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Plant Health Brief

First Report of Squash vein yellowing virus in Watermelon in Guatemala

Ayyamperumal Jeyaprakash, Carlye A. Baker, and Timothy S. Schubert, FDACS-DPI, Gainesville, FL 32608; Ismael E. Badillo-Vargas, University of Florida, NFREC, Quincy, FL 32351; Pamela D. Roberts, University of Florida, SWFREC, Immokalee, FL 34142; Joseph E. Funderburk, University of Florida, NFREC, Quincy, FL 32351; and Scott Adkins, USDA-ARS, Fort Pierce, FL 34945

Accepted for publication 14 July 2015. Published 15 July 2015.

Jeyaprakash, A., Baker, C. A., Schubert, T. S., Badillo-Vargas, I. E., Roberts, P. D., Funderburk, J. E., and Adkins, S. 2015. First report of Squash vein yellowing virus in watermelon in Guatemala. Plant Health Progress doi:10.1094/PHP-BR-15-0019.

Watermelon (Citrullus lanatus) and other cucurbits are important crops grown in Guatemala for local consumption and export. Several viruses are known to infect cucurbits in Guatemala, including the crinivirus Cucurbit yellow stunting disorder virus (CYSDV), begomovirus Melon chlorotic leaf curl virus (MCLCuV), and potyvirus Papaya ringspot virus (PRSV). During March and April 2015, watermelon plants growing on 2,000 ha in Zapaca in eastern Guatemala were observed with virus-like symptoms of leaf yellowing, mosaic, and deformation typical of those induced by CYSDV, MCLCuV, and PRSV. In addition, some plants were observed to wilt and collapse near harvest. Fruits exhibiting internal rind necrosis (Fig. 1) resembling symptoms of viral watermelon vine decline caused by Squash vein yellowing virus (SqVYV) (Adkins et al. 2013) were also observed. The whitefly (Bemisia tabaci) vector of CYSDV, MCLCuV, and SqVYV was observed in these fields with numbers increasing during the season.

Four samplings of crowns, peduncles, and/or leaves of symptomatic plants were made in March and April 2015. Total RNA was extracted (RNeasy Plant Mini Kit, Qiagen, Valencia, CA) from symptomatic plant tissue and tested by RT-PCR for SqVYV, CYSDV, PRSV, and/or begomoviruses as previously described (Adkins et al. 2008, 2009). Primers specific for the coat protein gene of SqVYV (1020 bp), CYSDV (707 bp), or PRSV (511 bp), and degenerate begomovirus primers (1159 or 533 bp) amplified products of the expected sizes from 15 of 24, 20 of 24, 4 of 24, or 8 of 8 plants, respectively. SqVYV amplicons from six individual plants from the fourth sampling, SqVYV and CYSDV amplicons from a pool of plants from the third sampling, and degenerate begomovirus amplicons from the second sampling were cloned in the pGEM-T vector (Promega, Madison, WI). Five clones of each amplicon were sequenced in both directions and representative consensus sequences were deposited in GenBank (Accession Nos. KT007178 to KT007183). Sequence analysis demonstrated that SqVYV coat protein gene sequences from Guatemala shared 99 to 100% nucleotide (nt) identity with each other, and 97 to 98% nt identity with divergent SqVYV isolates previously described from Florida (e.g., WM2005aHi, GenBank

Accession No. JF897974) (Webster and Adkins 2012) and California (GenBank Accession No. KP218061) (Batuman et al 2015), but only 90% nt identity with the predominant SqVYV isolate found in Florida (e.g., Sq2003Hi, GenBank Accession No. EU259611) (Webster and Adkins 2012). Tissue blots were prepared from crowns and peduncles from the first, third, and fourth samplings, and tested by tissue blot nucleic acid hybridization assay for SqVYV (Turechek et al. 2010). Tissue blots indicated SqVYV infection in an additional 48 of 102 watermelon samples, and cylindrical inclusions typical of SqVYV (Adkins et al. 2007) were observed in phloem tissue from the fourth sampling by light microscopy, confirming the identification of SqVYV.

Further analysis of virus sequences from Guatemala samples demonstrated that the CYSDV coat protein gene shared 89 to 100% nt identity with CYSDV sequences in GenBank, and that begomovirus sequences shared >94% nt identity with MCLCuV sequences in GenBank. Three mixed infections of CYSDV, MCLCuV, and PRSV were detected in eight plants from the second sampling. Twelve mixed infections of SqVYV and CYSDV were detected in 16 plants from the third and fourth

FIGURE 1 Internal rind necrosis in watermelon fruit harvested from a Squash vein yellowing virus-infected plant in Guatemala .

Corresponding author: Scott Adkins. Email: scott.adkins@ars.usda.gov

doi:10.1094 / PHP-BR-15-0019 This article is in the public domain and not copyrightable.

It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2015

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samplings. These results indicate that infection of cucurbits in Guatemala with two or more viruses was a common occurrence during this growing season.

Due to similarity of fruit symptoms to those induced by the tobamovirus Cucumber green mottle mosaic virus (CGMMV), 16 plants from the third and fourth samplings were also tested for CGMVV by RT-PCR using previously described primers (Tian et al. 2014) and commercially available lateral flow immunoassay reagents (Agdia, Inc., Elkhart, IN). No samples were found positive by either method. Furthermore, no ~300-nm, rigid, rod-shaped virions typical of CGMVV were observed by transmission electron microscopy of crown and peduncle samples, although numerous ~700-nm, flexuous, rod-shaped virions typical of CYSDV were present.

To the best of our knowledge, this is the first report of SqVYV infecting watermelon in Central America. Abandonment of fields that would normally have been harvested four times after a single harvest represents a major economic loss. Expansion of the geographic range of SqVYV to Guatemala provides further opportunity for spread of this emerging whitefly-transmitted virus to additional locations. The similarity of Guatemala SqVYV isolate sequences to those of divergent SqVYV isolates from Florida and California suggests that other more diverse SqVYV isolates may be detected in the future.

LITERATURE CITED

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Adkins, S., Webb, S. E., Achor, D., Roberts, P. D., and Baker, C. A. 2007. Identification and characterization of a novel whitefly-transmitted member of the family Potyviridae isolated from cucurbits in Florida. Phytopathology 97:145-154.

Adkins, S., Webb, S. E., Baker, C. A., and Kousik, C. S. 2008. Squash vein yellowing virus detection using nested polymerase chain reaction demonstrates that the cucurbit weed Momordica charantia is a reservoir host. Plant Dis. 92:1119-1123.

Adkins, S., Webster, C. G., Baker, C. A., Weaver, R., Rosskopf, E. N., and Turechek, W. W. 2009. Detection of three whitefly-transmitted viruses infecting the cucurbit weed Cucumis melo var. dudaim in Florida. Plant Health Progress doi:10.1094/PHP-2009-1118-01-BR.

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Turechek, W. W., Kousik, C. S., and Adkins, S. 2010. Distribution of four viruses in single and mixed infections within infected watermelon plants in Florida. Phytopathology 100:1194-1203.

Webster, C. G., and Adkins, S. 2012. Low genetic diversity of Squash vein yellowing virus in wild and cultivated cucurbits in the U.S. suggests a recent introduction. Virus Res. 163:520-527.