侵染花叶青木的病毒种类鉴定及其基因序列分析

doi:10.3864/j.issn.0578-1752.2023.10.007

摘要/Abstract

摘要：

【目的】探明采自云南和海南表现为黄化、皱缩等表型的花叶青木病毒种类。【方法】首先，利用转录组测序技术对来自云南省昆明市的花叶青木样品进行检测；然后，根据转录组测序结果，采用RT-PCR对采自云南省昆明市和海南省海口市的73份花叶青木样品开展转录组测序结果中发现的病毒种类检测验证，并扩增获得相关病毒基因片段，对这些病毒的基因序列进行比对分析。【结果】转录组测序结果发现，送测的花叶青木样品中含有蚕豆萎蔫病毒2号（broad bean wilt virus 2，BBWV2）、菜豆黄花叶病毒（bean yellow mosaic virus，BYMV）、黄瓜花叶病毒（cucumber mosaic virus，CMV）、豌豆耳突花叶病毒1号（pea enation mosaic virus 1，PEMV-1）和豌豆耳突花叶病毒2号（pea enation mosaic virus 2，PEMV-2）5种病毒的侵染。但随后对来自云南和海南的73份花叶青木样品的RT-PCR验证中，只检测到BBWV2、BYMV、CMV和PEMV-2 4种病毒。其中，云南和海南的样品中均能检测到BBWV2，总检出率为11.0%；而BYMV、CMV和PEMV-2仅在云南的样品中被检测到，检出率分别为5.5%、1.4%和1.4%。从两地采集的花叶青木样品中既未发现PEMV-1的侵染，也未发现BBWV2、BYMV、CMV和PEMV-2的复合侵染。为了进一步分析BBWV2、BYMV、CMV和PEMV-2花叶青木分离物与来自其他寄主植物相关病毒分离物的分子变异，选取检测到上述4种病毒的样品，分别对BBWV2、BYMV和CMV 3种病毒的cp及PEMV-2的RdRp序列进行扩增，并对扩增获得的BBWV2 490 nt的small cp（GenBank登录号：OQ137565、OQ137566）、BYMV 499 nt的cp核心区域（GenBank登录号：OQ137568）、CMV 880 nt的cp（GenBank登录号：OQ137567）和PEMV-2 800 nt的RdRp核心区域序列（GenBank登录号：OQ137569）进行分析。序列比对分析结果表明，BBWV2云南与海南花叶青木分离物间存在85.9%的核苷酸序列一致性，二者与GenBank中其他BBWV2分离物分别存在79.8%—94.5%和80.2%—92.0%的核苷酸序列一致性；BYMV、CMV和PEMV-2云南花叶青木分离物与GenBank中相应病毒的核苷酸序列一致性分别为79.8%—99.0%、71.5%—99.4%和84.9%—98.0%。利用以上病毒相应基因核苷酸序列构建系统发育树，发现BBWV2云南和海南花叶青木分离物分别属于I组中的a亚组和b亚组；BYMV云南花叶青木分离物与伊朗（GenBank登录号：MN241051，MN241060）和伊拉克（GenBank登录号：JQ026005）蚕豆分离物具有较近的亲缘关系；CMV云南花叶青木分离物属于I组中的IB亚组，但与IB亚组的其他成员具有较远的亲缘关系；PEMV-2云南花叶青木分离物与云南豌豆分离物亲缘关系最近并聚为一组，但与其他分离物存在较远的亲缘关系。【结论】首次报道了花叶青木被病毒感染，同时也是BBWV2、BYMV、CMV和PEMV-2在花叶青木乃至桃叶珊瑚属植物上的首次报道，BBWV2、BYMV、CMV和PEMV-2花叶青木分离物与来自其他植物的相关病毒分离物存在较大的分子变异。

关键词: 花叶青木, 病毒鉴定, 蚕豆萎蔫病毒2号, 菜豆黄花叶病毒, 黄瓜花叶病毒, 豌豆耳突花叶病毒2号

Abstract:

【Objective】The objective of this study is to identify the viruses infecting Aucuba japonica var. variegata plants with symptoms of yellowing and shrinking which collected from Yunnan and Hainan provinces.【Method】Firstly, RNA sequencing was used to detect the viruses infecting A. japonica var. variegata plants from Kunming, Yunnan Province. Then, based on the RNA sequencing results, RT-PCR was used to detect these viruses in 73 A. japonica var. variegata field samples collected in Kunming of Yunnan Province and Haikou of Hainan Province, and the related virus gene fragments were amplified, sequenced and analyzed.【Result】RNA sequencing results showed that 5 viruses were detected from the pooled diseased plants, such as broad bean wilt virus 2 (BBWV2), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pea enation mosaic virus 1 (PEMV-1), and pea enation mosaic virus 2 (PEMV-2). However, only BBWV2, BYMV, CMV and PEMV-2 were detected in the 73 A. japonica var. variegata samples from Yunnan and Hainan. Among them, BBWV2 was detected in the A. japonica var. variegata samples both from Yunnan and Hainan, with total detection rate of 11.0%, while BYMV, CMV and PEMV-2 were only detected in the Yunnan samples with detection rate of 5.5%, 1.4% and 1.4%, respectively. However, neither PEMV-1 nor the co-infections among BBWV2, BYMV, CMV and PEMV-2 was detected in these 73 A. japonica var. variegata samples from Yunnan or Hainan. In order to further analyze the molecular variation of BBWV2, BYMV, CMV and PEMV-2 aucuba isolates with the related virus isolates from other host plants, the 490 nt of small cp of BBWV2 (accession number OQ137565, OQ137566), 499 nt of the core cp region of BYMV (accession number OQ137568), 880 nt of the cp of CMV (accession number OQ137567), and 800 nt of the core RdRp of PEMV-2 (accession number OQ137569) were amplified from the A. japonica var. variegata samples infected with the above 4 viruses, and then the amplified gene sequences were analyzed, respectively. Sequence comparison analysis results showed that BBWV2 Yunnan aucuba isolate shared 85.9% nt identity with Hainan aucuba isolate, the BBWV2 Yunnan and Hainan aucuba isolates shared 79.8%-94.5% and 80.2%-92.0% nt identity with other BBWV2 isolates retrieved from GenBank, respectively. BYMV, CMV, and PEMV-2 aucuba isolates shared 79.8%-99.0%, 71.5%-99.4% and 84.9%-98.0% nt identity with other corresponding viruses in GenBank, respectively. Phylogenetic trees based on the gene nucleotide sequences of the above viruses revealed that the BBWV2 Yunnan and Hainan aucuba isolates were grouped into subgroup a and subgroup b in BBWV2 group I, respectively, and the BYMV Yunnan aucuba isolate showed a closest relationship with Iranian (accession number MN241051, MN241060) and Iraqi (accession number JQ026005) broad bean isolates. Moreover, the CMV Yunnan aucuba isolate was clustered into CMV IB subgroup of group I, but showed a distant relationship with other CMV isolates in the same subgroup. While PEMV-2 Yunnan aucuba isolate showed a closest relationship with Yunnan pea isolates and clustered into the same group, and had a distant relationship with other PEMV-2 isolates.【Conclusion】To our knowledge, this is the first record of virus infecting A. japonica var. variegata, and also the first report of BBWV2, BYMV, CMV and PEMV-2 infecting A. japonica var. variegata as well as the plants in genus Aucuba. Significant molecular variation was detected among the BBWV2, BYMV, CMV and PEMV-2 aucuba isolates with the related virus isolates from other plants worldwide.

Key words: Aucuba japonica var. variegata, virus identification, broad bean wilt virus 2, bean yellow mosaic virus, cucumber mosaic virus, pea enation mosaic virus 2

梁海文, 兰平秀, 柳勤海, 谭冠林, 陈小姣, 赵雁, 李凡. 侵染花叶青木的病毒种类鉴定及其基因序列分析[J]. 中国农业科学, 2023, 56(10): 1893-1904.

LIANG HaiWen, LAN PingXiu, LIU QinHai, TAN GuanLin, CHEN XiaoJiao, ZHAO Yan, LI Fan. Viruses Identification and Their Gene Sequences Analysis Infecting Aucuba japonica var. variegata[J]. Scientia Agricultura Sinica, 2023, 56(10): 1893-1904.

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病毒 Virus	引物名称 Primer name	序列 Sequence (5′-3′)	片段大小 Size (bp)	退火温度 Tm (℃)	来源 Source
BYMV	BYMVdF	ACTCTGAGGGGCAGAGTAGC	499	60	本研究This study
BYMV	BYMVdR	CCCATACCGTGGCATGTACC	499	60	本研究This study
PEMV-1	PEMVcpF	TGAATTGACATGGTTCCAGTCCG	806	57	本研究This study
PEMV-1	PEMVcpR	AAACGAGCTCCTACGGGAGTG	806	57	本研究This study
PEMV-2	PEMV-2dF	TGCGGATAACTGTGTCACC	800	57	本研究This study
PEMV-2	PEMV-2dR	GATCAAATCGGGAAGCGTCC	800	57	本研究This study
BBWV2	BBWV2dF	AGRTATATGCTTGGGCAAGCGCATG	490	57	文献[11] Reference [11]
BBWV2	BBWV2dR	CATGAACATTCCCCATCTCCACGTG	490	57	文献[11] Reference [11]
CMV	CMVCPuF	TCTCATGGATGCTTCTCCGCG	880	60	文献[11] Reference [11]
CMV	CMVCPuR	CCGTAAGCTGGATGGACAACC	880	60	文献[11] Reference [11]

重叠群 Contigs	长度 Length (bp)	参考序列 Reference sequence	登录号 Accession number	分类（属） Taxonomy (genus)	核苷酸序列一致性 Nucleotide sequence identity (%)
67547	1424	BBWV2 RNA1	JX183227	Fabavirus	99
71560	1609	BBWV2 RNA2	MW271032	Fabavirus	96
78575	753	BYMV	MT153866	Potyvirus	98
96531	3150	CMV RNA1	KC527787	Cucumovirus	98
94379	532	CMV RNA2	LC480454		96
41647	2016	CMV RNA3	MN340989		94
5899	5297	PEMV-1	MN497823	Enamovirus	98
8245	3873	PEMV-2	OK030736	Umbravirus	95

样品来源 Sample origin	样品数 Number of samples	阳性样品数 Numbers of virus positive samples
样品来源 Sample origin	样品数 Number of samples	BBWV2	BYMV	CMV	PEMV-1	PEMV-2
云南省昆明市 Kunming, Yunnan	58	4	4	1	-	1
海南省海口市 Haikou, Hainan	15	4	-	-	-	-
样品总数 Total samples	73	8	4	1	-	1
总检出率Total detection rate	19.2%	11.0%	5.5%	1.4%	-	1.4%