Search Results (32)

Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1–4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293–993 nt) and rep (1561–2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species. Full article

Tomato yellow leaf curl virus (TYLCV), tomato leaf curl New Delhi virus (ToLCNDV), and tomato chlorosis virus (ToCV) are emerging viruses that cause significant damage to tomato (Solanum lycopersicum). TYLCV and ToLCNDV are single-stranded DNA viruses from the genus Begomovirus, family Geminiviridae, while ToCV is an RNA virus from the genus Crinivirus (family Closteroviridae). These viruses share overlapping geographic ranges, vectors (the whitefly Bemisia tabaci), and host plants, making mixed infections common. This study investigated interactions between TYLCV and ToLCNDV and between ToLCNDV and ToCV in mixed infections of susceptible and TYLCV-resistant tomato genotypes. We evaluated infection, disease development, trans-replication of genome components, and genetic exchange. Our results showed no significant synergistic or antagonistic interactions, complementation, or interference between the viruses. TYLCV resistance in tomato genotypes remained stable. The DNA-B component of ToLCNDV exhibited impaired functionality and was not complemented by TYLCV. No evidence was found that the crinivirus tomato chlorosis virus (ToCV) enhances ToLCNDV infection, suggesting limited interactions despite shared vectors. Genetic exchange was detected in defective DNA (def-DNA) molecules using high-throughput sequencing (HTS), indicating potential genetic interactions between these viruses. These findings suggest that mixed infections do not pose immediate concerns for increased pathogenicity but highlight the ecological implications of genetic exchange, warranting further study of the evolutionary consequences of such interactions in mixed-virus environments. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) and tomato yellow leaf curl virus (TYLCV) are two important viral pathogens that severely affect Solanaceae and Cucurbitaceae plants. In order to reduce the further spread of these viruses, it is crucial to establish an efficient and reliable method to accurately detect the viruses. In this study, a multiplex PCR assay for the simultaneous detection of TYLCV and ToLCNDV was established. Three primer pairs designed from conserved regions within the coat protein or movement protein-encoding regions of the respective viruses were employed in the assay. The optimization of parameters such as primer concentration was set at 0.15 μM/0.15 μM, 0.25 μM/0.25 μM, and 0.50 μM/0.50 μM for ToLCNDV-DNA-A-F/R, TYLCV-F/R, and ToLCNDV-DNA-B-F/R primer pairs. At optimal primer concentrations, the multiplex PCR method demonstrates effective performance with an annealing temperature ranging from 51 °C to 66 °C. The specificity of the assay evaluated by testing against other begomoviruses showed no evidence of cross-amplification. Further sensitivity analysis performed using a serially diluted plasmid containing viral targets as templates demonstrated high sensitivity with a detection limit of 10³ copies/μL. Field surveys utilizing the multiplex PCR assay successfully identified the infection of TYLCV and ToLCNDV in field-collected samples. Full article

Among begomovirus species, tomato leaf curl New Delhi virus (ToLCNDV) is significant and stands out as a mechanically transmissible bipartite begomovirus originating from the Old World. However, the mechanisms underlying the mechanical transmission of different ToLCNDV strains remain understudied, as their natural transmission occurs via insect vectors. In this study, we investigated the mechanical transmissibility of two ToLCNDVs, one from Italy and another from Pakistan, in host plants. Several cucurbit species were screened, and symptom differences between the two ToLCNDV clones were observed only in zucchini when subjected to rubbing inoculation. The Italian isolate (ToLCNDV-ES) induced typical disease symptoms such as leaf curling, yellow mosaic, and internode stunting, whereas a normal phenotype was observed in zucchini mechanically infected with ToLCNDV-In (Pakistani isolate). Subsequently, a gene-swapping experiment between the two ToLCNDVs was conducted, and ToLCNDV-ES DNA-B was identified as a crucial factor in mechanical transmission. We then constructed chimeric mutant clones based on the DNA-B sequence and assessed their ability to induce symptoms in zucchini. These results indicated that the nuclear shuttle protein is a determinant of symptom development during ToLCNDV mechanical transmission. Moreover, several defense-related host genes showed significant changes in relative expression in different ToLCNDV clones, indicating their potential role in disease symptom development through the mechanical transmission of ToLCNDV. This is the first report comparing the mechanical transmissibility of two isolates of different ToLCNDV strains from the Mediterranean region and the Indian subcontinent in the same host plant, providing new insights into the virus’s pathogenicity across different geographic regions. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) is a begomovirus causing significant melon (Cucumis melo) crop losses globally. This study aims to map the ToLCNDV resistance in the PI 414723 melon accession, previously identified and characterized through phenotypic studies, thereby exploring shared genomic regions with the established resistant source WM-7. In the present study, WM-7 and PI 414723 were crossed with the susceptible accessions ‘Rochet’ and ‘Blanco’ respectively, to generate F₁ hybrids. These hybrids were self-pollinated to generate the populations for mapping the ToLCNDV resistance region and designing markers for marker-assisted selection. Disease evaluation included visual symptom scoring, viral-load quantification and tissue printing. Genotyping-by-sequencing and SNP markers were used for quantitative trait loci (QTL) mapping. For genetic analysis, qPCR and bulked segregant RNA-seq (BSR-seq) were performed. Gene expression was assessed using RNA-seq, and qRT-PCR was used for confirmation. The research narrows the candidate region for resistance in WM-7 and identifies overlapping QTLs on chromosome 11 in PI 414723, found in the region of the DNA primase large subunit. BSR-seq and expression analyses highlight potential regulatory roles of chromosome 2 in conferring resistance. Differential expression was confirmed for six genes in the candidate region on chromosome 2. This study confirms the existence of common resistance genes in PI 414723 and WM-7. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) is an emerging plant pathogen, fast spreading in Asian and Mediterranean regions, and is considered the most harmful geminivirus of cucurbits in the Mediterranean. ToLCNDV infects several plant and crop species from a range of families, including Solanaceae, Cucurbitaceae, Fabaceae, Malvaceae and Euphorbiaceae. Up to now, protection from ToLCNDV infection has been achieved mainly by RNAi-mediated transgenic resistance, and non-transgenic fast-developing approaches are an urgent need. Plant protection by the delivery of dsRNAs homologous to a pathogen target sequence is an RNA interference-based biotechnological approach that avoids cultivating transgenic plants and has been already shown effective against RNA viruses and viroids. However, the efficacy of this approach against DNA viruses, particularly Geminiviridae family, is still under study. Here, the protection induced by exogenous application of a chimeric dsRNA targeting all the coding regions of the ToLCNDV DNA-A was evaluated in zucchini, an important crop strongly affected by this virus. A reduction in the number of infected plants and a delay in symptoms appearance, associated with a tendency of reduction in the viral titer, was observed in the plants treated with the chimeric dsRNA, indicating that the treatment is effective against geminiviruses but requires further optimization. Limits of RNAi-based vaccinations against geminiviruses and possible causes are discussed. Full article

Begomoviruses (family Geminiviridae) cause serious diseases in many crop families. Since 2013, the Spanish isolate of tomato leaf curl New Delhi virus (ToLCNDV) has been a limiting factor for cucurbits production in the Mediterranean basin, forcing farmers to adapt new management and control techniques. Although it is well-known that begomoviruses are naturally transmitted by the whitefly Bemisia tabaci, the capacity of these viruses to be vertically transmitted through seeds remains controversial. Clarifying the potential ToLCNDV seed transmission is essential to understand the epidemiology of this threating-for-cucurbits virus and to design appropriate control strategies. We assessed ToLCNDV distribution in the leaves, flowers and seeds of the infected plants of susceptible Cucumis melo accessions and toleration to the infected genotypes of Cucurbita moschata by conventional and quantitative PCR. We analyzed whether the viral particle was transmitted to offspring. We also evaluated ToLCNDV presence in commercial seeds of cucurbits (zucchini (Cucurbita pepo), melon (C. melo), cucumber (Cucumis sativus) and watermelon (Citrullus lanatus)) and in their progenies. As the assayed seedlings remained symptomless, we increased the reliability and accuracy of detection in these samples by searching for replicative forms of ToLCNDV by combining Southern blot hybridization and rolling-circle amplification (RCA). However, integral genomic DNA was not identified in the plants of offspring. Although the seedborne nature of ToLCNDV was confirmed, our results do not support the transmission of this virus from contaminated seeds to progeny. Full article

The potato apical leaf curl disease is caused by tomato leaf curl New Delhi virus-potato (ToLCNDV-potato), which severely alters a plant’s starch metabolism, starch hydrolysing enzymes, and antioxidant mechanism. In this study, the result suggested that ToLCNDV-potato significantly (p < 0.01) affected the morphological parameters and photosynthetic pigment system in both the cultivars of potato, viz., Kufri Pukhraj (susceptible) and Kufri Bahar (tolerant). However, the impact of ToLCNDV-potato was lower in Kufri Bahar. Moreover, the viral infection in potato showed significant (p < 0.01) enhancement in the leakage of plant oxidative metabolites such as proline and malondialdehyde (MDA) which was further confirmed with higher electrolyte leakage. The viral infection imbalance of starch metabolism in the leaves ultimately affects the carbohydrate profile. ToLCNDV-potato significantly lowered starch synthesis, enhanced the accumulation of sucrose, glucose, fructose and—which was further validated by enzymatic estimation of β-amylase—α-amylase and phosphorylase activity in the leaves of both cultivars. The antioxidant enzymes, viz., catalase, ascorbate peroxidase, and superoxide dismutase, were reported to be enhanced in both the cultivars due to ToLCNDV-potato infection. The higher enhancement of antioxidant enzyme activity was observed in Kufri Bahar, which signifies its resistant attributes. These findings in the potato plant broaden our understanding of the regulatory mechanisms of starch metabolism and antioxidant activity and provide proof of concept for breeding potato for ToLCNDV-potato tolerance. Full article

The molecular characterization of a tomato leaf curl New Delhi virus (ToLCNDV) isolate, denoted ToLCNDV-Le, is reported. The virus was associated with severe and recurrent outbreaks in protected crops of zucchini squash grown in the Province of Lecce (Apulia, southern Italy). The fully sequenced genome of ToLCNDV-Le consists of two genomic components named DNA-A and DNA-B of 2738 and 2683 nt in size, respectively. Like other ToLCNDV isolates, ToLCNDV-Le DNA-A contains the AV2 and AV1 open reading frames (ORFs) in the virion-sense orientation and five additional ORFs named AC1, AC2, AC3, AC4 and AC5 in the complementary-sense orientation. The DNA-B contains BV1 ORF in the virion-sense orientation and BC1 ORF in the complementary-sense orientation. No DNA betasatellites were found associated with ToLCNDV-Le in naturally infected samples. Phylogenetic analysis clustered ToLCNDV-Le with the ToLCNDV-ES strain of western Mediterranean Basin isolates. Consequently, the ToLCNDV-ES-[IT-Zu-Le18] name is proposed as the descriptor for ToLCNDV-Le. Using recombination detection program RDP4, one putative recombination breakpoint (Rbp) was identified close to nucleotide positions 2197–2727, covering approximately half of the AC1 region, including the AC4 ORF and the 3′ UTR. RDP4 indicated the event represents an Rbp of an isolate similar to ToLCNDV [Pk-06] (Acc. No. EF620534) found in Luffa acutangula in Pakistan and identified as putative minor parent into the background of ToLCNDV [BG-Jes-Svr-05] (Acc. No. AJ875157), found in tomato in Bangladesh, and identified as putative major parent. To the best of our knowledge, this is the first report of a ToLCNDV-ES recombinant isolate in the AC1-AC4 region in Italy. Full article

Three primary species from the Begomovirus genus, Pepper yellow leaf curl Indonesia virus (PepYLCIV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), and Tomato leaf curl New Delhi virus (ToLCNDV), are suspected of spreading throughout pepper production centers, and plants are infected by a single species or a combination of two or three species. This study was conducted to provide complete information about the symptoms, incidence and severity, whitefly biotypes, as well as the dominance status of the three Begomovirus species in pepper-producing areas in Java. A DNA analysis was carried out on leaf samples to identify Begomovirus species and biotypes of B. tabaci collected from 18 areas (16 districts) in lowlands (<400 m asl) and highlands (>700 m asl). The DNA analysis showed that B. tabaci biotype B was the most commonly detected in all locations compared to the A, AN, and Q biotypes. The incidence of begomovirus infection was at a high level, 93% and 88.78% in the lowlands and highlands, respectively. However, the severity of begomovirus infection was significantly higher in the lowlands (54.50%) than in the highlands (38.11%). A single infection of PepYLCIV was most dominant in all locations sampled and caused severe infection, followed by a mixed infection with TYLCKaV. Therefore, the current status of begomovirus infection, especially PepYLCIV, can provide advice to farmers using more tolerant and resistant varieties as well as a breeding strategy for resistant pepper varieties. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (genus Begomovirus, family Geminiviridae) persistently transmitted, as with all other begomoviruses, by whiteflies (Hemiptera: Aleyrodidae) of the Bemisia tabaci cryptic species complex. The virus, originally from the Indian subcontinent, was recently introduced in the Mediterranean basin, where it is currently a major concern for protected and open-field horticulture. The Mediterranean ToLCNDV isolates belong to a novel strain named “Spain strain” (ToLCNDV-ES), which infects zucchini and other cucurbit crops but is poorly adapted to tomato. Recently, it has been reported that another whitefly, Trialeurodes vaporariorum, is able to transmit an isolate of ToLCNDV from India which infects the chayote plant, a cucurbit. The present work aimed to clarify some aspects of whitefly transmission of ToLCNDV-ES. It was shown that T. vaporariorum is not able to transmit ToLCNDV-ES between zucchini plants. In addition, Ecballium elaterium may not act as a relevant reservoir for this virus strain in the Mediterranean basin, as B. tabaci Mediterranean (MED), the most prevalent species of the complex in the region, is not an efficient vector of this begomovirus between cultivated zucchini and wild E. elaterium plants. Full article

Tomato leaf curl New Delhi virus (ToLCNDV) represents a threat to economically important horticultural crops. A real-time loop-mediated isothermal amplification (LAMP) assay for in-field ToLCNDV detection was developed, coupled to a rapid sample preparation method, and tested both in field and laboratory conditions on zucchini squash, tomato, and pepper samples. A set of six LAMP primers was designed for specific ToCLNDV detection, targeting a 218-nucleotide sequence within the AV1 gene. The sensitivity, specificity and accuracy of the real-time LAMP assay and comparison with canonical PCR were evaluated. The real-time LAMP assay developed was about one-thousand times more sensitive than the conventional PCR method, detecting a total of 4.41 × 10² genome copies as minimum target; no cross-reactivity was detected with the other geminiviruses used as the outgroup. The rapid sample preparation method allows for a reliable detection with a low reaction delay (≈2–3 min) compared to canonical DNA extraction, providing results in less than 45 min. Lastly, an increase in ToLCNDV-positive sample detection was observed compared to PCR, in particular for asymptomatic plants (85% and 71.6%, respectively). The real-time LAMP assay developed is a rapid, simple, specific, and sensitive technique for ToLCNDV detection, and it can be adopted as a routine test, for both in-field and laboratory conditions. Full article

Bitter gourd (Momordica charantia L.), one of the valued vegetable crops in India, is severely affected by yellow mosaic disease caused by two begomoviruses, tomato leaf curl New Delhi virus (ToLCNDV) and bitter gourd yellow mosaic virus (BgYMV). The symptoms are yellowing, distortion of leaf, puckering, and malformed fruits. Increased incidence of the disease and appearance of symptoms even in young emerging seedling stage were suggestive of seed transmission of the viruses, which was examined in detail. To study the seed transmission, two sources—seeds of elite hybrids H1, H2, H3, H4, and Co1 procured from a seed market; and seeds from infected plants in the farmer’s field were tested. Detection of the virus by DAS-ELISA using polyclonal antibody indicated embryo infection up to 63%, 26%, 20%, and 10% in hybrids H1, H2, H3, and H4, respectively, for market-procured seeds. In PCR analysis with primers specific for ToLCNDV and BgYMV, infection by ToLCNDV was as high as 76% and mixed infection was 24%. In contrast, in seeds derived from field-infected plants, the percentage detection was less. Grow-out tests with market-procured seeds revealed no transmission for BgYMV compared with 5% transmission for ToLCNDV. Whether seed-borne inocula could serve as an inoculum for new infection in a field and further progress of the disease was investigated in a microplot study. The study clearly revealed variation in seed transmission between different sources, lots, cultivars, and viruses. The virus present in symptomatic and asymptomatic plants was easily transmitted by whitefly. In another microplot experiment, the potential of seed-borne virus as inoculum was proved. There was 43.3% initial seed transmission in the microplot, increasing to 70% after release of 60 whiteflies. Full article

Tomato leaf curl New Delhi virus (ToLCNDV, family Geminiviridae, genus Begomovirus) is a whitefly-transmitted virus that causes widespread damage in Cucurbitaceae and Solaneceae crops worldwide. The Spanish strain, ToLCNDV-ES, affects mainly cucurbit crops and has spread through the Mediterranean basin since its first detection in 2013 in the south of Spain. The control of the virus has been based on the adoption of measures to control the vector, which have not been sufficient to reduce production losses. Therefore, the identification of key genes for ToLCNDV resistance is essential for the development of resistant plants. Regarding genetic control of resistance in cucurbit crops, one major locus on chromosome 11 and two additional regions in chromosomes 12 and 2 of C. melo linked to ToLCNDV resistance have been described recently. Concerning C. moschata, a major QTL was also identified on chromosome 8 that resulted in synteny with a QTL on chromosome 11 of C. melo. In this work, we investigated the molecular basis of ToLCNDV resistance in contrasting accessions of C. melo and C. moschata by transcriptional characterization of 10 different candidate genes controlling host factors related to proviral or antiviral mechanisms. Two proviral factor genes, ARP4 in C. melo and SYTA in C. moschata, showed clear differences in expression levels when the susceptible and resistant accessions were compared. The knowledge of proviral factors associated with resistance could be used to screen an active mutagenesis TILLING platform. This is the case of C. pepo, in which no ToLCNDV resistance has been described to date. The relationship between the regulation of the genes ARP4 and SYTA, as well as the genome position of the described loci related to ToLCNDV resistance, is also discussed. Full article

Tomato leaf curl New Delhi virus-ES (ToLCNDV-ES), a high threat to cucurbits in the Mediterranean Basin, is listed as a different strain from the Asian ToLCNDV isolates. In this study, the infectivity of two clones previously isolated from Italy and Pakistan were compared in cucumbers, which resulted in the opposite symptom appearance. The swapping subgenome was processed; however, the mechanisms related to the disease phenotype remain unclear. To identify the disease-associated genes that could contribute to symptom development under the two ToLCNDV infections, the transcriptomes of ToLCNDV-infected and mock-inoculated cucumber plants were compared 21 days postinoculation. The number of differentially expressed genes in ToLCNDV-India-infected plants was 10 times higher than in ToLCNDV-ES-infected samples. The gene ontology (GO) and pathway enrichment were analyzed using the Cucurbits Genomics Database. The flavonoid pathway-related genes were upregulated in ToLCNDV-ES, but some were downregulated in ToLCNDV-India infection, suggesting their role in resistance to the two ToLCNDV infections. The relative expression levels of the selected candidate genes were validated by qRT-PCR under two ToLCNDV-infected conditions. Our results reveal the different infectivity of the two ToLCNDVs in cucumber and also provide primary information based on RNA-seq for further analysis related to different ToLCNDV infections. Full article