First Report of Cucurbit aphid-borne yellows virus Infecting Cucurbita pepo in Oklahoma

Abstract

A survey of cucurbit fields was conducted in the September 2017 growing season in Oklahoma. A total of 121 symptomatic leaf samples (103 from pumpkin and 18 from squash) showing mosaic, mottling, and chlorosis and 10 asymptomatic samples from pumpkin (Cucurbita pepo) were collected from grower’s fields located in various counties. All samples were tested against the polyclonal antisera (AC Diagnostics, Fayetteville, AR) of 10 known cucurbit-infecting viruses including Papaya ringspot virus (PRSV), Watermelon mosaic virus (WMV), Zucchini mosaic virus (ZYMV), and Cucurbit aphid-borne yellows virus (CABYV, genus Polerovirus, family Luteoviridae) by dot-immunobinding assay (DIBA) as described previously (Ali et al. 2012). Eleven out of 121 symptomatic samples (eight samples from Blaine County and three from Payne County) of pumpkin reacted positively against the antisera of CABYV by DIBA, and the remaining samples were positive to WMV, PRSV, and ZYMV. None of the asymptomatic samples were positive against the 10 viruses in the DIBA. To further confirm the CABYV infection in these pumpkin samples, four of the eleven CABYV-DIBA-positive pumpkin samples (one from Payne County and three from Blaine County) were randomly selected, and total RNA was extracted using TRI reagent (Molecular Research). Reverse transcription polymerase chain reaction (PCR) was carried out using a pair of primers (CABYV-CP-F, 5′-ATGAATACGGCCGCGGCTAGAAATC-3′; CABYV-CP-R, 5′-CTATTTCGGGTTCTGGACCTGGCA-3′) that are specific to the coat protein (CP) gene of CABYV (Choi et al. 2015). Expected PCR products of approximately 600 bp were observed in all four Oklahoma samples (designated as isolates BL-4, BL-8, BL-45, and PY-46) when PCR products were analyzed on 1% agarose gel electrophoresis. An aliquot (5 µl) of PCR product from each sample was cleaned with 2 µl of ExoSAP-IT (Affymetrix). The cleaned PCR products were directly sequenced using ABI sequencer (3130XL genetic analyzer, Hitachi) at the core facility in the Department of Biological Science, University of Tulsa. The complete sequence of the CP gene of all four Oklahoma CABYV isolates (accession nos. MG590258, MG590259, MG590260, and MG590261) showed sequence identities from 98.0 to 98.3% with CABYV isolates from China (accession no. EU636992) and South Korea (accession no. KR231959) using the BLASTn algorithm, and the sequence identity among the four Oklahoma CABYV isolates ranged from 99.6 to 99.8%. Our results indicate the first report of CABYV infecting cucurbits in Oklahoma. CABYV was first described in France in 1992 (Lecoq et al. 1992), and in the United States it was first reported in California in 1993 (Lemaire et al. 1993). Cucurbits are economically important cash crops worldwide; therefore, the presence of CABYV poses a threat for cucurbit production in Oklahoma.