Study On Prunus Necrotic Ringspot Virus And Lily Symptomless Virus Occurred On LiLy

Lilium is an important economical crop which could be used as ornamental plant, food or medicine. Lilium has already become one of main international ornamental flowers in recent years.As the increasing acreage of Lily, the quantaty and batch of imported lily blubs have been increased tremendously.This trend leads to a prevalence of virus diseases in lily planting areas. This study focuses on Prunus necrotic ringspot virus (PNRSV) and Lily symptomless virus (LSV) occurred on lily. Molecular Characters of these tow viruses (PNRSV and LSV) were compared for analying the relations between origin and distribution. Based on this, several rapid identification methods included RT-PCR, multiple-PCR and RNA silde hybridization were developed for lily virus detection at port quarantine.The main result and conclusion were as follows.1. Study revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.According to nucleotide sequences of PNRSV coat protein open reading frame (ORF), we designed two paires of"strain-specific"primers and established a multiple-PCR method with optimized RT-PCR reaction conditions for discrimination of rugose from mild strains. Two RT-PCR products with different sizes were simutaneously amplified, which could distinguish rugose type from mild type. It revealed correlations between symptom type and the nucleotide sequences of 3b (coat protein) open reading frame.2. PNRSV from lily corm was obtained and identified on molecular levels.It shown that Lily is a new carrier of PNRSV.Lily corms were randomly selected and grown in a greenhouse. Total RNA was extracted from young leaves with RNA Extraction Kit (Sangon, China). Amplification of partial CP gene was performed by RT-PCR with the primer pairs specific for PNRSV. An expected 450 bp fragment was harvested, cloned into pGEM-T-easy vector, sequenced, and compared with 27 PNRSV isolates reported previously. This 450 bp fragment amplified from lily shared 84.5% to 99.1% homology with 27 PNRSV isolates reported previously at the nucleotide level.3. Comparison and identification of 7 LSV isolates were conducted on molecular levels for analying the relations between origin and distribution.Results shown that isolates are not regional relevant.Seven LSV isolates from secondary generations of imported Lily in different domestic areas were obtained by RT-PCR and sequenced. These sequences are corresponded to the 3'end region of LSV genome including a partial CP gene and a entire gene encoding a 16 kD protein. Compared with 12 other isolates reported on GenBank, the 7 isolates sequence homologies of partial CP and 16 kD protein genes were 87.4% to 100% and 97.9% to 100%, respectively. At the amino acid level, they shared 95.9% to 100% and 95.7% to 100% sequence similarity, respectively. Comparison of their phylogenetic tree of the CP gene sequences and amino caid sequences show that there are no apparent regional relationships among these isolates.4.An RNA slide hybridization assay was developed and used for detection of the natural occurrence of LSV in lily corm originated from both imported cultivars and from wild lily spp.Results show that LSV is one of the ordinary virues occurred on Lily in China.