| Cymbidium mosaic virus(CymMV) and Odontoglossum ringspot virus(ORSV) are the most prevalent and economically important two viruses which infect orchids and cause a lot of economic lost worldwide. In this study,the two viruses were identified with the methods of the biology, the serology and transmission electron microscopy(TEM). Furthermore,we prepared the monoclonal antibodies against CymMV.The presence of CymMV in field orchids tissues was investigated with ACP-ELISA that we established.1. Identification of CymMV and ORSVNine designation plants of Chenopidiaceae, Solanaceae and Cucurbitaceae were inoculated with orchids saps infected with linear virus or bacilliform virus which were selected by staining with 2% solution of phosphotungstic acid and indirect-ELISA with anti-CymMV antiserum or with anti-ORSV antiserum. The results indicated that there were no system symptoms on all the infected plants.Necrotic and chlorotic local lesions occurred on D. stramonium and C. quinoa inoculated with CymMV positive samples,but no symptoms were on C. amaranticolor.The leaves of C. amaranticolor and C. quinoa inoculated with ORSV positive samples had the same symptoms with D. stramonium. Two viruses didn't infect N. tabacum, N. tabacum.cv.Sam sum-NN, N. tabacum.cv.Xanthi-NC, N.glutinosa,N. benthamiana and Cucumis sativus.There were only one strip on 27kD and 17 kD of purified CymMV and ORSV by SDS-PAGE,respectively.The two viruses didn't reacted with MAbs against TuMV,PVX,PVY and TMV.Under the TEM,the strip inclusions were observed in the thin sections of leaves of P. amabilis and D. stramonium L.whichwas infected with CymMV andlayer aggregates and angled-layer aggregates were observed in the mesophyll in P. amabilis leaves infected with ORSV.Some nucleoluses were decomposed in infected samples. Also, immuno-colliodal gold label were used to detect the two viruses.The result showed that colliodal colloidal-gold granules were collected on the strip inclusions and the Layer aggregates in the leaves infected of CymMV and ORSV respectively.But there were almost no colliodal gold granules in the healthy samples.The above results showed that the two viruses separated from orchids were CymMV and ORSV.2. Production of monoclonal antibodies against Cymbidium mosaic virus and its applicationThree hybridoma cell lines,2C6,5B7 and 12G9, secreting MAbs against CymMV were produced by fusing mouse myeloma cells (Sp2/0) with spleen cells from BALB/c immunized by the CymMV particles.The three MAbs could react specifically with CymMV and didn't cross-react with other tested plant viruses such as BBWV-1, BBWV-2, CarMV,TMV,LSV, CMV,ToMV, ToYMV, TuMV, PVY.The results of Western-blot showed that three MAbs could react specifically with the 27 kD CymMV coat protein.The litres of ascitic fluids of 5B7 and 12G9 MAbs were up to 10-6 indirect-ELISA. Isotypes and subclasses of the the three MAbs were found to be allied to IgG1. Isotypes of light strains of the three MAbs all belonged to κ. A antigen-coated plate (ACP)-ELISA for CymMV detection was established with the MAbs against CymMV , and three MAbs could successfully detect purified CymMV at the amount as low as 0.487ng or virus in plant sap diluted to a ratio of l:10240.The presence of CymMV in 87 orchids samples from Haining Zhejiang,Chengdu Sichuan, Zhejiang university farm and Beijing was investigated with the ACP-ELISA and 54 samples were strong positive, the positive proportion of the detected orchids samples was 62.1%. The results obtained indicate that CymMV is widespread in field orchids.
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