BackgroundTomato yellow leaf curl virus (TYLCV), a monopartite begomovirus (family Geminiviridae) is responsible for heavy yield losses for tomato production around the globe. In Oman at least five distinct begomoviruses cause disease in tomato, including TYLCV. Unusually, TYLCV infections in Oman are sometimes associated with a betasatellite (Tomato leaf curl betasatellite [ToLCB]; a symptom modulating satellite). RNA interference (RNAi) can be used to develop resistance against begomoviruses at either the transcriptional or post-transcriptional levels.ResultsA hairpin RNAi (hpRNAi) construct to express double-stranded RNA homologous to sequences of the intergenic region, coat protein gene, V2 gene and replication-associated gene of Tomato yellow leaf curl virus-Oman (TYLCV-OM) was produced. Initially, transient expression of the hpRNAi construct at the site of virus inoculation was shown to reduce the number of plants developing symptoms when inoculated with either TYLCV-OM or TYLCV-OM with ToLCB-OM to Nicotiana benthamiana or tomato. Solanum lycopersicum L. cv. Pusa Ruby was transformed with the hpRNAi construct and nine confirmed transgenic lines were obtained and challenged with TYLCV-OM and ToLCB-OM by Agrobacterium-mediated inoculation. For all but one line, for which all plants remained symptomless, inoculation with TYLCV-OM led to a proportion (≤25%) of tomato plants developing symptoms of infection. For inoculation with TYLCV-OM and ToLCB-OM all lines showed a proportion of plants (≤45%) symptomatic. However, for all infected transgenic plants the symptoms were milder and virus titre in plants was lower than in infected non-transgenic tomato plants.ConclusionsThese results show that RNAi can be used to develop resistance against geminiviruses in tomato. The resistance in this case is not immunity but does reduce the severity of infections and virus titer. Also, the betasatellite may compromise resistance, increasing the proportion of plants which ultimately show symptoms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0263-y) contains supplementary material, which is available to authorized users.
Watermelon chlorotic stunt virus (WmCSV) is a bipartite begomovirus (genus Begomovirus , family Geminiviridae ) that causes economic losses to cucurbits, particularly watermelon, across the Middle East and North Africa. Recently squash ( Cucurbita moschata ) grown in an experimental field in Oman was found to display symptoms such as leaf curling, yellowing and stunting, typical of a begomovirus infection. Sequence analysis of the virus isolated from squash showed 97.6–99.9% nucleotide sequence identity to previously described WmCSV isolates for the DNA A component and 93–98% identity for the DNA B component. Agrobacterium -mediated inoculation to Nicotiana benthamiana resulted in the development of symptoms fifteen days post inoculation. This is the first bipartite begomovirus identified in Oman. Overall the Oman isolate showed the highest levels of sequence identity to a WmCSV isolate originating from Iran, which was confirmed by phylogenetic analysis. This suggests that WmCSV present in Oman has been introduced from Iran. The significance of this finding is discussed.
During the last few decades, Tomato yellow leaf curl disease (TYLCD) has resulted in heavy yield losses and increased incidences in tropical and subtropical regions. In Oman, six different species of begomoviruses were found to be associated with TYLCD. However, Tomato yellow leaf curl virus-Oman (TYLCV-OM) was found to be the most widely distributed species in the whole country. A real-time quantitative PCR (qPCR) assay with an internal control [tomato elongation factor 1(EF1)] was developed for TYLCV-OM, based on SYBRGreen chemistry for the rapid detection and quantification of the virus. This assay was carried out on field samples showing symptoms according to The World Vegetable Center (AVRDC) disease severity scales. The test was used to establish a relationship between virus load and phenotypic symptoms. Viral copies of 2.88 × 10 9 were detected in field infected tomato plants showing symptom severity according to AVRDC scale '3' (i.e. severe leaf curling and yellowing of leaves). The copy number of virus decreased with the relative decrease in symptom severity scale. Tomato plants exhibiting scale '1' and '2' have 7.7 × 10 4 and 7.47 × 10 6 viral copies, respectively. Very low copy number of the virus (564) was detected in tomato plants showing symptoms at a severity scale of '0', which were apparently symptomless. However, tomato plants developed by tissue culture in sterilized conditions, which were used as a negative control, did not show the presence of the virus. The developed qPCR assay could detect as low as 18 fg (femto gram) of virus from total nucleic acid equivalent to approximately 30 genomic units. This quantitative assay can be used to determine virus titer, in breeding programs for development of virus resistant plants and for epidemiological surveys to monitor viral populations and their intensities.
The influence of polyester cover on tomato crops was evaluated to determine its effectiveness in reducing the incidence of tomato yellow leaf curl disease and on tomato yield. Uncovered plots were compared in a randomised block design with 14 treatments having different durations of cover. The identification of begomovirus was carried out by polymerase chain reaction, cloning and complete nucleotide sequencing of viruses from infected plants. Disease incidence was high in plots covered for less than 3 weeks with no significant differences among these treatments and those that were left uncovered. In the three treatments with the longest duration of cover, no disease was recorded but yield was significantly decreased. The optimum covering period was 6-7 weeks, providing protection against disease (10% incidence) and maintaining high yields (3 kg per plant). Floating row covers can play a major role in reducing begomovirus-induced diseases by reducing exposure to whiteflies which transmit these viruses. The current work appears to be the first attempt to optimise the cover period by sequentially removing covers from crops to analyse the consequences in terms of crop biomass, yield and disease and to explore the interactions between these parameters.
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