| Grapevine virus A (GVA) is world-widely distributed in grape-growing areas. It forms grooves on the woody cylinder after infected grape, reduces the yield and quality of fruits, delays maturity of fruits, causes the tree decline and shortens the economic life. At present, there are no ideal chemical agent to prevent and control the virus disease. The cultivation of virus-free varieties is the effective way to reduce the losses caused by GVA. The virus detection is the crucial step for obtaining virus-free germplasm. In this study, the virus particles were extracted from the leaves of Nicotiana benthamiana inoculated with GVA. The Polyclonal antibody was prepared by immunizing rabbits with the purified virus solutions. The obtained results are summarized as follows.1. The plants of N. benthamiana were used as propagation hosts of GVA and were mechanically inoculated with a GVA isolate provided by an Italy professor Martelli G. P. The leaves of N. benthamiana showed the symptoms of chlorotic spot, crinkle and deformation five to seven days post-inoculation. The virus particles were extracted from the leaves of N. benthamiana by differential speed centrifugation, and then followed by a circle of sucrose density gradient centrifugation. The obtained virus extracts were analyzed by 10% SDS-PAGE and a clear protein bands about 23.0 kDa was detected.2. Virus particles in the extracted solution were observed under a transmission electron microscopy after negative staining with phosphotungstic acid. The results showed that there are a lot of filamentous particles about 800 nm long. The extract was also analyzed by western blot using polyclonal antibody purchased from Italy and specific immuno-reaction was observed between the purified virus protein and the GVA polyclonal antibody. The purified virus solution was used to immunize the rabbits and the titer of the produced polyclonal antibody was 1:6400.3. The results of Western-blot analysis showed that the prepared antibody can react with the purified virus particles. Five grapevine samples grown in greenhouse, which were previously proved to be infected by GVA, were tested by indirect ELISA with the prepared antibody. All these samples showed positive reactions and it indicate that the prepared antibody can be efficiently used to detect GVA grape samples. |
