First Report of Tobacco Streak Virus Infecting Summer Squash in Georgia, U.S.A

Summer squash (Cucurbita pepo) is an important vegetable crop in the southeastern United States. During August 2018, viral symptoms were observed in less than 5% of yellow summer squash ‘Gentry’ in a 10-acre field in Grady County, GA. Foliar symptoms such as mosaic and greening in leaves, and fruit symptoms of large green sunken spots that coalesced to form green islands that were necrotic with water-soaked spots, were observed. Symptomatic fruit samples (n = 3) were tested by enzyme-linked immunosorbent assay for 16 different viruses. Of these only tobacco streak virus (TSV) and zucchini yellow mosaic virus were detected. TSV (Ilarvirus, Bromoviridae) has a broad host range infecting more than 200 plant species in 30 plant families (EPPO 2005). TSV is efficiently transmitted by thrips (Frankliniella occidentalis, and Thrips tabaci) and is frequently observed in Georgia. The virus is also transmitted by seed, pollen, and mechanical injury. TSV has been reported in zucchini squash in the neighboring state’s Miami-Dade County of Florida (Padmanabhan et al. 2014). TSV is a nonenveloped, quasi-spherical virion with tripartite (RNA1, RNA2, and RNA3) segmented linear (+) sense RNA genome (Bag et al. 2008). Polymerase chain reaction (PCR) primers were designed targeting the movement protein (MP) and the coat protein (CP) gene encoded by RNA 3 and subgenomic RNA 4, respectively. The upstream primer SB162F, 5′-TCAGCCTGACTGTTGGGTTGT-3′, and downstream primer SB162R, 5′-AGCTATGCATGTTGTTCATAGG-3′, target ∼938 bp encoding the CP gene. Another set of primers, SB164F, 5′-ACGATTTCCAACTTTGAATTCCTACAA-3′, and 164R, 5′-ATCTATCTCTAGAATTCATCAACTTAATACT-3′, generate an amplicon size of ∼1,131 bp encoding the MP gene. Total RNA was extracted from symptomatic tissue (n = 3) using the RNeasy mini kit (Qiagen, U.S.A.) following manufacturer’s recommendation. The cDNA was synthesized using Superscript III (Invitrogen, U.S.A.) and downstream reverse primers 162R and 163R following manufacturer’s recommendation. The cDNA products were further used to amplify the CP and MP genes using specific primer combinations stated above. Expected amplicons of size ∼900 bp for CP and ∼1,100 bp for MP were obtained from symptomatic tissue but not from RNase-free water. Reverse transcription (RT) PCR products were purified using a gel extraction kit (Qiagen) and sequenced using Sanger sequencing (GenScript, U.S.A.). Three independent RT-PCR products were sequenced, and the consensus sequences were analyzed using MEGA 7. The CP and MP gene sequences from this study (GenBank accession nos. MK307507 and MK307506) were compared against those available in GenBank and showed 99% sequence identity for both CP and MP genes of TSV isolate FL13-07 (GenBank accession no. KM504248.1) from Florida, confirming the presence of the virus in summer squash in Georgia. To our knowledge, this is the first report of TSV infecting summer squash in Georgia, U.S.A. The existence of diverse genotypes of TSV that are distinctive serologically with variable symptomatology in different plant species makes it difficult to accurately diagnose. The whitefly-transmitted viruses have been causing severe yield loss for vegetable growers in Georgia. The occurrence of a new thrips-transmitted virus in cucurbits with broad host range calls for an extensive survey of vector-transmitted viruses in Georgia and for further research in epidemiology, virus-virus, virus-vector, and virus-host interactions in commercial vegetable production.