Watermelon Chlorotic Stunt Virus Research Articles

Identification and molecular characterization of cotton leaf curl Gezira betasatellite and two distinct begomoviruses infecting papaya trees in the Kingdom of Saudi Arabia

Begomovirus is the largest genus in the family Geminiviridae and constitutes more than 445 virus species. Begomoviruses are characterized by single-stranded circular genomes with monopartite or bipartite components and transmitted by whitefly (Bemisia tabaci). Begomoviruses cause severe diseases in many economically important crops throughout the world. Typical symptoms of a begomovirus infection including severe leaf curling, vein thickening, vein darkening and reduced leaf size were observed in papaya plants in the Dammam district of the Eastern Province of Saudi Arabia during the growing season in 2022. A total of 10 samples were collected, and total genomic DNA was isolated from naturally infected papaya tree samples and subjected to PCR amplification using universal diagnostic primers for begomoviruses and associated satellites. Three PCR-amplified genomic components of begomoviruses and betasatellite namely P61Begomo (645 bp), P62Begomo (341 bp) and P62Beta (563 bp) were sent for Sanger DNA sequencing to Macrogen Inc. These partial viral genome sequences were submitted to Genbank database and accession numbers ON206051, ON206052 and ON206050 were assigned to P61Begomo, P62Begomo and P62Beta respectively. Phylogenetic analysis and pairwise nucleotide sequence identity studies identified P61Begomo was identified as Tomato yellow leaf curl virus, P62Begomo as DNA A component of a bipartite begomovirus Watermelon chlorotic stunt virus and P62Beta as begomovirus associated betasatellite; Cotton leaf curl Gezira betasatellite. To the best of our knowledge, this is the first report of a begomovirus complex infecting papaya (Carica papaya) in the Kingdom of Saudi Arabia.

Identification of the Begomoviruses Squash Leaf Curl Virus and Watermelon Chlorotic Stunt Virus in Various Plant Samples in North America.

Geminiviruses are a group of plant-infecting viruses with single-stranded DNA genomes. Within this family, viruses in the genus Begomovirus are known to have a worldwide distribution causing a range of severe diseases in a multitude of dicotyledonous plant species. Begomoviruses are transmitted by the whitefly Bemisia tabaci, and their ssDNA genomes can be either monopartite or bipartite. As part of a viral survey, various plants including those in the families Alliaceae, Amaranthaceae, Apiaceae, Asteraceae, Brassicaceae, Cactaceae, Cucurbitaceae, Lamiaceae, Lauraceae, Malvaceae, Oleaceae and Solanaceae were sampled and screened for begomoviruses using both a high-throughput sequencing and a begomovirus-specific primer pair approach. Based on the sequences derived using these approaches, the full-length genome of various begomoviruses were amplified from plants using abutting primers. Squash leaf curl virus (SLCV) and watermelon chlorotic stunt virus (WCSV) were identified in Cactaceae (n = 25), Solanaceae (n = 7), Cucurbitaceae (n = 2) and Lamiaceae (n = 1) samples. WCSV is an Old World bipartite begomovirus that has only recently been discovered infecting watermelons in the Americas. Our discovery of WCSV in the USA is the first indication that it has reached this country and indicates that this virus might be widespread throughout North America. Phylogenetic analysis suggests WCSV was introduced to the New World twice. The detection of begomoviruses in cactus plants suggests possible spillover events from agricultural areas into native vegetation. Since WCSV and SLCV have previously been found in mixed infections, pseudo-recombination infection experiments were conducted. We demonstrate that WCSV DNA-B is successfully trans-replicated by SLCV DNA-A despite very low degree of similarity between the replication-associated iterative sequences present in their common region, an essential feature for binding of the replication associated protein. This study highlights the importance of viral surveys for the detection of spillover events into native vegetation, but also suggests the need for more surveillance of WCSV in the USA, as this virus is a serious threat to watermelon cultivation in the Middle East.

English

Cucumber (Cucumis sativus; family Cucurbitaceae) plants exhibiting begomovirus-like symptoms such as yellowing, mosaics and stunting were studied using cloning, sequencing, Species Demarcation Tool followed by phylogenetic clustering. The complete genome of DNA-A showed maximum sequence identity of 98.7% with the corresponding DNA-A of an isolate from “Iran” strain of Watermelon chlorotic stunt virus (WmCSV). The DNA-B displayed 97.5% nt identity with the component of DNA-B of WmCSV from Iran, too. Our results confirmed that yellowing and mosaic symptoms of cucumber are associated with a bipartite begomovirus (WmCSV). This study is the first characterization of WmCSV in association with described symptoms in cucumber from Oman. © 2021 Friends Science Publishers

Cucurbit leaf crumple virus (CuLCrV) associated to watermelon disease in Campeche state, Mexico.

An annual recurrent disease causing yield reduction in cultivated watermelon (Citrullus lanatus) was documented by the growers in different farms of Campeche state, Mexico. In April 2019 and March 2020 open field grown watermelon plants showed symptoms such as leaf curling, crumpling, and leaf basal or apical necrosis (Figure S1), with an incidence ranging from 30 up to 80%. These plants also presented high populations of whitefly, especially in the most affected fields. In order to identify the causal agent of the disease, a total of 22 symptomatic watermelon plants were collected in four locations from Campeche state. Total nucleic acids (DNA and RNA) were extracted from these leaf samples. Initially, RT-PCR analysis was performed with specific primers (Table S1) for cucurbit-infecting Crinivirus transmitted by whitefly but the expected size PCR product for those viruses was not amplified in any of these samples. To investigate the presence of cucurbit-infecting begomoviruses, PCR was performed by using specific primers for those begomoviruses reported in Mexico and north/central America including Squash leaf curl virus (SLCV), Watermelon chlorotic stunt virus (WmCSV), Melon chlorotic leaf curl virus (MCLCuV), and Cucurbit leaf crumple virus (CuLCrV) (Table S1). Only the expected amplicon size of ~1089 bp for CuLCrV was amplified from DNA extracts from all 22 watermelon samples, suggesting a single cucurbit-associated virus. The putative complete genome of the CuLCrV Campeche isolate was amplified by circular DNA enrichment using a Rolling Circle Amplification (RCA) procedure from two representative samples, followed by enzymatic digestion using BamHI, EcoRI, KpnI, and SacI enzymes (Inoue-Nagata et al., 2004). Expected linearized full-length viral components (~2.7 kb) were obtained with EcoRI and SacI, and both products, from one selected sample, were cloned in to pGreen0029 vector and were fully sequenced. Sequence analysis of the EcoRI clone, designated as LV2019Camp_A (deposited in GenBank accession no. MW273384) revealed the highest identity of 97.52% to CuLCrV DNA-A isolate Baja California Sur isolate (GeneBank accession no. MN625831.1), whereas the KpnI clone, designated as LV2019Camp_B (deposited in GenBank accession no. MW273385), shared 94.87% identity with DNA B of CuLCrV isolate Arizona (GeneBank accession no. AF327559.1). Subsequently, CuLCrV isolate Campeche-derived agroinfectious clone, was obtained by constructing a partial dimeric tandem repeat of both DNA-A and DNA-B components (Bang et al., 2014). Twelve watermelon plants were agroinfiltrated with the infectious clone at the fourth true leaf stage, resulting in symptomatic plants (11/12) exhibiting leaf yellowing, curling, and crumpling 15 days after agroinfiltrated (Figure S1), and CuLCrV infection was confirmed by PCR specific detection using DNA extract from non-inoculated leaves. Previously CuLCrV has been detected in the USA (Arizona, Texas, California, Florida, South Carolina, and Georgia), and north Mexico (Coahuila) infecting cucurbits including squash, cucumber, cantaloupe, pumpkin, and watermelon (Brown et al., 2000., Keinath et al., 2018), in both single and mixed infection with other whitefly transmitted RNA viruses (CYSDV, genera Crinivirus), and DNA viruses (SLCV, genera Begomovirus) (Kuo et al., 2007). To our knowledge, this is the first report of CuLCrV infecting a cucurbit crop in the Campeche state from the Yucatán peninsula, in Mexico.

Interaction of watermelon chlorotic stunt virus with satellites

Pumpkin (Cucurbita maxima) plants showing begomovirus-like yellowing, curling and stunting symptoms were observed in the Barka Governate, Oman in December 2016. Cloning and sequencing of rolling circle amplified circular DNA molecules showed the plants to be infected with watermelon chlorotic stunt virus (WmCSV); a bipartite Old World begomovirus that is a serious pathogen of cucurbit crops across the Middle East, Arabian Peninsula and North Africa. Although not known to interact with satellites, the risk of WmCSV possibly interacting with betasatellites and alphasatellites to agriculture in Oman, and beyond, was assessed. Tomato leaf curl betasatellite (ToLCB) and Ageratum enation alphasatellite (AEA) are commonly associated with other begomoviruses in Oman. A cognate pair of WmCSV DNA-A and DNA-B clones was highly infectious to Nicotiana benthamiana by Agrobacterium-mediated inoculation, inducing severe leaf curling and crumpling symptoms. Inoculation of only the DNA-A component with ToLCB or AEA did not result in symptoms or significant levels of the DNA-A or satellites in plants. Inoculation of both DNA-A and DNA-B with ToLCB or AEA resulted in a slight change in symptom phenotype and reduced DNA-A and DNA-B titers. Although ToLCB DNA levels in co-infected plants were low, the levels of AEA were high. This is the first identification of WmCSV infecting pumpkin in Oman and the isolates identified were typical of the virus previously identified in Oman, being distinct from the isolates occurring in the western Arabian Peninsula. Additionally the infectivity analyses suggest that WmCSV interaction with satellites, in contrast to some other bipartite begomoviruses, is unlikely to result in more serious disease in the field.

Identification of watermelon chlorotic stunt virus from watermelon and zucchini in Saudi Arabia

Symptomatic and non-symptomatic leaf samples of cucumber, zucchini, melon and watermelon plants were collected from three locations (Al-Ahsa in the East, Jizan in the South and Tabuk in the North) in the Kingdom of Saudi Arabia during 2013–2014. The detection of begomovirus infection was commenced with serological assay, rolling circle amplification and PCR amplification with universal begomovirus primers. The zucchini and watermelon samples tested positive for the presence of begomovirus infection in 53 and 39.6%, and 54.5 and 41.5% samples using serological and PCR-based detection, respectively. Based upon the core coat protein sequence, the full-length genomes of DNA-A and DNA-B were amplified and subsequently sequenced. The sequence analysis revealed that the two clones of DNA-A shared 96.9% nucleotide (nt) sequence identity with each other while they shared 95.9–98.4% nt sequence identity with watermelon chlorotic stunt virus (WmCSV) isolates reported so far. Similarly, the two DNA-B clones showed 91.8% mutual nt sequence identity, while sharing 95.2–95.5% nt sequence identity with all other reported DNA-B sequences. The phylogenetic dendrogram grouped both DNA-A clones into separate sub-groups, suggesting that both isolates were introduced separately into Saudi Arabia. The identification of WmCSV from zucchini in Saudi Arabia and other hosts in the neighbouring countries suggests that this virus is becoming an emerging threat to cucurbits in this region.

Molecular Detection of Begomoviruses Infecting Some Vegetable Crops in Saudi Arabia

Begomoviruses have a huge impact on crop production worldwide. Investigations of begomviruses in four vegetable crops namely cucumber, squash, tomato, and watermelon were conducted by visual symptoms and molecular analysis using PCR with universal primers for begomoviruses. Obtained sequencing results of the core Coat Protein confirmed the presence of three already reported begomoviral species. TYLCV was detected on tomato samples exhibiting leaf curling and yellowing. Squash and greenhouse-grown cucumber plants were infected with the Oman strain TYLCV-OM. Watermelon chlorotic stunt virus (WmCSV) isolates were detected in watermelon and greenhouse cucumbers. The begomoviral species ToLCPMV shared 99% identity with the Iranian isolates that are known to be very destructive. Integrated pest management should be implemented against whitefly (Bemisia tabaci) to prevent the spread of begomoviruses on vegetable crops.

Insights Into a Watermelon Virome Contribute to Monitoring Distribution of Whitefly-Borne Viruses

The composition of a plant virome may represent spatiotemporal patterns of plant virus abundance. Using next generation sequencing, we investigated the viromes of watermelon fruits grown in two adjacent open fields located in Eastern Israel: Kalia and Mitzpe-Shalem. The two viromes were comprised of distinct virus species and genera. Studying spatial and temporal effects on virus occurrence, we detected the crinivirus Cucurbit yellow stunting disorder virus in Kalia and not in Mitzpe-Shalem, irrespective of collection time. A spatial effect was also observed regarding the occurrence of the begomovirus Watermelon chlorotic stunt virus, which was not detected in watermelons from Kalia, but rather in watermelon fruit in Northern Israel in 2018. A temporal effect was observed on the appearance of the ipomovirus Cucumber vein yellowing virus, which was detected in watermelons from Kalia in 2017 and was absent in the 2016 virome analysis. Importantly, regardless of temporal and spatial effects, the crinivirus Cucurbit chlorotic yellows virus, which was new to Israeli landscape, was detected in all the tested watermelon plants. These changes in viral abundance were associated with an early whitefly (Bemisia tabaci) occurrence, which might be the cause for the severe disease spread in watermelons.

Insights into the Incidence of Watermelon chlorotic stunt virus Causing Yellowing Disease of Watermelon in Western and Southwestern Regions of Saudi Arabia.

During the spring season of 2014, a total of 148 melon and watermelon leaf samples were collected from symptomatic and asymptomatic plants in the western and southwestern regions of Saudi Arabia and were tested for the presence of Watermelon chlorotic stunt virus (WmCSV) and other suspected cucurbit viruses by double antibody sandwich enzyme-linked immunosorbent assays. Ninety-eight samples were found to be positive for the presence of WmCSV, nine samples were positive for the presence of Cucurbit yellow stunting disorder virus (CYSDV), and 22 showed a mixed infection with both WmCSV and CYSDV. No other cucurbit viruses were detected in any of the samples. Host range experiments revealed that eight out of fourteen tested plant species were susceptible to WmCSV. PCR products of approximately 1.2 kb were obtained after amplification using primers specifically targeting the coat protein region of WmCSV. Positive PCR results were confirmed by dot blot hybridization. Coat protein gene sequences from eleven WmCSV isolates indicated that the highest identity was between the 104WMA-SA isolate from the Wadi Baish location and a previously reported isolate from the AL-Lith location in Saudi Arabia. The lowest identity was observed between the 42WMA-SA isolate and an isolate from Palestine.

English

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.

Turnip leaf curl disease associated with two begomoviruses in south-eastern Iran

A handful of geminiviruses have been associated with turnip leaf curl disease. As part of our turnip surveys in Kerman province (south-eastern Iran), turnip samples showing mild leaf curl disease were collected and screened for turncurto- and begomoviruses by PCR. In four out of seven samples, we identified co-infection with begomoviruses of the species Tomato yellow leaf curl virus and Watermelon chlorotic stunt virus. Neither of these two begomovirus species have been described to be associated with turnip leaf curl disease in Iran. Using infectious clones and agroinoculation of turnip, tomato and watermelon seedlings, our results indicated that single and/or co-agroinoculation of plants with the two begomovirus constructs result in symptoms with different severity in turnip, tomato and watermelon.

Molecular and biological characterization of Watermelon chlorotic stunt virus (WmCSV): An Eastern Hemisphere begomovirus introduced in the Western Hemisphere

Watermelon chlorotic stunt virus (WmCSV) is a bipartite begomovirus that causes severe diseases to cucurbits, particularly watermelon, across Eastern Mediterranean countries. Watermelon plants showing begomovirus-associated symptoms and virus vector whitefly infestation were collected from a cultivated area in Sonora, Mexico. Using rolling circle amplification (RCA) and sequencing, the full-length genome of an isolate of WmCSV was obtained. Agrobacterium-mediated virus inoculation to Solanaceae (Nicotiana benthamiana and tomato) and Cucurbitaceae (Squash, melon and watermelon) plants, fulfilled Koch's postulates confirming the disease causal agent. To the best of our knowledge, this is the first report of WmCSV associated to cucurbit diseases in the Western Hemisphere.

Charlock mustard (Sinapis arvensis): a weed reservoir for begomoviruses and associated betasatellite in Jordan

Four crop-infecting begomoviruses were found naturally affecting Charlock mustard (Sinapis arvensis L.) plants in Jordan and characterized at the molecular level. Symptoms included leaf curling and stunting. PCR analysis showed the presence of Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV). These viruses were associated with Cotton leaf curl Gezira betasatellite (CLCuGβ). Sequence analysis revealed that TYLCV and TYLCSV had 98.5% and 99.3% nucleotide (nt) sequence identities with a previously described TYLCV strain from Florida and TYLCSV from Sardinia, respectively. The other two viruses, SLCV and WmCSV, shared 99.7% and 99.5% nt identities with previously described strains from Jordan. Sequence analysis also showed that CLCuGβ had 98.8% nt identity with a strain from Israel. To our knowledge, this is the first report of S. arvensis as a natural host of SLCV, WmCSV, TYLCSV and CLCuGβ.

First Report of Datura innoxia as a Natural Host of Watermelon chlorotic stunt virus in Sudan

First Report of <i>Datura innoxia</i> as a Natural Host of <i>Watermelon chlorotic stunt virus</i> in Sudan

Inoculation of cucumber, melon and zucchini varieties with Tomato leaf curl New Delhi virus and evaluation of infection using different detection methods

AbstractThe disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post‐inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.

Watermelon chlorotic stunt virus. [Distribution map

Abstract A new distribution map is provided for Watermelon chlorotic stunt virus. Geminiviridae: Begomovirus. Hosts: Cucurbitaceae including watermelon ( Citrullus lanatus ), melon ( Cucumis melo ) and cucumber ( Cucumis sativus ) and Solanaceae including tomato ( Solanum lycopersicum ). Information is given on the geographical distribution in Asia (Iran, Israel, Jordan, Lebanon, Oman, Saudi Arabia and Yemen) and Africa (Sudan).

Analysis of watermelon chlorotic stunt virus and tomato leaf curl Palampur virus mixed and pseudo-recombination infections.

Watermelon chlorotic stunt virus (WmCSV) and tomato leaf curl Palampur virus (ToLCPMV) are limiting factors for cucurbit production in south and southeastern Iran. ToLCPMV infects all cucurbit crops (except watermelons) whereas WmCSV is somewhat limited to watermelon, causing detrimental effects on fruit production. In a survey, we detected WmCSV in all watermelon growing farms in Fars province (southern Iran). Given that WmCSV and ToLCPMV are present in the same geographical location in Iran, we studied the interaction of two viruses. Co-infection using agroinfectious clones of WmCSV and ToLCPMV caused severe symptoms in watermelon and zucchini in comparison to symptoms observed from individual infections. Interestingly, inoculation of zucchini with WmCSV DNA-A and ToLCPMV DNA-B agroinfectious clones or vice versa produced a viable pseudo-recombinant and induced systemic symptoms. This demonstrates that replication-associated protein of DNA-A of each virus is able to bind to cis elements of the DNA-B molecules of another virus.

Impact of cis-acting elements' frequency in transcription activity in dicot and monocot plants.

The production of new cultivars via recombinant DNA technology is important in applied agriculture. Promoters play fundamental roles in successful transformation and gene expression. Fragments of the upstream regulatory region of the movement protein gene of the Tomato yellow leaf curl virus (TYLCV; two fragments) and Watermelon chlorotic stunt virus (WmCSV, two fragments) and one fragment of the coat protein putative promoter of TYLCV (CPTY-pro) were isolated to assess their abilities to drive expression in monocot and dicot plants. We used bioinformatic analyses to identify tentative motifs in the fragments. The five promoter fragments were isolated, fused with the GUS reporter gene, and transformed into tomato, watermelon, and rice plantlets via Agrobacterium infiltration. GUS expression driven by each putative promoter was analysed using histochemical and fluorometric analyses. In both dicots and the monocots, the highest level of GUS expression was obtained using a truncated regulatory region from TYLCV (MMPTY-pro) followed by a truncated regulatory region from WmCSV (MMPWm-pro). However, the corresponding full-length fragments from TYLCV and WmCSV showed essentially equivalent expression levels in the fluorometric GUS assay compared with the enhanced Cauliflower mosaic virus e35S-pro. In addition, CPTY-pro showed no expression in either the dicots or the monocot. This study demonstrated that MMPTY-pro and MMPWm-pro may be useful as plant promoters.

Quantification and localization of Watermelon chlorotic stunt virus and Tomato yellow leaf curl virus (Geminiviridae) in populations of Bemisia tabaci (Hemiptera, Aleyrodidae) with differential virus transmission characteristics.

Bemisia tabaci (Gennadius) is one of the economically most damaging insects to crops in tropical and subtropical regions. Severe damage is caused by feeding and more seriously by transmitting viruses. Those of the genus begomovirus (Geminiviridae) cause the most significant crop diseases and are transmitted by B. tabaci in a persistent circulative mode, a process which is largely unknown. To analyze the translocation and to identify critical determinants for transmission, two populations of B. tabaci MEAM1 were compared for transmitting Watermelon chlorotic stunt virus (WmCSV) and Tomato yellow leaf curl virus (TYLCV). Insect populations were chosen because of their high and respectively low virus transmission efficiency to compare uptake and translocation of virus through insects. Both populations harbored Rickettsia, Hamiltonella and Wolbachia in comparable ratios indicating that endosymbionts might not contribute to the different transmission rates. Quantification by qPCR revealed that WmCSV uptake and virus concentrations in midguts and primary salivary glands were generally higher than TYLCV due to higher virus contents of the source plants. Both viruses accumulated higher in insects from the efficiently compared to the poorly transmitting population. In the latter, virus translocation into the hemolymph was delayed and virus passage was impeded with limited numbers of viruses translocated. FISH analysis confirmed these results with similar virus distribution found in excised organs of both populations. No virus accumulation was found in the midgut lumen of the poor transmitter because of a restrained virus translocation. Results suggest that the poorly transmitting population comprised insects that lacked transmission competence. Those were selected to develop a population that lacks virus transmission. Investigations with insects lacking transmission showed that virus concentrations in midguts were reduced and only negligible virus amounts were found at the primary salivary glands indicating for a missing or modified receptor responsible for virus attachment or translocation.

Frequent migration of introduced cucurbit-infecting begomoviruses among Middle Eastern countries.

BackgroundIn the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine.MethodsWe explored this unique situation by assessing the diversity and biogeography of the DNA-A component of SLCV and WmCSV in these five countries.ResultsThere was fairly low sequence variation in both begomovirus species (SLCV π = 0.0077; WmCSV π = 0.0066). Both viruses may have been introduced only once into the eastern Mediterranean basin, but once established, these viruses readily moved across country boundaries. SLCV has been introduced at least twice into each of all five countries based on the absence of monophyletic clades. Similarly, WmCSV has been introduced multiple times into Jordan, Israel and Palestine.ConclusionsWe predict that uncontrolled movement of whiteflies among countries in this region will continue to cause SLCV and WmCSV migration, preventing strong genetic differentiation of these viruses among these countries.