Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology

doi:10.16420/j.issn.0513-353x.2021-0450

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (7): 1415-1428.doi: 10.16420/j.issn.0513-353x.2021-0450

• Research Papers • Previous Articles Next Articles

Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology

XIA Yan¹, HUANG Song¹^,², WU Xueli¹, LIU Yiqi¹, WANG Miaomiao¹, SONG Chunhui¹, BAI Tuanhui¹, SONG Shangwei¹, PANG Hongguang¹, JIAO Jian¹(), ZHENG Xianbo¹()

¹College of Horticulture,Henan Agricultural University,Zhengzhou 450002,China
²Xinyang Agriculture and Forestry University,Xinyang,Henan 464001,China

Received:2022-03-11 Revised:2022-05-30 Online:2022-07-25 Published:2022-07-29

Abstract

Abstract:

Virome Sequencing Technology was used for virus detection and analysis of virus-infected apple trees in Henan Province,a total of 13 viruses were detected from apple leaves in the sequenced database,including five apple viruses:apple stem grooving virus（ASGV）,apple stem pitting virus（ASPV）,apple chlorotic leaf spot virus（ACLSV）,apple necrotic mosaic virus（ApNMV）and apple green crinkle associated virus（AGCaV）. RT-PCR was used to verify the detection results of five apple viruses,and objective bands were obtained after amplification,it was consistent with virome sequencing results,indicating that the virome sequencing detection results were accurate and reliable. Based on the sequencing results,specific primers were designed to amplify the full length of ASPV,a length of 9 246 nt ASPV-HN isolate genome was obtained and shared sequence identities 75.48% to 79.85% with complete genome of 19 ASPV isolates published in NCBI database;the two assembly sequences of ASGV genomes ASGV-HN1 and ASGV-HN2 based on virome sequencing results were both 6 475 nt in length and shared sequence identities 79.98% to 98.08% with complete genome of 18 ASGV isolates published in NCBI database. Detection of apple mosaic virus（ApMV）and ApNMVwere carried out in 53 apple leaf samples collected from different apple producing areas in Henan Province. ApNMV was detected in all samples with mosaic disease,while ApMV was not detected in 53 samples. It indicated that ApNMV may be the main pathogen which can cause apple mosaic disease in Henan Province.

Key words: apple, virus disease, Virome Sequencing Technology, RT-PCR, detection, apple mosaic disease

CLC Number:

S661.1

XIA Yan, HUANG Song, WU Xueli, LIU Yiqi, WANG Miaomiao, SONG Chunhui, BAI Tuanhui, SONG Shangwei, PANG Hongguang, JIAO Jian, ZHENG Xianbo. Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology[J]. Acta Horticulturae Sinica, 2022, 49(7): 1415-1428.

Figures/Tables 15

References 30

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引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增大小/bp Amplification length	退火温度/℃ Annealing temperature	来源 Source
ASGV-F	ATGAGTTTGGAAGACGTGCTTC	745	58	Hye-Kyung et al.,2006
ASGV-R	CTAACCCTCCAGTTCCAAGTTA	745	58	Hye-Kyung et al.,2006
ASPF1CP	GGGTGTACTTTGAGGCAGTATT	235	56	Komorowska et al.,2010
ASPR3CP	GAGCGGATGCGGTACATCTGTAT	235	56	Komorowska et al.,2010
ACLSV-F	CCATCTTCGCGAACATAGC	660	51	本研究合成 Synthesized in this study
ACLSV-R	GTAGTAAAATATTTAAAAGTCTAC	660	51	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
AGCaV-F	TCGGAAAGACTGTCCCGTTG	647	58	本研究合成 Synthesized in this study
AGCaV-R	ACTCTTGCCCCCGATTCTTG	647	58	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
ApMV-F1	CAACATGGTCTGCAAGTACT	640	50	Noda et al.,2017
ApMV-R1	GGAGCTAATCGCTCCATCAT	640	50	Noda et al.,2017

引物名称 Primer name	引物序列（5′-3′） Primer sequence	扩增大小/bp Amplification length	退火温度/℃ Annealing temperature	来源 Source
ASGV-F	ATGAGTTTGGAAGACGTGCTTC	745	58	Hye-Kyung et al.,2006
ASGV-R	CTAACCCTCCAGTTCCAAGTTA	745	58	Hye-Kyung et al.,2006
ASPF1CP	GGGTGTACTTTGAGGCAGTATT	235	56	Komorowska et al.,2010
ASPR3CP	GAGCGGATGCGGTACATCTGTAT	235	56	Komorowska et al.,2010
ACLSV-F	CCATCTTCGCGAACATAGC	660	51	本研究合成 Synthesized in this study
ACLSV-R	GTAGTAAAATATTTAAAAGTCTAC	660	51	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
AGCaV-F	TCGGAAAGACTGTCCCGTTG	647	58	本研究合成 Synthesized in this study
AGCaV-R	ACTCTTGCCCCCGATTCTTG	647	58	本研究合成 Synthesized in this study
ApNMV-F	CTTGCGTGCAATCGATATGG	676	54	Noda et al.,2017
ApNMV-R	TCATCTCAACCTAGACATCC	676	54	Noda et al.,2017
ApMV-F1	CAACATGGTCTGCAAGTACT	640	50	Noda et al.,2017
ApMV-R1	GGAGCTAATCGCTCCATCAT	640	50	Noda et al.,2017

引物名称 Primer name	用途 Amplification	引物序列（5′-3′） Primer sequence	扩增位置 Amplification position
5'RACE外引物 Outer Primer	5′端扩增 5′ RACE	TGTTCCTCAGAAGTGAATGA	NC003462（101 ~ 121）
5'RACE内引物 Inner primer	5′端扩增 5′ RACE	TCCATGCATCCAGGAATGAG	NC003462（553 ~ 571）
ASPV-1F	ASPV	GGATACGCAAACAAACTCTGAA	NC003462（1 ~ 22）
ASPV-1R	ASPV	CATGCADGGYTTRAARTGRTTRTC	NC003462（3627 ~ 3650）
ASPV-2F	ASPV	AATCAYTTYAARCCHTGCATGCC	NC003462（3627 ~ 3650）
ASPV-2R	ASPV	CAAAYTTBGTGCAVAGYTGRGACT	NC003462（5725 ~ 5748）
ASPV3-F	ASPV	AGTCYCARCTBTGCACVAARTTTG	NC003462（5725 ~ 5748）
ASPV-3R	ASPV	AATATTAAACAGATACGCCCTGACC	NC003462（7862 ~ 7886）
ASPV-4F	ASPV	GGTCAGGGCGTATCTGTTTAATATT	NC003462（7862 ~ 7886）
ASPV-4R	ASPV	GAAAATCTAGTTAAAACAAAAATAAG	NC003462（9307 ~ 9332）
3'RACE外引物 Outer Primer	3′端扩增 3′ RACE	AGCATGTCTGGAACCTCATGCT	NC003462（8896 ~ 8917）
3'RACE内引物 Inner primer	3′端扩增 3′ RACE	GACTTCTTCTTTGGAGTTGAG	NC003462（8988 ~ 9008）

引物名称 Primer name	用途 Amplification	引物序列（5′-3′） Primer sequence	扩增位置 Amplification position
5'RACE外引物 Outer Primer	5′端扩增 5′ RACE	TGTTCCTCAGAAGTGAATGA	NC003462（101 ~ 121）
5'RACE内引物 Inner primer	5′端扩增 5′ RACE	TCCATGCATCCAGGAATGAG	NC003462（553 ~ 571）
ASPV-1F	ASPV	GGATACGCAAACAAACTCTGAA	NC003462（1 ~ 22）
ASPV-1R	ASPV	CATGCADGGYTTRAARTGRTTRTC	NC003462（3627 ~ 3650）
ASPV-2F	ASPV	AATCAYTTYAARCCHTGCATGCC	NC003462（3627 ~ 3650）
ASPV-2R	ASPV	CAAAYTTBGTGCAVAGYTGRGACT	NC003462（5725 ~ 5748）
ASPV3-F	ASPV	AGTCYCARCTBTGCACVAARTTTG	NC003462（5725 ~ 5748）
ASPV-3R	ASPV	AATATTAAACAGATACGCCCTGACC	NC003462（7862 ~ 7886）
ASPV-4F	ASPV	GGTCAGGGCGTATCTGTTTAATATT	NC003462（7862 ~ 7886）
ASPV-4R	ASPV	GAAAATCTAGTTAAAACAAAAATAAG	NC003462（9307 ~ 9332）
3'RACE外引物 Outer Primer	3′端扩增 3′ RACE	AGCATGTCTGGAACCTCATGCT	NC003462（8896 ~ 8917）
3'RACE内引物 Inner primer	3′端扩增 3′ RACE	GACTTCTTCTTTGGAGTTGAG	NC003462（8988 ~ 9008）

分类 Classify	病毒名称 Virurs name	相关Contig数量 Quantity of related contig
植物病毒 Plant virus	苹果茎沟病毒Apple stem grooving virus,ASGV	25
	苹果茎痘病毒Apple stem pitting virus,ASPV	71
	苹果褪绿叶斑病毒Apple chlorotic leaf spot virus,ACLSV	8
	苹果坏死花叶病毒Apple necrotic mosaic virus,ApNMV	7
	苹果绿皱缩相关病毒Apple green crinkle associated virus,AGCaV	6
	柑橘叶斑病毒Citrus leaf blotch virus,CLBV	1
真菌病毒 Mycovirus	树孢链格菌丝裂病毒1 Alternaria arborescens mitovirus 1	1
	球孢白僵菌分体病毒2 Beauveria bassiana partitivirus 2	1
	葡萄座腔菌镰刀病毒1 Botryosphaeria dothidea fusarivirus 1	5
	葡萄座腔菌分体病毒1 Botryosphaeria dothidea partitivirus 1	2
	葡萄座腔菌病毒1 Botryosphaeria dothidea virus 1	2
	白粉病菌相关病毒11 Erysiphe necator associated ourmia-like virus 11	1
	葡萄霜霉病菌相关病毒Plasmopara viticola lesion associated ourmia-like virus	8

Identification and Analysis of Apple Viruses Diseases Based on Virome Sequencing Technology

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序号 No.	NCBI登录号 NCBI accession No.	寄主 Host	国家 Country	核苷酸 Genome	RdRp	CP	基因盒 Triple gene block
序号 No.	NCBI登录号 NCBI accession No.	寄主 Host	国家 Country	核苷酸 Genome	RdRp	CP	TGB1	TGB2	TGB3
1	KF321966.1	苹果 Malus × domestica	英国 United Kingdom	77.92	80.47	75.23	78.87	80.06	80.31
2	KY702580.1	苹果 Malus × domestica	中国 China	79.85	79.83	81.16	83.63	83.20	89.47
3	KF321967.1	苹果 Malus × domestica	德国 Germany	76.83	76.47	69.21	80.51	75.41	80.70
4	FR694186.1	苹果 Malus × domestica	印度 India	79.43	80.53	76.93	78.12	78.63	85.53
5	KY242757.1	苹果 Malus × domestica	中国 China	76.72	79.76	66.93	78.42	80.02	77.92
6	MK923754.1	苹果 Malus × domestica	巴西 Brazil	78.38	81.15	78.52	77.53	78.79	89.91
7	KF319056.1	苹果 Malus × domestica	德国 Germany	78.24	78.24	75.72	79.17	80.06	83.33
8	MK923753.1	苹果 Malus × domestica	巴西 Brazil	76.52	76.52	68.21	77.98	77.53	77.87
9	KY702581.1	苹果 Malus × domestica	中国 China	79.57	79.52	76.29	78.84	76.78	83.41
10	KF915809.1	苹果 Malus × domestica	中国 China	78.49	78.44	75.71	77.38	78.41	84.65
11	KJ522472.1	苹果 Malus × domestica	中国 China	79.55	79.52	81.50	77.53	81.82	90.35
12	MK923756.1	苹果 Malus × domestica	巴西 Brazil	77.10	78.28	74.48	76.93	77.96	83.77
13	MN887352.1	苹果 Malus × domestica	印度 India	75.72	77.61	76.77	77.38	76.86	90.35
14	KU308398.1	梨 Pyrus communis	中国 China	79.13	79.80	74.81	78.12	76.03	85.53
15	JF946775.1	梨 Pyrus communis	中国 China	75.68	78.12	68.43	79.32	76.58	78.61
16	JF946772.1	梨 Pyrus communis	中国 China	75.48	77.94	68.49	79.32	76.31	81.58
17	LM999967.1	枇杷Eriobotrya japonica	印度 India	76.02	79.06	67.58	76.79	76.22	81.14
18	MW045213.1	无宿主信息 No host imformation	西班牙 Spain	77.58	79.89	76.62	76.64	79.49	84.21
19	NC_003462.2 （D21829）	苹果 Malus × domestica	德国 Germany	79.37	79.62	72.52	78.72	80.97	84.21

序号 No.	重组分离物 Recombinant	主要亲本 Major parent	次要亲本 Minor parent	检测方法 Detection method
序号 No.	重组分离物 Recombinant	主要亲本 Major parent	次要亲本 Minor parent	RDP	GENECONV	Bootscan	MaxChi	Chimaera	SiScan	3Seq
1	MK923753.1	MN887352.1	Unknown	+	-	-	-	-	-	-
2	FR694186.1	MK923753.1	JF946772.1	+	-	-	-	-	-	-
3	FR694186.1	Unknown	JF946775.1	-	+	-	-	-	-	-
4	KF319056.1	KF321966.1	KJ552472.1	-	-	+	-	-	-	-
5	KF321966.1	Unknown	KU308398.1	-	-	+	-	-	-	-
6	KF321967.1	KU308398.1	JF946772.1	+	-	-	-	-	-	-
7	KF321967.1	KU308398.1	Unknown	+	+	-	+	-	-	-
8	KF915809.1	Unknown	MK923756.1	-	-	-	+	-	-	-
9	KF915809.1	Unknown	KY702581.1	+	+	+	+	+	-	-
10	KJ552472.1	Unknown	JF946772.1	-	-	+	-	-	-	-
11	KJ552472.1	Unknown	FR694186.1	+	-	-	-	-	-	-
12	KJ552472.1	KF319056.1	FR694186.1	+	-	-	-	-	-	-
13	KY242757.1	FR694186.1	Unknown	-	-	+	-	-	-	-
14	KY702580.1	KF915809.1	KF321967.1	-	-	+	-	-	-	-
15	KY702580.1	Unknown	KF321966.1	+	-	-	-	-	-	-
16	LM999967.1	ASPV-HN	KJ552472.1	-	-	+	-	-	-	-
17	LM999967.1	MW045213.1	ASPV-HN	+	-	-	-	-	-	-
18	MK923753.1	NC_003462.2	MK923754.1	+	-	-	-	-	-	-
19	MK923753.1	MK923756.1	MN887352.1	+	-	-	-	-	-	-
20	MK923756.1	Unknown	JF946772.1	+	+	-	+	-	-	-
21	MN887352.1	MK923756.1	MK923754.1	-	-	+	-	-	-	-
22	NC_003462.2	KY702581.1	ASPV-HN	-	-	-	-	+	-	-
23	NC_003462.2	KF915809.1	Unknown	+	-	-	-	-	-	-
24	NC_003462.2	FR694186.1	Unknown	-	+	-	-	-	-	-

序号 No.	NCBI登录号 NCBI accession No.	寄主 Host	国家 Country	核苷酸 Genome	RdRp	外壳蛋白 CP
1	KU947036.1	苹果 Malus × domestica	中国 China	79.98	79.83	83.61
2	MF616381.1	苹果 Malus × asiatica	中国 China	82.93	82.51	85.63
3	KF434636.1	苹果 Malus × domestica	中国 China	83.81	83.45	84.96
4	JX080201.1	苹果 Malus × domestica	德国 Germany	83.34	82.89	85.98
5	KX686100.1	苹果 Malus × domestica	中国 China	82.60	82.22	85.73
6	KX686111.1	苹果 Malus × domestica	巴西 Brazil	83.94	83.51	85.15
7	KX668488.1	苹果 Malus × domestica	巴西 Brazil	83.81	83.42	85.73
8	KJ579253.1	苹果 Malus × domestica	中国 China	83.91	83.55	85.80
9	JQ308181.1	苹果 Malus × domestica	中国 China	81.86	81.51	83.13
10	MN786531.1	苹果 Malus × domestica	印度 India	82.54	82.19	83.95
11	MK929791.1	苹果 Malus × domestica	巴西 Brazil	83.29	82.84	85.46
12	HE978837.1	苹果 Malus × domestica	印度 India	82.86	82.16	84.94
13	MK929792.1	苹果 Malus × domestica	巴西 Brazil	83.08	82.75	85.46
14	MK923757.1	苹果 Malus × domestica	巴西 Brazil	83.12	82.69	85.46
15	KU198289.1	梨 Pyrus pyrifolia	中国 China	82.48	82.16	84.16
16	KU605672.1	梨 Pyrus communis	中国 China	83.41	83.00	84.99
17	MH108986.1	柑橘 Citrus	中国 China	82.73	82.34	85.67
18	NC_001749.2	苹果 Malus × domestica	日本 Japan	82.92	82.53	84.96

序号 No.	重组分离物 Recombinant	主要亲本Major parent	次要亲本Minor parent	检测方法 Detection methods
序号 No.	重组分离物 Recombinant	主要亲本Major parent	次要亲本Minor parent	RDP	GENECONV	Bootscan	MaxChi	Chimaera	SiScan	3Seq
1	ASGV-HN2	MK979792.1	Unknown	-	+	+	-	-	-	-
2	HE978837.1	ASGV-HN1	MK979791.1	-	-	-	-	-	-	+
3	JQ308181.1	NC_001749.2	Unknown	+	-	+	+	+	-	+
4	JX080201.1	MH108986.1	Unknown	-	-	-	+	-	-	-
5	KJ579253.1	JQ308181.1	Unknown	+	-	+	+	+	+	+
6	KJ579253.1	KX686100.1	JQ308181.1	-	-	-	-	-	+	-
7	KX668488.1	KX686111.1	KX686100.1	+	+	+	+	+	-	+
8	KX668488.1	Unknown	KX686111.1	+	+	+	+	+	+	+
9	KX668488.1	JQ308181.1	Unknown	-	-	-	+	+	-	+
10	KX681118.1	NC_001749.2	KX686100.1	+	+	+	+	+	-	+
11	KX686100.1	NC_001749.2	KU198289.1	-	+	-	-	-	-	-
12	KX686100.1	Unknown	KJ579253.1	-	-	-	+	+	-	+
13	MF616381.1	MH108986.1	Unknown	+	-	-	+	+	-	-
14	MF616381.1	MH108986.1	MK979792.1	-	+	-	-	-	-	-
15	MH108986.1	KF434636.1	KU198289.1	-	+	-	-	-	-	-
16	MH108986.1	KU605672.1	MF616381.1	-	-	-	+	+	-	+
17	MK929792.1	ASGV-HN2	HE978837.1	+	-	-	-	-	-	-
18	NC_001749.2	JQ308181.1	Unknown	+	-	+	+	+	+	+

采集地点 Collection site	品种 Cultivar	序号 No.	花叶症状 Symptoms of mosaic disease	检测结果Detection results
采集地点 Collection site	品种 Cultivar	序号 No.	花叶症状 Symptoms of mosaic disease	ApNMV	ApMV
洛宁县Luoning	金冠Golden Delicious	1	有 Positive	有 Positive	无 Negative
		2	无 Negative	无 Negative	无 Negative
		3	无 Negative	无 Negative	无 Negative
		4	有 Positive	有 Positive	无 Negative
		5	有 Positive	有 Positive	无 Negative
		6	无 Negative	无 Negative	无 Negative
	富士Fuji	7	有 Positive	有 Positive	无 Negative
		8	无 Negative	无 Negative	无 Negative
		9	有 Positive	有 Positive	无 Negative
		10	无 Negative	无 Negative	无 Negative
		11	有 Positive	有 Positive	无 Negative
		12	有 Positive	有 Positive	无 Negative
		13	有 Positive	有 Positive	无 Negative
		14	有 Positive	有 Positive	无 Negative
		15	有 Positive	有 Positive	无 Negative
		16	有 Positive	有 Positive	无 Negative
	秦冠Qinguan	17	有 Positive	有 Positive	无 Negative
		18	无 Negative	无 Negative	无 Negative
		19	有 Positive	有 Positive	无 Negative
		20	有 Positive	有 Positive	无 Negative
		21	有 Positive	有 Positive	无 Negative
		22	有 Positive	有 Positive	无 Negative
		23	无 Negative	无 Negative	无 Negative
		24	有 Positive	有 Positive	无 Negative
		25	有 Positive	有 Positive	无 Negative
灵宝市Lingbao	富士Fuji	26	有 Positive	有 Positive	无 Negative
		27	有 Positive	有 Positive	无 Negative
		28	有 Positive	有 Positive	无 Negative
		29	有 Positive	有 Positive	无 Negative
		30	有 Positive	有 Positive	无 Negative
		31	有 Positive	有 Positive	无 Negative
	红星Starking	32	无 Negative	无 Negative	无 Negative
		33	有 Positive	有 Positive	无 Negative
		34	有 Positive	有 Positive	无 Negative
		35	有 Positive	有 Positive	无 Negative
		36	无 Negative	无Negative	无 Negative
		37	有 Positive	有 Positive	无 Negative
		38	有 Positive	有 Positive	无 Negative
		39	有 Positive	有 Positive	无 Negative
		40	无 Negative	无 Negative	无 Negative
陕州区 Shanzhou	富士Fuji	41	有 Positive	有 Positive	无 Negative
		42	无 Negative	无 Negative	无 Negative
		43	有 Positive	有 Positive	无 Negative
		44	有 Positive	有 Positive	无 Negative
		45	有 Positive	有 Positive	无 Negative
		46	有 Positive	有 Positive	无 Negative
		47	有 Positive	有 Positive	无 Negative
		48	有 Positive	有 Positive	无 Negative
		49	有 Positive	有 Positive	无 Negative
		50	有 Positive	有 Positive	无 Negative
		51	有 Positive	有 Positive	无 Negative
		52	无 Negative	无 Negative	无 Negative
郑州市Zhengzhou	金冠Golden Delicious	53	有 Positive	有 Positive	无 Negative

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