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Peer Reviewed

© 2013 Plant Management Network.
Accepted for publication 23 April 2013. Published 29 July 2013.

Identification of Two Tobacco streak virus Capsid Protein Variants Associated With Leaf Mottle and Necrosis Symptoms on Astilbe

John R. Fisher, Ohio Department of Agriculture, Plant Health Diagnostic Laboratory, Plant Health Division, Reynoldsburg, OH 43068

Corresponding author: John R. Fisher.

Fisher, J. R. 2013. Identification of two Tobacco streak virus capsid protein variants associated with leaf mottle and necrosis symptoms on Astilbe. Online. Plant Health Progress doi:10.1094/PHP-2013-0729-02-BR.

Tobacco streak virus (TSV) is the type species of the Ilarvirus genus of the family Bromoviridae (5), and is transmitted mechanically, through thrips feeding, by pollen (6), and by seed (4). The virus has a tripartite genome of single-stranded messenger-sense RNA, which encodes four non-structural proteins and a single structural capsid protein (5). The movement protein and capsid protein encoding genes are located on the 5' half and 3' half, respectively, of RNA 3 (5).

In the spring of 2013, seventeen dormant bare-root Astilbe × arendsii (false spirea) plants of mixed cultivars were submitted to the Ohio Plant Diagnostic Network for virus screening as part of a Farm Bill-funded survey of viruses infecting ornamental hosts. The plants were potted in sterile potting mix and grown for several weeks in an indoor growth room until there was sufficient top growth for observation and sampling. The leaves developed a mottle symptom, and in several cases partial or complete necrosis of the top growth (Fig. 1). All seventeen plants tested positive for TSV and negative for the Potyvirus group, Alfalfa mosaic virus, Apple mosaic virus, Arabis mosaic virus, Broad bean wilt virus, Carnation latent virus, Carnation mottle virus, Carnation necrotic fleck virus, Carnation ringspot virus, Cowpea mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Lily symptomless virus, Peanut stunt virus, Pelargonium flower break virus, Prunus necrotic ringspot virus, Tobacco etch virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato aspermy virus, Tomato bushy stunt virus, Tomato mosaic virus, Tomato ringspot virus, Tomato spotted wilt virus, and Watermelon mosaic virus by enzyme-linked immunosorbent assays (ELISA) using commercially available antibodies (Agdia Inc., Elkhart, IN).


Fig. 1. Necrosis symptom (A) and leaf mottle symptom (B) associated with TSV infection of Astilbe.

Double-stranded ribonucleic acid (dsRNA) was purified from symptomatic leaf tissue and used as a template for cDNA synthesis as previously described (2,7). Immunocapture reverse transcription (IC-RT) using sheep anti-rabbit conjugated magnetic beads incubated with polyclonal rabbit anti-TSV IgG was also performed as previously described (1,3). TSV specific primers were used to amplify the movement protein (MP) and capsid protein (CP) genes from cDNAs synthesized from dsRNA template and immunocaptured virions (1). Both sets of primers amplified distinct products (Fig. 2). The amplicons were excised, purified from the agarose, and cloned into pGEM-T vector as previously described (2, 3). Colonies were screened for inserts which were subsequently sequenced. Sequences were assembled and subjected to pairwise and multiple sequence alignments (1,2). Finally, open reading frames (ORFs) were translated using Genedoc (Genedoc v. 2.6.001).


Fig. 2. PCR detection of TSV from cDNAs synthesized from dsRNA template with MP-specific primers (Lane 1) and CP-specific primers (Lane 2). Water controls with MP primers (Lane 3) and CP primers (Lane 4). TSV-Hosta MP clone with MP primers (Lane 5) and TSV-Hosta CP clone with CP primers (Lane 6) used as positive PCR controls. M=1 Kb DNA ladder (250, 500, 750, 1000, and 1500 bp markers indicated). Electrophoresis was performed in 0.8% agarose at 100 volts for 60 min in 1X TAE buffer. Gel was stained with ethidium bromide. MP and CP amplicons are 1036 bp and 947 bp, respectively.


Ten MP and nine CP clones were sequenced (The Ohio State University Plant Microbe Genomics Facility) and the ORF sequences deposited in GenBank (accession numbers KC776130- KC776148). The TSV-Astilbe MP ORF is 870 nucleotides (nt) long and encodes a predicted 289 amino acid (aa) protein. The clones had 99.4-100% nt sequence identity to one another with a 99.8% mean, and a 99.0-100% predicted aa identity to one another with a 99.7% mean. The TSV-Astilbe CP ORF is 714 nt long and encodes a predicted 237 aa protein. When aligned, two distinct populations of CP clone nt sequences were identified. Six of nine clones were 99.4-100% identical to one another with a 99.7% mean, and three of nine clones were 99.7-99.9% identical to one another with a 99.8% mean. However, the two populations were only 91.9-92.4% identical to each other with a 92.1% mean. The translated CP ORFs could also be divided into two distinct populations of predicted aa sequences. Six of nine clones were 98.7-100% identical with a 99.2% mean, and three of nine clones were 99.6-100% identical with a 99.7% mean. The two populations were only 92.8-93.2% identical to each another with a 93.1% mean. The two CP populations could also be distinguished based on the source of the cDNA used for amplification. Five of five clones from cDNAs from immunocaptured virions were all of the same genotype, but clones from cDNAs from dsRNA template were of two distinct genotypes. One of the four clones belonged to the same population as those from immunocaptured virions. The other three were distinctly different. These results demonstrate that at least two TSV RNA 3 variants are associated with virus-like symptoms in Astilbe × arendsii. The MP nt and predicted aa sequences are highly conserved among all of the sequences analyzed here, but there are clearly two CP nt and aa sequence variants. Further, when compared to a TSV isolate we recently reported from Hosta (1) the TSV-Astilbe CP ORF is three nt shorter and the predicted protein is two aa shorter. The two TSV-Astilbe CP variants had only 88.7% and 90.0% nt sequence identity, and 90.3% and 92.4% aa identity to TSV-Hosta. BLASTn searches of the NCBI database using default settings with the TSV-Astilbe MP ORF sequences (100% query coverage) resulted in an 82% match with accession numbers X00435.1, JX073658.1, and FJ403377.1. BLASTn searches using default settings with the two TSV-Astilbe CP ORF variants resulted in reciprocal matches: 98% (accession number X00435.1) and 92% (accession number AM933669.1) with one variant and 98% (accession number AM933669.1) and 92% (accession number X00435.1) with the second variant.

To our knowledge, the results presented here represent the first confirmed report of TSV infecting Astilbe × arendsii. They also demonstrate that there are two CP nt sequence variants that translate into two CP aa sequence variants. It is interesting to note that only one of the variants was detected by IC-RT-PCR but both were detected from cDNAs from dsRNA template. That we only detected the second CP ORF variant using a strictly nucleic acid based detection method may be significant and might suggest that the antibodies used in this study aren’t detecting the second variant, but an analysis of a larger sampling of clone sequences would be necessary to rule out that possibility. Commercial perennial growers should benefit from this work by gaining awareness of TSV as an emerging threat to ornamentals in general, and a new threat to Astilbe production in particular.

Literature Cited

1. Fisher, J. R. 2013. Identification of Tobacco streak virus associated with a virus-like mottle symptom on Hosta. Online. Plant Health Progress doi:10.1094/PHP-2013-122-01-BR.

2. Fisher, J. R. 2012. First report of Tobacco rattle virus associated with ring spot and line pattern disease of peony in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2012-0711-01-BR.

3. Fisher, J. R. 2012. Identification of three distinct classes of satellite RNAs associated with two Cucumber mosaic virus serotypes from the ornamental groundcover Vinca minor. Online. Plant Health Progress doi:10.1094/PHP-2012-0412-01-RS.

4. Fulton, R. W. 1985. Tobacco streak virus. CMI/AAB Descriptions of Plant Viruses No. 307. Association of Applied Biologists, Wellsbourne, Warwick, UK.

5. King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J. 2012. Bromoviridae. Pages 965-976 in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, MA.

6. Sdoodee, R., and Teakle, D. S. 1987. Transmission of tobacco streak virus by Thrips tabaci: a new method of plant virus transmission. Plant Pathol. 36:377–380. doi: 10.1111/j.1365-3059.1987.tb02247.x.

7. Valverde, R. A., Nameth, S. T., and Jordan, R. L. 1990. Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis. 74:255-258.