新德里番茄曲叶病毒侵染性克隆构建及甜瓜品种抗病性鉴定

doi:10.16420/j.issn.0513-353x.2023-0733

摘要/Abstract

摘要：

以新德里番茄曲叶病毒（tomato leaf curl New Delhi virus，ToLCNDV）甜瓜分离物Haimen 6（HM6）为研究对象，采用串联重复片段法构建该分离物的侵染性克隆，分析其在不同作物中的侵染性，同时对25份甜瓜品种进行抗性评价。结果显示，用分别含有1.57个DNA-A串联重复（1.57A）和1.43个DNA-B串联重复（1.43B）的侵染性克隆共浸润接种及用同时含有1.57A和1.43B的侵染性克隆单独接种甜瓜，植株的侵染率均达100%。将分别含有1.57A、1.43B的侵染性克隆共浸润接种其他作物，发现可以侵染本氏烟草、番茄、南瓜、西葫芦、瓠瓜、黄瓜、丝瓜，不能侵染西瓜。通过抗性评价，25份供试甜瓜材料中有3份表现为抗病，3份表现为感病，19份表现为高感病。

关键词: 甜瓜, 新德里番茄曲叶病毒, 侵染性克隆, 侵染性, 抗性

Abstract:

In this study，a tomato leaf curl New Delhi virus（ToLCNDV）melon isolate Haimen 6 （HM6）was used as template to obtain its infectious clone by tandem repeat fragment method，and its infectivity was examined in different crops. Meanwhile，the resistance of 25 Cucumis melo cultivars were evaluated. The results showed that whatever infectious clones containing 1.57-mer DNA-A tandem repeat （1.57A）and 1.43-mer DNA-B tandem repeat（1.43B），respectively，were co-infiltrated or the clone containing both 1.57A and 1.43B was directly inoculated with melon，and the infection rate of the plants could reach 100%. Infectious clones containing 1.57A and 1.43B，respectively，were co-infiltrated into other crops，and it was found that it could infect Nicotiana benthamiana，Solanum lycopersicum，Cucurbita moschata，Cucurbita pepo，Lagenaria siceraria，Cucumis sativus，and Luffa aegyptiaca，but could not infect Citrullus lanatus. Through resistance evaluation，it was found that 3 of the 25 tested melon materials showed resistance，3 materials showed susceptibility and 19 materials showed high susceptibility.

Key words: melon, tomato leaf curl New Delhi virus, infectious clone, infectivity, resistance

刘莉铭, 耿艳飞, 郝芳敏, 康保珊, 吴会杰, 古勤生. 新德里番茄曲叶病毒侵染性克隆构建及甜瓜品种抗病性鉴定[J]. 园艺学报, 2024, 51(11): 2555-2564.

LIU Liming, GENG Yanfei, HAO Fangmin, KANG Baoshan, WU Huijie, GU Qinsheng. Construction of Tomato Leaf Curl New Delhi Virus Infectious Clone and Its Application in Evaluation for Melon Disease Resistance[J]. Acta Horticulturae Sinica, 2024, 51(11): 2555-2564.

图/表 7

参考文献 17

[1]	Gong P, Tan H, Zhao S, Li H, Liu H, Ma Y, Zhang X, Rong J, Fu X, Lozano-Durán R, Li F, Zhou X. 2021. Geminiviruses encode additional small proteins with specific subcellular localizations and virulence function. Nature Communications, 12 (1):4278. doi: 10.1038/s41467-021-24617-4 pmid: 34257307
[2]	Gu Q, Yan L, Liu L, Bao W, Fang H, Xu J, Li J, Kang B, Wu H, Wang K, Tao X, Peng B. 2023. First report of tomato leaf curl New Delhi virus infecting several cucurbit plants in China. Plant Disease,doi:10.1094/PDIS-01-23-0180-PDN.
[3]	Gu Qinsheng, Yan Leiyan, Liu Liming, Tao Xiaorong, Bao Weihong, Fang Hui, Xu Jinhua, Li Jufen, Kang Baoshan, Wang Shaoli, Wang Kang, Peng Bin, Wu Huijie. 2023. A new virus of cucurbits-tomato leaf curl New Delhi virus. Chinese Cucurbits and Vegetables, 36 (8):1-4. (in Chinese)
	古勤生, 严蕾艳, 刘莉铭, 陶小荣, 包卫红, 方辉, 徐锦华, 李菊芬, 康保珊, 王少丽, 王康, 彭斌, 吴会杰. 2023. 瓜类作物新病毒——新德里番茄曲叶病毒. 中国瓜菜, 36 (8):1-4.
[4]	Hussain M, Mansoor S, Iram S, Fatima A N, Zafar Y. 2005. The nuclear shuttle protein of tomato leaf curl New Delhi virus is a pathogenicity determinant. Journal of Virology, 79 (7):4434-4439. doi: 10.1128/JVI.79.7.4434-4439.2005 pmid: 15767443
[5]	Lee C H, Zheng Y X, Chan C H, Ku H M, Chang C J, Jan F J. 2020. A single amino acid substitution in the movement protein enables the mechanical transmission of a geminivirus. Molecular Plant Pathology, 21 (4):571-588.
[6]	Li F, Xu X, Huang C, Gu Z, Cao L, Hu T, Ding M, Li Z, Zhou X. 2015. The AC 5 protein encoded by mungbean yellow mosaic india virus is a pathogenicity determinant that suppresses RNA silencing-based antiviral defenses. The New Phytologist, 208 (2):555-569.
[7]	Li P, Su F, Meng Q, Yu H, Wu G, Li M, Qing L. 2021. The C 5 protein encoded by ageratum leaf curl Sichuan virus is a virulence factor and contributes to the virus infection. Molecular Plant Pathology, 22 (9):1149-1158.
[8]	Li R, Liu Y, Yin C, Sun K, Zhang P. 2022. Occurrence of tomato leaf curl New Delhi virus in tomato(Lycopersicon esculentum)in China. Plant Disease,doi:10.1094/PDIS-06-22-1427-PDN.
[9]	López C, Ferriol M, Pico M B. 2015. Mechanical transmission of tomato leaf curl New Delhi virus to cucurbit germplasm:selection of tolerance sources in Cucumis melo. Euphytica, 204:679-691.
[10]	Moriones E, Praveen S, Chakraborty S. 2017. Tomato leaf curl New Delhi virus:an emerging virus complex threatening vegetable and fiber crops. Viruses, 9 (10):264.
[11]	Romay G, Pitrat M, Lecoq H, Wipf-Scheibel C, Millot P, Girardot G, Desbiez C. 2019. Resistance against melon chlorotic mosaic virus and tomato leaf curl New Delhi virus in melon. Plant Disease, 103 (11):2913-2919. doi: 10.1094/PDIS-02-19-0298-RE pmid: 31436474
[12]	Sáez C, Esteras C, Martínez C, Ferriol M, Dhillon N P S, López C, Picó B. 2017. Resistance to tomato leaf curl New Delhi virus in melon is controlled by a major QTL located in chromosome 11. Plant Cell Reports, 36 (10):1571-1584.
[13]	Sáez C, Martínez C, Ferriol M, Manzano S, Velasco L, Jamilena M, López C, Picó B. 2016. Resistance to tomato leaf curl New Delhi virus in Cucurbita spp. Annals of Applied Biology, 169:91-105.
[14]	Venkataravanappa V, Ashwathappa K V, Reddy C N L, Shankarappa K S, Reddy M K. 2020. Characterization of tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of watermelon and development of LAMP assay for its detection. 3 Biotech, 10 (6):282.
[15]	Vo T T B, Lal A, Ho P T, Troiano E, Parrella G, Kil E J, Lee S. 2022. Different infectivity of mediterranean and southern asian tomato leaf curl New Delhi virus isolates in cucurbit crops. Plants, 11 (5):704.
[16]	Zaidi S S, Martin D P, Amin I, Farooq M, Mansoor S. 2017. Tomato leaf curl New Delhi virus:a widespread bipartite begomovirus in the territory of monopartite begomoviruses. Molecular Plant Pathology, 18 (7):901-911.
[17]	Zeng R, Gu H, Fan J, Zhu P, Xu L, Gao S G, Gao P, Song Z, Zhang K, Zhang C, Dai F. 2023. Occurrence of tomato leaf curl New Delhi virus in cucurbit plants in China. Plant Disease,doi:10.1094/PDIS-01-23-0059-PDN.

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
DNA-A扩增及侵染性克隆构建 Amplication and infectious clones construction of DNA-A	A-362F	ATTAGAGCTCGTGCAGTTGTCCCCATTG
	A-367R	TGACCATGATTACGAATTCGAGCTCCATAGGGGCTGTCGAAGTTG
	A-1555F	TAAAACGACGGCCAGTGCCAAGCTTGACCGGAATAGAGTGGTTCT
DNA-B扩增及侵染性克隆构建 Amplication and infectious clones construction of DNA-B	B-2020F	GCAGAAGCTTCTGCTCGAACATGG
	B-2060F	TGACCATGATTACGAATTCGAGCTCTGCAGCATCGTTTGTGAG
	B-2025R	TAAAACGACGGCCAGTGCCAAGCTTAGCCAGTTGAGAAATAG
	B-1520F	CGTTCGGACCTATAGCCAG
	B-518R	CTTGAGCTCTTGTTTCCGTTGTACGT
pToLCNDV-HM6构建 pToLCNDV-HM6 construction	1.43BF	AACCACTCTATTCCGGTCAAGCTTAATTCGAGCTCTTGTTTCCGTT
pToLCNDV-HM6构建 pToLCNDV-HM6 construction	1.43BR	ACGACGGCCAGTGCCAAGCTT
DNA-A检测Detection of DNA-A	ToLCNDV-A-61F	CCTATTTAATGCGTGGGGGT
DNA-A检测Detection of DNA-A	ToLCNDV-A-1622R	AGCACTGAAGCAGTGGGCAA
DNA-B检测Detection of DNA-B	ToLCNDV-B-62F	GCCGTTATCCGTCCGATTTG
DNA-B检测Detection of DNA-B	ToLCNDV-B-1598R	CGTCTTGTAGATGGTTCAAGG

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Primer sequence
DNA-A扩增及侵染性克隆构建 Amplication and infectious clones construction of DNA-A	A-362F	ATTAGAGCTCGTGCAGTTGTCCCCATTG
	A-367R	TGACCATGATTACGAATTCGAGCTCCATAGGGGCTGTCGAAGTTG
	A-1555F	TAAAACGACGGCCAGTGCCAAGCTTGACCGGAATAGAGTGGTTCT
DNA-B扩增及侵染性克隆构建 Amplication and infectious clones construction of DNA-B	B-2020F	GCAGAAGCTTCTGCTCGAACATGG
	B-2060F	TGACCATGATTACGAATTCGAGCTCTGCAGCATCGTTTGTGAG
	B-2025R	TAAAACGACGGCCAGTGCCAAGCTTAGCCAGTTGAGAAATAG
	B-1520F	CGTTCGGACCTATAGCCAG
	B-518R	CTTGAGCTCTTGTTTCCGTTGTACGT
pToLCNDV-HM6构建 pToLCNDV-HM6 construction	1.43BF	AACCACTCTATTCCGGTCAAGCTTAATTCGAGCTCTTGTTTCCGTT
pToLCNDV-HM6构建 pToLCNDV-HM6 construction	1.43BR	ACGACGGCCAGTGCCAAGCTT
DNA-A检测Detection of DNA-A	ToLCNDV-A-61F	CCTATTTAATGCGTGGGGGT
DNA-A检测Detection of DNA-A	ToLCNDV-A-1622R	AGCACTGAAGCAGTGGGCAA
DNA-B检测Detection of DNA-B	ToLCNDV-B-62F	GCCGTTATCCGTCCGATTTG
DNA-B检测Detection of DNA-B	ToLCNDV-B-1598R	CGTCTTGTAGATGGTTCAAGG

编号 No.	甜瓜品种 Melon cultivar	病情指数 Disease index	抗性评价 Evaluation	编号 No.	甜瓜品种 Melon cultivar	病情指数 Disease index	抗性评价 Evaluation
1	绿秀 Lüxiu	100.0	高感 High sensitivity	14	众天翠宝 Zhongtian Cuibao	92.5	高感 High sensitivity
2	新密25号 Xinmi 25	100.0	高感 High sensitivity	15	博洋9号 Boyang 9	100.0	高感 High sensitivity
3	白玫 Baimei	100.0	高感 High sensitivity	16	甘甜3号 Gantian 3	77.5	高感 High sensitivity
4	黄梦脆 Huangmengcui	100.0	高感 High sensitivity	17	福甜一号 Futian 1	91.7	高感 High sensitivity
5	西州密25号 Xizhoumi 25	100.0	高感 High sensitivity	18	福甜二号 Futian 2	75.0	感病 Sensitivity
6	纳斯密 Nasimi	87.5	高感 High sensitivity	19	绿冠 Lüguan	100.0	高感 High sensitivity
7	雪蜜 Xuemi	100.0	高感 High sensitivity	20	早雪 Zaoxue	55.0	感病 Sensitivity
8	网络时代4号 Wangluo Shidai 4	100.0	高感 High sensitivity	21	锦烽 Jinfeng	75.0	感病 Sensitivity
9	玉姑 Yugu	100.0	高感 High sensitivity	22	Charentais	100.0	高感 High sensitivity
10	农友金香玉 Nongyou Jinxiangyu	80.6	高感 High sensitivity	23	PI414723	0	抗病 Resistance
11	新雅典 Xinyadian	100.0	高感 High sensitivity	24	PI124112	0	抗病 Resistance
12	众天翠玉 Zhongtian Cuiyu	100.0	高感 High sensitivity	25	PI179901	0	抗病 Resistance
13	皇妃 Huangfei	100.0	高感 High sensitivity	对照 Control	新密杂11号 Xinmiza 11	100.0	高感 High sensitivity

编号 No.	甜瓜品种 Melon cultivar	病情指数 Disease index	抗性评价 Evaluation	编号 No.	甜瓜品种 Melon cultivar	病情指数 Disease index	抗性评价 Evaluation
1	绿秀 Lüxiu	100.0	高感 High sensitivity	14	众天翠宝 Zhongtian Cuibao	92.5	高感 High sensitivity
2	新密25号 Xinmi 25	100.0	高感 High sensitivity	15	博洋9号 Boyang 9	100.0	高感 High sensitivity
3	白玫 Baimei	100.0	高感 High sensitivity	16	甘甜3号 Gantian 3	77.5	高感 High sensitivity
4	黄梦脆 Huangmengcui	100.0	高感 High sensitivity	17	福甜一号 Futian 1	91.7	高感 High sensitivity
5	西州密25号 Xizhoumi 25	100.0	高感 High sensitivity	18	福甜二号 Futian 2	75.0	感病 Sensitivity
6	纳斯密 Nasimi	87.5	高感 High sensitivity	19	绿冠 Lüguan	100.0	高感 High sensitivity
7	雪蜜 Xuemi	100.0	高感 High sensitivity	20	早雪 Zaoxue	55.0	感病 Sensitivity
8	网络时代4号 Wangluo Shidai 4	100.0	高感 High sensitivity	21	锦烽 Jinfeng	75.0	感病 Sensitivity
9	玉姑 Yugu	100.0	高感 High sensitivity	22	Charentais	100.0	高感 High sensitivity
10	农友金香玉 Nongyou Jinxiangyu	80.6	高感 High sensitivity	23	PI414723	0	抗病 Resistance
11	新雅典 Xinyadian	100.0	高感 High sensitivity	24	PI124112	0	抗病 Resistance
12	众天翠玉 Zhongtian Cuiyu	100.0	高感 High sensitivity	25	PI179901	0	抗病 Resistance
13	皇妃 Huangfei	100.0	高感 High sensitivity	对照 Control	新密杂11号 Xinmiza 11	100.0	高感 High sensitivity