First report of Coleus blumei viroid 5 and molecular confirmation of Coleus blumei viroid 1 in commercial Coleus blumei in Canada

Plectranthus scutellarioides (synonym: Coleus blumei, and hereinafter referred to as coleus) is a popular ornamental plant grown worldwide. Six viroids, namely Coleus blumei viroid 1 through 6 (CbVd-1 to CbVd-6), belonging to the genus Coleviroid, family Pospiviroidae, have been found to date in coleus (Nie and Singh 2017). Previously, a viroid was detected in commercial seeds of coleus in Canada (Singh et al. 1991); although the exact species of the viroid was unknown, the presence of CbVd-1 has been inferred. From May to July 2017, leaf tissue was collected from 56 coleus plants of 6 cultivars (Black Dragon, Wizard Select Mix, Pineapple Surprise, Wizard Coral Sunrise, Premium Sun Chocolate Covered Cherry, and Wizard Rose) at a local nursery in Fredericton, New Brunswick, Canada. No clear-cut symptoms were observed in the plants. Total RNA was extracted from pooled tissue from all individual plants. Return-polyacrylamide gel electrophoresis (Singh et al 1991) analysis detected a viroid-like fragment in the sample. When the purified viroid-like fragment was subjected to RT-PCR using the universal coleviroid primers CbVds-P1 (5’-GCAGCGCTGCAACGGAAT-3’) and CbVds-P2 (5’-GCAGCGCTGCCAGGGAACCCAGGT-3’) (Jiang et al. 2011), a CbVd-specific fragment (~250 bp) was produced, confirming the presence of a coleviroid(s) in the sample. The resulting amplicon was cloned into pCR™-Blunt vector (Thermo Fisher Scientific, Waltham, MA) and 10 clones were sequenced by the Sanger method. BLAST analysis of the sequences revealed the presence of CbVd-1 and CbVd-5, with frequencies of 9/10 and 1/10, respectively. The 249-nt-long CbVd-1 (accession number MG767212) exhibited a 100 % sequence identity with most CbVd-1 sequences deposited in GenBank whereas the 274-nt-long CbVd-5 (accession number MG767213) showed a single substitution of G for C at position 54 with other CbVd-5 isolates. RNA was then extracted from pooled tissue from all plants within each cultivar, and subject to RT-PCR using the universal coleviroid primers as well as newly designed CbVd-1-specific primer pair CbVd1-F1 (5'-GAGCTTGGCTCGAACTGACT-3')/CbVd1-R1 (5'-CGAAGAAGAAGCCGAAGCTA-3') (amplicon size, 144 bp) and CbVd-5-specific primer pair CbVd5-F1 (5'-GAAAAGGCAGGTTGCTTACC-3')/CbVd5-R1 (5'-AGCAGGAACCCGGAAGAC-3') (amplicon size, 171 bp). These tests indicated that the cultivar Wizard Coral Sunrise was not infected with any CbVds, the cultivar Black Dragon was infected with both CbVd-1 and -5, and that the four other cultivars were infected solely with CbVd-1. To further study the prevalence and disease symptoms of CbVd infections in coleus plants, sixty commercial seeds each of cultivars Black Dragon, Pinto Mix and Wizard Mix were germinated, and the resulting seedlings were transplanted and grown in the greenhouse at 20-25 oC with a light/dark cycle of 16/8 h and humidity of 75 %. RT-PCR tests of the plants using the universal coleviroid primer pair indicated that the cultivars had different CbVd-infection rates: 0 % in Pinto Mix, 20 % in Black Dragon and 35 % in Wizard Mix. Further testing of the CbVd-positive samples using the CbVd-1- and CbVd-5-specific primer pairs showed that all the CbVd-positive samples were infected solely with CbVd-1. Interestingly, most CbVd-1 infected Wizard plants were significantly lighter in colour than those that were not infected with the viroid (Supplementary Figure 1). However, in Black Dragon, no visible differences were observed between the infected and the uninfected plants, suggesting that host genotype plays a role in CbVd-induced symptom development. To our knowledge, this is the first report of CbVd-5 and the first confirmed detection of CbVd-1 in coleus in Canada. Considering the apparent propensity for coleviroids to recombine and evolve (Nie and Singh 2017), and the ability of viroids in general to cause disease symptoms in not only ornamental plants, but economically important crop species, precautions may be necessary to minimize the spread of these viroids.