Detection of Cucurbit leaf crumple virus in Florida Cucurbits

Abstract

In October of 2006, yellow straightneck and zucchini squash plants (Cucurbita pepo L.) with crumpled, curled, thickened leaves were found in St. Johns and Marion counties in central Florida, respectively. Both locations had high populations of the whitefly, Bemisia tabaci. Incidences of symptomatic plants were greater than 95% in three squash fields (33 ha total) in St. Johns County and 35% in an experimental plot in Marion County. Twenty-three samples were collected from symptomatic plants (two from St. Johns County and 21 from Marion County). DNA was extracted for PCR and tested for the presence of begomoviruses using the following pairs of degenerate primers: AC1048/AV494, which amplifies a conserved region of the coat protein gene (2), PAR1c496/PAL1v1978, which amplifies a region of the begomovirus A component, and PBL1v2040/PCRc154, which amplifies a hypervariable region of the begomovirus B component (1). All squash samples yielded amplicons of sizes expected for a bipartite begomovirus: 1,159 nt with PAR1c496/PAL1v1978, 550 nt with AC1048/AV494, and 493 nt with PBL1v2040/PCRc154. The 1,159- and 493-nt amplicons obtained from two squash plants were cloned and sequenced. The 1,159 nt sequences from both plants shared 98% sequence identity with each other and 97% identity with equivalent regions of the A component of Cucurbit leaf crumple virus (CuLCrV) from Arizona and California (GenBank Accession Nos. AF256200 and AF224760, respectively). The 493-nt sequences amplified with PBL1v2040/PCRc154 were identical and shared a 96% identity with CuLCrV sequence (GenBank Accession No. AF327559) from Arizona and 97% identity with CuLCrV B component sequence (GenBank Accession No. AF224761) from California. Leaves were collected from eight symptomatic squash plants from Citra, FL and used for whitefly transmission assays. Approximately 100 adults of Bemisia tabaci biotype B were released onto each caged leaf and given a 24-h acquisition access period, after which a healthy squash seedling was introduced. Symptoms developed within 10 days on all test plants, and the presence of CuLCrV was confirmed by PCR assays, (primer pairs PAR1c496/PAL1v1978 and PBL1v2040/PCRc154) followed by sequencing. In 2007, similar symptoms were seen in several locations around the state. The same assays confirmed the presence of CuLCrV in watermelon (Citrullus lanatus L.) and squash in the following counties: Collier and Hendry in southwest Florida and Hillsborough, Manatee, and Sarasota in west-central Florida. To our knowledge, this is the first report of CuLCrV, and the first report of any begomovirus in cucurbits in Florida. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Development of Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Cucurbit Leaf Crumple Virus

“…CuLCrV is able to infect most cucurbits, including cucumber, cantaloupe, squash (yellow, zucchini, and winter squash), pumpkin, and watermelon [1,2] and has been reported to infect bean plants [3]. CuLCrV has been identified in several states of the United States, including Arizona, Texas, California [2,4], Florida [1,5], South Carolina [6] and Georgia [7], as well as in north-central Mexico [4]. Georgia and Florida are major cucurbit (squash, watermelon, pumpkin, cucumber and cantaloupe) producing states [8].…”

“…Several methods have been used to detect and distinguish CuLCrV from other begomoviruses. Among those, polymerase chain reaction (PCR) is the most commonly used molecular method [5,[11][12][13]. Degenerate primers are also widely used for PCR amplification to distinguish the identity of the virus from other begomoviruses [3,5,6,12,14].…”

Spatial and Temporal Analysis of Squash vein yellowing virus Infections in Watermelon

“…CuLCrV is a begomovirus that was discovered in cucurbit crops in the southwestern United States in 1998 (10,14). In October 2006, the virus was found in central Florida on yellow straightneck and zucchini squash (Cucurbita pepo L.) and then in watermelon in southwest and west-central Florida in 2007 (6), in green bean plants adjacent to infected watermelon plants (2), and in some common cucurbit weeds (4,5). CYSDV is a crinivirus first found in melon (Cucumis melo) in southern Texas and northern Mexico in 1999 and in Florida in watermelon and squash in 2007 (16,24).…”

Semipersistent Whitefly Transmission of Squash vein yellowing virus, Causal Agent of Viral Watermelon Vine Decline

“…However, until 2003, no whitefly-transmitted viruses had been found in cucurbit vegetables in Florida. Now, growers face the challenge of managing three such viruses, which have appeared in rapid succession: Squash vein yellowing virus (SqVYV) (1), Cucurbit leaf crumple virus (4), and Cucurbit yellow stunting disorder virus (22). Of these, SqVYV appears to be the most economically damaging (1,11).…”