Identification of Two Tobacco rattle virus Sequence Variants Associated with Virus-like Mottle Symptom on Hosta in Ohio

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1 2013 Plant Management Network. Accepted for publication 21 December Published. Identification of Two Tobacco rattle virus Sequence Variants Associated with Virus-like Mottle Symptom on Hosta 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 sequence variants associated with virus-like mottle symptom on Hosta in Ohio. Online. Plant Health Progress doi: /php rs. Abstract Two Hosta sp. So Sweet plants and one Hosta sieboldii (labeled as Albomarginata ) plant showing a suspected virus-like leaf mottle symptom tested negative for the Potyvirus group, Hosta virus X, Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato ringspot virus, and Tomato spotted wilt virus by ELISA. DsRNA analysis produced a banding profile suggestive of a viral infection, and dsrna was used as template to synthesize cdnas for use with tobravirus group and Tobacco rattle virus (TRV) specific PCR primers. Amplicons were cloned and sequenced, and results showed two distinct populations of sequences: the two So Sweet isolates were ~99% identical to each other but only ~92% identical to the Albo-marginata isolate. These results represent the first confirmed report of TRV in Hosta in Ohio, and further demonstrate that there are at least two nucleotide sequence variants of the virus infecting Ohio Hosta. 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, RNA 1 and RNA 2. RNA 1 encodes a replicase, intercellular movement, and pathogenicity proteins. RNA 2 encodes the coat protein and those required for nematode transmission (1,4). TRV has a broad host range which includes ornamental, vegetable, and weed hosts, and is transmitted by nematodes in the genera Trichodorus and Paratrichodorus (1,4). In 2009 two Hosta sp. So Sweet samples (designated SoS6 and SoS8) and one Hosta sieboldii (labeled by the nursery as Albo-marginata ) sample (designated Alb6) exhibiting a suspected virus-like leaf mottle symptom (Fig. 1) were submitted to the Ohio Plant Diagnostic Network for analysis. All three samples tested negative for the Potyvirus group, Hosta virus X, Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato ringspot virus, and Tomato spotted wilt virus by enzyme-linked immunosorbent assay (ELISA) using commercially available antibodies (Agdia Inc., Elkhart, IN). Double stranded ribonucleic acid (dsrna) was purified from symptomatic leaf tissue as previously described (5) with results suggestive of a viral infection (Fig. 2), although the banding profile differed from the expected 6.8 Kb and Kb bipartite TRV genome (1,4). DsRNA was used as template for first strand cdna synthesis as previously described (2). 5 µl of cdna or sterile water was used for PCR with published tobravirus group and primer pairs (Integrated DNA Technologies Inc., Coralville, IA) to amplify regions in the 194 K RNA polymerase gene (nt and nt , respectively) as described (2,3). Amplification was done in 25 µl reactions using the following cycling parameters: 94 C (2 min); 30 cycles of 94 C (45 sec), 52 C (30 sec), 72 C (60 sec); 72 C (10 min). Gel purified PCR products were ligated into pgem-t Easy

2 plasmid (Promega Inc., Madison, WI), E. coli JM109 cells were transformed, and colonies were screened for an insert using M13 primers. Plasmid DNA was purified, sequenced (Plant Microbe Genomics Facility, The Ohio State University), vector was trimmed from raw sequences (Chromas v. 2.33), sequences were assembled and subjected to pairwise and multiple sequence alignments (Vector NTI Advance 11, Invitrogen Inc., Carlsbad, CA) as described (2), and clones were translated using Genedoc (Genedoc v , 2000). A Fig. 1. Asymptomatic Hosta sp. So Sweet leaf (A) and Hosta So Sweet 8 leaves showing virus-like mottle symptom (B). B Fig. 2. dsrna profiles from Hosta isolates So Sweet 8 (Lane 1), So Sweet 6 (Lane 2), and Albo-marginata 6 (Lane 3) testing positive for TRV by PCR. M = 1 Kb DNA ladder (250-10,000 bp markers indicated). Electrophoresis was done in 0.8% agarose at 100 volts for 60 min in 1X TAE buffer. Putative dsrnas are indicated by arrows. The tobravirus group primers and primers both amplified clear products of expected size with the two So Sweet and the Albo-marginata isolates; approximately 830 bp and 780 bp respectively (Fig. 3). Four clones each from SoS6, SoS8, and Alb6 of the tobravirus group amplicons, and five clones each from SoS6 and SoS8, and four from Alb6 of the TRV-specific amplicons were sequenced and the sequences deposited in GenBank (accession numbers JX JX267289). Clones generated with the tobravirus group primers were 830 bp for all three isolates and corresponded to nucleotides (nt)

3 (amino acids ) of the TRV open reading frame (ORF) 1 on RNA 1. Clones generated with the primers were 779 bp for all three isolates and corresponded to nt (amino acids ) of the TRV ORF 1. There were two distinct populations of sequences of each amplicon: the SoS6 and SoS8 clones (tobravirus group amplicon) had only 91.0 and 91.2% mean nt identity, respectively, with the Alb6 clones. Similarly, the SoS6 and SoS8 clones (TRV-specific amplicon) shared only 91.8 and 92.0% mean nt identity, respectively, with the Alb6 clones (TRV-specific amplicon). A comparison is shown in Table 1. BLASTn searches of the NCBI database identified sequences with ~91% nt identity (100% query coverage) with the SoS6 and SoS8 isolates and ~96% identity with the Alb6 isolate (tobravirus group amplicon). Similarly, BLASTn searches identified sequences with ~94-95% nt identity (100% query coverage) with the SoS6 and SoS8 isolates and 96% identity with the Alb6 isolate (TRV-specific amplicon). A summary is shown in Table 2. Fig. 3. PCR detection of TRV from Hosta spp. So Sweet 8 cdnas synthesized from dsrna template with tobravirus group (Lane 1) and (Lane 2) primers, water controls with tobravirus group (Lane 3) and (Lane 4) primers, and TRV positive control with tobravirus group (Lane 5) and (Lane 6) 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. Table 1. Comparison of percent nucleotide sequence identities between TRV-Hosta isolates So Sweet 6, So Sweet 8, and Albo-marginata 6 tobravirus group and TRVspecific amplicons. Tobra group SoS6 a SoS8 b Alb6 c SoS SoS Alb SoS SoS Alb

4 Table 2. NCBI database accessions having the greatest percent nucleotide identities to TRV-Hosta isolates So Sweet 6, So Sweet 8, and Albo-marginata 6 tobravirus group and TRV-specific amplicons. Tobra group Geographic region SoS6 a SoS8 b Alb6 c AF AF AF North America GQ Michigan AF Netherlands AJ Netherlands JX Ohio AF AF AF North America GQ Michigan AF Netherlands AJ Netherlands JQ HM JX Ohio 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 (4). These results show that the TRV isolates from Ohio Hosta sp. So Sweet and Hosta sieboldii ( Albo-marginata ) are distinctly different from each other in the nt and nt regions of ORF1 spanned by the tobravirus group and TRV-specific primers, respectively. Further, BLASTn searches identified sequences with maximum nt identity of 96% (Alb6) and 92% (SoS6, SoS8) for the tobravirus group amplicons and ~96% (Alb6) and ~94-95% (SoS6, SoS8) for the TRV-specific amplicons suggesting the two TRV sequence variants from Ohio Hosta are unique. When translated, the SoS6, SoS8, and Alb6 isolates (tobravirus group amplicons) had the greatest predicted mean a.a. identities to TRV isolates from Ohio peony (2) and Michigan potato. Likewise, the SoS6, SoS8, and Alb6 isolates (TRV-specific amplicon) had the greatest predicted mean amino acid (aa) identities to the same TRV isolate from Ohio peony (2). A comparison of percent predicted a.a. identities to GenBank accessions is shown in Table 3.

5 Table 3. NCBI database accessions having the greatest predicted percent amino acid identities to TRV-Hosta isolates So Sweet 6, So Sweet 8, and Albo-marginata 6 tobravirus group and TRV-specific amplicons. Geographic Tobra group region SoS6 a SoS8 b Alb6 c JX Ohio AF GQ Michigan JX Ohio AF JQ The results presented here demonstrate that at least two TRV ORF 1 nucleotide sequence variants exist in Ohio Hosta sp. So Sweet and Hosta sieboldii (Albo-marginata) cultivars, but the differences do not translate into significant variability in the predicted amino acid sequences for that region of the genome. These results represent the first confirmed report of TRV in Hosta spp. in Ohio and expand the known host range of the virus in the state. This report also serves to increase awareness of the potential threat this virus may pose to Ohio s home gardens and Hosta producing nurseries. Literature Cited 1. Fauquet, C. M., Mayo, M. A., Maniloff, J., Desselberger, U., and Ball, L. A Tobacco rattle virus. 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. 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, Waltham, MA. 5. Valverde, R. A., Nameth, S. T., and Jordan, R. L Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis. 74: