Molecular Detection And Elimination Technique Of Lily Viruses

Lilium spp.is an important economic crop with ornamental, edible and officinal value.Virus diseases affect the quality of the cut flowers and bulbs of lily which limited the development of lily production. How to establish a system of rapid and sensitive detection and useful virus-free technique is the key question in reducing the harm to lily production and recover the quality of species.In this study, three viruses which do greatest harm to lilies are chosen. They are: Cucumber mosaic virus(CMV), Lily symptomless virus(LSV) and Lily mosettle virus(LMoV). A multiplex RT-PCR detection system has been established according to the analysis of coat protein sequences of these viruses. The whole sequences of the virus CP gene in Beijing samples have been cloned and determined. The detection technique was used to detect occurrence of the three viruses in lily of cultivars, tissue culture and wild group. Also we did exploratory research on the virus-free plants to get rid of the virus infected Lily longiflorum'Snow Queen'. All results of the study were summarized as follows:According to the CP gene of CMV, LMoV and LSV in the GenBank, 3 sets of specific primers were designed. Samples infected by the three viruses could be amplified by the optimized multiplex RT-PCR, and yielding three special bands of CMV (657bp), LMoV (428bp) and LSV (171bp). The CP genes were cloned after multiplex RT-PCR and the homology between the production in this study and the other isolates in Gen Bank was over 90%. It indicated that the CP sequences were highly conserved without district difference.CP gene complete sequences of three viruses were obtained from Lily longiflorum'Snow Queen'.The fragment sizes were CMV(657bp),LMoV(825bp),LSV(844bp)and the three above were submitted to NCBI separately with corresponding sequence number: EF158108,EF158109,EF158110.Cultivars, tissue culture plants and wild species Lily regale were detected by the established multiplex RT-PCR system. The result indicated that all the 9 cultivars were detected by LMoV and LSV. The two viruses infected all the other lilies in the same field. Three samples in eight tissue culture plants were detected positive LSV suggested that the system could be used in virus detection of tissue culture plants. The LSV was detected even in the wild L. regale in Sichuan province which was reported for the first time in nation. Its sequence homology of CP gene is up to 98.5-99% compared with other isolates reported.Virus-free plants were obtained by the combination of stem tip and virazole . The best combination of the Ravizole concentration is 4mg/L, the size extent between 0.5-0.7mm. Under this condition stem tips could grow quickly and in high rate of survival and virus-free. We are trying to obtain virus-free plant by shoot tips tissue culture combined with ribavirin, however, the result was not ideal. So we need to do more research.