATAACTGCTTGTGTACG 2879-2855
301-YoMV-F1 AAGTTCATTTCATTTGGAGAGGGTT
TTATTTTTATTGCAACAACAACAA		 1-27 3405 66
301-YoMV-R1 TGAACACTGTGTCGATCTGTAATT
GCTATTGGGTCCCCGA		 3405-3366
301-YoMV-F2 AACAGCGACTCCCTTGGAGATCAT
ATCAAGAGCGTCACCTCATG		 3194-3237 3111 70
301-YoMV-R2 GGTGGAGATGCCATGCCGACCCTT
GGGCCCCTACCCGGGGTTA		 6304-6284

Fig. 1

Symptoms of diseased radish plants in the field A—C：发病植株1 Sample 1。A：发病植株与健康植株对比,左边为感病植株,右边为健康植株Comparison between diseased and symptomless plant. The left is diseased plant, and the right is symptomless plant;B、C：A图中感病植株放大图Magnification of the diseased plant in figure A。D：发病植株2 Samples 2。E：发病植株3 Samples 3"

Table 2

Small RNA deep sequencing result"

基因登录号
GenBank
number		 长度
Length
(bp)		 覆盖度
Coverage
(%)		 拼接序
列条数
Contig		 测序
深度
Depth		 平均测
序深度
Depth
(Norm)		 同源性
Identity
(%)		 最高
同源性
Iden.
Max (%)		 最低
同源性
Iden. Min
(%)		 科
Family		 属
Genus		 种
Species
FJ461349 1609 1544 (96.0) 1 82.0 6.8 99.42 99.42 99.42 Partitiviridae maybe Alphapartitivirus RsCV3
segment 1
FJ461350 1581 1404 (88.8) 12 19.9 1.7 99.50 100.00 95.56 Partitiviridae maybe Alphapartitivirus RsCV3
segment 2
JQ045335 3638 3411 (93.8) 12 28.7 2.4 98.83 100.00 97.62 Chrysoviridae Alphachrysovirus RasCV1
segment 1
JQ045336 3517 3419 (97.2) 6 21.5 1.8 98.86 99.38 97.73 Chrysoviridae Alphachrysovirus RasCV1
segment 2
JQ045337 3299 3218 (97.5) 9 31.6 2.6 99.25 100.00 97.91 Chrysoviridae Alphachrysovirus RasCV1
segment 3
EU734433 9833 9833 (100) 127 2343.0 194.7 95.33 100.00 89.17 Potyviridae Potyvirus TuMV
isolate TANX2
AY318866 6304 6302 (100) 1 980.2 81.4 99.21 99.21 99.21 Virgaviridae Tobamovirus YoMV

Fig. 2

RT-PCR verification of small RNA deep sequencing A：RT-PCR验证萝卜样品中RsCV3和RasCV1 RT-PCR detection of RsCV3 and RasCV1 in radish samples;B：RT-PCR验证萝卜样品中YoMV和TuMV RT-PCR detection of YoMV and TuMV in radish samples。CK：无症状对照Control sample without symptom"

Fig. 3

Phylogenetic analysis of YoMV-GD The genomes of YoMV isolates and other tobamoviruses were downloaded from NCBI and used to construct the phylogenic tree by neighbor-joining method with MEGA7 WMoV: wasabi mottle virus; TVCV: turnip vein-clearing virus; RMV: ribgrass mosaic virus; TMV: tobacco mosaic virus"

Fig. 4

Symptoms and detection of N. tabacum and N. benthamiana plants inoculated with pCB301-YoMV-GD infectious cDNA clone A：YoMV-GD侵染性克隆载体示意图Schematic presentation of YoMV-GD infectious cDNA clone。LB：左臂Left border;RB：右臂Right border;35S：35S启动子35S promoter;RT：通读Read-through;ORF1、ORF2：开放阅读框1、2 Open reading frame 1, 2;MP：运动蛋白Movement protein;CP：外壳蛋白Coat protein;RZ：核糖剪切酶Ribozyme;NOS：NOS终止子NOS terminator。B：普通烟和本生烟发病症状Disease symptoms of N. tabacum and N. benthamiana。Mock：未接种对照植株Control plant;普通烟和本生烟分别于接种后第7天和第5天拍照Photos of N. tabacum and N. benthamiana were taken at 7 dpi and 5 dpi, respectively。C：RT-PCR验证YoMV-GD的侵染RT-PCR verification of YoMV-GD infection;普通烟和本生烟接种YoMV-GD后第7天和第5天,RT-PCR检测上部叶片发病情况RT-PCR detection of YoMV-GD in the upper leaves of N. tabacum and N. benthamiana at 7 dpi and 5 dpi, respectively;M：DL2000 DNA Marker"

Fig. 5

Pathogenicity analyses of YoMV-GD isolate in radish and Chinese flowering cabbage Mock：接种空载体农杆菌对照Control sample agroinfiltrated with empty vector。A：YoMV-GD分离物侵染不同萝卜品种植株症状 Symptoms of different radish cultivars infected by YoMV-GD;下图为上图的中心位置放大图The image below is an enlarged view of the center position of the above image。B：YoMV-GD分离物侵染不同菜心品种植株症状 Symptoms of different Chinese flowering cabbage cultivars infected by YoMV-GD;下图为上图的中心位置放大图The image below is an enlarged view of the center position of the above image。C：RT-PCR检测YoMV-GD在萝卜和菜心上的发病情况RT-PCR detection of YoMV-GD in radish and Chinese flowering cabbage;NT：未加任何cDNA模板阴性对照Negative control without cDNA template;M：DL2000 DNA Marker"

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