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

Abstract

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

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.

Figures/Tables 10

Table 1

Fig. 1

Fig. 2

Fig. 3

Table 2

Nucleotide and amino acid sequence identities between the isolate Tomato-2022 and other 33 begomoviruses"

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

Table 2

Fig. 4

Fig. 5

Fig. 6

Table 3

Fig. 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

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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