广东省番茄白尖曲叶病病原鉴定及其致病性分析

doi:10.3864/j.issn.0578-1752.2024.12.007

中国农业科学 ›› 2024, Vol. 57 ›› Issue (12): 2350-2363.doi: 10.3864/j.issn.0578-1752.2024.12.007

广东省番茄白尖曲叶病病原鉴定及其致病性分析

王愿¹^,²(), 都梦丹¹^,², 李正刚², 佘小漫², 于琳², 蓝国兵², 丁善文², 何自福²(), 汤亚飞²()

¹ 华南农业大学植物保护学院，广州 510642
² 广东省农业科学院植物保护研究所/农业农村部华南果蔬绿色防控重点实验室/广东省植物保护新技术重点实验室，广州 510640

收稿日期:2024-01-05 接受日期:2024-01-27 出版日期:2024-06-25 发布日期:2024-06-25
通信作者:
何自福，E-mail：hezf@gdppri.com。
汤亚飞，E-mail：tangyafei@gdppri.com
联系方式: 王愿，E-mail：y1875357469@163.com。
基金资助:
国家自然科学基金(32072392); 广东省现代农业产业技术体系创新团队项目(2020KJ134); 广东省农业科学院“十四五”学科团队建设项目(202105TD); 广东省农业科学院协同创新中心项目(XT202210)

Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province

WANG Yuan¹^,²(), DU MengDan¹^,², LI ZhengGang², SHE XiaoMan², YU Lin², LAN GuoBing², DING ShanWen², HE ZiFu²(), TANG YaFei²()

¹ College of Plant Protection, South China Agricultural University, Guangzhou 510642
² Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences/South China Key Laboratory for Green Prevention and Control of Fruits and Vegetables, Ministry of Agriculture and Rural Affairs/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640

Received:2024-01-05 Accepted:2024-01-27 Published:2024-06-25 Online:2024-06-25

摘要/Abstract

摘要：

【目的】洋桔梗耳突曲叶病毒（lisianthus enation leaf curl virus，LELCV）是2015年发现的菜豆金色黄花叶病毒属（Begomovirus）病毒新种。论文旨在探究入侵中国大陆的首个LELCV分离物Tomato-2022的分子特征、亲缘关系、致病性及有效传播介体，为防控该病毒病提供理论依据。【方法】 2022年，在广东省广州市增城区发生一种番茄新病害，病株表现叶小、卷曲、叶尖白化症状，采集两份病样提取总DNA，利用Begomovirus通用引物AV₄₉₄/CoPR进行PCR检测。选取PCR检测为阳性的样品进行RCA扩增、酶切、克隆及测序，获得分离物Tomato-2022的基因组全长序列。采用BLASTn程序对病毒序列进行相似性检索并下载相似性较高的代表分离物序列，进一步用SDTv1.2的MUSCLE alignment方法进行序列相似性比对；利用软件MEGA7.0对获得的Tomato-2022基因组序列及已报道的相似性较高的代表分离物进行系统发育分析。应用酶切连接法构建Tomato-2022的侵染性克隆pGreenII0229-1.6A，通过农杆菌介导注射接种本氏烟叶片和番茄茎秆，测定其致病性。采取室内人工传毒方法，测定烟粉虱MEAM1隐种对分离物Tomato-2022的传播效率。利用LELCV的PCR特异引物对2021—2023年采集于广东省番茄主产区的135份番茄病样进行检测，明确该病毒在广东省的分布。【结果】 PCR检测结果表明，两份表现叶小、卷曲、叶尖白化症状的番茄病样感染了菜豆金色黄花叶病毒属病毒。基因克隆与序列分析显示，该病毒分离物Tomato-2022基因组仅含DNA-A，大小为2 757 nt，含有6个ORF。序列相似性比较表明，分离物Tomato-2022基因组序列与已登录GenBank的LELCV各分离物相似性较高，其中与LELCV中国台湾洋桔梗分离物（LC091539、LC091538）相似性最高，为98.33%。系统发育分析显示，Tomato-2022与LELCV的19个分离物聚在一个独立的分支，亲缘关系较近。利用构建的Tomato-2022侵染性克隆pGreenII0229-1.6A，农杆菌介导注射接种本氏烟和番茄，10 dpi，本氏烟上部叶叶缘轻微上卷，1株番茄的新叶轻微卷曲；18 dpi，本氏烟和番茄植株均表现较为明显的卷曲症状；随着接种时间的延长，接种植株的症状越来越明显，30 dpi，本氏烟表现叶脉肿大、叶片严重卷曲，番茄植株的叶片表现卷曲、叶尖白化；PCR检测表明，在接种发病本氏烟和番茄植株中均能检测到LELCV。室内传毒试验证明，烟粉虱MEAM1隐种能高效传播Tomato-2022，当每株番茄接1、5、10头饲毒48 h的烟粉虱时，MEAM1的平均传毒效率分别为26.67%、93.33%、96.67%。PCR检测结果表明，采集的135份番茄病样中，8份样品检测到LELCV，说明LELCV已在广东省定殖。【结论】 Tomato-2022是洋桔梗耳突曲叶病毒的一个新分离物，也是在中国大陆发现的第一个分离物，其基因组为单链环状DNA，全长为2 757 nt，编码6个ORF；该病毒分离物是引起广东省番茄曲叶、叶尖白化病的病原，可被烟粉虱MEAM1隐种高效传播。

关键词: 洋桔梗耳突曲叶病毒, 番茄, 侵染性克隆, 致病性, 介体传毒效率, 烟粉虱

Abstract:

【Objective】 Lisianthus enation leaf curl virus (LELCV) is a new species of the genus Begomovirus discovered in 2015. The objective of this study is to explore the molecular characteristics, genetic relationship, pathogenicity and effective transmission vector of the first isolate Tomato-2022 of LELCV invading mainland China, and to provide a theoretical basis for the prevention and control of the virus disease.【Method】In 2022, a new disease of tomato occurred in Zengcheng District, Guangzhou City, Guangdong Province. The diseased plants exhibited small leaf, curling and leaf tips whitening symptoms. Total DNA was extracted from two diseased samples, respectively, and used as template for PCR detection with degenerate Begomovirus primers AV₄₉₄/CoPR. The full genome sequence of isolate Tomato-2022 was obtained by RCA amplification, gene cloning and sequencing from positive samples by PCR detection. The virus sequence was analyzed with BLASTn program and the representative isolate sequences with high similarity were downloaded. Further the sequence similarity was compared using MUSCLE alignment of SDTv1.2 software. Phylogenetic analysis between isolate Tomato-2022 and the reported representative isolates with high similarity was performed using MEGA7.0. Infectious clone pGreenII0229-1.6A of isolate Tomato-2022 was constructed using restriction enzyme digestion and ligation. The infectious clone was inoculated to Nicotiana benthamiana leaves and tomato stems by Agrobacterium-mediated injection to determine its pathogenicity. The efficiency of Bemisia tabaci MEAM1 cryptic species transmitting isolate Tomato-2022 was determined by indoor artificial virus transmission method. PCR specific primers of LELCV were used to detect 135 diseased tomato samples collected from the main tomato producing areas of Guangdong Province from 2021 to 2023 to clarify the distribution of LELCV in Guangdong Province.【Result】The PCR detection result showed that two diseased tomato samples with small leaf, curling and leaf tips whitening symptoms were infected by begomoviruses. Gene cloning and sequence analysis indicated that the genome of the virus isolate Tomato-2022 contained only DNA-A, with a size of 2 757 nt, and encoded six ORFs. The comparison of sequence similarity showed that the genomic sequence of isolate Tomato-2022 had high similarity with LELCV isolates registered in GenBank, and had the highest similarity with LELCV Taiwan, China Eustoma grandiflorum isolates (LC091539, LC091538) at 98.33%. Phylogenetic analysis indicated that Tomato-2022 and 19 isolates of LELCV were clustered in a separate branch and had a close relationship. The constructed Tomato-2022 infectious clone pGreenII0229-1.6A was inoculated into N. benthamiana and tomato by Agrobacterium-mediated injection. At 10 days post-inoculation (dpi), the upper leaf margins of N. benthamiana showed slight upward curling symptoms, and new leaves of one tomato plant showed slight curling symptoms. At 18 dpi, N. benthamiana and tomato plants showed more obvious curling symptoms. As time went by, the symptoms of the inoculated plants became more and more obvious. At 30 dpi, N. benthamiana showed veins swelling and leaves severe curling symptoms, and the leaves of tomato plants showed curling and leaf tips whitening symptoms. The results of PCR showed that LELCV could be detected in both the inoculated plants. The indoor virus transmission experiments proved that B. tabaci MEAM1 cryptic species could efficiently transmit Tomato-2022. When each tomato plant was inoculated with 1, 5 and 10 MEAM1 B. tabaci fed LELCV for 48 h, the average virus transmission efficiency was 26.67%, 93.33% and 96.67%, respectively. LELCV was detected from 8 of 135 diseased tomato samples by PCR using specific primer, indicating that LELCV had been colonized in Guangdong Province【Conclusion】 Tomato-2022 is a new isolate of LELCV, and also the first isolate found in Chinese mainland. Its genome is a single-stranded circular DNA with a total length of 2 757 nt, encoding six ORFs. The virus isolate is the pathogen of tomato curl leaf and white tip disease in Guangdong Province, and can be efficiently transmitted by B. tabaci MEAM1 cryptic species.

Key words: lisianthus enation leaf curl virus (LELCV), tomato, infectious clone, pathogenicity, virus transmission efficiency of vector, Bemisia tabaci

王愿, 都梦丹, 李正刚, 佘小漫, 于琳, 蓝国兵, 丁善文, 何自福, 汤亚飞. 广东省番茄白尖曲叶病病原鉴定及其致病性分析[J]. 中国农业科学, 2024, 57(12): 2350-2363.

WANG Yuan, DU MengDan, LI ZhengGang, SHE XiaoMan, YU Lin, LAN GuoBing, DING ShanWen, HE ZiFu, TANG YaFei. Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province[J]. Scientia Agricultura Sinica, 2024, 57(12): 2350-2363.

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https://www.chinaagrisci.com/CN/Y2024/V57/I12/2350

图/表 10

表1

图1

图2

图3

表2

分离物Tomato-2022与其他33个菜豆金色黄花叶病毒属病毒分离物的相似性比较"

病毒名 Virus	分离物 Isolate	登录号 Accession number	一致性Identity (%)
			全序列 Total sequence	基因间隔区 IR	AV1		AV2		AC1		AC2		AC3		AC4
						Nt	Nt	Nt	AA	Nt	AA	Nt	AA	Nt	AA	Nt	AA	Nt	AA
			LELCV	BG-9	LC091539	98.33	96.27	99.48	100.00	98.45	98.44	98.34	97.22	98.53	97.78	97.53	94.78	98.63	97.92
BG-1	LC091538	98.33		96.27	99.61	100.00	98.71	98.44	98.15	98.33	99.02	98.52	97.78	96.27	98.97	98.96
HT19T347	MW248580	98.30		95.02	99.35	100.00	98.71	96.09	98.34	98.33	99.26	99.26	98.02	96.27	98.97	97.92
HT19T242	MW248577	98.26		96.67	99.48	99.61	97.93	96.09	98.25	98.61	98.77	98.52	97.28	94.78	99.31	98.96
HT19T340	MW248579	98.22		95.02	99.35	100.00	98.71	96.09	98.15	98.06	99.26	99.26	98.02	96.27	98.97	97.92
Japan: Tomigusuku: T326	LC642632	98.19		94.58	98.96	99.61	98.19	96.88	98.61	97.78	99.26	99.26	97.78	96.27	98.97	97.92
HT19T346	MW248578	98.11		94.61	99.22	99.61	98.71	96.09	98.06	98.33	99.26	99.26	98.02	96.27	98.63	96.88
CY19T09-3	MW248581	98.01		95.85	99.35	100.00	97.93	96.09	98.17	97.97	98.28	96.30	97.28	93.28	98.63	96.88
TN19T143	MW248582	97.13		95.44	99.33	99.50	97.42	96.09	96.12	95.83	98.28	97.04	97.28	94.78	95.88	93.75
ML19T484	MW248584	93.75		95.44	99.61	100.00	98.45	96.88	91.57	91.39	78.77	75.37	88.40	82.84	98.97	97.92
YL19T38	MW248583	93.71		97.03	99.35	100.00	98.71	96.88	91.11	91.39	80.49	76.87	88.64	82.09	98.97	97.92
CH19T127	MW248589	92.97		80.51	98.96	99.61	98.45	97.66	89.20	88.06	98.77	97.78	98.02	95.52	86.94	71.88
CH19T442	MW248592	92.97		82.56	99.48	99.61	98.23	96.43	89.10	87.78	98.53	97.04	97.78	96.27	85.57	66.67
TC19T115	MW248591	92.93		81.03	99.22	99.61	97.94	96.43	89.20	87.50	98.28	96.30	97.78	96.27	85.91	68.75
DC-1	MN692224	93.00		77.19	99.35	99.22	98.53	97.32	89.01	87.50	98.53	97.04	97.78	96.27	85.57	66.67
KH19T321	MW248588	92.93		84.10	99.22	99.61	98.45	96.09	88.74	87.78	98.53	97.04	97.53	94.78	85.57	68.75
TC19T111	MW248594	92.86		82.05	99.35	99.22	97.94	96.43	89.20	87.50	97.55	96.24	97.28	96.24	85.91	67.71
CY19T213	MW248590	92.86		82.56	99.22	99.61	98.23	96.43	89.38	87.78	98.53	97.04	96.54	94.03	86.94	71.88
IL19T405	MW248593	92.82		81.54	99.35	99.61	98.53	97.32	89.10	87.50	98.04	97.04	97.78	95.52	85.91	67.71
HT1528	MW248586	92.75		83.59	99.36	95.19	98.19	96.09	88.46	87.50	98.28	96.30	97.28	94.03	86.25	69.79
KH19T328	MW248587	92.53		83.59	99.09	99.61	98.19	96.09	88.18	87.22	97.79	95.56	97.28	94.03	85.91	69.79
HC19T477	MW248585	88.91		84.30	99.61	100.00	98.45	96.09	83.06	83.33	79.01	76.12	88.64	82.84	87.29	73.96
TYLCTHV	Chiang Mai	AY514630	85.25	78.17	95.60	99.22	96.46	95.54	77.25	78.61	80.00	76.87	87.65	79.10	74.31	43.75
	Y72	AJ495812	84.09	77.39	92.61	98.83	95.28	94.64	77.04	78.89	80.49	78.36	88.89	81.34	72.22	39.58
	Sakon Nakhon	AY514632	83.67	75.55	91.31	93.75	88.20	83.04	77.04	78.61	80.00	76.87	87.65	79.85	73.52	42.71
ToLCTV	T2-4	GU723730	76.62	69.27	73.80	79.69	74.93	70.54	80.07	83.33	81.13	69.63	81.39	79.10	76.66	47.92
ToLCTV	NS1	JQ867093	76.45	76.15	73.80	78.91	73.45	70.54	79.54	82.78	80.15	69.63	80.15	73.88	74.65	46.88
PaLCuCNV	G8	AJ558124	77.02	77.58	75.10	81.64	75.52	70.54	79.30	81.67	80.15	71.11	78.02	72.39	71.72	41.67
PaLCuCNV	GX4	FN297834	76.93	73.52	74.84	81.64	76.40	69.64	79.87	78.65	79.41	70.37	79.01	73.13	74.91	45.83
TYLCV	Almeria	AJ489258	75.73	75.11	73.15	73.44	74.04	72.32	79.83	79.27	76.30	63.70	77.04	70.90	78.82	46.88
ToLCGxV	GX-1	AM236784	81.60	76.36	83.40	91.41	73.75	67.86	84.86	86.11	81.37	74.07	79.26	73.88	86.94	72.92
TYLCGdV	G3	AY602166	78.14	73.87	75.62	81.25	76.40	72.32	80.26	80.28	82.35	77.04	83.37	74.63	75.26	44.79
ToLCGdV	G2	AY602165	77.48	73.49	74.71	81.25	76.11	72.32	79.26	80.56	82.60	77.04	84.29	73.88	79.07	58.82

表2

图4

图5

图6

表3

图7

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引物名称 Primer name	引物序列 Primer sequence (5′-3′)	结合位置 Binding site	目的片段长度Product size (bp)
pz11-Xho I-F1（引入Xho I位点Introduction of Xho I restriction enzyme site）	CTCGAGCTCAAGCAGAGAATGGCGTATATATC	1407-1432	2757
pz11-R1（含Pst I位点Contained Pst I restriction enzyme site）	CTGCAGTGATGGGATCCCCTGTGCGTGAATC	1433-1463	2757
pz11-F2（含Pst I位点Contained Pst I restriction enzyme site）	AA CTGCAGCTCAAGCAGAGAATGGCGTATATATC	1407-1438	1664
0.6pz11-Xba I-R2（引入Xba I位点Introduction of Xba I restriction enzyme site）	GC TCTAGAGTAAGCGAACAGTGTGGATAAG	2532-2553	1664
LELCV-F	TAATGATGCCTGGTACAACGTCATTG	1822-1847	816
LELCV-R	CAGAAGTTATTTCTATGACTCCGTGACG	1032-1059	816
LE-F1	GTGGCAGTTAATGGATACAAAG	1471-1492	1126
LE-R1	CCACCCAGAAAATTCCAAATAAACGCC	2570-2596	1126

广东省番茄白尖曲叶病病原鉴定及其致病性分析

Identification of Pathogen Causing Tomato White Tip and Curl Leaf Disease and Its Pathogenicity in Guangdong Province

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接虫量 Insect inoculation amount (/plant)	处理I Treatment I				处理II Treatment II				平均传毒效率 Average virus transmission efficiency (%)
接虫量 Insect inoculation amount (/plant)	接种株数 Number of inoculated plants	病株数 Number of diseased plants	PCR检测阳性株数 Number of PCR positive plants	传毒效率 Virus transmission efficiency (%)	接种株数 Number of inoculated plants	病株数 Number of diseased plants	PCR检测阳性株数 Number of PCR positive plants	传毒效率 Virus transmission efficiency (%)
1	15	5	5	33.33	15	3	3	20.00	26.67
5	15	14	14	93.33	15	14	14	93.33	93.33
10	15	14	14	93.33	15	15	15	100.00	96.67

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