First Report of Watermelon chlorotic stunt virus Infecting Watermelon in Saudi Arabia

Abstract

In the Saudi Arabian deserts, watermelon [Citrullus lanatus (Thunb.)] is cultivated in the lowlands and wadis (washes) where water accumulates following rainfall, and where heat, salt, and drought stress are common constraints on production. During the spring of 2014, watermelon leaves exhibited yellowing and severe chlorotic mottling symptoms. The foliar symptoms were reminiscent of Watermelon chlorotic stunt virus (WmCSV), a bipartite begomovirus previously reported in several neighboring countries (1,3). Ten samples were collected from three farms in the Leith region, where 100% of the watermelon plants were symptomatic. Total nucleic acids were extracted from the symptomatic watermelon plants and were subjected to PCR using WmCSV DNA-A specific primers designed based on a complete genome sequence (GenBank Accession No. AJ012081), WmCSVF-3′-CGTGCTGTTGCCCCCACTGT-5′ and WmCSVR-3′-CCTGCATATCTCGTGCCAGAATC-5′ to obtain an expected size fragment of 1,111 bp located between the nucleotide (nt) coordinates 400-1510. The amplicons (one per sample) were cloned, and the DNA sequence was determined for each and used to search the NCBI database. The top hits for sequences obtained from all 10 samples were to WmSCV sequences, with shared nt identity values of 97 to 98%. To clone the full-length begomoviral DNA-A and DNA-B components, nucleic acids were subjected to rolling circle amplification (RCA) (2). The RCA products were cloned into the pGEM7 plasmid vector using the unique restriction sites, identified as HindIII for DNA-A, and as EcoRI for DNA-B, respectively. Ten DNA-A clones and two DNA-B component clones were sequenced. The DNA-A components ranged in size from 2,751 (KM066100) to 2,752 bp (KJ939448), whereas the DNA-B components were 2,757 bp in size (KJ939447). Analysis of the viral sequences from the DNA-A clones indicated they had the characteristics of a typical genome of a begomovirus DNA-A component that consist of a hairpin stem-loop structure in the intergenic region, two tandem copies of the iteron (TGGAGAC) located between the nt coordinates 2675 and 2680, 2682 and 2688 predicted to be involved in Rep binding, and six predicted genes encoding proteins with high sequence identity to those encoded by other WmCSV isolates. The 10 DNA-A component sequences were aligned with sequences for previously described WmCSV isolates available in GenBank using Muscle, followed by pairwise comparisons using SDT software (4). The analysis revealed that the cloned DNA-A components shared 99 to 100% nt sequence identity with each other, and 97 to 98% nt identity with WmCSV isolates reported from Yemen (AJ012081), Jordan (EU561237), Iran (AJ245652), and Sudan (AJ245650). Further, the WmCSV DNA-B from Saudi Arabia shared the highest nt identity with sequences from Yemen (AJ012082) at 96%, Iran (AJ245653) at 95%, and only 94% with DNA-B from both Sudan (AJ245651) and Jordan (EU561236). To our knowledge, this is the first report of WmCSV in Saudi Arabia. WmCSV poses a serious threat to the production of this highly valued crop in Saudi Arabia and considerably reduces crop yield (1). References: (1) I. D. Bedford et al. Eur. J. Plant Pathol. 100:243, 1994. (2) A. Idris et al. Plant Dis. 97:910, 2007. (3) A. Kheyr-Pour et al. Phytopathology 90:629, 2000. (4) B. Muhire et al. Arch. Virol. 158:1411, 2013.