Zucchini tigre mosaic virus Infection of Cucurbits in Florida
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2016, American Phytopathological Society. All rights reserved. A zucchini squash (Cucurbita pepo) with foliar chlorosis and mosaic was collected in Miami-Dade County in April 2002 as part of a 931-sample Florida cucurbit virus survey (Webb et al. 2003). Zucchini yellow mosaic virus (ZYMV) was readily identified by enzyme-linked immunoassay. However, systemic symptoms in squash cv. Prelude II (resistant to ZYMV) mechanically inoculated with the original sample indicated the presence of another virus. Foliar tiger stripe symptoms appeared 7 days postinoculation in squash and pumpkin plants (C. pepo; multiple cultivars) that were mechanically inoculated with sap from systemically infected Prelude II. Immunodiffusion assays with antiserum to what was then known as Papaya ringspot virus type tigr (PRSV-T; Quiot-Douine et al. 1986) demonstrated a reaction of identity between PRSV-T antigen and extracts from squash and pumpkin plants inoculated with Prelude II sap. PRSV-T has subsequently been recognized as a distinct potyvirus species: Zucchini tigr mosaic virus (ZTMV; Romay et al. 2014). Total RNA was extracted from a pumpkin plant inoculated with Prelude II sap and tested by RT-PCR with degenerate potyvirus primers as previously described (Gibbs and Mackenzie 1997). Amplicons of the expected size (1.8 kb) were cloned and sequenced, and 293 aa of the coat protein showed 96% identity with a ZTMV isolate from Guadeloupe (GenBank Accession No. AGY36170) representing the first detection of this virus in Florida. In September 2015, six butternut (C. moschata) and two spaghetti (C. pepo) squash samples with foliar tiger stripe symptoms were collected in Palm Beach County. Initial testing with commercial lateral flow immunoassay reagents for potyviruses (Agdia, Inc., Elkhart, IN) gave positive results for 6 of 8 samples. At this time, total RNA was extracted from 2002 (pumpkin cv. Small Sugar inoculated with Prelude II sap) and 2015 samples using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and tested by RT-PCR with ZTMV-6K1-5 and ZTMV-6K2-3 primers (Romay et al. 2014) that amplify the ZTMV cylindrical inclusion (CI) coding region. Amplicons of the expected size (2.1 kb) were produced from the 2002 sample and 6 of 8 2015 samples and cloned in pGEM-T (Promega, Madison, WI). Five clones of each amplicon were sequenced in both directions. Consensus sequences were deposited in GenBank (KT992780 to KT992783). The CI coding regions of 2002 and 2015 isolates showed the highest identity to ZTMV isolates from Guadeloupe (95%; Accession No. KC345605) and Venezuela (92%; Accession No. KC345606), respectively. The 2002 isolate was 89% identical to all 2015 isolates, and 2015 isolates were 99% identical to each other. The diversity of CI coding region of 2002 and 2015 isolates may indicate evolution of ZTMV in, or multiple introductions to, Florida. All 2015 samples were also tested for whitefly-transmitted viruses as previously described (Adkins et al. 2009) and found positive for Cucurbit yellow stunting disorder virus. Collectively, these results demonstrate that ZTMV has been present in Florida since at least 2002, often in mixed infections, although determining its prevalence in the state awaits further study.
