Identification of Two Tobacco rattle virus Variants Associated with Line Pattern Disease of Bleeding Heart in Ohio

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1 2013 Plant Management Network. Accepted for publication 19 December Published. Identification of Two Tobacco rattle virus Variants Associated with Line Pattern Disease of Bleeding Heart in Ohio John R. Fisher, Ohio Department of Agriculture, Plant Health Diagnostic Laboratory, Plant Health Division, Reynoldsburg, OH Corresponding author: John R. Fisher. Fisher, J. R Identification of two Tobacco rattle virus variants associated with line pattern disease of bleeding heart in Ohio. Online. Plant Health Progress doi: /php RS. Abstract Tobacco rattle virus (TRV) is a member of the Tobravirus genus and has a broad host range including weeds, vegetables, and ornamentals. Several bleeding heart (Lamprocapnos spectabilis) plants showing a yellow line pattern symptom were negative for the Potyvirus group, Alfalfa mosaic, Arabis mosaic, Cucumber mosaic, Impatiens necrotic spot, Tobacco mosaic, Tobacco ringspot, Tomato ringspot, and Tomato spotted wilt viruses by ELISA. cdnas synthesized from dsrna template and immunocaptured virions from symptomatic and asymptomatic bleeding heart amplified clear products with Tobravirus group and TRV specific primers targeted at regions in the 194 kda open reading frame 1 on RNA 1. Cloned amplicon sequences demonstrated two distinct populations of sequences for both primer pairs and the two populations correlated with symptoms, or lack thereof, on bleeding heart. These results represent the first confirmed report of TRV associated with line pattern disease on bleeding heart in Ohio. Introduction Tobacco rattle virus (TRV) is the type species of the Tobravirus genus (family Virgaviridae), which also includes Pea early browning virus and Pepper ringspot virus. The TRV genome is linear, single stranded, and has a positive sense RNA divided into two segments (5). RNA 1 encodes the replicase, intercellular movement, and pathogenicity proteins. RNA 2 encodes the coat protein and those required for nematode transmission (5). TRV has a broad host range which includes vegetable, weed, and ornamental hosts, and is transmitted by nematodes in the genera Trichodorus and Paratrichodorus (5). Virus-like yellow ring spotting and line pattern symptoms on bleeding heart have been previously attributed to TRV in Minnesota, Michigan, and Massachusetts (6) but this is the first confirmed report of the disease in Ohio. In the spring of 2010 several bleeding heart (Lamprocapnos spectabilis, formerly Dicentra spectabilis) plants displaying yellow line patterns (Fig. 1) were submitted to the Ohio Plant Diagnostic Network. The samples tested negative for the Potyvirus group, Alfalfa mosaic, Arabis mosaic, Cucumber mosaic, Impatiens necrotic spot, Tobacco mosaic, Tobacco ringspot, Tomato ringspot, and Tomato spotted wilt viruses by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies (Agdia Inc., Elkhart, IN). Double stranded ribonucleic acid (dsrna) was purified from symptomatic and asymptomatic tissue as described (7) resulting in a gel electrophoretic profile suggestive of a viral infection.

2 A B C Fig. 1. Line patterns observed on TRV infected bleeding heart (A), localized ring spot and line pattern symptoms observed on inoculated Nicotiana tabacum Glurk leaves after approximately 30 days (B), and systemic oak leaf (C) and line pattern (D) symptoms observed on inoculated Lycopersicon esculentum Rutgers after approximately 30 days. TRV may produce local or systemic infections depending on host (5) so transmission studies were done to verify presence of a virus. One field isolate was inoculated onto lambsquarter (Chenopodium quinoa), tobacco (Nicotiana tabacum Glurk ), and tomato (Lycopersicon esculentum Rutgers ) by triturating symptomatic bleeding heart tissue (1:5 ratio) in 100 mm sodium phosphate buffer (ph 7.0) containing 1% celite and rubbing the sap extract on plants at the four leaf stage (lambsquarter and tobacco) or the fully expanded cotyledon stage (tomato). Two plants each of lambsquarter and tobacco, and four tomato were inoculated with the extracted plant sap solution and one with buffer alone. The plants were held in indoor growth chambers at room temperature under natural light ( ~12 h day/night) and observed over a 90-day period. The Chenopodium plants developed chlorotic local lesions approximately 7 days post-inoculation (dpi). Tobacco Glurk developed line pattern and ring spot symptoms on inoculated leaves (Fig. 1) approximately 30 dpi but never developed systemic symptoms. Tomato Rutgers plants developed systemic oak leaf and line pattern symptoms (Fig. 1) approximately dpi, and all four inoculated plants remained symptomatic over the course of more than a year. The fruit, however, never developed viral symptoms. The TRV isolate from symptomatic bleeding heart produced only a local infection on inoculated tobacco Glurk leaves but produced a systemic infection in tomato Rutgers. DsRNA analysis of symptomatic tomato Rutgers tissue (Fig. 2) produced a banding profile indistinguishable from the profile obtained from symptomatic bleeding heart tissue (not shown). Asymptomatic (presumed uninfected) bleeding heart tissue produced a similar, but not identical, profile. cdnas were synthesized from dsrna template as previously described and used with Tobravirus group and TRV specific primers to amplify regions in the TRV 194K RNA polymerase gene (2,4). For immunocapture (IC) RT-PCR, magnetic beads conjugated with sheep anti-rabbit IgG (Dynabeads M-280, Dynal Biotech/Invitrogen, Carlsbad, CA) were incubated with polyclonal rabbit anti- TRV IgG. Tissue was ground in phosphate sucrose buffer, cdnas synthesized as previously described (3), and used with the same primers as above. The cdnas D

3 synthesized from dsrna isolated from symptomatic and asymptomatic bleeding heart tissue (Fig. 3), as well as tomato Rutgers inoculated with bleeding heart extract (not shown), amplified a single clear product of expected size with both sets of primers. The cdnas from IC-RT also amplified a single product of expected size with both sets of primers, but the amplification intensity was weaker (not shown). The amplicons were cut from the gels, purified from the agarose, ligated into pgem-t vector, and transformed into E. coli JM109 cells. Clones were screened for an insert by PCR using the M13 vector forward and reverse primers, and subsequently plasmid DNA was purified from positive colonies as previously described (2,3). Fifteen and nineteen clones of the Tobravirus group and TRV specific amplicons, respectively, were sequenced (Plant Microbe Genomics Facility, The Ohio State University, Columbus, OH.). Vector was trimmed from raw sequences (Chromas v. 2.33, Technelysium Pty Ltd., Australia), contigs assembled, and pairwise plus multiple sequence alignments were performed (Vector NTI Advance 11, Invitrogen). The resulting sequences were edited and open reading frames were translated (Genedoc v , 2000). The processed sequences were deposited in GenBank under accession numbers JX JX Fig. 2. dsrna analysis of buffer mock inoculated tomato Rutgers (Lane 1), symptomatic tomato Rutgers inoculated with sap extract from symptomatic bleeding heart (Lane 2), and N. tabacum Glurk infected with a Cucumber mosaic virus isolate as an extraction control (Lane 3). M = 1 Kb DNA ladder (250-10,000 bp markers indicated). Electrophoresis was done in 1% agarose at 100 volts for 90 min in 1X TAE buffer. Arrows indicate putative dsrnas.

4 Fig. 3. PCR detection of TRV from cdnas synthesized from dsrna template from symptomatic (Lanes 1, 2) and asymptomatic (Lanes 3, 4) bleeding heart with Tobravirus group (Lanes 1, 3) and TRV specific (Lanes 2, 4) primers. Water controls with Tobravirus group (Lane 5) and TRV specific (Lane 6) primers, and TRV positive control with Tobravirus group (Lane 7) and TRV specific (Lane 8) primers. M = 1 Kb DNA ladder (250, 500, 750, 1000 bp markers indicated). Electrophoresis was done in 0.8% agarose at 100 volts for 60 min in 1X TAE buffer. The Tobravirus group clones were 830 nucleotides (nt) and corresponded to nt of the TRV ORF 1 on RNA 1. The TRV specific clones were 779 nt and corresponded to nt of the TRV ORF 1. When aligned, two distinct populations of sequences were evident for both sets of amplicons; the clones from symptomatic bleeding heart and inoculated tomato Rutgers comprising one population and those from asymptomatic bleeding heart comprising the other. A comparison of the percent nt identities is presented in Table 1. When translated, the Tobravirus group clones corresponded to amino acids (aa) and the TRV specific clones to aa of the TRV ORF 1. A comparison of the predicted percent aa identities is also presented in Table 1. Table 1. Comparison of percent nucleotide (blue shaded cells) and percent predicted amino acid (yellow shaded cells) identities of Tobravirus group and TRV specific clones generated from symptomatic and asymptomatic bleeding heart. Clones generated from a symptomatic bleeding heart (Bh), b tomato Rutgers inoculated with symptomatic bleeding heart, and c asymptomatic bleeding heart. Mean of uvwz 5, y 4, and x 10 clones.

5 The Eighth Report of the International Committee on Taxonomy of Viruses (ICTV) indicated that strains of TRV are more than 99% identical with respect to their RNA 1 nt sequences (1), although the Ninth Report of the ICTV dropped that distinction from the description (5). The results presented here suggest at least two TRV lineages are infecting bleeding heart in Ohio. Further, one variant is associated with line pattern symptoms observed on bleeding heart and inoculated tomato Rutgers while the other is associated with an absence of symptoms on bleeding heart. A single consistent amino acid residue difference between clones from symptomatic and asymptomatic bleeding heart was noted. Amino acid 163 is a serine in clones from symptomatic bleeding heart and inoculated Rutgers, but a glycine in clones from asymptomatic bleeding heart. However, the nt sequence differences between the two TRV lineages do not translate into significant variability in the predicted amino acid sequences for the region of the genome studied here. A BLASTn search of the NCBI database identified sequences that were 95.6 to 97.0% identical to the symptomatic bleeding heart and inoculated Rutgers variant and 95.0 to 96.4% identical to the asymptomatic bleeding heart variant. It is interesting to note that the accessions (JX JX ) with the greatest percent nt identity (95.6 to 97.0%) to the Ohio bleeding heart TRV variants are from Ohio peony (2), but distinctly different from the Ohio peony strain. It is also interesting to note that the TRV dsrna profile obtained from Ohio peony (2) differs from that obtained from Ohio bleeding heart (Fig. 2). The symptomatic bleeding heart variant produced a single major genomic dsrna (RNA 1) >10 Kb while RNA 1 of the Ohio peony strain is just over 6 Kb (2). The symptomatic bleeding heart variant produced a minor dsrna (presumably RNA 2) just over 1 Kb while RNA 2 of the Ohio peony strain is approximately 2.5 Kb (2). These results suggest that the two TRV variants infecting bleeding heart in Ohio are previously undescribed, and are different from the TRV strain previously reported in Ohio peony. These results also represent the first confirmed report of TRV infecting bleeding heart in Ohio and increase awareness of the disease among Ohio s perennial growers. Acknowledgements We thank Dr. Ben Lockhart (University of Minnesota) for supplying purified Tobacco rattle virus IgG. Literature Cited 1. Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., and Ball, L. A Tobravirus. Pages in: Virus Taxonomy, Eighth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, MA. 2. Fisher, J. R First report of Tobacco rattle virus associated with ring spot and line pattern disease of peony in Ohio. Online. Plant Health Progress doi: /php br. 3. Fisher, J. R 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: /php rs. 4. Jones, D., Farreyrol, K., Clover, G. R. G., and Pearson, M. N Development of a generic PCR detection method for tobraviruses. Australas. Plant Pathol. 37: King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J Virgaviridae. Pages in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press. Elsevier Academic Press, Waltham, MA. 6. Lockhart, B. E. L Dicentra, Epimedium, and Heuchera: New Perennial Ornamental Hosts of Tobacco rattle virus in the United States. Plant Dis. 84: Valverde, R. A., Nameth, S. T., and Jordan, R. L Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis. 74: