The first report of Coleus blumei viroid from coleus in China

Viroids have been found in coleus in Brazil, Germany, Canada and Japan. Since coleus is a common ornamental plant in China, plants were examined for viroid infection. In January 2005, 11 symptomless Coleus blumei cv. “Wizard”, with pink, scarlet or purple colour, were purchased from Huaxiang flower market in Haidian district in Beijing, Peoples Republic of China. Low molecular weight RNAs were extracted (Li et al., 1995) and were identified as Coleus blumei viroid (CbVd). All of the samples were CbVd positive by return-polyacrylamide gel electrophoresis (Return-PAGE) and dot-blot hybridisation using a digoxigenin-labeled CbVd RNA probe (Ishiguro et al., 1996). A 254-bp DNA fragment was amplified from all the samples by reverse transcription-polymerase chain reaction (RT-PCR) using forward (5'-TGGATCCAGC GCTGCAACGGAATCCA-3') and reverse (5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3') primers, based on the first reported sequence of CbVd1 (Genbank accession X52960; Spieker et al., 1990). To confirm the sequence of the primer regions, an additional set of primers, forward (5'-TTGGATCCTCCTGGTTCGCTGACCCG-3' and reverse (5'-TTGGATCCCC TTTGCAATCGCTCGCG-3'), were also used for RT-PCR. The amplified products from five of the 11 coleus samples were digested with Bam HI (the underlined sequence in the primers) and cloned into Bam HI site of pGEM3zf (+) (Promega, Madison, USA) for sequencing. Alignment of five sequences (Genbank DQ178395-178399) showed 99.2%, 99.2%, 98%, 97.6% and 98.4% identity to CbVd1 accession X52960, and 87.6%, 88.0%, 85.6%, 85.2% and 86.0% to a tentative member CbVd1-RG (Spieker 1996) respectively.

Further investigations were carried out in October, with 25 out of the 27 samples collected from 14 coleus producers in Beijing and Tianjin, found to be infected with viroid using return-PAGE analysis. These results indicated that coleus in commercial markets in Beijing and Tianjin, China, are highly infected with CbVd1. These results stress the need for a certification program to help control the spread of flower viroids in China.