First Report of Cotton leaf curl Gezira virus and Its Associated Alphasatellite and Betasatellite from Disease Affected Okra Plants in the United States

Okra (Abelmoschus esculentus [L.] Moench) is an important fresh market vegetable crop in southern Texas, U.S.A. In fall 2018, an investigation was conducted to identify the agent(s) of a virus-like disease outbreak on a 4.9-ha okra (cv. Clemson Spineless) farm in Hidalgo Co., Texas. By mid-growing season, disease incidence was 100%, which led to total crop loss. Symptoms—foliar yellow mosaic, bleaching, and vein clearing—were reminiscent of those caused by begomoviruses (Begomovirus, Geminiviridae). Leaves collected from 10 randomly distributed symptomatic plants were subjected to total DNA isolation (Dellaporta et al. 1983) and rolling cycle amplification (RCA) (TempliPhi 100 Amplification Kit, GE Healthcare Biosciences). The RCA product was used as a template for PCR amplification of a diagnostic fragment of the DNA-A (Wyatt and Brown 1996) and DNA-B (Rojas et al. 1993) components and full-length alphasatellites (Bull et al. 2003) and betasatellites (Briddon et al. 2002). The expected-size amplicon corresponding to each of the four targets was obtained from all 10 samples, except the betasatellite, for which half of the expected ∼1,350-bp product resulted. Amplicons were cloned into the pJET1.2 plasmid vector, and inserts of the expected sizes were Sanger sequenced (n ≥ 3 per event). BLASTn searches of GenBank revealed an unexpectedly complex begomovirome, consisting of the monopartite cotton leaf curl Gezira virus (CLCuGV), cotton leaf curl Gezira alphasatellite (CLCuGA; Colecusatellite, Alphasatellitidae), cotton leaf curl Gezira betasatellite (CLCuGB; Betasatellite, Tolecusatellitidae), and the bipartite okra yellow mosaic Mexico virus (OkYMMV). Viral genome-, component-, and satellite-specific abutting primers were designed and used to PCR amplify the corresponding molecule from two RCA products in two different labs, followed by cloning and bidirectional Sanger sequencing of ≥3 clones per event. The complete genome sequences were analyzed comparatively with corresponding GenBank sequences of each genomic DNA segment using SDT1.2 (Muhire et al. 2014). The OkYMMV DNA-A of 2,613 nt (GenBank MN027195 to 96) and DNA-B of 2,594 nt (MN027197 to 98), each shared 99.5 and 99.7% maximum identity, respectively, with the DNA-A (HM035059) and -B (HM035060) of OkYMMV-[US:TX:09] from okra plants in Texas (Hernandez-Zepeda et al. 2010). The complete monopartite genome sequence of the CLCuGV okra isolates (MN027199 to 202) was 2,764 nt and shared 99.5 to 99.6% maximum identity with an isolate of CLCuGV from hollyhock plants in Jordan (CLCuGV-[JO:Hol:09]: GU945265). The closest relative of the CLCuGA okra isolate, of 1,354 nt (MN027203 to 05), was CLCuGA-[IL:IsSq-C171:11] (KT099176) from whiteflies collected from squash plants in Israel, at 99% identity. Several approaches by two independent laboratories to clone and sequence a full-length CLCuGB, involving restriction digestion from total DNA or RCA (n = 2) and/or PCR amplification with abutting primers designed based on the full-length CLCuGB sequences (AF397217, KT099178), yielded only “defective-sized” CLCuGB molecules of 676 to 730 nt (MN027206 to 220). The betasatellite half-mers lacked the βC1 coding region while sharing 96 to 99% identity with CLCuGB-[EG:OK03:02] (AF397217) from okra plants in Egypt and CLCuGB-[IL:IsSq-C9:11] (KT099178) from whiteflies. This is the first U.S. report of CLCuGV and its associated alphasatellite and betasatellite, which are endemic to Africa (Idris and Brown 2000). More recently, members of the CLCuGV-satellite complex have been reported in the African Sahel, Arabian Peninsula and Middle East, and Pakistan (Idris et al. 2014; Tahir et al. 2011; Tiendrebeogo et al. 2010). The source of this Old World CLCuGV-satellite complex into New World okra plants is unknown, but its introduction via infected plants or viruliferous whiteflies transported into southern Texas, with its intensive cotton and vegetable production, is plausible. Spread of this complex poses a serious threat because U.S. cotton and vegetable varieties harbor no resistance to CLCuGV.