13种类病毒RNA/DNA基因组对番茄的侵染性鉴定

doi:10.16420/j.issn.0513-353x.2024-0050

摘要/Abstract

摘要：

选取13种在园艺植物上报道较多的类病毒，将其人工合成的cDNA通过Sac I和Spe I酶切后，经T7酶体外转录为常规RNA基因组，同时利用PCR合成各类病毒DNA全长基因组，将RNA和DNA基因组机械摩擦接种于Ailsa Craig番茄苗上，并通过RT-PCR和RT-qPCR检测不同类病毒对番茄的侵染效率。分别以RNA和DNA形式进行类病毒基因组人工接种，结果表明，除了常规的RNA基因组外，类病毒DNA基因组亦具有侵染性，少量类病毒RNA和DNA基因组侵染性存在差异。总体来看，13种类病毒均可侵染番茄，并引起植株矮化、落叶、叶片卷曲、革质和黄化等病症。其中侵染性较强的为CChMVd、ELVd-1、PSTVd、TCDVd、TPMVd和HSVd，其在感染植株中滴度较高，引起的病情指数较高。PLMVd在植株内平均滴度较低，但致病性较高。侵染后RT-PCR检测产物与人工合成cDNA序列测序基本一致。

关键词: 番茄, 类病毒, RNA基因组, cDNA基因组, 侵染性评价

Abstract:

Thirteen types of viroids that have been widely reported in horticultural plants were selected. Their artificially synthesized cDNA was digested by Sac I and Spe I enzymes and transcribed into conventional RNA genomes in vitro by T7 enzyme. At the same time，PCR was used to synthesize full-length genomes of various viroids DNA genome. The RNA and DNA genomes were mechanically rubbed and inoculated onto Ailsa Craig tomato seedlings，and the infection efficiency of different types of viroids on tomatoes was detected by RT-PCR and RT-qPCR. Artificial inoculation of viroid genomes was conducted in both RNA and DNA forms，and the results showed that in addition to the conventional RNA genomes，the viroid DNA genomes also have infectivity，with differences in infectivity between a small amount of viral RNA and DNA genomes. Overall，all 13 types of viroids can infect tomatoes and cause diseases such as plant dwarfism，leaf shedding，leaf curling，leatheriness，and yellowing. Among them，CChMVd，ELVd-1，PSTVd，TCDVd，TPMVd，and HSVd have stronger infectivity，with higher titers in infected plants and higher disease indices. PLMVd has a lower average titer within the plant，but higher pathogenicity. The RT-PCR detection products after infection are basically consistent with the sequencing of artificially synthesized cDNA sequences.

Key words: tomato, viroid, RNA genome, cDNA genome, infectivity evaluation

陆秀萍, 汤智超, 唐文琨, 毛妃凤, 张万萍, 李经纬. 13种类病毒RNA/DNA基因组对番茄的侵染性鉴定[J]. 园艺学报, 2025, 52(3): 749-760.

LU Xiuping, TANG Zhichao, TANG Wenkun, Mao Feifeng, ZHANG Wanping, LI Jingwei. Identification of Infectivity of 13 Viroid RNA and DNA Genomes of Tomato[J]. Acta Horticulturae Sinica, 2025, 52(3): 749-760.

图/表 10

参考文献 23

[1]	Abrahamian P, Avina-Padilla K, Kovalskaya N, Hammond R W. 2022. Tomato planta macho viroid targets the tomato genome through viroid small interfering RNA. Phytopathology, 112 (11):156.
[2]	Batuman O, Osei M K, Mochiah M B, Lamptey J N, Miller S, Gilbertson R L. 2013. The first report of tomato apical stunt viroid(TASVd)and potato spindle tuber viroid(PSTVd)in tomatoes in Ghana. Phytopathology, 103 (6):12.
[3]	Carbonell A, Martínez de Alba, Flores R, Gago S. 2008. Double-stranded rna interferes in a sequence-specific manner with the infection of representative members of the two viroid families. Virology, 371 (1):44-53. doi: 10.1016/j.virol.2007.09.031 pmid: 18028975
[4]	Chen Haiyan. 2018. Cryopreservation of potato leaf roll virus,potato virus S and potato spindle tuber viroid in shoot tips[M. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	陈海燕. 2018. 马铃薯卷叶病毒,S病毒与马铃薯纺锤形块茎类病毒的茎尖超低温保存研究[硕士论文]. 杨凌: 西北农林科技大学.
[5]	Constable F, Chambers G, Penrose L, Daly A, Mackie J, Davis K, Rodoni B, Gibbs M. 2019. Viroid-infected tomato and capsicum seed shipments to Australia. Viruses-Basel, 11 (2):98.
[6]	Dong Yanna, Zheng Yinying, Xu Wenxing. 2016. Identification of five citrus viruses using the herbaceous plant tomato. Scientia Agricultura Sinica, 49 (4):784-790. (in Chinese) doi: 10.3864/j.issn.0578-1752.2016.04.017
	董艳娜, 郑银英, 徐文兴. 2016. 用草本植物番茄鉴定五种柑橘类病毒. 中国农业科学, 49 (4):784-790. doi: 10.3864/j.issn.0578-1752.2016.04.017
[7]	Faggioli F, Luigi M, Sveikauskas V, Olivier T, Marn M V, Plesko I M, de Jonghe K, van Bogaert N. 2015. An assessment of the transmission rate of four pospiviroid species through tomato seeds. European Journal of Plant Payhology, 143 (3):613-617.
[8]	Flores R, Gago-Zachert S, Serra P, Peña M D L, Navarro B. 2017. Chrysanthemum chlorotic mottle viroid//Hadidi A,Flores R,Randles J W,Palukaitis P. Viroids & salellites. London:Elsevier:331-338.
[9]	Flores R, Hernández C, Martinez de Alba A E, Serio F D. 2005. Viroids and viroid-host interactions. Ann Rev Phytopathol, 43:117-139.
[10]	Huo Jianyong. 2016. Status quo and safety precautions of Chinese tomato industry. Vegetables,(6):1-4. (in Chinese)
	霍建勇. 2016. 中国番茄产业现状及安全防范. 蔬菜,(6):1-4.
[11]	Li Junming, Xiang Chaoyang, Wang Xiaoxuan, Guo Yanmei, Huang Zejun, Liu Lei, Li Xin, Du Yongchen. 2021. Current situation and prospect of China’s tomato industry in the 13th Five-Year Plan period. Chinese Vegetables,(2):13-20. (in Chinese)
	李君明, 项朝阳, 王孝宣, 国艳梅, 黄泽军, 刘磊, 李鑫, 杜永臣. 2021. “十三五”我国番茄产业现状及展望. 中国蔬菜,(2):13-20.
[12]	Liu Qian. 2019. Infection,replication,and transmission of viriods in chestnut disease pathogens[M. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	刘茜. 2019. 类病毒在栗疫病菌中的侵染,复制和传播[硕士论文]. 杨凌: 西北农林科技大学.
[13]	Lü Dianqiu, Qiu Cailing, Wang Shaopeng, Li Yong, Gao Yunfei. 2009. Development of cDNA dineric probes and its application in diagnosis of potato spindle tuber viroid. Acta Horticulturae Sinica, 36 (10):1538-1544. (in Chinese)
	吕典秋, 邱彩玲, 王绍鹏, 李勇, 高云飞. 2009. 马铃薯类病毒cDNA双体探针的研制及其在检测上的应用. 园艺学报, 36 (10):1538-1544.
[14]	Matsuura S, Matsushita Y, Kozuka R, Shimizu S, Tsuda S. 2010. Transmission of tomato chlorotic dwarf viroid by bumblebees(Bombus ignitus)in tomato plants. European Journal of Plant Payhology, 126 (1):111-115.
[15]	Matsushita Y, Tsuda S. 2015. Host ranges of potato spindle tuber viroid,tomato chlorotic dwarf viroid,tomato apical stunt viroid,and columnea latent viroid in horticultural plants. Eur J Plant Pathol, 141:193-197.
[16]	Nie Ruihan, Sun Manli, Feng Di, Song Xietian, Gao Junyan, Guo Lina, Deng Xiuxin, Chai Lijun, Xie Zongzhou, Ye Junli. 2024. An investigation on occurrence and distribution patterns of five common citrus diseases in Yunnan. Acta Horticulturae Sinica, 51 (11):2685-2700. (in Chinese) doi: 10.16420/j.issn.0513-353x.2023-0766 URL
	聂睿涵, 孙曼丽, 冯迪, 宋谢天, 高俊燕, 郭莉娜, 邓秀新, 柴利军, 谢宗周, 叶俊丽. 2024. 云南柑橘5种常见病害发生分布调查及其规律分析. 园艺学报, 51 (11):2685-2700. doi: 10.16420/j.issn.0513-353x.2023-0766 URL
[17]	Pea-Iglesias A, Vecino B. 1987. Cytological studies of grapevine leafroll infected tissue:further evidence of viroid etiology and improvement of diagnosis. Vitis, 26:37-41.
[18]	Sofy A, Mahfouze S, Enany M E. 2013. Isozyme markers for response of wild potato species to potato spindle tuber viroid egyptian isolate. World Applied Sciences Journal, 27 (8):1010-1022.
[19]	Verhoeven J T J, Botermans M, Meekes E T M, Roenhorst J W. 2012. Tomato apical stunt viroid in the Netherlands:most prevalent pospiviroid in ornamentals and first outbreak in tomatoes. European Journal of Plant Pathology, 133 (4):803-810.
[20]	Wang Jin, Ou Yi, Wu Zheng, Dai Zhenglin, Liu Shengwei. 2011. Effect of high temperature stress on physiological effects and early defoliation in precocious pear. Southwest Agricultural Journal, 24 (2):6. (in Chinese)
	王进, 欧毅, 武峥, 代正林, 刘圣维. 2011. 高温胁迫对早熟梨生理效应和早期落叶的影响. 西南农业学报, 24 (2):6.
[21]	Zhang Z B, Lee Y K, Sivertsen A, Skjeseth G, Haugslien S, Clarke J H L, Wang Q C, Blystad D R. 2016. Low temperature treatment affects concentration and distribution of chrysanthemum stunt viroid in Argyranthemum. Front Microbiol, 7:224.
[22]	Zhang Zhibo. 2015. Effects and mechanisms of cryopreservation and removal of chrysanthemum stunt viroid(CSVd)in shoot tips of Argyranthemum[Ph. D. Dissertation]. Yangling: Northwest A & F University. (in Chinese)
	张知博. 2015. 木茼蒿(Argyranthemum)茎尖超低温保存与脱除菊花矮化类病毒(CSVd)的效应与机理研究[博士论文]. 杨凌: 西北农林科技大学.
[23]	Zhang Zhixiang, Ge Beibei, Pan Song, Zhao Zhe, Wang Hongqing, Li Shifang. 2011. Molecular detection and sequences analysis of chrysanthemum stunt viroid. Acta Horticulturae Sinica, 38 (12):2349-2356. (in Chinese)
	张志想, 葛蓓孛, 潘嵩, 赵哲, 王红清, 李世访. 2011. 菊花矮化类病毒的分子检测与序列分析. 园艺学报, 38 (12):2349-2356.

序号 Number	科 Family	类病毒名称 Viroid name	大小/bp Size	基因编号 GenBank number	缩写 Abbreviation
1	鳄梨日斑类病毒科 Avsunviroidae	类苹果锤头类病毒 Apple hammerhead viroid-like	434	KR605506.1	AHVd-like
2	鳄梨日斑类病毒科 Avsunviroidae	鳄梨日斑类病毒 Avocado sunblotch viroid	247	J02020.1	ASBVd
3	鳄梨日斑类病毒科Avsunviroidae	菊花黄化花叶类病毒 Chrysanthemum chlorotic mottle viroid	399	Y14700.1	CChMVd
4	鳄梨日斑类病毒科 Avsunviroidae	茄潜隐类病毒-1 Eggplant latent viroid-1	333	AJ536613.1	ELVd-1
5	鳄梨日斑类病毒科 Avsunviroidae	茄潜隐类病毒-2 Eggplant latent viroid-2	335	AJ536612.1	ELVd-2
6	鳄梨日斑类病毒科 Avsunviroidae	桃潜隐花叶类病毒 Peach latent mosaic viroid	337	M83545.1	PLMVd
7	马铃薯块茎纺锤体类病毒科 Pospiviroidae	柑橘类病毒VII Citrus viroid VII	368	KX013549.1	CVd VII
8	马铃薯块茎纺锤体类病毒科 Pospiviroidae	马铃薯块茎纺锤体类病毒 Potato spindle tuber viroid	359	V01465.1	PSTVd
9	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄顶矮缩类病毒 Tomato apical stunt viroid	360	K00818.1	TASVd
10	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄褪绿矮缩类病毒 Tomato chlorotic dwarf viroid	360	AF162131.1	TCDVd
11	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄雄植株类病毒 Tomato planta macho viroid	360	K00817.1	TPMVd
12	马铃薯块茎纺锤体类病毒科 Pospiviroidae	啤酒花矮化类病毒 Hop stunt viroid	302	X06719.1	HSVd
13	鳄梨日斑类病毒科 Avsunviroidae	苹果锈皮类病毒 Apple scar skin viroid	329	X17696.1	ASSVd

序号 Number	科 Family	类病毒名称 Viroid name	大小/bp Size	基因编号 GenBank number	缩写 Abbreviation
1	鳄梨日斑类病毒科 Avsunviroidae	类苹果锤头类病毒 Apple hammerhead viroid-like	434	KR605506.1	AHVd-like
2	鳄梨日斑类病毒科 Avsunviroidae	鳄梨日斑类病毒 Avocado sunblotch viroid	247	J02020.1	ASBVd
3	鳄梨日斑类病毒科Avsunviroidae	菊花黄化花叶类病毒 Chrysanthemum chlorotic mottle viroid	399	Y14700.1	CChMVd
4	鳄梨日斑类病毒科 Avsunviroidae	茄潜隐类病毒-1 Eggplant latent viroid-1	333	AJ536613.1	ELVd-1
5	鳄梨日斑类病毒科 Avsunviroidae	茄潜隐类病毒-2 Eggplant latent viroid-2	335	AJ536612.1	ELVd-2
6	鳄梨日斑类病毒科 Avsunviroidae	桃潜隐花叶类病毒 Peach latent mosaic viroid	337	M83545.1	PLMVd
7	马铃薯块茎纺锤体类病毒科 Pospiviroidae	柑橘类病毒VII Citrus viroid VII	368	KX013549.1	CVd VII
8	马铃薯块茎纺锤体类病毒科 Pospiviroidae	马铃薯块茎纺锤体类病毒 Potato spindle tuber viroid	359	V01465.1	PSTVd
9	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄顶矮缩类病毒 Tomato apical stunt viroid	360	K00818.1	TASVd
10	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄褪绿矮缩类病毒 Tomato chlorotic dwarf viroid	360	AF162131.1	TCDVd
11	马铃薯块茎纺锤体类病毒科 Pospiviroidae	番茄雄植株类病毒 Tomato planta macho viroid	360	K00817.1	TPMVd
12	马铃薯块茎纺锤体类病毒科 Pospiviroidae	啤酒花矮化类病毒 Hop stunt viroid	302	X06719.1	HSVd
13	鳄梨日斑类病毒科 Avsunviroidae	苹果锈皮类病毒 Apple scar skin viroid	329	X17696.1	ASSVd

类病毒Viroid	引物序列Primer sequence	类病毒Viroid	引物序列Primer sequence
AHVd-like	5′-GAAGGCTATTAGCCTTCCTGATGAGT-3′ 5′-GACCGTCACGGGGGTGTTAAAAC-3′	PSTVd	5′-CGGAACTAAACTCGTGGTTCCTGTG-3′ 5′-AGGAACCAACTGCGGTTCCAAG-3′
ASBVd	5′-TTTATTAGAACAAGAAGTGAGGATATGAT-3′ 5′-CAAGATTTTGTAAAAAAACAATGAAGAT-3′	TASVd	5′-CGGGATCTTTCGTGAGGTTCCT-3′ 5′-AGGGACCATTCAGGGTTCC-3′
CChMVd	5′-GGCACCTGACGTCGGTGTC-3′ 5′-GACCTCTTGGGGGTTTCAAACC-3′	TCDVd	5′-CGGAACTAAACTCGTGGTTCCTGTG-3′ 5′-AGGAACCAACTGCGGTTCCAAG-3′
ELVd-1	5′-GGGTGGTGTGTGCCACC-3′ 5′-TATGGGGAGAGGTCGTCCTCTATCT-3′	TPMVd	5′-CGGGATCTTTTCCTTGTGGTTCC-3′ 5′-AGGGACCCTTTTCGAAGGGT-3′
ELVd-2	5′-GGGTGGTGTGTACCACCCCT-3′ 5′-TATGGGGAGAGGCCGTCCT-3′	HSVd	5′-CTGGGGAATTCTCGAGTTGC-3′ 5′-AGGGGCTCAAGAGAGGATC-3′
PLMVd	5′-GTCATAAGTTTCGTCGCATTTCAGC-3′ 5′-AAGAAGAGTTTCGTCTCATCTCAGAG-3′	ASSVd	5′-GGTAAACACCGTGCGGTTC-3′ 5′-GGGAAACACCTATTGTGTTTTACCC-3′
CVd VII	5′-TCGAGGACACTTCTGGTTCCTAT-3′ 5′-GGGACGACTCTAGGTCTCCCTT-3′

类病毒Viroid	引物序列Primer sequence	类病毒Viroid	引物序列Primer sequence
AHVd-like	5′-GAAGGCTATTAGCCTTCCTGATGAGT-3′ 5′-GACCGTCACGGGGGTGTTAAAAC-3′	PSTVd	5′-CGGAACTAAACTCGTGGTTCCTGTG-3′ 5′-AGGAACCAACTGCGGTTCCAAG-3′
ASBVd	5′-TTTATTAGAACAAGAAGTGAGGATATGAT-3′ 5′-CAAGATTTTGTAAAAAAACAATGAAGAT-3′	TASVd	5′-CGGGATCTTTCGTGAGGTTCCT-3′ 5′-AGGGACCATTCAGGGTTCC-3′
CChMVd	5′-GGCACCTGACGTCGGTGTC-3′ 5′-GACCTCTTGGGGGTTTCAAACC-3′	TCDVd	5′-CGGAACTAAACTCGTGGTTCCTGTG-3′ 5′-AGGAACCAACTGCGGTTCCAAG-3′
ELVd-1	5′-GGGTGGTGTGTGCCACC-3′ 5′-TATGGGGAGAGGTCGTCCTCTATCT-3′	TPMVd	5′-CGGGATCTTTTCCTTGTGGTTCC-3′ 5′-AGGGACCCTTTTCGAAGGGT-3′
ELVd-2	5′-GGGTGGTGTGTACCACCCCT-3′ 5′-TATGGGGAGAGGCCGTCCT-3′	HSVd	5′-CTGGGGAATTCTCGAGTTGC-3′ 5′-AGGGGCTCAAGAGAGGATC-3′
PLMVd	5′-GTCATAAGTTTCGTCGCATTTCAGC-3′ 5′-AAGAAGAGTTTCGTCTCATCTCAGAG-3′	ASSVd	5′-GGTAAACACCGTGCGGTTC-3′ 5′-GGGAAACACCTATTGTGTTTTACCC-3′
CVd VII	5′-TCGAGGACACTTCTGGTTCCTAT-3′ 5′-GGGACGACTCTAGGTCTCCCTT-3′

类病毒Viroid	引物序列Primer sequence	类病毒Viroid	引物序列Primer sequence
AHVd-like	5′-CTATTAGCCTTCCTGATG-3′ 5′-GTGTAAGTCGTAGTCTATT-3′	PSTVd	5′-CGGTGGAAACAACTGAAG-3′ 5′-GTTCCAAGGGCTAAACAC-3′
ASBVd	5′-TCTGTTCCGACTTTCCGACTC-3′ 5′-TTCTTCCCATCTTTCCCTGA-3′	TASVd	5′-TTTCTTCTGGTTTCCTTC-3′ 5′-AGAGCAACAAAGATAGAG-3′
CChMVd	5′-CAGGAAACCCACTTCAGGTC-3′ 5′-TCCGAGGAGAATATCCAACG-3′	TCDVd	5′-GCTTGTGGAAGGCGAAAC-3′ 5′-AAGGAAGGAAACCGCAGAA-3′
ELVd-1	5′-AGACCGAAAGGTCGAAATGG-3′ 5′-AAGGGACTCTTGGAGGAACG-3′	TPMVd	5′-CTGACCTCCTGACCAGAA-3′ 5′-AGTTCGCTCCAGGTTTCC-3′
ELVd-2	5′-TACCACCCCTGATGAGTC-3′ 5′-GCTCTTGGAGGAACGATT-3′	HSVd	5′-AACAAGGCAGGGAGGAGACT-3′ 5′-GAAGAGCCAGGAGAAGGTAAAA-3′
PLMVd	5′-CCGTAGAAACTGGATTAC-3′ 5′-TTAGCACAGACTCTTCAT-3′	ASSVd	5′-CCGTGCGGTTCCTGTGGTTC-3′ 5′-GGTTAGTGCTGGCAGCTCCTTT-3′
CVd VII	5′-ACTGGAACTGCTTGCCTTGA-3′ 5′-CGGTCCATTGCCTTAGAATA-3′