ToLCNDV广东冬瓜分离物的分子特征及其遗传演化规律

doi:10.3864/j.issn.0578-1752.2025.19.007

摘要/Abstract

摘要：

【目的】新德里番茄曲叶病毒（tomato leaf curl New Delhi virus，ToLCNDV）是近年来危害我国瓜类蔬菜最严重的病毒性病原之一。目前，有关中国流行株的基因组序列报道有限，且国内尚未报道ToLCNDV在冬瓜（Benincasa hispida）上的发生。本研究旨在明确ToLCNDV在广东冬瓜中流行规律，分析ToLCNDV中国分离物的序列特异性、致病性、遗传多样性以及时空迁移特征，为ToLCNDV的防控提供理论依据。【方法】通过高通量测序、PCR扩增技术对79份冬瓜样品进行ToLCNDV的检测和全基因组序列扩增，并进行系统发育、序列一致性、遗传多样性、贝叶斯系统发育及谱系地理分析。此外，通过汁液摩擦接种技术对12种ToLCNDV潜在寄主进行致病性验证。【结果】高通量测序结果表明，所采集冬瓜样品中存在ToLCNDV。PCR检测发现79份疑似病毒样本中58份感染了ToLCNDV，检出率高达73.4%；ToLCNDV冬瓜分离物可通过汁液摩擦接种侵染西瓜（Citrullus lanatus）、丝瓜（Luffa aegyptiaca）、西葫芦（Cucurbita pepo）、菜豆（Phaseolus vulgaris）、黄瓜（Cucumis sativus）、瓠瓜（Lagenaria siceraria）、克利夫兰烟（Nicotiana clevelandii）以及本氏烟（N. benthamiana）8种植物；ToLCNDV冬瓜分离物23GD_BeHi1的DNA A片段核苷酸序列全长为2 739 bp，DNA B片段核苷酸序列全长为2 693 bp；序列一致性分析结果显示，ToLCNDV冬瓜分离物与国内已知的ToLCNDV分离物DNA A和DNA B完整序列一致性分别在98.7%—99.7%和98.7%—99.3%；系统发育分析显示，基于ToLCNDV中国分离物DNA A和DNA B序列所重建的系统发育树均被分为两个明显的分支，且本研究所获的分离物均在Clade I分支；ToLCNDV中国分离物之间不存在重组事件；遗传多样性分析结果显示，ToLCNDV中国分离物群体内部遗传多样性相对较低，群体大小保持稳定且未发现重组事件，但江苏和上海分离物之间存在显著遗传分化；贝叶斯系统发育分析结果显示，基于DNA A和DNA B数据集所构建的MCC树的根均为浙江分离物，推断ToLCNDV的出现时间分别为2020年4月25日和2020年11月30日；谱系地理分析结果显示，浙江是我国ToLCNDV的起源中心，在广东发生了种群扩张。【结论】获得了我国ToLCNDV冬瓜分离物的全基因组序列，其DNA A和DNA B片段核苷酸序列全长分别为2 739和2 693 bp。通过摩擦接种证明其亦可侵染西葫芦、西瓜、丝瓜、黄瓜、菜豆、克利夫兰烟、瓠瓜以及本氏烟，并引起卷曲、褪绿等症状。

关键词: 新德里番茄曲叶病毒, 冬瓜, 全基因组序列, 遗传多样性, 谱系地理分析

Abstract:

【Objective】Tomato leaf curl New Delhi virus (ToLCNDV) is one of the most severe viral pathogens affecting cucurbit crops in recent years. Reports on the genome sequences of ToLCNDV isolates from China remain limited. ToLCNDV infection in wax gourd (Benincasa hispida) has not been reported in China. The objective of this study is to investigate the occurrence patterns of ToLCNDV in B. hispida in Guangdong Province, analyze sequence characteristics, pathogenicity, genetic diversity, and spatiotemporal dynamics of ToLCNDV isolates from China, and to provide a theoretical basis for the prevention and control of ToLCNDV.【Method】The complete genome of ToLCNDV was amplified using high-throughput sequencing and PCR. PCR assays were performed to detect ToLCNDV in 79 B. hispida samples. The obtained full-length genome sequences were subjected to phylogenetic analysis, sequence identity assessment, genetic diversity evaluation, Bayesian phylogenetics, and phylogeographic analysis. Pathogenicity was tested on 12 potential host species using mechanical sap-inoculation.【Result】High-throughput sequencing revealed that ToLCNDV was present in the collected B. hispida samples. PCR assays confirmed ToLCNDV infection in 58 of 79 suspected virus samples, representing a detection rate of 73.4%. The B. hispida isolate of ToLCNDV was mechanically transmissible through sap-inoculation to eight plant species. These included Citrullus lanatus, Luffa aegyptiaca, Cucurbita pepo, Phaseolus vulgaris, Cucumis sativus, Lagenaria siceraria, Nicotiana clevelandii, and N. benthamiana. All sap-inoculated plants developed characteristic symptoms. The complete nucleotide sequences of the DNA A and B components of the B. hispida isolate 23GD_BeHi1 were 2 739 and 2 693 bp, respectively. Pairwise sequence comparison showed 98.7%-99.7% identity for DNA A and 98.7%-99.3% for DNA B compared to representative Chinese ToLCNDV isolates. Phylogenetic trees based on complete DNA A and DNA B sequences of Chinese ToLCNDV isolates revealed two distinct clades. All isolates obtained in this study clustered within Clade I. No recombination events were detected among these isolates. Genetic diversity analyses indicated that the Chinese ToLCNDV population exhibited relatively low genetic diversity. The adequate population size remained stable, and no evidence of recombination was observed. However, significant genetic differentiation was detected between isolates from Jiangsu and Shanghai. Bayesian maximum clade credibility (MCC) trees constructed from DNA A and DNA B datasets placed Zhejiang isolates at the basal position. The estimated emergence times were April 25, 2020, for DNA A and November 30, 2020, for DNA B. Phylogeographic reconstruction suggested that Zhejiang served as the origin center of ToLCNDV in China, and the population expansion occurred in Guangdong.【Conclusion】The complete genome sequence of ToLCNDV isolate from B. hispida in China was obtained. The DNA A and B components comprise 2 739 and 2 693 bp, respectively. Mechanical sap-inoculation demonstrates that this isolate can infect C. pepo, C. lanatus, L. aegyptiaca, C. sativus, P. vulgaris, N. clevelandii, L. siceraria, and N. benthamiana. Infected plants develop leaf curling, chlorosis, and other typical symptoms.

Key words: tomato leaf curl New Delhi virus (ToLCNDV), wax gourd (Benincasa hispida), complete genome sequence, genetic diversity, phylogeographic analysis

郭孟泽, 张磊, 孙平平, 江彪, 闫晋强, 李正男. ToLCNDV广东冬瓜分离物的分子特征及其遗传演化规律[J]. 中国农业科学, 2025, 58(19): 3890-3904.

GUO MengZe, ZHANG Lei, SUN PingPing, JIANG Biao, YAN JinQiang, LI ZhengNan. Molecular Characterization and Evolutionary Dynamics of Tomato Leaf Curl New Delhi Virus Isolate from Wax Gourd (Benincasa hispida) in Guangdong[J]. Scientia Agricultura Sinica, 2025, 58(19): 3890-3904.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2025.19.007

https://www.chinaagrisci.com/CN/Y2025/V58/I19/3890

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表4

不同ToLCNDV亚群体之间的遗传分化"

比较Comparison	Ks*	Kst*	P value	Z	P value	Z*	P value	S_nn	P value	F_ST	N_m
广东Guangdong (n=25) vs. 江苏Jiangsu (n=3)	3.59050	0.01339	0.0020**	176.30097	0.0190*	4.79813	0.0010**	1.00000	P<0.001***	0.281	1.28
广东Guangdong (n=25) vs. 山东Shandong (n=3)	3.61758	0.00563	0.1060ns	181.84139	0.1200ns	4.89036	0.1070ns	0.91071	0.0330ns	0.059	7.92
广东Guangdong (n=25) vs. 上海Shanghai (n=12)	3.49517	0.02367	P<0.001***	304.62982	P<0.001***	5.23252	P<0.001***	0.97297	P<0.001***	0.132	3.26
广东Guangdong (n=25) vs. 浙江Zhejiang (n=11)	3.52988	0.02868	P<0.001***	278.73554	P<0.001***	5.16733	P<0.001***	0.88889	P<0.001***	0.164	2.56
江苏Jiangsu (n=3) vs. 山东Shandong (n=3)	3.41363	0.05378	0.0940ns	4.91667	0.0940ns	1.42466	0.0940ns	0.66667	0.1920ns	0.220	1.77
江苏Jiangsu (n=3) vs. 上海Shanghai (n=12)	3.21326	0.03059	0.0100*	43.07300	0.0100*	3.40610	0.0040**	0.86667	0.0500ns	0.223	1.74
江苏Jiangsu (n=3) vs. 浙江Zhejiang (n=11)	3.29615	0.03585	0.0180*	39.19818	0.0340*	3.31263	0.0100*	0.78571	0.1640ns	0.271	1.34
山东Shandong (n=3) vs. 上海Shanghai (n=12)	3.27235	0.02922	0.0130*	44.25069	0.0200*	3.44929	0.0090**	0.80000	0.2510ns	0.071	6.49
山东Shandong (n=3) vs. 浙江Zhejiang (n=11)	3.36115	0.03228	0.0380*	39.12727	0.0440*	3.33703	0.0340*	0.78571	0.2120ns	0.111	4.01
上海Shanghai (n=12) vs. 浙江Zhejiang (n=11)	3.27000	0.03913	P<0.001***	106.61994	P<0.001***	4.20364	P<0.001***	0.78261	0.0110*	0.148	2.88
冬瓜B. hispida (n=5) vs. 西瓜C. lanatus (n=3)	3.53104	0.00964	0.1460ns	13.20833	0.4390ns	2.29216	0.2000ns	0.81250	0.1250ns	0.084	5.48
冬瓜B. hispida (n=5) vs. 甜瓜C. melo (n=13)	3.61523	0.00894	0.0700ns	72.24615	0.0680ns	3.95727	0.0670ns	0.91667	0.0020**	0.063	7.44
冬瓜B. hispida (n=5) vs. 南瓜C. moschata (n=7)	3.61913	-0.01784	0.9980ns	35.44345	0.9750ns	3.41699	0.9980ns	0.16667	0.9700ns	-0.094	-5.81
冬瓜B. hispida (n=5) vs. 黄瓜C. sativus (n=7)	3.54832	-0.00045	0.4900ns	30.90893	0.1330ns	3.15826	0.21100ns	0.66667	0.1960ns	0.026	19.07
冬瓜B. hispida (n=5) vs. 丝瓜L. aegyptiaca (n=14)	3.66197	-0.00604	0.9040ns	88.52385	0.9200ns	4.22878	0.8780ns	0.55263	0.6940ns	-0.033	-15.49
冬瓜B. hispida (n=5) vs. 番茄S. lycopersicum (n=6)	3.46208	-0.01724	0.9130ns	28.80000	0.8660ns	3.19820	0.8960ns	0.40909	0.6390ns	-0.088	-6.21
西瓜C. lanatus (n=3) vs. 甜瓜C. melo (n=13)	3.60395	-0.00045	0.5070ns	60.79327	0.6520ns	3.83590	0.5320ns	0.71875	0.5040ns	0.052	9.17
西瓜C. lanatus (n=3) vs. 南瓜C. moschata (n=7)	3.59786	0.00822	0.1640ns	21.46429	0.3470ns	2.72573	0.1140ns	0.90000	0.0520ns	0.101	4.46
西瓜C. lanatus (n=3) vs. 黄瓜C. sativus (n=7)	3.50344	0.00740	0.2650ns	21.63492	0.3830ns	2.77007	0.1850ns	0.90000	0.0380*	0.070	6.68
西瓜C. lanatus (n=3) vs. 丝瓜L. aegyptiaca (n=14)	3.65875	0.00277	0.2910ns	68.45323	0.5840ns	3.89677	0.2360ns	0.94118	0.0230*	0.093	4.87
西瓜C. lanatus (n=3) vs. 番茄S. lycopersicum (n=6)	3.37373	0.02037	0.0420*	15.17333	0.0930ns	2.40958	0.0590ns	0.88889	0.0520ns	0.108	4.15
甜瓜C. melo (n=13) vs. 南瓜C. moschata (n=7)	3.62976	0.01302	0.0080**	87.52810	0.0070**	4.12911	0.0140*	1.00000	P<0.001***	0.073	6.32
甜瓜C. melo (n=13) vs. 黄瓜C. sativus (n=7)	3.59436	-0.00115	0.5280ns	95.23867	0.5550ns	4.28371	0.5660ns	0.55000	0.4370ns	-0.003	-145.54
甜瓜C. melo (n=13) vs. 丝瓜L. aegyptiaca (n=14)	3.65447	0.01116	P<0.001***	165.11085	P<0.001***	4.75332	0.0040**	0.85185	P<0.001***	0.058	8.09
甜瓜C. melo (n=13) vs. 番茄S. lycopersicum (n=6)	3.55718	0.00891	0.0730ns	81.53650	0.0580ns	4.07607	0.0590ns	0.68421	0.1620ns	0.070	6.60
南瓜C. moschata (n=7) vs. 黄瓜C. sativus (n=7)	3.58495	0.00802	0.1470ns	42.53571	0.1090ns	3.42859	0.1100ns	0.78571	0.0510ns	0.044	10.91
南瓜C. moschata (n=7) vs. 丝瓜L. aegyptiaca (n=14)	3.67015	-0.00744	0.9620ns	107.58382	0.9050ns	4.44414	0.9560ns	0.38095	0.9070ns	-0.034	-15.30
南瓜C. moschata (n=7) vs. 番茄S. lycopersicum (n=6)	3.52195	-0.01071	0.9670ns	40.81323	0.9330ns	3.49448	0.8570ns	0.42308	0.6120ns	-0.057	-9.23
黄瓜C. sativus (n=7) vs. 丝瓜L. aegyptiaca (n=14)	3.63683	0.00187	0.3010ns	103.33603	0.2540ns	4.34873	0.3240ns	0.61905	0.2840ns	0.026	18.63
黄瓜C. sativus (n=7) vs. 番茄S. lycopersicum (n=6)	3.45900	0.00457	0.4120ns	36.48042	0.1060ns	3.31631	0.2100ns	0.57692	0.3250ns	0.046	10.48
丝瓜L. aegyptiaca (n=14) vs. 番茄S. lycopersicum (n=6)	3.60463	-0.00764	0.9840ns	100.08773	0.9960ns	4.32873	0.8890ns	0.42500	0.8910ns	-0.036	-14.56

表4

图7

表5

表6

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	退火温度 Tm (℃)	位置 Region	位点 Site (nt)
DG1-1	CTAGAACGTCTCCGTCTTTGT	58	DNA A	2251-2541
DG1-2	TCCTCACATATCCAAAGTGCT	58	DNA A	2251-2541
DG1-3	ACCGAATGGCCGCGCAAATT	60	DNA A	1-2739
DG1-4	AATATTATACGAATGGCCGCTT	60	DNA A	1-2739
DG1-5	CTTTAGGGAATTTGAATTAAAGTAAT	60	DNA B	1-2675
DG1-6	TACCGAAAGGCCGCGAAAATTT	60	DNA B	1-2675

编码区 Coding region	ENC	dN	dS	dN/dS	归一化dN-dS值 Normalized dN-dS		负选择密码子数 Number of negatively selected codon			正选择密码子位点 Site of positively selected codon
编码区 Coding region	ENC	dN	dS	dN/dS	Log (L)	Mean (dN/dS)	FEL	SLAC	FUBAR	FEL	SLAC	FUBAR	MEME
AC1	361	0.00406	0.01514	0.268	-2325.48	0.307	23	4	9	0	0	0	0
AC2	139	0.00469	0.00566	0.829	-809.73	0.946	1	0	1	0	0	0	0
AC3	136	0.00363	0.01068	0.340	-782.48	0.548	2	1	2	0	0	0	0
AC4	58	0.00652	0.00480	1.358	-325.65	5.16	1	0	1	0	0	10	0
AC5	162	0.00447	0.00288	1.552	-742.11	2.12	2	1	1	0	0	0	0
AV1	256	0.00781	0.00193	4.047	-1429.51	0.284	7	0	6	0	0	0	0
AV2	122	0.00322	0.00729	0.442	-644.11	0.399	5	1	3	0	0	112	0
BC1	281	0.00223	0.01783	0.125	-1383.69	0.133	30	4	20	0	0	0	0
BV1	258	0.00468	0.01069	0.438	-1780.71	0.549	14	2	5	0	0	48, 193	193

群体 Population	序列数 Number of sequences	单倍型多样性 Haplotype diversity	核苷酸多态性 Nucleotide diversity	Tajima’s D	Fu and Li’s D*	Fu and Li’s F*
All	56	1.000±0.003	0.00708±0.00095	-2.52445***	-4.66339**	-4.58633**
广东Guangdong	25	1.000±0.011	0.00727±0.00087	-2.15107*	-3.15544**	-3.33509**
江苏Jiangsu	3	1.000±0.272	0.00444±0.00075	NA	NA	NA
山东Shandong	3	1.000± 0.272	0.00786±0.00100	NA	NA	NA
上海Shanghai	12	1.000±0.034	0.00509±0.00062	-1.04203ns	-0.99753ns	-1.15086ns
浙江Zhejiang	11	1.000±0.039	0.00587±0.00072	-1.38448ns	-1.58347ns	-1.74005ns
冬瓜B. hispida	5	1.000±0.126	0.00679±0.00082	-0.70558ns	-0.68630ns	-0.74972ns
西瓜C. lanatus	3	1.000±0.272	0.00564±0.00084	NA	NA	NA
甜瓜C. melo	13	1.000±0.030	0.00742±0.00085	-1.77037ns	-1.93222ns	-2.16362ns
南瓜C. moschata	7	1.000±0.076	0.00729±0.00082	-0.94402ns	-0.91144ns	-1.01852ns
黄瓜C. sativus	7	1.000±0.076	0.00658±0.00080	-1.03788ns	-1.12559ns	-1.12983ns
丝瓜L. aegyptiaca	14	1.000±0.027	0.00767±0.00089	-1.98427*	-2.53216*	-2.73777**
番茄S. lycopersicum	6	1.000±0.096	0.00558±0.00074	-1.11658ns	-1.17026ns	-1.27258ns

片段 Segment	分子钟模型 Molecular clock model	溯祖树先验模型 Coalescent tree prior	边际似然值的对数 Log marginal likelihood
DNA A	严格分子钟Strict clock	贝叶斯天际线Bayesian skyline	-7047.037
	严格分子钟Strict clock	恒定种群大小Constant size	-7063.477
	严格分子钟Strict clock	指数增长种群Exponential growth	-7048.470
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	贝叶斯天际线Bayesian skyline	-7054.126
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	恒定种群大小Constant size	-7055.758
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	指数增长种群Exponential growth	-7065.284
DNA B	严格分子钟Strict clock	贝叶斯天际线Bayesian skyline	-7021.287
	严格分子钟Strict clock	恒定种群大小Constant size	-7056.605
	严格分子钟Strict clock	指数增长种群Exponential growth	-7041.550
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	贝叶斯天际线Bayesian skyline	-7030.064
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	恒定种群大小Constant size	-7040.607
	非相关对数正态宽松分子钟 Uncorrelated lognormal relaxed clock	指数增长种群Exponential growth	-7024.184

事件Event	输出From	输入To	DNA A	DNA B
1	广东Guangdong	上海Shanghai	+**	+**
2	上海Shanghai	浙江Zhejiang	+***	+*