First report of chilli leaf curl virus and tomato leaf curl betasatellite infecting watermelon (Citrullus lanatus) in Oman

Watermelon (Citrullus lanatus; family Cucurbitaceae) is an important food crop in Oman. In November 2015, C. lanatus plants exhibiting leaf curl as well yellowing and crumpling of leaves were observed at the Agricultural Experimental Station at Sultan Qaboos University Oman. Disease incidence in the fields was in the range of 70 to 80%. Due to the presence of large numbers of whiteflies (Bemisia tabaci, the vector of begomoviruses) on the plants, a begomovirus (genus Begomovirus, family Geminiviridae) was suspected to be the causal agent. Four symptomatic and two nonsymptomatic samples were collected. Crude sap extracted from symptomatic leaves was examined using electron microscopy and twinned-icosahedral particles (18 to 20 nm), typical of geminiviruses, were observed. DNA was extracted from symptomatic and apparently healthy plants using the CTAB method (Doyle and Doyle 1987). The DNA was subjected to rolling-circle amplification (RCA) using TempliPhi 100 Amplification System (GE Healthcare). Digestion of the concatameric RCA products with BamHI released an ∼2.7 kb fragment, which was cloned into a pGEM-3zf (+) (Promega) and sequenced. The complete nucleotide sequences of seven clones, with at least one from each plant, were determined and found to have >99% sequence identity For this reason, only one full-length clone, of 2,760 bp, was submitted to the databases (accession no. KX787939). A Blastn comparison with sequences available in the databases showed the sequence to have the highest levels of nucleotide sequence identity (99%) to a Chilli leaf curl virus isolate (ChiLCV; HG969264) originating from Oman. No potential recombination events were detected for the ChLCV isolate using the recombination detection program (Martin et al. 2015). This is the first report of ChiLCV infecting C. lanatus. PCR was used to amplify betasatellites using primers Sat101(5′-GTAGGTACCACTACGCTACGCAGCAGCC-3′)/Sat102 (5′-AGTGGTACCTACCCTCCCAGGGGTACACAC-3′). The amplified PCR products were cloned in PTZ257R/T (Thermo Scientific) and sequenced. The complete nucleotide sequences of four clones, one from each plant, were obtained and shown to have 99 to 100% identity. The sequence of one clone (1,378 bp) was submitted to the databases (KX787940). The sequence showed the highest levels of nucleotide sequence identity (97%) to a Tomato leaf curl betasatellite (ToLCB) isolate from Capsicum annuum originating from Oman (HE800540). No alphasatellite or possible DNA-B component was detected, either by digestion of RCA products or by PCR using specific primers (Bull et al. 2003; Rojas et al. 1993). To our knowledge, this is the first report of a monopartite begomovirus associated with betasatellite infecting C. lanatus in Oman. ChiLCV has been reported to infect several important horticulture crops in South Asia and Oman; the addition of another host, C. lanatus, indicates the importance of ChiLCV and ToLCB and likely facilitates the spread of the virus in Oman.