Characterization And Detection Of Sorghum Mosaic Virus Of Sugarcane In Hainan

Hainan is a very important province of producing sugarcane in China,and sugarcane is an important economic crop in Hainan. However,the viral disease was one of the most important constraint in sugarcane industry.Before this study,no preliminary molecular identification was conducted on these viruses which infected sugarcane in Hainan.It was lacked of system identification at the molecular level.In this study is as follows:An isolate of sorghum mosaic virus (SrMV-HN) was isolated from hainan of south China.It was identified sorghum mosaic virus(SrMV) with biological, serological, biochemical,electron microscope and so on.The electron microscope study revealed that the purified virus was bending line virus, whose size about800±50nm×13±2nm.The study of host responses to virus infection suggested that the SrMV-HN could infect corn and sugarcane.But no obvious symptoms of sugarcane. The virus RNA was extracted from purified viral particles and used as a template.The RT-PCR was carried out with two mutagenesis primers flanking the coat protein gene. A DNA fragment about987bp was obtained after30PCR amplification cycles.DNA sequence analysis showed the fragment that included the whole SrMV coat protein gene, comparing with three reported SrMV coat protein gene sequences. The homology of nucleotide sequences between them were95%,94%and93%.It showed that positive reaction of the SrMV-HN was detected via the SrMV antiserum by ELISA. Western Blot showed that the protein bands with SrMV antibodies have specific reaction. The molecular weight of the coat protein (CP) subunit was about36kDa as evaluated by SDS-PAGE. The purified SrMV antigen was used to prepare SrMV highly specific antiserum by injecting rabbits,and the titer of antiserum reached1:25000, which can use this antiserum to SrMV ELISA detectionThe viral disease survey was carried out for sugarcane cultivated area in the research, in which69samples were collected and their disease symptom were observed and described. Perfect detection system for SrMV was established through experiment. The detection system is as follows. First, triple antibody sandwich-ELISA was established and optimized, then the sensitivity and stability of SrMV were tested by diluted antigens. Second, five primers were designed according to sequences registered in GenBank, from which one pair of routine primers and one pair of degenerate primers were screened through the sensitivity and specificity experiment. Third, two multiplex PCR assays were developed and subsequently evaluated for their effectiveness as means to simultaneously detect multiple viral infections of sugarcane. Two multiplex PCR assays, SrMV ScYLV multiplex RT-PCR were established, respectively. The relative efficiency and sensitivity of the two multiplex PCRs developed in this study seemed to meet their potential application for routine molecular diagnostic purposes.In the study reported here, two multiplex PCR methods can be used to effectively detect and differentiate SrMV. Single or co-infection in samples from diseased of sugarcane, thus perfect detection system for SrMV and ScYLV was laid for the research and application of sugarcane virus detection in Hainan. Virus-free seedling cultivation,virus epidemiology and basic virology in the future.