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  

The Complex of Cotton leaf curl Multan virus and Its Associated Betasatellite Molecule Causing Cotton Leaf Curl Disease in Guangdong Province

作　　者：汤亚飞[1] 何自福[1] 杜振国[1] 佘小漫[1] 蓝国兵[1] 

机构地区：[1]广东省农业科学院植物保护研究所/广东省植物保护新技术重点实验室,广州510640

出　　处：《中国农业科学》2015年第16期3166-3175,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31171817);广东省自然科学基金(2014A030313571);广东省农业科学院院长基金(201424)

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p GreenⅡ049-1.6A、p GreenⅡ049-2.0β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 737nt,且与在中国发现的木尔坦棉花曲叶病毒(Cotton leaf curl Multan virus,CLCu Mu V)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,CLCu Mu B-[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p GreenⅡ049-1.6A、p GreenⅡ049-2.0β,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CLCu Mu V第一个分离物朱槿分离物G6相同,CLCu Mu V-[GD01]及其伴随的CLCu Mu 【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 p GreenⅡ049-1.6A of GD01 and p GreenⅡ049-2.0β 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（CLCu Mu V） isolate G6 from Hibiscus rosa-sinensis, which was the first isolate of CLCu Mu V 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（CLCu Mu B-[G6]）. The infectious clones p GreenⅡ049-1.6A and p GreenⅡ049-2.0β were inoculated to Nicotiana benthamiana by agro-inoculation. At 15 dpi, the new leaves of the N. benthamiana inoculated with p GreenⅡ049-1.6A and p GreenⅡ049-2.0β 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 p GreenⅡ049-1.6A and p GreenⅡ049-2.0β 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

关 键 词：棉花曲叶病 木尔坦棉花曲叶病毒 β卫星分子 侵染性克隆 致病性 

分 类 号：S435.62[农业科学—农业昆虫与害虫防治]