Detection Of Lily Symptomless Virus In Lilium Species By RT-PCR Technique

Abstract: Lily symptomless virus (LSV), a species of the genus Carlavirus, is the most prevalent virus infecting lily plants throughout the world. The host range of the virus is restricted to the Liliaceae. Leaves of lilies infected with LSV alone remain symptomless or show very mild mosaic. However, LSV frequently occurs with Cucumber mosaic virus or Lily mottle virus in cultivated lily cultivars, and complex infection results in severe leaf mosaic and dwarfing, which threatens the yield and commercial production of lily. So studing on LSV has great theoretical and actual significances. It can lay a theoretical foundation for producing of lily virus-free seedlings, and provides technologies for LSV detection and identification. In this study, forms of viruses particles and inclusions of cells infected with LSV and other virues were studied by electron microscope techniques. An ELISA system for detection of LSV was established. Study on the extracting of plant total RNA, establishing of optimum RT-PCR detection system of LSV . All result of the studies were summarized as follows: 1. Enzyme –linked immumnosorbent assay (ELISA) detection indicated that Cucumber mosaic virus (CMV) and lily symptomless virus (LSV) were important viral pathogens for infecting Gansu lily. The rate of lily mosaic diseases infected by CMV was 40%, by LSV was 80%, mixed by CMV and LSV was 40%. The sensibility of DAS-ELISA detection LSV was the highest, it was four times as high as ones of I-ELISA and DIBA-ELISA. LSV was detected in 17 out of 21 lily samples, so the infection rate of 80.95%, mixed infection rate was 57.14%.The concentration of LSV on lily leaf was higher than ones of flower and bulb. 2. Typical elongated particles (593.29⁶39.97×12.15¹4.12nm) of LSV were observed in PTA preparations and in transverse and cross section in the cytoplasm of cells of necrotic fleck diseased Lilium longiflorum leaves. Particles were scattered or in parallel aggregates in cell cytoplasm. Cells containing LSV were found in leaf mesophyll, epidermis, and phloem. But inclusion body in cells infected by LSV was not found by electron microscope. 3. Using lily leaves and bulb as materials, total RNA was extracted by using three methods. The results showed that Trizol methods was the fittest for obtaining high purity and integrity total RNA. Establishing of optimum RT-PCR detection system of LSV. Based on the effectual RT-PCR detection system of LSV, concentration of RT reaction ingredients dNTPs, primer, AMV, template and RNasin were optimized, and concentration of PCR reaction ingredients dNTPs, primer, Mg²⁺, Taq E and cDNA were optimized too. The results indicated that concentration of dNTPs, RNasin, primer, AMV, template is not lower than 0.4mmol/L, 0.3U/μl　,　0.4μmol/L, 0.1U/μl, 0.005μg/μl in RT system,and RT time was no less than 40 minutes. Concentration of Taq E , dNTPs and primer should be 0.020U/μl , 0.2mmol/L and 0.4μmol/L in PCR system respectively. The proper concentration Mg2+ of was more than 1.2mmol/L. A comparison of DAS-ELISA, RT-PCR and IC-RT-PCR methods showed that LSV was detected from dilutions equivalent to 2ng and 200ng of lily leaf material by RT-PCR and IC-RT-PCR, respectively. IC-RT-PCR and RT-PCR were 10 to1000 times as sensitive as DAS-ELISA in detection LSV. Different sampling parts of lily bulb affected the PCR result. IC-RT-PCR could detect LSV from scale and root, but RT-PCR could not detect.