Detection And Molecular Characterization Of Coleus Blumei Viroid In China

Coleus blumei viroid was discovered firstly in commercially field-grown yellow coleus in Brazil (Fonseca et al, 1989), and then Germany, Canada, Japan etc reported it. Coleus blumei viroid (CBVd) was only found in coleus currently. The genus consists of three members: CBVd1, CBVd2 and CBVd3, and nucleotide sequence lengths are about from 248 to 373nt. The results come from the research on coleus plants purchased from Huaxiang flower market in Fengtai district in Beijing, coleus plants purchased from flower markets of Beijing and Tianjin and coleus seeds purchased from Xinfadi and Dasenlin in Beijing. This is the first reported of Coleus blumei viroid in China. It means that CBVd infected China coleus earlier. These results stress the need for a certification program to help study the pathogenesis of CBVd and control the spread of CBVd in China.Return-PAGE and Two-dimensional electrophoresis are used to detect whether CBVd infected coleus of Huaxiang flower market by the novel circular molecular of viroids. It shows that CBVd infected all the 11-coleus plants that collected from Huaxiang flower market. Equivalent to 0.15g fresh leaves also can be detected by Return-PAGE. It can embody the characterization of circular viroid RNA though it shows lower sensitivity by Return-PAGE .Low molecular weight RNAs were extracted from coleus and about 250bp DNA fragment was amplified by RT-PCR using primers designed by the registered sequences in GenBank according the result of detection by Return-PAGE and Two-dimensional PAGE. The amplifiedproducts of RT-PCR were cloned into vector pGEM-3Zf (+) and sequenced. Sequences variants DQ178395, DQ178396, DQ178397, DQ178398 and DQ178399 were obtained and registered in GenBank, compared with reported sequences of CBVdl in GenBank show 85.23%-99.20% identity. Comparing with X95291 showed 85.23%-87.92% identity and with X52960 showed 97.60%-99.20% identity. Equivalent to 45 n% fresh leaves can be detected by RT-PCR. A sensitivity of DIG-labled RNA dot-blot hybridization was experimented by diluting denatured solutions. The denaturing solutions were diluted 10°-10*6, andlO0 Equivalent to 5-10mg fresh leaves. As a result, we also detected the CBVdl even the denaturing solutions were diluted 10"3. It means a high-viroid-concentration. 27 coleus plants have purchased from other flower markets of Beijing and Tianjin. We found 25 of 27 coleus plants were infected by Coleus blumei viroid using Return-PAGE and dot-blot hybridization. Viroid-infect rate reach to 92.59%There is a high rate of viroid transmission through coleus seeds (Singh, 1991). We have detected CBVd in coleus plants, and we further want to know if CBVd also exit in coleus seeds. Coleus seeds were purchased from Xinfadi and Dasenlin seeds markets. We get 44- coleus seedlings, 20-30cm heights, by greenhouse culture. But we can't detect CBVd from coleus seedlings by Return-PAGE (NO. 1 -44 ) , RT-PCR (NO. 1 -21) and dot-blot hybridization (NO. 1-22) .This is the first report of Coleus blumei viroid in China. We can't know clearly the distribution of Coleus blumei viroid in China because of limited detection quantities and areas. It needs much work to further detect CBVd in other areas, and especially if CBVd2 and CBVd3 also have exist in China.