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© 2009 Plant Management Network.
Accepted for publication 2 January 2009. Published 13 March 2009.


Report of a Watermelon mosaic potyvirus Strain in Kentucky Undetected by ELISA


Paul Vincelli and Kenneth Seebold, Department of Plant Pathology, University of Kentucky, Lexington, KY 40546


Corresponding author: P. Vincelli.  pvincell@uky.edu


Vincelli, P., and Seebold, K. 2009. Report of a Watermelon mosaic potyvirus strain in Kentucky undetected by ELISA. Online. Plant Health Progress doi:10.1094/PHP-2009-0313-01-BR.


During the 2007 and 2008 growing seasons, several cucurbit crops in Kentucky exhibiting stunting, mosaic, and foliar distortion typical of virus infection provided anomalous reactions in ELISA tests. Symptomatic plants reacted positively using an AgDia Inc., potyvirus group (PVG) ELISA test but reacted negatively to all viruses included in AgDia’s ELISA-based cucurbit virus screen (CVS). Similar results were reported from a virus survey of 13 states conducted in 2008 by the cucurbit IPM PIPE project (G. Holmes, personal communication). The AgDia Inc. CVS includes tests for Cucumber mosaic cucumovirus, Impatiens necrotic spot tospovirus, Papaya ringspot potyvirus, Squash mosaic comovirus, Tobacco mosaic tobamovirus, Tobacco ringspot nepovirus, Tomato mosaic tobamovirus, Tomato ringspot nepovirus, Tomato spotted wilt tospovirus, Watermelon mosaic potyvirus (WMV) (formerly Watermelon mosaic virus 2) (1), and Zucchini yellow mosaic potyvirus.

We selected two plants with representative symptoms from each of two pumpkin (Cucurbita pepo) fields in western and central Kentucky. Plants were collected in August 2008 and October 2008, respectively. Plants collected in western Kentucky gave a positive reaction using the PVG ELISA test, but were negative for all viruses in the CVS. Plants collected in central Kentucky also reacted positively in the PVG test; in addition, they gave a positive reaction in the WMV ELISA test in the CVS but were negative for all other viruses tested for in the CVS.

RNA was extracted from all four plant samples using a QIAGEN RNeasy Mini Kit, and reverse-transcribed using a QIAGEN Omniscript RT Kit. The resulting cDNA was PCR-amplified using Titanium Taq DNA polymerase and either the primer pairs POT1 and POT2 or Potyvirid2 and POT2 to yield a 1218-or 1019-bp sequence, respectively (2,3). Amplicons were gel-purified, ligated into Promega pGEM-T Easy vectors, and cloned in E. coli JM109 cells. Plasmid DNA was extracted using a QIAGEN QIAprep Miniprep Kit; the presence of the insert was verified by EcoR1 restriction digest, and sequencing of the insert was performed in both directions by the University of Kentucky’s Advanced Genetic Technology Center. After removing primer sequences, nucleotide sequences obtained were assembled using Contig Express in the Vector NTI Advance software suite, and assembled sequences were compared to those in GenBank using the BlastN algorithm.


   

Fig. 1. Nucleotide sequence alignment (5’ to 3’) with a Watermelon mosaic potyvirus isolate (GenBank accession DQ399708) with two potyviral strains that gave negative reactions (Pumpkin-1 and Pumpkin-3) and two potyviral isolates that gave positive reactions (Pumpkin-4 and Pumpkin-5) when tested with an AgDia Inc. Watermelon mosaic potyvirus (= WMV2) ELISA assay. The portion of the coat protein-coding region of DQ399708 represented in this figure spans bases 813-1212. A dot (.) indicates that the base matches that of DQ399708; a dash (—) indicates a gap in the alignment. (Download figure as PDF.)


Sequences obtained from all four plants had a 95 to 97% identity to multiple accessions of WMV over 98 to 100% of their length (for example, see Fig. 1). Sequence alignments revealed the presence of several single nucleotide polymorphisms in the coat protein-coding region between the samples that reacted positively to the WMV ELISA test and those that did not.

Our results indicate that a strain of WMV is present in Kentucky which does not react to the current AgDia Inc. ELISA test for WMV, a widely used assay for detection of this economically important virus. Prior to this report, it has been unknown whether the recent, widespread occurrence of PVG-positive/CVS-negative cucurbit samples was due to a new variant of a previously recognized potyvirus or a new potyvirus. This report identifies the causal agent for at least some of these anomalous cases. Further work is needed in order to fully characterize this new strain and to determine its prevalence. Significantly, this strain does not go completely undetected, since all four plants gave a strong positive reaction in the AgDia Inc. PVG test.

Tissue samples from the PVG-positive, CVS-negative plants analyzed in this study were provided to AgDia Inc., in order to facilitate the development of a WMV-specific ELISA test that will detect this new strain.


Literature Cited

1. Desbiez, C., and Lecoq, H. 2004. The nucleotide sequence of Watermelon mosaic virus (WMV, Potyvirus) reveals interspecific recombination between two related potyviruses in the 5’ part of the genome. Arch Virol 149:1619-1632.

2. Gibbs, A., and Mackenzie, A. 1997. A primer pair for amplifying part of the genome of all potyvirids by RT-PCR. J. Virol. Methods 63:9-16.

3. Ndunguru, J., Taylor, N. J., Yadav, J., Aly, H., Legg, J. P., Aveling, T., Thompson, T., and Fauquet, C. M. 2005. Application of FTA technology for sampling, recovery, and molecular characterization of viral pathogens and virus-derived transgenes from plant tissue. Virol. J. 2:45 doi:10.1186/1743-422X-2-45.