Research On Fast Detection Of Citrus Exocortis Viroid And Clone Of CEVd-HB Isolate

Viroids are the smallest known pathogens of plants. They consist of circular and single-stranded RNA. In most cases, they adopt rod-like compact conformations. In contrast to virus, viroids do not code for any protein. A lot of plants can be infected by viroids causing diseases, especially citrus plants. Viroids found in citrus plants can be classified into seven citrus viroids and citrus exocortis viroid (CEVd) is the most serious one of them. CEVd which is a number of Pospiviroidae is the casual agent of citrus exocortis disease, the symptoms of which are bark scaling on trifoliate orange root-stocks, foliar distortion and stunting of the entire. It distributes all over the world, induces economical loss and confines the development of production of citrus plants.In order to set up rapid detection system of CEVd, three methods including bi-directional PAGE, RT-PCR and hybridization were used in our studies. A CEVd strain was isolated from Heibei province in China and its complete nucleotide sequences were identified.Accounting to the characteristic of structure of viroid, the nucleic acid from the positive material was detected by bidirectional PAGE. Through silver-stained after electrophoresis, a special bend of CEVd was observed, which was behind of other bends. The special bend didn't be found in the result of negative material. The research showed that bi-directional PAGE was a simple, rapid and economical method for detection of CEVd.Based on the reportorial sequences of CEVd, we designed the primers to detect CEVd by RT-PCR. Amplified fragments specific to CEVd were obtained from the two positive materials. The length of amplified fragments was about 370bp. For the negative material, there was no nonspecific amplification. RT-PCR was a sensitive and stable method. It would be useful for large-scale diagnosis.A length cDNA of CEVd had been cloned into plasmid vector. Biotinylated probes were prepared from these plasmid DNAs. CEVd were examined from the positive and netative materials by Dot-blotting hybridization and imprint-hybridization. The result was exposed to x-ray film. Singles were detected for positive materials' spots probed with probe, indicating that under the reaction conditions the probe hybridized with the target viroid specifically. The results showed that it was a feasible way to detect CEVd.In the research, a CEVd strain from Citrus sinensis Osbeck cv. Robertson was isolated in Heibei province and named CEVd-HB, which was identified by bidirectional PAGE RT-PCR, and Dot-blotting. The isolate could infect the indication plant and show the typesymptoms of CEVd. The complete nucleotide sequences of the isolate were identified and logged on GenBank (AY456136). After comparative analysis of the sequences of CEVd-HB and other published isolates, CEVd-HB shared the highest homology with CEVd-de26. The identity of nucleotide between CEVd-HB and CEVd-de26 was 99.2%. Phylogenetic tree of nucleotide sequences of 58 strains of CEVd indicated that CEVd-HB had same origin with three strains isolated from USA.