四川成都地区复合侵染南瓜4种病毒的分子鉴定分析

doi:10.16420/j.issn.0513-353x.2020-0770

摘要/Abstract

摘要：

南瓜近年来受到病毒病的危害日益严重。对从四川成都地区采集的典型病毒症状南瓜叶片,进行了small RNA（sRNA）深度测序分析,发现有4种病毒复合侵染,分别是瓜类蚜传黄化病毒（cucurbit aphid-borne yellows virus,CABYV）、番木瓜环斑病毒（papaya ringspot virus,PRSV）、西瓜花叶病毒（watermelon mosaic virus,WMV）和小西葫芦黄花叶病毒（zucchini yellow mosaic virus,ZYMV）。依据sRNA测序结果设计病毒的特异引物,对提取的病毒样本RNA进行反转录PCR扩增,得到了预期大小的特异DNA片段,这些片段经Sanger测序分析,进一步证实了上述4种病毒的存在。将这些病毒序列与GenBank中相应序列进行比对分析发现,CABYV和ZYMV与近来在国内流行的病毒类型相似,而PRSV和WMV与国内其他地区的病毒相近,而且也与国外报道病毒相似;系统分析还发现,CABYV和PRSV在四川可能分别有两种病毒株系同时在流行。这些结果表明,在四川成都地区南瓜受到多种病毒复合侵染,病毒的传播和流行种类较为复杂,在生产上需要高度重视南瓜病毒病的防控。

关键词: 南瓜, 病毒, 复合侵染, CABYV, PRSV, WMV, ZYMV

Abstract:

In recent years the disease caused by virus has become more and more serious issue in pumpkin field production. In this study,samples from the field in Chengdu,Sichuan Province of China were collected in May,2020. Deep sequencing of small RNA（sRNA）from the pumpkin sample revealed that there were four viruses of cucurbit aphid-borne yellows virus（CABYV）,watermelon mosaic virus（WMV）,papaya ringspot virus（PRSV）and zucchini yellow mosaic virus（ZYMV）. All the viruses were further confirmed by reverse transcriptional PCR（RT-PCR）and Sanger sequencing of the RT-PCR fragments. Alignments and phylogenetic analyses with sequences of the viruses in GenBank showed that CABYV and ZYMV were more similar with the viruses recently reported in China,whereas WMV and PRSV were similar with the other viruses in China and abroad,as well. Besides,the sequence alignments and phylogenetics further suggested that two isolates for both CABYV and PRSV might be currently infecting pumpkin in Sichuan Province. Discovery of the complicated mixed virus-infection in pumpkin might be of potential significance for the virus disease control in pumpkin production.

Key words: pumpkin, virus, mixed infection, CABYV, PRSV, WMV, ZYMV

中图分类号:

S642.1

林晓敏, 彭梅芳, 范晓丽, 陈克贵. 四川成都地区复合侵染南瓜4种病毒的分子鉴定分析[J]. 园艺学报, 2021, 48(11): 2299-2310.

LIN Xiaomin, PENG Meifang, FAN Xiaoli, CHEN Kegui. Molecular Identification of Four Viruses that Cause Mixed Infection of Pumpkin in Chengdu,Sichuan Province of China[J]. Acta Horticulturae Sinica, 2021, 48(11): 2299-2310.

图/表 7

表1 本研究中所使用的PCR引物

Table 1 PCR primers used in this study

扩增的病毒 Virus	引物名称 Primer name	引物序列（5′-3′） Sequence
CABYV	CABYV-F	ACATTATTAGCGCAGAGAGTCCC
CABYV	CABYV-R	CCACTCGCAATATTTCTCCGTGTA
ZYMV	ZYMV-F	GATAGTTCTTTAACTCCAGCCTTACTG
ZYMV	ZYMV-R	TCCGTCCATCATGAACCACA
WMMV	WMV-F	TACACAAGTTAGCCATACCACATCA
WMMV	WMV-R	CACTCGCTCTTTAGCTCTTGG
PRSV	PRSV-F	CTCTCATTCCAAGAGGCTCGA
PRSV	PRSV-R	GTGTCTGGGTTATGATGGATGG

图1 感染病毒南瓜植株的症状 A:植株叶片;B:植株顶端幼叶;C:叶背面及蚜虫;D:叶背面及烟粉虱。

Fig. 1 Symptoms of the pumpkin plants with virus disease A:Plant leaves;B:Young leaves;C:Aphids on the abaxial surface;D:Whiteflies on the abaxial surface.

图2 感病样品的RT-PCR扩增 M:Marker;-:未加模板扩增的负对照。

Fig. 2 RT-PCR amplification from the sample RNA M:Marker;-:Negative control without template.

图3 基于瓜类蚜传黄化病毒（CABYV）核酸分子序列（MW002437）的系统进化树

Fig. 3 Neighbor-joining phylogram of CABYV obtained from alignment of nucleotide sequence（MW002437）of this isolate with others from GenBank

图4 基于番木瓜环斑病毒（PRSV）核酸分子序列（MW002438）的系统进化树

Fig. 4 Neighbor-joining phylogram of PRSV obtained from alignment of nucleotide sequence（MW002438）of this isolate with others from GenBank

图5 基于西瓜花叶病毒（WMV）核酸分子序列（MV002439）的系统进化树

Fig. 5 Neighbor-joining phylogram of WMV obtained from alignment of nucleotide sequence（MV002439）of this isolate with others from GenBank

图6 基于小西葫芦黄花叶病毒（ZYMV）核酸分子序列（MW002440）的系统进化树

Fig. 6 Neighbor-joining phylogram of ZYMV obtained from alignment of nucleotide sequence（MW002440）of this isolate with others from GenBank

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