First Report of a Novel Potyvirus from Florida Causing Chlorotic Mottling in Squash (Cucurbita pepo)

Abstract

During the 2010 to 2011 growing seasons, nine cucurbit leaf samples including cantaloupe, cucumber, pumpkin, squash, and watermelon, showing mosaic and mottling, were collected from fields in the Homestead and Tampa areas in Florida (1). Eight of the nine samples were positive by dot-immunobinding assay (DIBA) and reverse transcription (RT)-PCR for either Watermelon mosaic virus (WMV), Papaya ringspot virus (PRSV-W), or mixed infection of both viruses. One squash sample from the Homestead area showing unique symptoms including chlorotic spots, yellowing, mottling, vein clearing, and mild mosaic was negative by RT-PCR against PRSV-W, Squash vein yellowing virus (SqVYV), WMV, and Zucchini yellow mosaic virus (ZYMV).The presence of virus-like particles (VLP) from symptomatic squash leaves (1) was prepared as described previously (2). Typical potyvirus-like particles ~700 nm long and 12 to 14 nm wide were observed by electron microscope from VLP preparations. Analysis of VLP on SDS-PAGE demonstrated a slightly larger coat protein (CP) (37 kDa compared with PRSV-W [35 kDa]). Sap from symptomatic squash leaf samples or VLP was mechanically inoculated to 10 squash seedlings at cotyledon stage using 0.1 M K2HPO4 buffer. Chlorotic spots were observed on the first true leaf 7 days post inoculation. However, symptoms became more severe by 2 to 3 weeks post inoculation and systemically infected leaves showed chlorosis and mottling similar to the original symptoms when tissues were collected from the field. Mock-inoculated control squash seedlings did not produce any symptoms. Symptomatic leaves from mechanically infected squash plants were used for VLP preparations and virus particles and size of the CP on SDS-PAGE was observed as before. Total RNA was extracted from VLP (2) and tested by RT-PCR using universal Potyviridae primers (forward primer 5′-CACGGATCCCGGG (T)17AGC and reverse primer 5′-GGBAAYAAYAGYGGDCARCC (3) to amplify a fragment from the 3′ end of the genome (including part of NIb gene, whole CP). A band of 1.2 kb was observed when the PCR product was analyzed on 1% agarose gel. PCR product was purified using QIAquick PCR Purification Kit (QIAGEN, USA), cloned (pGEM-T Easy Vector, Promega, USA), and sequenced in both directions. Consensus sequence was obtained from at least five clones and submitted to GenBank (KC522968). A BLASTn comparing the sequence from the squash potyvirus to others in GenBank found the highest similarity was 72.0% at nucleotide level and 64.8% at amino acid level with PRSV-W (JN831646), and less than 70% nucleotide similarity with WMV (NC_006262) and SqVYV (NC_010521). Based on the particle morphology, CP size on SDS-PAGE, nucleotide identity with other cucurbit potyviruses, and unique symptoms, it is concluded that this could be a new potyvirus. The threshold for classifying distinct species in Potyviridae is less than 76% identity at nucleotide level for either CP gene or the whole genome (4). This virus has been tentatively named as Squash chlorosis mottling virus (SqCMV). Florida is one of the leading states in acreage and production of cucurbits in the United States. The emergence of this new virus could be a potential future threat to cucurbits production. References: (1) A. Ali et al. Plant Health Progress. Online publication. doi:10.1094/PHP-2012-0824-01-RS, 2012. (2) A. Ali et al. Plant Dis. 96:243, 2012. (3) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (4) A. M. Q. King et al. Virus Taxonomy-ICTV 9th Report:1071, 2012.