First report of squash vein yellowing virus naturally infecting cucumber, squash and melon in Jordan.

Abstract

Plant viruses are major restrictive pathogens to cucurbits production in Jordan. During field surveys conducted in September 2022 in the main cucurbit growing areas (desert area, Jordan Valley, and highlands), virus-like symptoms such as vein clearing, mosaic patterns, interveinal chlorosis, and fruit malformation were observed, in the presence of high whitefly populations (Bemisia tabaci MEAM1, Mdanat et al., 2022). A total of 80 leaf samples from different species [48 cucumber (Cucumis sativus), 11 squash (Cucurbita pepo), 14 melon (Cucumis melo) and 7 watermelon (Citrullus lanatus)], including 70 symptomatic and 10 asymptomatic samples, were collected for further investigations. Total RNA and DNA were extracted using RNeasy and DNeasy plant mini kit (QIAGEN), respectively, and molecular detection against an array of cucurbit-infecting viruses was conducted using protocols available in the literature (Suppl. Table). Squash vein yellowing virus (SqVYV; Potyviridae, Ipomovirus) was detected in combination with other cucurbit-infecting viruses, in 10 of 70 symptomatic samples with primers (SqYVV-v4762; SqYVV-c5512) targeting a portion of the cylindrical inclusion (CI) gene (Hernandez et al., 2021), including 3 cucumber, 4 squash and 3 melon samples, however, it was not detected in watermelon. Among other RNA viruses, cucumber green mottle mosaic virus, cucurbit yellow stunting disorder virus, cucurbit aphid-borne yellows virus, cucumber vein yellowing virus, and cucurbit chlorotic yellows virus were detected in 9, 34, 7, 18, and 23 samples, respectively. The DNA viruses, squash leaf curl virus and watermelon chlorotic stunt virus, were detected in 36 and 30 samples. None of the detected viruses were present in asymptomatic samples. All 80 samples were negative for watermelon mosaic virus and tomato leaf curl New Delhi virus. Cloning (pGEM T-Easy Vector; Promega), sequencing, and BLASTn analysis of 4 CI-specific cloned amplicons (~ 0.75 kb, GenBank Acc no. PP908660-PP908663) confirmed the identification of SqVYV, with highest BLASTn identity of ~91% and ~99% to isolates ESF3 from USA (MW584342) and SqVYV-IL from Israel (KT721735), respectively. To reconstruct the complete genome sequence of the SqVYV isolate from Jordan, total RNA from a pool of squash and melon plants was used to construct a cDNA library with the Illumina DNA Prep kit, which was sequenced on a NextSeq2000 instrument as paired reads (2x150 bp) at Leibniz Institute DSMZ, to generate 18,723,252 total reads. Bioinformatic analysis in Geneious (Biomatters) resulted in the assembly of a single genome sequence of 9,831 nt (GenBank Acc. no. PP916052), covered by 83,995 reads, with mean coverage of 1,197 (Geneious mapper, 10% maximum mismatches per read). The complete genome sequence and the deduced polyprotein sequence shared over 99% identity with SqVYV-IL from Israel. Mechanical inoculation of 10 cucumber (cv. Giant Global) and 10 squash (cv. Lebanese) plants with inoculum from infected cucumber, resulted in vein clearing and mosaic symptoms, after three weeks from inoculation, while no symptoms were observed in the six negative controls. Symptomatic plants were confirmed by PCR to be infected by SqVYV as described earlier. In this study, we report the occurrence of SqVYV for the first time in cucurbit crops in Jordan. SqVYV has been reported to cause large economic losses in cucurbits in USA and Israel, posing a major threat to watermelon growers (Adkins et al., 2007; 2008 a & b; 2013; Reingold et al., 2016; Webster et al., 2013). Our findings should encourage further studies on the incidence and prevalence of SqVYV in cucurbit and non-cucurbit crops, including weeds, to understand its epidemiology in Jordan, including its natural host range.