新疆北疆葡萄病毒和类病毒组分析与分子鉴定

doi:10.16420/j.issn.0513-353x.2025-0642

摘要/Abstract

摘要：

为明确新疆北疆地区葡萄病毒和类病毒的发生情况，采集葡萄幼嫩叶片，用LncRNA测序技术从4个混合和6个单株样本中获得病毒及类病毒序列，共鉴定出分属于7个病毒科、9个病毒属的11种植物病毒和1个类病毒科、2个类病毒属的3种类病毒，首次证实了葡萄蚕豆萎蔫病毒（grapevine fabavirus，GFabV）、葡萄双生病毒A（grapevine geminivirus A，GGVA）、葡萄叶脉羽化病毒（grapevine rupestris vein feathering virus，GRVFV）和黄瓜花叶病毒（cucumber mosaic virus，CMV）在新疆葡萄产区的发生。采用RT-PCR技术，检测北疆主要葡萄种植区石河子和昌吉5个葡萄栽培品种共117个样本。结果表明，采集葡萄样本中葡萄浆果内坏死病毒（grapevine berry inner necrosis virus，GINV）的检出率最高，达73.5%，检出率最高的类病毒是啤酒花矮化类病毒（hop stunt viroid，HSVd），高达88.9%。不同品种感染的病毒种类及优势病毒不同，其中鲜食葡萄‘无核白’感染病毒种类最多，感染了本研究中发现的全部11种病毒和3种类病毒。同时，复合感染在葡萄样本中普遍存在，95.7%的样本存在病毒与类病毒的复合感染，其中感染5种以上（类）病毒的样品有60份，最主要的复合侵染类型为GINV + 葡萄灰比诺病毒（grapevine Pinot gris virus，GPGV）+ 沙地葡萄茎痘相关病毒（grapevine rupestris stem pitting-associated virus，GRSPaV）+ HSVd，占比为35.0%。近几年新发现的病毒GPoV-1在石河子鲜食和酿酒葡萄中均能检测到，在昌吉酿酒葡萄中暂未检测到。全长或近全长病毒序列的系统进化发育表明GINV、GRSPaV、GPGV和葡萄黄斑类病毒1（grapevine yellow speckle viroid 1，GYSVd-1）存在较多的分离株，GINV和GPGV存在地理相关性，其余病毒分离株序列差异较大，无地理相关性。单核苷酸多态性（single nucleotide polymorphism，SNP）与插入缺失变异（insertion-deletion，Indel）分析表明，所有病毒与类病毒都存在较高的遗传变异，在新疆葡萄园内类病毒偏好于Indel，而病毒偏好于单碱基变异。

关键词: 葡萄, 高通量测序, RT-PCR, 系统进化分析, 序列变异分析

Abstract:

In order to systematically investigate the occurrence and infection status of grapevine viruses and viroids in Northern Xinjiang. LncRNA sequencing technology was used to obtain virus and viroid sequences from four mixed and six individual young grape leaf samples. Eleven plant viruses belonging to 7 viral families and 9 genera，along with 3 viroids from 1 viroid family and 2 genera were identified. This study provides the first molecular evidence confirming the presence of grapevine fabavirus（GFabV），grapevine geminivirus A（GGVA），grapevine rupestris vein feathering virus（GRVFV）and cucumber mosaic virus（CMV）in Xinjiang’s viticultural areas. Subsequent RT-PCR testing of 117 samples from five major grape cultivars in the primary viticultural regions of Shihezi and Changji，revealed grapevine berry inner necrosis virus（GINV）as the most prevalent virus（73.5%）infection rate and Hop stunt viroid（HSVd） as the dominant viroid（88.9% infection rate）. Viral infection profiles exhibited significant variation across grapevine cultivars，with distinct dominant virus species observed in each cultivar. Among them，the table grape cultivar‘Thompson Seedless’demonstrated the most comprehensive infection virus，harboring all 11 virus and 3 viroid species identified in this investigation. Notably，mixed infections were widespread in the grape samples, with 95.7% of samples showing co-infection by viruses and viroids. High prevalence of complex infections was observed，with 60 samples harboring co-infections by more than five distinct viral pathogens，and the most common mixed infection type was GINV + grapevine pinot gris virus（GPGV）+ grapevine rupestris stem pitting associated virus（GRSPaV）+ HSVd，accounting for 35%. The presence of the newly identified GPoV-1 in both table and wine grapes cultivars from Shihezi vineyards but was not detected in wine grapes collected from Changji. Phylogenetic analysis of full-length or near full-length viral sequences revealed that GINV，GRSPaV，GPGV and grapevine yellow speckle viroid 1（GYSVd-1）had multiple isolates. Notably，GINV and GPGV exhibited geographic correlations. In contrast，other viral isolates displayed high sequence divergence，with scattered distribution and no geographic correlation. Single nucleotide polymorphism and insertion-deletion analyses revealed that all viruses and viroids exhibited high genetic variability. In Xinjiang vineyards，viroids exhibited a significant preference for Indel，while viral genomes showed a predominant tendency toward single-nucleotide variations.

Key words: grape, high-throughput sequencing, RT-PCR, phylogenetic analysis, sequence variation analysis

刘琳, 赛小玲, 桑海洋, 卢如霞, 向本春, 郑银英. 新疆北疆葡萄病毒和类病毒组分析与分子鉴定[J]. 园艺学报, 2026, 53(6): 1765-1778.

LIU Lin, SAI Xiaoling, SANG Haiyang, LU Ruxia, XIANG Benchun, ZHENG Yinying. Analysis and Molecular Identification of Grapevine Virus and Viroid Genomes in Northern Xinjiang[J]. Acta Horticulturae Sinica, 2026, 53(6): 1765-1778.

图/表 9

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病毒或类病毒 Virus or Viroid	引物名称 Primer name	引物序列（5'-3'） Sequence of primer	产物大小/bp Size	退火温度/℃ Annealing temperature
GFabV	GFabV4300F GFabV4750R	GGGTTCCCTTTGACGGTGAT ACTCCCTCAAAACCGACTCA	451	55
GFkV	GFkV6445F GFkV6964R	CCGTCTGCTGACCAGCCTG CGACGCAGGCGTCATTGCG	520	57
GINV	GINV6178F GINV6583R	TAGGTGATGCTCTGGCGT TGCCTTATCGACATCTCGGC	406	57
GLRaV-3	GLaV4320F GLaV 4692R	GAGGGCTAGCGCGAATACTT TTCCCCGTTGGTGCTAATCC	373	57
GPGV	GPGV6661F GPGV7000R	TTCATGGGCTGACAGAAGGC TCACACAACTTGGGGTGCTT	340	57
GRSPaV	GRSPaV8236F GRSPaV8605R	ATCCATCTGGAGACGGGGAT CAGGCTTAACCCAGCCTTGA	370	58
GRVFV	GRVFV6156F GRVFV6600R	ACTCWCYATCCCCTTCCAGT GCTGACCATGCCACGAATCA	445	60
GVE	GV-E7155F GV-E7448R	TGGGTGAGTCTAGAAGTGCT CTGGAGTTTCCTCTGGGCTG	294	55
CMV	CMV3D/SP CMV1DA/SP	AGTTCCTGCCTCCTCGGA CCGACGTCCTTTATCATGAC	394	57
GGVA	GGVA2510F GGVA2863R	CGGACGAGGATTTAGCTCCC ATTTCACCCCATTCACCCCC	354	58
AGVd	AGVd70F AGVd229R	AGCTGGGTAAGACTCACCTG CAAGCGCAACAGGGATTCCA	160	60
GYSVd-1	GYSVd92F GYSVd271R	TCGTCGTCGACGAAGGGGTG GCTCGACTAGCGGAGGCAT	180	60
HSVd	HSVd5F HSVd179R	GGAATTCTCGAGTTGCCGC AGGGACGATCGATGGTGTTTC	175	60

病毒或类病毒 Virus or Viroid	引物名称 Primer name	引物序列（5'-3'） Sequence of primer	产物大小/bp Size	退火温度/℃ Annealing temperature
GFabV	GFabV4300F GFabV4750R	GGGTTCCCTTTGACGGTGAT ACTCCCTCAAAACCGACTCA	451	55
GFkV	GFkV6445F GFkV6964R	CCGTCTGCTGACCAGCCTG CGACGCAGGCGTCATTGCG	520	57
GINV	GINV6178F GINV6583R	TAGGTGATGCTCTGGCGT TGCCTTATCGACATCTCGGC	406	57
GLRaV-3	GLaV4320F GLaV 4692R	GAGGGCTAGCGCGAATACTT TTCCCCGTTGGTGCTAATCC	373	57
GPGV	GPGV6661F GPGV7000R	TTCATGGGCTGACAGAAGGC TCACACAACTTGGGGTGCTT	340	57
GRSPaV	GRSPaV8236F GRSPaV8605R	ATCCATCTGGAGACGGGGAT CAGGCTTAACCCAGCCTTGA	370	58
GRVFV	GRVFV6156F GRVFV6600R	ACTCWCYATCCCCTTCCAGT GCTGACCATGCCACGAATCA	445	60
GVE	GV-E7155F GV-E7448R	TGGGTGAGTCTAGAAGTGCT CTGGAGTTTCCTCTGGGCTG	294	55
CMV	CMV3D/SP CMV1DA/SP	AGTTCCTGCCTCCTCGGA CCGACGTCCTTTATCATGAC	394	57
GGVA	GGVA2510F GGVA2863R	CGGACGAGGATTTAGCTCCC ATTTCACCCCATTCACCCCC	354	58
AGVd	AGVd70F AGVd229R	AGCTGGGTAAGACTCACCTG CAAGCGCAACAGGGATTCCA	160	60
GYSVd-1	GYSVd92F GYSVd271R	TCGTCGTCGACGAAGGGGTG GCTCGACTAGCGGAGGCAT	180	60
HSVd	HSVd5F HSVd179R	GGAATTCTCGAGTTGCCGC AGGGACGATCGATGGTGTTTC	175	60

样本类型 Type of sample	文库 Libraries	原始读数 Raw reads	高质量读数 Clean reads	Q30/%	GC含量/% GC content	总重叠群数 Total contigs
单株Simple	V1	41 397 343	41 112 905	92.13	42.95	33 236
	V2	40 263 391	39 903 268	92.64	43.39	46 239
	V3	42 547 703	42 059 119	92.31	43.36	58 095
	V4	39 967 782	39 604 897	92.22	42.79	36 978
	V5	45 127 671	44 669 559	92.88	42.61	31 350
	V6	46 588 381	46 228 193	92.75	42.48	46 144
混样Mixed	PT191	25 465 372	25 093 079	92.11	43.71	29 242
	PT192	38 331 384	37 963 812	92.23	44.16	42 079
	PT193	42 164 523	41 799 420	93.14	42.98	56 496
	PT20	43 585 928	43 220 174	93.45	46.31	31 813

样本类型 Type of sample	文库 Libraries	原始读数 Raw reads	高质量读数 Clean reads	Q30/%	GC含量/% GC content	总重叠群数 Total contigs
单株Simple	V1	41 397 343	41 112 905	92.13	42.95	33 236
	V2	40 263 391	39 903 268	92.64	43.39	46 239
	V3	42 547 703	42 059 119	92.31	43.36	58 095
	V4	39 967 782	39 604 897	92.22	42.79	36 978
	V5	45 127 671	44 669 559	92.88	42.61	31 350
	V6	46 588 381	46 228 193	92.75	42.48	46 144
混样Mixed	PT191	25 465 372	25 093 079	92.11	43.71	29 242
	PT192	38 331 384	37 963 812	92.23	44.16	42 079
	PT193	42 164 523	41 799 420	93.14	42.98	56 496
	PT20	43 585 928	43 220 174	93.45	46.31	31 813

（类）病毒 Virus and Viroid	不同品种检测数量 Number of different cultivars						总检出率% Total detection rate
	石河子 Shihezi					昌吉Changji
	无核白 Thompson Seedless	无核紫 Black Monukka	红地球 Red Globe	夏黑 Summer Black	赤霞珠 Cabernet Sauvignon	赤霞珠 Cabernet Sauvignon
GFabV	9/15	1/15	0/15	0/15	13/20	3/37	22.2
GFkV	13/15^*	0/15	5/15	2/15	0/20	0/37	17.0
GGVA	11/15	0/15	1/15	13/15^*	3/20	20/37	41.0
GINV	7/15	6/15	12/15^*	12/15	17/20^*	32/37	73.5
GLRaV-3	1/15	1/15	1/15	4/15	7/20	3/37	11.1
GPGV	9/15	2/15	5/15	5/15	14/20	36/37^*	60.6
GPoV-1	4/15	3/15	3/15	3/15	7/20	0/37	17.0
GRSPaV	3/15	7/15^*	8/15	1/15	11/20	19/37	41.9
GRVFV	7/15	2/15	0/15	0/15	—	—	15.0
GVE	7/15	0/15	0/15	0/15	6/20	7/37	17.0
CMV	2/15	1/15	0/15	0/15	—	—	5.0
AGVd	1/15	11/15	0/15	1/15	6/20	4/37	19.7
GYSVd-1	12/15	0/15	0/15	0/15	18/20	22/37	44.4
HSVd	15/15	12/15	13/15	13/15	15/20	36/37	88.9