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

Abstract

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

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

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