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© 2013 Plant Management Network.
Accepted for publication 22 May 2013. Published 13 August 2013.


Identification of Poinsettia mosaic virus Associated with a Virus-like Mottle Symptom on Poinsettia in Ohio


John R. Fisher, Ohio Department of Agriculture, Plant Health Diagnostic Laboratory, Plant Health Division, Reynoldsburg, OH 43068; and John Bell, The Ohio State University, C. Wayne Ellett Plant and Pest Diagnostic Clinic, Reynoldsburg, OH 43068


Corresponding author: John R. Fisher.  jfisher@agri.ohio.gov


Fisher, J.áR.,áandáBell, J. 2013. Identification of Poinsettia mosaic virus associated with a virus-like mottle symptom on poinsettia in Ohio. Online. Plant Health Progress doi:10.1094/PHP-2013-0813-01-BR.


Poinsettia mosaic virus (PnMV) is a member of the family Tymoviridae currently unassigned to a genus. The virus has a 6099-nucleotide (nt) single-stranded, positive-sense RNA genome containing a single long open reading frame (ORF) which includes fused replication and coat protein domains (2). In the fall of 2010 several poinsettia samples exhibiting a virus-like mottle symptom (Fig. 1) were submitted to the Ohio Plant Diagnostic Network for disease analysis. The samples tested negative for Alfalfa mosaic virus, Arabis mosaic virus, Cucumber mosaic virus, Impatiens necrotic spot virus, Tobacco mosaic virus, Tobacco ringspot virus, Tomato spotted wilt virus, and Tomato ringspot virus by ELISA using commercially available antibodies (Agdia, Inc., Elkhart, IN).


 

Fig. 1. Virus-like mottle symptom observed on Poinsettia leaves.

 

Turnip yellow mosaic virus (TYMV), Erysimum latent virus (ErLV), Eggplant mosaic virus (EPMV), Physalis mottlevirus (PhMV), Grapevine fleck virus (GFV), and PnMV genome sequences (accession numbers X16378.1, AF098523.1, J04374.1, Y16104.1, AJ309022.1, and NC_002164.1, respectively) were used to design two sets of degenerate primers (TYMOfwd290: 5'-SCACAAGAYMATCGARACHHWTCTC-3'; TYMOrev1073: 5'-CCAGGCRKWSGAVGTGACCCARKMG-3'; TYMOfwd4381: 5'-CSCARCACAARRTSAAYGAMGSSTC-3'; TYMOrev4759: 5'-GCAYGTKAGRGGSCCRAACTGGGTG-3') corresponding to the regions spanning approximately nt 290-1073 and 4381-4759 of the TYMV genome. Additionally, two sets of PnMV-specific primers (PnMVfwd264: 5'-GGTCATCAAGCCCACCCTCATC-3'; PnMVrev1098: 5'-GGAGAGAAAAGTGCTGGAGATTG-3'; PnMVfwd1023: 5'-CGCACACAGTCCAACAAGCCAG-3'; PnMVrev2195: 5'-CGAAGTCTGACCGCCCATAG-3') were designed corresponding to nt 264-1098 and nt 1023-2195 of the PnMV genome, respectively. Double-stranded RNA (dsRNA) was purified from symptomatic tissue from one of the samples and used as a template for cDNA synthesis as previously described (1,3). cDNAs and sterile water were used as template for PCR amplification with the above primer pairs in 25 Ál reactions (5 Ál 5X GoTaq Flexi buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.2 ÁM primer pair, 0.625 units GoTaq polymerase; Promega Inc., Madison, WI) using the cycling conditions: 94°C (2 min), 40 cycles of 94°C (45 sec), 52°C (30 sec), 72°C (1 min), and 72°C final extension (10 min).

The TYMOfwd290 + rev1073 and TYMOfwd4381 + rev4759 primers both amplified a single, distinct product of expected size (~780 and 380 bp, respectively). The PnMVfwd264 + rev1098 primers amplified two products; one strong product of expected size (~830 bp) and a smaller, unexpected product (<250 bp). The PnMVfwd1023 + rev2195 amplified a product of expected size (~1170 bp) but it was much weaker (Fig. 2). The DNA was cut from the gel, cloned as previously described (1), and sequenced in both directions using M13 primers (Plant Microbe Genomics Facility, The Ohio State University). Vector was trimmed from raw sequences (Chromas v. 2.33), contigs were assembled, subjected to pairwise and multiple sequence alignments with reference sequences (Vector NTI Advance 11, Invitrogen, Inc.), and the ORFs were translated (Genedoc v. 2.6.001, 2000).


 

Fig. 2. PCR amplification of PnMV ORF 1 regions from cDNAs synthesized from dsRNA template with degenerate TYMOfwd290 + rev1073 and TYMOfwd4381 + rev4759 (Lanes 1 and 2, respectively) tymovirus and PnMVfwd264 + rev1098 and PnMVfwd1023 + rev2195 (Lanes 3 and 4, respectively) PnMV primers. Water controls with TYMOfwd290 + rev1073, TYMOfwd4381 + rev4759 (Lanes 5 and 6, respectively), PnMVfwd264 + rev1098, PnMVfwd1023 + rev2195 (Lanes 7 and 8, respectively), and Tobacco rattle virus (TRV) specific primers (Lane 9). A TRV clone with TRV specific primers was used as a positive PCR control (Lane 10) due to identical reaction and cycling conditions to those used for the PnMV and degenerate tymovirus primers. M = 1 Kb DNA ladder (250, 500, 750, 1000, 1500 bp markers indicated). Electrophoresis was performed in 0.8% agarose at 100 volts for 60 min in 1X TAE buffer.

 

Five and six of the degenerate TYMOfwd290 + rev1073 and TYMOfwd4381 + rev4759 amplicon clones were sequenced and all were 784 and 354 bp, respectively. The primers were trimmed, and the resulting 736 and 338 nt sequences were deposited in GenBank (accession numbers KC533856- KC533866). Five of the PnMVfwd264 + rev1098 amplicon clones were also sequenced. All were 835 nt and the processed sequences were deposited in GenBank (accession numbers KC533851- KC533855). No clones were obtained from the PnMVfwd1023 + PnMVrev2195 amplicon. The TYMOfwd290 + rev1073 clones corresponded to nt 315-1050 of the PnMV genome and when translated, the predicted amino acid (aa) sequence corresponded to aa 78-322 of the PnMV ORF 1 protein. The TYMOfwd4381 + rev4759 clones corresponded to nt 4407-4744 of the PnMV genome and when translated, the predicted aa sequence corresponded to aa 1442-1553 of the PnMV ORF 1 protein. The PnMVfwd264 + rev1098 clones corresponded to nt 264-1098 of the PnMV genome and when translated, the predicted aa sequence corresponded to aa 61-338 of the PnMV ORF 1 protein. A summary of percent nt and aa sequence identities of the cloned amplicons is presented in Table 1. BLASTn searches of the NCBI database were performed using the sixteen clone sequences with default settings (100% query coverage) to identify sequences with the greatest percent nt identity with the PnMV isolate from Ohio. These results are also summarized in Table 1.


Table 1. Summary of percent nucleotide (nt) sequence identities, percent amino acid (aa) sequence identities, and BLASTn maximum identities of cloned amplicons obtained from degenerate tymovirusA,B and PnMVC specific primers.

     TYMOfwd290/
TYMOrev1073A
TYMOfwd4381/
TYMOrev4759B
PnMVfwd264/
PnMVrev1098C
% nt sequence identity
(mean)
98.5-99.6
(99.0)
97.0-100
(98.7)
96.4-100
(97.6)
% aa sequence identity
(mean)
99.1-100
(99.5)
95.3-100
(97.2)
99.3-100
(99.7)
BLASTn maximum % nt
identity (accession no.)
98
(AB550788.1)
99
(AJ271595.1)
98
(AB550788.1)

The results presented here demonstrate that the virus-like mottle symptom observed on poinsettia can be associated with PnMV infection. Both the degenerate tymovirus primers and the PnMV specific primers amplified products whose nt and predicted aa sequences confirmed the amplicons were from PnMV. The PnMVfwd264 + rev1098 primer pair also amplified a second low MW product. That product was cloned and sequenced and was found to be a 187 bp fragment corresponding to the 3' end of the full length PnMVfwd264 + rev1098 amplicon, indicating the 5' primer is sufficiently complementary to a second site with the annealing temperature used for cycling. These results represent the first confirmed report of PnMV in poinsettia in Ohio. They also serve to increase awareness of the virus among poinsettia growers so they may more effectively scout nursery stock for infected plants.


Literature Cited

1. 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.

2. King, A. M. Q., Adams, M. J., Carstens, E. B., and Lefkowitz, E. J. 2012. Tymoviridae. Pages 944-952 in: Virus Taxonomy, Ninth Report of the International Committee on Taxonomy of Viruses. Elsevier Academic Press, Waltham, MA.

3. 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.