木尔坦棉花曲叶病毒及其伴随的β卫星分子复合侵染引起广东棉花曲叶病

doi:10.3864/j.issn.0578-1752.2015.16.007

摘要/Abstract

摘要： 【目的】明确引起广东棉花曲叶病的病原，为该病害的防控提供理论依据。【方法】应用PCR、滚环扩增（rolling circle amplification，RCA）、基因克隆及测序等技术获得病毒分离物GD01基因组及卫星分子的序列，并对序列进行分析；通过构建GD01及其β卫星分子的侵染性克隆pGreenⅡ049-1.6A、pGreenⅡ049 -2.0β和利用农杆菌介导的注射接种方法，测定GD01及其β卫星分子对棉花的致病性；进一步通过Southern blot检测对接种植株进行分析与验证。【结果】侵染广东棉花的病毒分离物GD01基因组仅含A组分（DNA-A），其全长为2 737 nt，且与在中国发现的木尔坦棉花曲叶病毒（Cotton leaf curl Multan virus, CLCuMuV）第一个分离物朱槿分离物G6序列相似性为100%。该病毒分离物也伴随有β卫星分子，其全长为1 345 nt，与G6伴随的β卫星分子（Cotton leaf curl Multan betasatellite isolate G6, CLCuMuB-[G6]）序列相似性为99.9%。构建了GD01 DNA-A及其β卫星分子侵染性克隆pGreenⅡ049-1.6A、pGreenⅡ049-2.0β，农杆菌注射接种本氏烟，接种后15 d，二者混合接种的本氏烟植株新出的叶片表现明显的皱缩、卷曲等症状；随着时间的推移，本氏烟的症状越来越明显。农杆菌注射接种棉花，接种后30 d，二者混合接种的棉花植株新出叶片开始出现叶脉变深绿色，叶片卷曲等症状；接种后90 d，二者混合接种的植株大部分叶片表现为叶片向上卷曲、叶脉深绿色、叶脉肿大等典型棉花曲叶症状，且与田间自然病株症状相同，而二者各自单独接种的棉花植株没有产生明显的症状。Southern blot检测结果显示,表现典型曲叶症状的接种棉花植株均含有GD01 DNA-A及其β卫星分子，进一步支持这些症状是由GD01 DNA-A及其β卫星分子的共同侵染引起。【结论】病毒分离物GD01 DNA-A及其伴随的β卫星分子与入侵中国的CLCuMuV第一个分离物朱槿分离物G6相同，CLCuMuV -[GD01]及其伴随的CLCuMuB -[GD01]复合侵染引起广东棉花曲叶病，利用农杆菌介导的侵染性克隆接种法，完成了CLCuMuV及其CLCuMuB复合侵染引起棉花曲叶病的柯赫氏法则(Koch's Postulates)。

关键词: 棉花曲叶病, 木尔坦棉花曲叶病毒, &beta, 卫星分子, 侵染性克隆, 致病性

Abstract: 【Objective】The objective of this study is to identify the pathogen of cotton leaf curl disease in Guangdong province, and to provide a theoretical basis for controlling this disease. 【Method】The sequences of the genome of virus isolate GD01 and its satellite molecule isolated from cotton plants exhibiting leaf curl symptoms were obtained by PCR, rolling circle amplification (RCA), cloning and sequencing techniques. The infectious clones pGreenⅡ049-1.6A of GD01 and pGreenⅡ049-2.0β of its betasatellite were constructed, respectively. Their pathogenicity on cotton was determined by agro-inoculation method, and further detected by Southern blot assay.【Result】The genome of the begomovirus isolate GD01 infecting cotton only contains A component (DNA-A). The full length of DNA-A was 2 737 nucleotides. It has 100% sequence identity with that of Cotton leaf curl Multan virus (CLCuMuV) isolate G6 from Hibiscus rosa-sinensis, which was the first isolate of CLCuMuV in China. The virus was also associated with betasatellite molecule, which was determined to be 1 345 nucleotides. It has 99.9% nucleotide sequence identity with that of Cotton leaf curl Multan betasatellite isolate G6 (CLCuMuB-[G6]). The infectious clones pGreenⅡ049-1.6A and pGreenⅡ049-2.0β were inoculated to Nicotiana benthamiana by agro-inoculation. At 15 dpi, the new leaves of the N. benthamiana inoculated with pGreenⅡ049-1.6A and pGreenⅡ049-2.0β displayed severe crumple and leaf curling symptoms. As time went by, the symptoms of the inoculated N. benthamiana plants become more and more obvious. The infectious clones pGreenⅡ049-1.6A and pGreenⅡ049-2.0β were inoculated to cotton plants by agro-inoculation. At 30 dpi, the new leaves of the inoculated cotton plants displayed vein deep-greening and leaf curling symptoms. At 90 dpi, most of the leaves of the inoculated cotton plants exhibited typical leaf curl disease symptoms including leaves curling upward, vein deep-greening and vein swelling, similar to those observed in the fields. However, neither the plants inoculated alone with pGreenⅡ049-1.6A nor pGreenⅡ049-2.0β showed visible symptoms. The Southern blot assay showed that these cotton plants with typical leaf curling symptoms contained GD01 DNA-A and its betasatellite molecules, further supported that these symptoms caused by co-infection of GD01 DNA-A and its betasatellite molecule.【Conclusion】The isolate GD01 and its betasatellite were same as the first isolate G6 of CLCuMuV from Hibiscus rosa-sinensis invading China. Cotton leaf curl disease in Guangdong was caused by co-infection of CLCuMuV isolate GD01 and its associated CLCuMuB isolate GD01. This is the first time to fulfill Koch’s Postulates on cotton for CLCuMuV and its betasatellite by agro-inoculation technique.

Key words: cotton leaf curl disease, Cotton leaf curl Multan virus, betasatellite, infectious clone, pathogenicity

汤亚飞，何自福，杜振国，佘小漫，蓝国兵. 木尔坦棉花曲叶病毒及其伴随的β卫星分子复合侵染引起广东棉花曲叶病[J]. 中国农业科学, 2015, 48(16): 3166-3175.

TANG Ya-fei, HE Zi-fu, DU Zhen-guo, SHE Xiao-man, LAN Guo-bing. The Complex of Cotton leaf curl Multan virus and Its Associated Betasatellite Molecule Causing Cotton Leaf Curl Disease in Guangdong Province[J]. Scientia Agricultura Sinica, 2015, 48(16): 3166-3175.

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参考文献

[1] Briddon R W, Markham P G. Cotton leaf curl virus disease. Virus Research, 2000, 71(1/2): 151-159.

[2] Rishi N, Chauhan M S. Appearance of leaf curl disease of cotton in Northern India. Journal of Cotton Research and Development, 1994, 8(1): 179-180.

[3] Idris A M, Brown J K. Identification of a new, monopartite begomovirus associated with leaf curl disease of cotton in Gezira, Sudan. Plant Disease, 2000, 84(7): 809.

[4] Abdel-Salam A M. Isolation and characterization of a whitefly- transmitted geminivirus associated with leaf curl and mosaic symptoms in cotton in Egypt. Arabian Journal of Biotechnology, 1999, 2: 193-218.

[5] Briddon R W. Cotton leaf curl disease, a multicomponent begomovirus complex. Molecular Plant Pathology, 2003, 4(6): 427-434.

[6] 毛明杰, 何自福, 虞皓, 李华平. 侵染朱槿的木尔坦棉花曲叶病毒及其卫星DNA全基因组结构特征. 病毒学报, 2008, 24(1): 64-68.

Mao M J, He Z H, Yu H, Li H P. Molecular characterization of Cotton leaf curl Multan virus and its satellite DNA that infects Hibiscus rosa-sinensis. Chinese Journal of Virology, 2008, 24(1): 64-68. (in Chinese)

[7] Cai J H, Xie K, Lin L, Qin B X, Chen B S, Meng J R, Liu Y L. Cotton leaf curl Multan virus newly reported to be associated with cotton leaf curl disease in China. Plant Pathology, 2010, 59(4): 794-795.

[8] Farquharson C O. Report of mycologist//Annual Report of the Agricultural Department of Nigeria. 1912.

[9] Kirkpatrick T W. Further studies on leaf curl of cotton in the Sudan. Bulletin of Entomological Research, 1931, 22: 323-363.

[10] Hussain T, Mahmood T. A note on leaf curl disease of cotton. The Pakistan Cottons, 1988, 32(4): 248-251.

[11] Zhou X P, Liu Y L, Robinson D J, Harrison B D. Four DNA-A variants among Pakistani isolates of cotton leaf curl virus and their affinities to DNA-A of geminivirus isolates from okra. Journal of General Virology, 1998, 79(4): 915-923.

[12] Mansoor S, Briddon R W, Bull S E, Bedford I D, Bashir A, Hussain M, Saeed M, Zafar Y, Malik K A, Fauquet C, Markham P G. Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β. Archives of Virology, 2003, 148(10): 1969-1986.

[13] Idris A M, Briddon R W, Simon E, Brown J K. Cotton leaf curl Gezira virus-satellite DNAs represent a divergent, geographically isolated Nile Basin lineage: predictive identification of a satDNA REP-binding motif. Virus Research, 2005, 109(1): 19-32.

[14] Kirthi N, Priyadarshini C G P, Sharma P, Maiya S P, Hemalatha V, Sivaraman P, Dhawan P, Rishi N, Savithri H S. Genetic variability of begomoviruses associated with cotton leaf curl disease originating from India. Archives of Virology, 2004, 149(10): 2047-2057.

[15] Chowda Reddy R V, Muniyappa V, Colvin J, Seal S. A new begomovirus isolated from Gossypium barbadense in Southern India. Plant Pathology,2005, 54: 570.

[16] Amrao L, Amin I, Shahid M S, Briddon R W, Mansoor S. Cotton leaf curl disease in resistant cotton is associated with a single begomovirus that lacks an intact transcriptional activator protein. Virus Research, 2010, 152(1/2): 153-163.

[17] Amrao L, Akhter S, Tahir M N, Amin I, Briddon R W, Mansoor S. Cotton leaf curl disease in Sindh province of Pakistan is associated with recombinant begomovirus components. Virus Research, 2010, 153(1): 161-165.

[18] Fauquet C M, Briddon R W, Brown J K, Moriones E, Stanley J, Zerbini M, Zhou X P. Geminivirus strain demarcation and nomenclature. Archives of Virology, 2008, 153(4): 783-821.

[19] 林林, 蔡健和, 罗恩波, 秦碧霞, 胡冬梅, 蒙姣荣, 陈保善, 农恒志. 南宁市朱槿曲叶病毒病病原分子鉴定和寄主范围研究. 植物保护, 2011, 37(4): 44-47.

Lin L, Cai J H, Luo E B, Qin B X, Hu D M, Meng J R, Chen B S, Nong H Z. Molecular identification and host range of a geminivirus infecting Hibiscusin Nanning City. Plant Protection, 2011, 37(4): 44-47. (in Chinese)

[20] 章松柏, 夏宣喜, 张洁, 张友军, 吴祖建. 福州市发生由木尔坦棉花曲叶病毒引起的朱槿曲叶病. 植物保护, 2013, 39(2): 196-200.

Zhang S B, Xia X X, Zhang J, Zhang Y J, Wu Z J. A leaf curl disease on Hibiscus rosa-sinensis in Fuzhou caused by Cotton leaf curl Multan virus. Plant Protection, 2013, 39(2): 196-200. (in Chinese)

[21] 董迪, 朱艳华, 何自福, 柴兆祥, 佘小漫, 罗方芳. 侵染广东黄秋葵的木尔坦棉花曲叶病毒及伴随卫星DNA的分子特征. 华南农业大学学报, 2012, 33(1): 33-39.

Dong D, Zhu Y H, He Z F, Chai Z X, She X M, Luo F F. Molecular characterization of Cotton leaf curl Multan virus and the associated satellite DNA infecting okra in Guangdong. Journal of South China Agricultural University, 2012, 33(1): 33-39. (in Chinese)

[22] 董迪, 何自福, 柴兆祥. 广东黄秋葵黄脉曲叶病样中检测到烟粉虱传双生病毒. 植物保护, 2010, 36(1): 65-68.

Dong D, He Z F, Chai Z X. Detection of whitefly transmitted geminivirus associated with the okra yellow vein leaf curl disease in Guangdong. Plant Protection, 2010, 36(1): 65-68. (in Chinese)

[23] 汤亚飞, 何自福, 杜振国, 韩利芳, 佘小漫, 罗方芳. 侵染垂花悬铃花的木尔坦棉花曲叶病毒分子特征研究. 植物病理学报, 2013, 43(2): 120-127.

Tang Y F, He Z F, Du Z G, Han L F, She X M, Luo F F. Molecular characterization of the Cotton leaf curl Multan virus infecting Malvaiscus arboreus. Acta Phytopathologica Sinica, 2013, 43(2): 120-127. (in Chinese)

[24] 周雪平, 戚益军, 李德葆. 从棉花组织中提取棉花曲叶病毒基因组DNA之方法. 农业生物技术学报, 1999, 7(2): 169-172.

Zhou X P, Qi Y J, Li D B. Extraction methods of cotton leaf curl virus from infected cotton leaves. Journal of Agricultural Biotechnology, 1999, 7(2): 169-172. (in Chinese)

[25] Wyatt S D, Brown J K. Detection of subgroup Ⅲ geminivirus isolates in leaf extracts by degenerate primers and polymerase chain reaction. Phytopathology, 1996, 86(12): 1288-1293.

[26] 何自福, 虞皓, 罗方芳. 番茄烟粉虱传双生病毒PCR检测. 中国病毒学, 2004, 19(1): 67-69.

He Z F, Yu H, Luo F F. Detection of whitefly-transmitted Geminiviruses from tomato by PCR. Virologica Sinica, 2004, 19(1): 67-69. (in Chinese)

[27] Haible D, Kober S, Jeske H. Rolling circle amplification revolutionizes diagnosis and genomics of geminiviruses. Journal of Virological Methods, 2006, 135(1): 9-16.

[28] 胡高洁. 木尔坦棉花曲叶病毒的分子鉴定与致病性研究[D]. 杭州: 浙江大学, 2010.

Hu G J. Molecular identification and pathogenicity research of Cotton leaf curl Multan virus[D]. Hangzhou: Zhejiang University, 2010. (in Chinese)

[29] 余云舟, 杜娟, 王罡, 季静. 重组质粒导入根癌农杆菌冻融法的研究. 吉林农业大学学报, 2003, 25(3): 257-259, 262.

Yu Y Z, Du J, Wang G, Ji J. Studies on the Freeze-Thaw method of transforming recombinant plasmid DNA into Agrobacterium tumefaciens. Journal of Jilin Agricultural University, 2003, 25(3): 257-259, 262. (in Chinese)

[30] Hanley-Bowdoin L, Settlage S B, Orozco B M, Nagar S, Robertson D. Geminiviruses: models for plant DNA replication, transcription, and cell cycle regulation. Critical Reviews in Plant Sciences, 1999, 18(1): 71-106.

[31] Briddon R W, Bull S E, Amin I, Idris A M, Mansoor S, Bedford I D, Dhawan P, Rishi N, Siwatch S S, Abdel-Salam A M, Brown J K, Zafar Y, Markham P. Diversity of DNA β, a satellite molecule associated with some monopartite begomoviruses. Virology, 2003, 312(1): 106-121.

[32] Tahir M N, Amin I, Briddon R W, Mansoor S. The merging of two dynasties identification of an African cotton leaf curl disease- associated begomovirus with cotton in Pakistan. PLoS ONE, 2011, 6(5): e20366.

[33] Briddon R W, Mansoor S, Bedford I D, Pinner M S, Saunders K, Stanley J, Zafar Y, Malik K A, Markham P G. Identification of DNA components required for induction of cotton leaf curl disease. Virology, 2001, 285(2): 234-243.

[34] 何自福, 董迪, 李世访, 佘小漫, 罗方芳. 木尔坦棉花曲叶病毒已对中国棉花生产构成严重威胁. 植物保护, 2010, 36(2): 147-149.

He Z F, Dong D, Li S F, She X M, Luo F F. Threat of Cotton leaf curl Multan virus to China cotton production. Plant Protection, 2010, 36(2): 147-149. (in Chinese)