Abstract
Cucurbit yellow stunting disorder virus (CYSDV) and Watermelon chlorotic stunt virus (WmCSV) are the most widespread and damaging viruses to cucurbits in the Middle East. CYSDV and WmCSV are cucurbit-infecting bipartite whitei¬‚y-transmitted viruses. Post-transcriptional gene silencing (PTGS) is a universal mechanism by which plants are able to systemically switch off the expression of targeted genes via the reduction of steady-state levels of specific RNAs. PTGS was used in this study to control the two viruses. In this study, the efficiency of the dsRNA for the ability to trigger resistance against the CYSDV and WmCSV was investigated. Three regions of three genes of CYSDV genome were selected; the coat protein gene (CP), heat shock gene (Hsp70) and ORF3, while the two regions of two genes of WmCSV genome were selected; CP gene and rep gene. Bioassay, dot-blot hybridization and polymerase chain reaction (PCR) methods were capable to evaluate the resistance against viruses. Clear symptoms on tobacco plants took two to three weeks to appear and all non-infiltrating tobacco plants (positive control) showed viral symptoms after inoculation. Most of the agro-infiltrating sense/antisense constructs did not yield symptoms of the viruses. Dot-blot hybridization, showed that negative hybridization was obtained with infiltrating tobacco plants with prepared constructs compared to those non-infiltrating tobacco plants used as the control. Only one out of five gave positive signals with the construct pasCYSDV-Hsp70. Using PCR, positive reactions of the expected size of 500 bp fragment with WmCSV and 800 bp with CYSDV were obtained with the infiltrating tobacco plants with sense constructs, which pointed out the existence of viral genome in challenging tobacco plants. Infiltrating tobacco plants with sense/antisense constructs gave negative PCR pointed out the lack of the viral genome. Key words: Cucurbit yellow stunting disorder virus (CYSDV), watermelon chlorotic stunt virus (WmCSV), Post-transcriptional gene silencing (PTGS), coat protein (CP), Hsp70, ORF3, Rep, dot-blot, hybridization.
Highlights
Cucurbit crops are widely grown in the Middle East and consumed in large quantities in the traditional diet
Using polymerase chain reaction (PCR), positive reactions of the expected size of 500 bp fragment with Watermelon chlorotic stunt virus (WmCSV) and 800 bp with Cucurbit yellow stunting disorder virus (CYSDV) were obtained with the infiltrating tobacco plants with sense constructs, which pointed out the existence of viral genome in challenging tobacco plants
WmCSV was first identified in Yemen (Jones et al, 1988) and after that, in the following years, it was reported through the Middle East and in North Africa as the following: Sudan (Kheyr-Pour et al, 2000), Jordan (Al-Musa et al, 2011), Oman (Khan et al, 2012) and Palestine (Ali-Shtayeh et al, 2014)
Summary
Cucurbit crops are widely grown in the Middle East and consumed in large quantities in the traditional diet. During 2001, two new viral diseases were detected in cucurbits: watermelon (Citrullus lanatus), squash (Cucurbita pepo) and cucumber (Cucumis sativus L.) in the Middle East region. Sequence analysis revealed that the diseases were caused by the native Watermelon chlorotic stunt virus and Squash leaf curl virus (Abdel-Salam et al, 2006; Abudy et al, 2010; Ali-Shtayeh et al, 2014; Idris et al, 2006). WmCSV was first identified in Yemen (Jones et al, 1988) and after that, in the following years, it was reported through the Middle East and in North Africa as the following: Sudan (Kheyr-Pour et al, 2000), Jordan (Al-Musa et al, 2011), Oman (Khan et al, 2012) and Palestine (Ali-Shtayeh et al, 2014). The symptoms caused by WmCSV are chlorotic mottling, vein yellowing, stunting and severe decrease of yield, generally in watermelon (Citrullus species) crops. WmCSV host range covers the most cucurbits including squash, cucumber, melons, and pumpkin
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