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

Abstract

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

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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References 28

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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%

Viruses Identification and Their Gene Sequences Analysis Infecting Aucuba japonica var. variegata

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