Genus Coleviroid Research Articles

First report of Coleus blumei viroid 1 in commercial Coleus blumei in the United States.

Coleus scutellarioides (syn. Coleus blumei) is a widely grown evergreen ornamental plant valued for its highly decorative variegated leaves. Six viroids, named Coleus blumei viroid 1 to 6 (CbVd-1 to -6) have been identified in coleus plants in many countries of the world (Nie and Singh 2017), including Canada (Smith et al. 2018). However there have been no reports of Coleus blumei viroids occurring in the U.S.A. (Nie and Singh 2017). In April 2021, leaf tissue samples from 27 cultivars of C. blumei, one plant of each, were submitted to the University of Idaho laboratory from a commercial nursery located in Oregon to screen for the presence of viroids. The sampled plants were selected randomly and no symptoms were apparent in any of the samples. Total nucleic acids were extracted from each sample (Dellaporta et al. 1983) and used in reverse-transcription (RT)-PCR tests (Jiang et al. 2011) for the CbVd-1 and CbVd-5 with the universal primer pair CbVds-P1/P2, which amplifies the complete genome of all members in the genus Coleviroid (Jiang et al. 2011), and two additional primer pairs, CbVd1-F1/R1 and CbVd5-F1/R1, specific for CbVd-1 and CbVd-5, respectively (Smith et al. 2018). Five C. blumei plants (cvs Fire Mountain, Lovebird, Smokey Rose, Marrakesh, and Nutmeg) were positive for a coleviroid based on the observation of the single 250-nt band in the RT-PCR test with CbVds-P1/P2 primers. Two of these CbVd-1 positive plants (cvs Lovebird and Nutmeg) were also positive for CbVd-1 based on the presence of a single 150-nt band in the RT-PCR assay with CbVd1-F1/R1 primers. One plant (cv Jigsaw) was positive for CbVd-1, i.e. showing the 150-nt band in RT-PCR with CbVd1-F1/R1 primers, but did not show the ca. 250-bp band in RT-PCR with primers CbVds-P1/P2. None of the tested plants were positive for CbVd-5, either with the specific, or universal primers. All coleviroid- and CbVd-1-specific PCR products were sequenced directly using the Sanger methodology, and revealed whole genomes for five isolates of CbVd-1 from Oregon, U.S.A. The genomes of the five CbVd-1 isolates displayed 96.9-100% identity among each other and 96.0-100% identity to the CbVd-1 sequences available in GenBank. Because the sequences from cvs Lovebird, Marrakesh, and Nutmeg, were found 100% identical, one sequence was deposited in GenBank (MZ326145). Two other sequences, from cvs Fire Mountain and Smokey Rose, were deposited in the GenBank under accession numbers MZ326144 and MZ326146, respectively. To the best of our knowledge, this is the first report of CbVd-1 in the United States.

Coleus blumei viroid 7: a novel viroid resulting from genome recombination between Coleus blumei viroids 1 and 5.

The genus Coleviroid, family Pospiviroidae, comprises six known viroids, all infecting Plectranthus scutellarioides (Coleus blumei; coleus). In 2017, a novel viroid-like RNA sequence that shares ca. 65% identity with Coleus blumei viroid 1 (CbVd-1) was identified in a coleus cultivar infected by multiple coleviroids. Further sequence and secondary structure analyses are consistent with the discovery of a seventh viroid in the genus Coleviroid: tentatively named "Coleus blumei viroid 7" (CbVd-7). The viroid appears to be the product of a natural recombination event between CbVd-1 and Coleus blumei viroid 5. We prove CbVd-7 to be infectious and in turn demonstrate the ability of all known coleviroid left- and right-arm segments to recombine. With a length of 234 nucleotides, this is the smallest viroid described to date.

Molecular characterization of Coleus blumei viroids 1 and 3 in Plectranthus scutellarioides in Croatia

Asymptomatic Plectranthus scutellarioides (syn. Coleus blumei) plants of four different cultivars were investigated for the presence of Coleus blumei viroid (CbVd) species (genus Coleviroid, family Pospiviroidae). Cloned and sequenced amplicons obtained by generic coleviroid primers were analyzed for their primary and secondary structure. CbVd-1 was found in one of the coleus cultivars in a single infection and in another cultivar in a mixed infection with CbVd-3. Additionally, CbVd-3 was found in a third coleus cultivar in a single infection. In singly and doubly infected cultivars, both CbVd-1 and CbVd-3 variants displayed higher variability regarding both length and nucleotide composition in a doubly infected cultivar. Sequences from different cultivars showed different phylogenetic grouping suggesting distinct infection sources, probably introduced infected seeds. For all phylogenetic subgroups, characteristic nucleotide changes were identified and their influence on the secondary structure was predicted. The most noticeable structural changes were observed in nucleotide positions 25–26 in CbVd-1 and in the position 138 in CbVd-3 variants. This work includes the first report of coleviroids in Croatia, the second reported coleviroids sequences from Europe and the third report of CbVd-3 in the world enlarging the pool of knowledge on the variability and distribution of viroids in horticultural crops.

High-Throughput Sequencing Analysis of Small RNAs Derived from Coleus Blumei Viroids.

Characterization of viroid-derived small RNAs (vd-sRNAs) is important to understand viroid–host interactions; however, vd-sRNAs belonging to the genus Coleviroid are yet to be identified and characterized. Herein, we used coleus plants singly infected with coleus blumei viroid (CbVd)-1, -5, or -6 and doubly infected with CbVd-1 and -5 to identify and analyze their vd-sRNAs. We found sense and antisense vd-sRNAs for CbVd-1, -5 and -6, and 22-nt vd-sRNAs were the most abundant; moreover, the 5′-terminal nucleotides (nts) of CbVd-1, -5, and -6 were biased toward U and C, and sRNAs derived from these three viroids were unevenly distributed along their genomes. We also noted that CbVd-5 and -6 share a fragment that forms the right half of the rod-like secondary structure of these viroids, which implied that they generated almost the same type of vd-sRNAs. This finding indicated that vd-sRNA biogenesis is mainly determined by the primary sequence of their substrates. More importantly, we found two complementary vd-sRNAs (22 nt) that were generated from the central conserved region (CCR) of these three viroids, suggesting an important role of CCR in vd-sRNA biogenesis. In conclusion, our results provide novel insight into the biogenesis of vd-sRNAs and the biological roles of CCR.

FIRST REPORT OF COLEUS BLUMEI VIROID IN MALAYSIA

Plectranthus scutellarioides (synonym: Coleus blumei) is a popular home garden plant in Malaysia. It is susceptible to infection of six variants of Coleus blumei viroid (CbVd) (genus Coleviroid; family Pospiviroidae). A total of 49 samples from cultivars Saturn and Dipt in Wine showing symptoms such as dwarfing, faded color and reduction in leaf size were collected from commercial nurseries and residential areas from different states in Malaysia from February to June 2016. Total nucleic acid was extracted from the leaves and tested by RT-PCR using three sets of primers: CbVdR/CbVdF, CbVd5F/CbVd5R (Hou et al., 2009) and CbVd-1F/CbVd-1R (Chung and Choi, 2008). Altogether 39 out of 49 samples were positive to CbVd (primers CbVdR/CbVdF) (250-300 bp), CbVd-1 (primers CbVd-1F/CbVd-1R) (249 bp) and CbVd-5 (primers CbVd5F/CbVd5R) (274 bp). The amplicons of 249 bp and 274 bp from CbVd-1 and CbVd-5 specific primers, respectively, were cloned into pCR 2.1 TOPO® vector. Sequence analysis of both the 249 nt and 274 nt clones revealed 99-100% identity to CbVd-1 clone 1 (GenBank accession No. DQ178399.1) and CbVd-5 clone 1 (FJ151370.1) from China, respectively, thus confirming the presence of CbVd-1 (MF176948-51) and CbVd-5 (MF176952-55) in all of the tested samples. CbVd-1 and CbVd-5 were found together in both cultivars without particular symptom correlation. The cultivation of P. scutellarioides in Malaysia is mainly through imported seeds, which could explain the transmission of CbVd in Malaysia. To the best of our knowledge, this is the first report of CbVd-1 and -5 in Malaysia.

First report of Coleus blumei viroid 5 infection in vegetatively propagated clonal coleus cv. ‘Aurora black cherry’ in Japan

Plectranthus scutellarioides (synonym: Coleus blumei ) is a popular garden plant, cultivated worldwide and is known to be infected with several viroids of the genus Coleviroid , family Pospiviroidae . Coleus blumei viroid 1 (CbVd-1) is the…

First Report of Coleus blumei viroid 5 from Coleus blumei in India and Indonesia.

Coleus blumei, which was found originally in Indonesia, is an ornamental plant grown worldwide. It can be infected by several viroids of the genus Coleviroid, family Pospiviroidae. Six main viroids that infect coleus have been reported: Coleus blumei viroid 1 through 6 (CbVd-1 ~ CbVd-6). Although CbVd-1 was first reported in a commercial coleus in Brazil in 1989 (1), and then in Germany, Japan, Canada, Korea, China, and India, CbVd-5 was reported only in China in 2009 (2). Symptoms caused by CbVd-5 varied depending on different cultivars, and in case of an unknown cultivar of "Red with dark green edge," are very clear albino symptoms. From 2010 to 2011, 60 and 3 leaf samples of coleus were collected from Hyderabad, India, and Java, Indonesia, respectively, and subjected to low molecular weight RNA extraction according to Li et al. (3). The results of dot-blot hybridization using CbVd-5 cRNA probes and RT-PCR using CbVd-5 specific primers (CbVd-5-PF: 5'-TGACTAGAACAGTAGTAAAG-3' / CbVd-5-PR: 5'-AATTGAGGTCAAACCTCTTT-3') demonstrated that 28 out of the 60 samples from India and all three samples from Indonesia were positive for CbVd-5. The resulting RT-PCR fragments from one sample selected randomly from each country were cloned into the pMD18-T vector (Takara) and transformed into E. coli DH5α competent cells. Five positive clones of each sample were sequenced. The result of sequence analysis revealed that the similarities of CbVd-5 between the sequences we obtained and the reference sequence (GenBank Accession No. NC003683) were 97.8 to 100%. Bioassay using nine viroid-free coleus plants from three cultivars (three from each cultivar), inoculated with CbVd-5 infectious clones by stem slashing, demonstrated that CbVd-5 could induce albino symptom on the leaves of the unknown cultivar "Red with dark green edge" 2 months after inoculation. To our knowledge, this is the first report of CbVd-5 from India and Indonesia, and the second report of CbVd-5 in the world. Considering the effect of CbVd-5 on the appearance of coleus and its recombination ability, a certification program may be needed to control the spread of this viroid.

First Report of Coleus blumei viroid Infecting Coleus in India.

Members of the genus Coleviroid (Coleus blumei viroid [CbVd]), family Pospiviroidae have been reported to infect Coleus (Solenostemon sp.). CbVd-1 was first reported from Brazil, CbVd-2, -3, and -4 were first reported from Germany, whereas CbVd-5 and -6 were recently identified in China (2). In India, Coleus is extensively cultivated as an ornamental plant in home gardens. In March to June 2012, Coleus leaf samples with irregular chlorotic spots/patches were collected from home gardens of two different districts of Karnataka (Kodagu and Mysore districts), India, suspecting the presence of Coleus blumei viroids (CbVd 1 to 6). Low molecular weight RNAs were extracted using 2% CTAB buffer containing 1.4 M NaCl, 20 mM EDTA, pH 8.0 and 100 mM Tris-Cl, pH 8.0 (1). Viroid-like RNA was enriched by fractionation 2M LiCl soluble nucleic acids (4). A DNA fragment with the expected size of CbVd-1 was detected in 10 (including both districts) of 14 analyzed (incident rate of 71%) from reverse transcription (RT)-PCR assay using Coleviroid specific primers (forward 5'-TGGATCCAGCGCTGCAACGGAATCCA-3' and reverse 5'-TTGGATCCGCCAGGGAACCCAGGTAAG-3'). RT was performed at 37°C for 60 min in 25 μl reaction mix containing 5 μl RNA extracts, 1 μl reverse primer, 1× first strand buffer, 10 mM dNTPs, and 200U M-MuLV-RT (Invitrogen, USA). For PCR, 2 μl RT was mixed in 25 μl PCR mix containing 0.2 μM each forward and reverse primers and 2U LA Taq (Takara-Bio, Japan) according to the manufacturer's instruction. PCR parameter was one cycle at 94°C for 2 min, 35 cycles at 94°C for 45 s, 53°C for 30 s, and 72°C for 60 s, followed by final extension at 72°C for 10 min (4). Sequence analysis of cloned amplicons detected CbVd-1 in India. To confirm the sequence of the primer regions, an additional set of tail-tail primers were designed, CbVd1-136F (5'-CTTCGTGGAACGGCTCCGCG-3') and CbVd1-136Rev (5'-GAAGAAGCCGAAGCAACTCTC-3') and were used for RT-PCR. Amplified products were cloned, sequenced and compared with previously obtained data. Further, the presences of CbVd-1 in Coleus samples was confirmed by a RNA gel blot assay using digoxigenin-labeled CbVd-1 cRNA probe (3). Alignment of 19 sequences obtained from four representative Coleus samples found the presence of two sequence variants of CbVd-1, namely Ind-1 (GenBank Accession No. AB740017) and Ind-2 (AB740018). Ind-1 was found to differ from Ind-2 by two nucleotide substitutions at position 40 (C to T) and 211 (T to C). BLAST analysis of Ind-1 showed 100% sequence similarity with CbVd-1 isolates from China (DQ178399) and South Korea (EU 410620), whereas Ind-2 was 99% identical to these two Chinese and Koreans isolates. Furthermore, 97% and 96% sequence identity with CbVd 1-RL RNA (Accession no. X95366) was observed for Ind-1 and Ind-2, respectively. Isolates from India were 88% similar with Coleus blumei viroid 1-RG (X95291). To the best of our knowledge, this is the first molecular evidence for the presence of CbVd-1 infecting Coleus in India. Coleus harbors various viroid species and CbVd-1, reported widely, can transmit efficiently through seed and also could infect the other herbaceous plants (3). This report from India will contribute further understanding of a potential risk of Coleus viroids in ornamental species.

Rapid detection and identification of viroids in the genus Coleviroid using a universal probe

A simple, low-cost hybridization assay using a universal DIG-labeled riboprobe for the rapid detection and identification of coleus viroids is presented. An octamer of 32-nucleotide sequence derived from the central conserved region (CCR) of viroids in the genus Coleviroid was used to develop a universal cRNA probe (8-central-conserved-region probe, 8CCR probe) for coleus viroids. Dot-blot hybridization assays demonstrated that the sensitivity of this probe was similar to specific probes for each CbVd, and Northern hybridization results revealed that at least four coleus viroids could be distinguished readily and simultaneously using the 8CCR probe. Batch detection assay showed that hybridization using the 8CCR probe can identify coleus viroids rapidly and effectively. This rapid and low-cost molecular hybridization technique is an effective way to survey the occurrence of coleus viroids, and has reference for the detection of other viroids and possibly viruses.

First report of Alfalfa mosaic virus in eggplant in Turkey.

Coleus (Coleus blumei) is an ornamental plant hosting several species of the genus Coleviroid [type member Coleus blumei viroid 1 (CbVd-1)], family Pospiviroidae. CbVd-3, a recognized member of the family, has only been reported from Germany in 1996 (Spieker et al., 1996). According to their proposed rod-like secondary structure, the left portion of CbVd-3 is also preserved in CbVd-2 and CbVd-6, which are viroid chimeras (Spieker, 1996; Hou et al., 2009). Fifty leaf samples of coleus, including 30 from Beijing and five each from Tianjin, Nanjing, Fujian and Hainan (China), were collected from 2009 to 2010. Low molecular weight RNAs were extracted as previously reported (Li et al., 1995). RNA extraction from viroid-free coleus was used as negative control. Results of dot-blot hybridization using DIG-labeled CbVd-3 cRNA probe and RT-PCR using CbVd-3 specific primers (PF: 5’-CGGGTACCCAGCAACCTGCT-3’; PR: 5’-TGGGTACCCGCGAAGAGC-3’) showed that one sample from Beijing and one from Hainan were positive for CbVd-3. Products of the expected size were amplified only from the hybridization-positive samples. After cloning, sequence analysis of five selected clones revealed that the similarities between the sequences we obtained and the CbVd-3 sequence reported previously (GenBank accession No. X57294) varied from 99.72% to 100%. The dominant sequence has been submitted to GenBank (accession No. HQ727548). These results provide experimental evidence that the two tested plants were infected by CbVd-3. To our knowledge, this is the first report of CbVd-3 from China and the only report of this viroid outside Germany.

First Report of Coleus blumei viroid 2 from Commercial Coleus in China.

Coleus (Coleus blumei) is an ornamental plant that is susceptible to infection by several viroids of the genus Coleviroid, which is a member of the family Pospiviroidae. Coleus blumei viroid (CbVd) -1 was first reported in commercial yellow coleus fields in Brazil in 1989 (1). In addition, CbVd-2, CbVd-3, and CbVd-4 have only been detected from coleus in Germany in 1996 (4). CbVd-5 and CbVd-6 were recently identified in China (2). In March 2010, leaves were collected from 50 symptomless coleus plants from a commercial nursery in Hainan Province, China. Total RNA was extracted from the leaves (3). Reverse transcription (RT)-PCR using CbVd-2 specific primers (forward: 5'-AGCTTACCTGGGTTCCCT-3' and reverse: 5'-CTCTCCTCTATTTACTCTCTTCTC-3') corresponding to positions 76 to 93 and 52 to 75 on the CbVd-2 reference sequence, respectively (GenBank Accession No. NC003682). Amplification of a 301-bp product was obtained from one sample. This PCR product was then cloned into pMD18-T (Takara, Dalian, China). Twelve positive clones were sequenced and the results were subjected to BLAST analysis. Sequence analysis showed that two sequences (GenBank Accessions Nos. HQ727542 and HQ727544) shared 99% identity with the reference sequence of CbVd-2 (NC003682), and four sequences (HQ727541, HQ727543, HQ727545 and HQ727547) had 99.34% identity with the reference sequence of CbVd-2 (NC003682). The proposed secondary structures of these variants have approximately 75% paired nucleotides. Results suggested the presence of CbVd-2, which is a member of the Coleviroid genus, Pospoviroidae family. To our knowledge, this is the first report of CbVd-2 from commercial coleus in China.

Sap-direct RT-PCR for the rapid detection of coleus blumei viroids of the genus Coleviroid from natural host plants

A simple and fast sap-direct RT-PCR (reverse transcription-polymerase chain reaction) for the rapid detection of 3 viroids of the genus Coleviroid is presented. The templates for cDNA synthesis were obtained directly from the sap of coleus using a pipettor, a common tool in molecular biology laboratories, and 3 coleus blumei viroids (CbVds) were detected simultaneously using a pair of universal primers designed according to sequences in the central conserved region (CCR) of CbVds. RT-PCR results demonstrated that CbVd-1, CbVd-5, and CbVd-6 can be detected accurately in viroid-infected plants but not in viroid-free plants. The results of RT-PCR, dot-blot, sequencing, and batch-detection revealed that this method can be used to identify CbVds rapidly. The method also reduces cross-contamination among different samples to a minimum. It is considered that this rapid and simple technique is an effective method for the identification and cloning of CbVds.

Coleus blumei viroid 6: a new tentative member of the genus Coleviroid derived from natural genome shuffling

Coleus blumei can be infected by several viroids of the genus Coleviroid. One year after detecting a mixed infection of coleus blumei viroid 1 (CbVd-1) and 5 (CbVd-5) in coleus seedlings inoculated with these two viroids, we found an additional viroid-like RNA. Sequence analysis revealed a viroid of 342 nucleotides that contains the central conserved region of coleviroids and is a chimera of the left half of CbVd-3 and the right half of CbVd-5. This new viroid, tentatively referred to as coleus blumei viroid 6 (CbVd-6), appears to have arisen from a natural recombination event or genome shuffling.

Identification and characterization of a new coleviroid (CbVd-5)

A viroid-like RNA was detected from coleus (Coleus blumei) in China. It consisted of 274 nucleotides and had 66% sequence identity with a member of the closest known viroid species. The predicted secondary structure is rod-shaped with extensive base pairing, and it has the conserved region characteristic of the genus Coleviroid. Two terminal sequences that are highly conserved among some members of the genus were also identified. The viroid-like RNA was successfully transmitted to coleus by slash-inoculation. This viroid was identified as a new member of the genus Coleviroid, and we tentatively propose the name Coleus blumei viroid 5 (CbVd-5).