Cy-1, a major QTL for tomato leaf curl New Delhi virus resistance, harbors a gene encoding a DFDGD-Class RNA-dependent RNA polymerase in cucumber (Cucumis sativus).

BACKGROUND: Tomato leaf curl New Delhi virus (ToLCNDV) (family Geminiviridae, genus Begomovirus) is a significant threat to cucumber (Cucumis sativus) production in many regions. Previous studies have reported the genetic mapping of loci related to ToLCNDV resistance, but no resistance genes have been identified. RESULTS: We conducted map-based cloning of the ToLCNDV resistance gene in cucumber accession No.44. Agroinfiltration and graft-inoculation analyses confirmed the resistance of No.44 to ToLCNDV isolates from the Mediterranean and Asian countries. Initial mapping involving two rounds of phenotyping with two independent F2 populations generated by crossing the begomovirus-susceptible cultivar SHF and No.44 consistently detected major quantitative trait loci (QTLs) on chromosomes 1 and 2 that confer resistance to ToLCNDV. Fine-mapping of Cy-1, the dominant QTL on chromosome 1, using F3 populations narrowed the candidate region to a 209-kb genomic segment harboring 24 predicted genes. Among these genes, DFDGD-class RNA-dependent RNA polymerase (CsRDR3), an ortholog of Ty-1/Ty-3 of tomato and Pepy-2 of capsicum, was found to be a strong candidate conferring ToLCNDV resistance. The CsRDR3 sequence of No.44 contained multiple amino acid substitutions; the promoter region of CsRDR3 in No.44 had a large deletion; and the CsRDR3 transcript levels were greater in No.44 than in SHF. Virus-induced gene silencing (VIGS) of CsRDR3 using two chromosome segment substitution lines harboring chromosome 1 segments derived from No.44 compromised resistance to ToLCNDV. CONCLUSIONS: Forward and reverse genetic approaches identified CsRDR3, which encodes a DFDGD-class RNA-dependent RNA polymerase, as the gene responsible for ToLCNDV resistance at the major QTL Cy-1 on chromosome 1 in cucumber. Marker-assisted breeding of ToLCNDV resistance in cucumber will be expedited by using No.44 and the DNA markers developed in this study.

Genetic Dissection of ToLCNDV Resistance in Resistant Sources of Cucumis melo.

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.

Genomic distinctiveness and recombination in tomato leaf curl New Delhi virus (ToLCNDV-BG) isolates infecting bitter gourd.

There are two begomoviruses, tomato leaf curl New Delhi virus (ToLCNDV) and bitter gourd yellow mosaic virus (BgYMV) infecting bitter gourd in India. An extensive survey conducted from 2019 to 2022 clearly established that infection by ToLCNDV is more predominant (92.43%) than BgYMV (44%). The ToLCNDV isolates infecting bitter gourd shared only 88% identity in the DNA-A component with other ToLCNDV isolates and were found to be a distinct variant. The predicted amino acid sequence of the viral proteins, replication initiation protein, coat protein, and the symptom determinant protein in the study isolates are markedly different. Especially the RCR motif I and RCR motif III are different from other geminiviruses. Infectivity of cloned components of one of the isolates ToLCNDV-BG NP was demonstrated in bitter gourd. Recombination analysis clearly revealed that the study isolates are recombinants with the major parent predicted as squash leaf curl Yunnan virus (GenBank Accession Number: MK064241) and the minor parent as ToLCNDV from Pakistan (GenBank Accession Number: AM747291). Due to distinct genomic features and less than 90% identity with the majority of ToLCNDV isolates, the study isolates deserve to be raised to the status of a distinct strain, designated as ToLCNDV-BG. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-024-04009-3.

Identification of new stable resistant sources and assessing agro-morphological performance of sponge gourd germplasm against Tomato Leaf curl New Delhi Virus incidence.

Tomato leaf curl New Delhi virus (TolCNDV) causes yellow mosaic disease, which poses a significant biotic constraint for sponge gourd cultivation, potentially resulting in crop loss of up to 100%. In the present investigation, 50 diverse genotypes were screened for 3 years under natural epiphytotic conditions. A subset of 20 genotypes was further evaluated across four different environments. The combined analysis of variance revealed a significant genotype × environment interaction. Eight genotypes consistently exhibited high and stable resistance in the preliminary screening and multi-environment testing. Furthermore, genotype plus genotype × environment interaction biplot analysis identified DSG-29 (G-3), DSG-7 (G-2), DSG-6 (G-1), and DSGVRL-18 (G-6) as the desirable genotypes, which have stable resistance and better yield potential even under diseased conditions. The genotype by yield × trait biplot analysis and multi-trait genotype-ideotype distance index analysis further validated the potential of these genotypes for combining higher yield and other desirable traits with higher resistance levels. Additionally, resistant genotypes exhibited higher activities of defense-related enzymes as compared to susceptible genotypes. Thus, genotypes identified in our study will serve as a valuable genetic resource for carrying out future resistance breeding programs in sponge gourd against ToLCNDV.

Emerging evidence of seed transmission of begomoviruses: implications in global circulation and disease outbreak.

Begomoviruses (family Geminiviridae) are known for causing devastating diseases in fruit, fibre, pulse, and vegetable crops throughout the world. Begomoviruses are transmitted in the field exclusively through insect vector whitefly (Bemisia tabaci), and the frequent outbreaks of begomoviruses are attributed largely due to the abundance of whitefly in the agri-ecosystem. Begomoviruses being phloem-borne were known not be transmitted through seeds of the infected plants. The recent findings of seed transmission of begomoviruses brought out a new dimension of begomovirus perpetuation and dissemination. The first convincing evidence of seed transmission of begomoviruses was known in 2015 for sweet potato leaf curl virus followed by several begomoviruses, like bhendi yellow vein mosaic virus, bitter gourd yellow mosaic virus, dolichos yellow mosaic virus, mungbean yellow mosaic virus, mungbean yellow mosaic India virus, pepper yellow leaf curl Indonesia virus, tomato leaf curl New Delhi virus, tomato yellow leaf curl virus, tomato yellow leaf curl Sardinia virus, and okra yellow mosaic Mexico virus. These studies brought out two perspectives of seed-borne nature of begomoviruses: (i) the presence of begomovirus in the seed tissues derived from the infected plants but no expression of disease symptoms in the progeny seedlings and (ii) the seed infection successfully transmitted the virus to cause disease to the progeny seedlings. It seems that the seed transmission of begomovirus is a feature of a specific combination of host-genotype and virus strain, rather than a universal phenomenon. This review comprehensively describes the seed transmitted begomoviruses reported in the last 9 years and the possible mechanism of seed transmission. An emphasis is placed on the experimental results that proved the seed transmission of various begomoviruses, factors affecting seed transmission and impact of begomovirus seed transmission on virus circulation, outbreak of the disease, and management strategies.

Further Molecular Diagnosis Determines Lack of Evidence for Real Seed Transmission of Tomato Leaf Curl New Delhi Virus in Cucurbits.

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.

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

Coat protein is responsible for tomato leaf curl New Delhi virus pathogenicity in tomato.

Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite Begomovirus belonging to the family Geminiviridae, causes severe damage to many economically important crops worldwide. In the present study, pathogenicity of Asian (ToLCNDV-In from Pakistan) and Mediterranean isolates (ToLCNDV-ES from Italy) were examined using infectious clones in tomato plants. Only ToLCNDV-In could infect the three tomato cultivars, whereas ToLCNDV-ES could not. Genome-exchange of the two ToLCNDVs revealed the ToLCNDV DNA-A segment as the main factor for ToLCNDV infectivity in tomato. In addition, serial clones with chimeric ToLCNDV-In A and ToLCNDV-ES A genome segments were generated to identify the region determining viral infectivity in tomatoes. A chimeric clone carrying the ToLCNDV-In coat protein (CP) exhibited pathogenic adaptation in tomatoes, indicating that the CP of ToLCNDV is essential for its infectivity. Analyses of infectious clones carrying a single amino acid substitution revealed that amino acid at position 143 of the CP is critical for ToLCNDV infectivity in tomatoes. To better understand the molecular basis whereby CP function in pathogenicity, a yeast two-hybrid screen of a tomato cDNA library was performed using CPs as bait. The hybrid results showed different interactions between the two CPs and Ring finger protein 44-like in the tomato genome. The relative expression levels of upstream and downstream genes and Ring finger 44-like genes were measured using quantitative reverse transcription PCR (RT-qPCR) and compared to those of control plants. This is the first study to compare the biological features of the two ToLCNDV strains related to viral pathogenicity in the same host plant. Our results provide a foundation for elucidating the molecular mechanisms underlying ToLCNDV infection in tomatoes.

Investigating the Interplay between Tomato Leaf Curl New Delhi Virus Infection, Starch Metabolism and Antioxidant Defence System in Potato (Solanum tuberosum L.).

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 ß-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.

Size variations of mesoporous silica nanoparticle control uptake efficiency and delivery of AC2-derived dsRNA for protection against tomato leaf curl New Delhi virus.

KEY MESSAGE: We report the size dependent uptake of dsRNA loaded MSNPs into the leaves and roots of Nicotiana benthamiana plants and accessed for their relative reduction in Tomato leaf curl New Delhi viral load. A non-GMO method of RNA interference (RNAi) has been recently in practice through direct delivery of double stranded RNA into the plant cells. Tomato leaf curl New Delhi virus (ToLCNDV), a bipartitie begomovirus, is a significant viral pathogen of many crops in the Indian subcontinent. Conventional RNAi cargo delivery strategies for instance uses viral vectors and Agrobacterium-facilitated delivery, exhibiting specific host responses from the plant system. In the present study, we synthesized three different sizes of amine-functionalized mesoporous silica nanoparticles (amino-MSNPs) to mediate the delivery of dsRNA derived from the AC2 (dsAC2) gene of ToLCNDV and showed that these dsRNA loaded nanoparticles enabled effective reduction in viral load. Furthermore, we demonstrate that amino-MSNPs protected the dsRNA molecules from nuclease degradation, while the complex was efficiently taken up by the leaves and roots of Nicotiana benthamiana. The real time gene expression evaluation showed that plants treated with nanoparticles of different sizes ~ 10 nm (MSNPDEA), ~ 32 nm (MSNPTEA) and ~ 66 nm (MSNPNH3) showed five-, eleven- and threefold reduction of ToLCNDV in N. benthamiana, respectively compared to the plants treated with naked dsRNA. This work clearly demonstrates the size dependent internalization of amino-MSNPs and relative efficacy in transporting dsRNA into the plant system, which will be useful in convenient topical treatment to protect plants against their pathogens including viruses. Mesoporous silica nanoparticles loaded with FITC, checked for its uptake into Nicotiana benthamiana.

RNA-seq analysis reveals an early defense response to tomato leaf curl New Delhi virus in potato cultivar Kufri Bahar.

Potatoes in India are very susceptible to apical leaf curl disease, which causes severe symptoms and greater yield losses. Because the majority of potato cultivars are susceptible to the virus, it is crucial to discover sources of resistance and investigate the mechanism of resistance/susceptibility in potato cultivars. In this study, the gene expression profile of two potato cultivars, Kufri Bahar (resistant) and Kufri Pukhraj (susceptible), varying in their level of resistance to ToLCNDV, was analyzed using RNA-Seq. The Ion ProtonTM system was used to sequence eight RiboMinus RNA libraries from inoculated and uninoculated potato plants at 15 and 20 days after inoculation (DAI). The findings indicated that the majority of differentially expressed genes (DEGs) were cultivar-or time-specific. These DEGs included genes for proteins that interact with viruses, genes linked with the cell cycle, genes for proteins involved in defense, transcription and translation initiation factors, and plant hormone signaling pathway genes. Interestingly, defense responses were generated early in Kufri Bahar, at 15 DAI, which may have impeded the replication and spread of ToLCNDV. This research provides a genome-wide transcriptional analysis of two potato cultivars with variable levels of ToLCNDV resistance. At an early stage, we observed suppression of genes that interact with viral proteins, induction of genes associated with restriction of cell division, genes encoding defense proteins, AP2/ERF transcription factors, and altered expression of zinc finger protein genes, HSPs, JA, and SA pathway-related genes. Our findings add to a greater comprehension of the molecular basis of potato resistance to ToLCNDV and may aid in the development of more effective disease management techniques.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant.

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Development of an In-Field Real-Time LAMP Assay for Rapid Detection of Tomato Leaf Curl New Delhi Virus.

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.

DNA primase large subunit is an essential plant gene for geminiviruses, putatively priming viral ss-DNA replication.

The family of Geminiviridae consists of more than 500 circular single-stranded (ss) DNA viral species that can infect numerous dicot and monocot plants. Geminiviruses replicate their genome in the nucleus of a plant cell, taking advantage of the host's DNA replication machinery. For converting their DNA into double-stranded DNA, and subsequent replication, these viruses rely on host DNA polymerases. However, the priming of the very first step of this process, i.e. the conversion of incoming circular ssDNA into a dsDNA molecule, has remained elusive for almost 30 years. In this study, sequencing of melon (Cucumis melo) accession K18 carrying the Tomato leaf curl New Delhi virus (ToLCNDV) recessive resistance quantitative trait locus (QTL) in chromosome 11, and analyses of DNA sequence data from 100 melon genomes, showed a conservation of a shared mutation in the DNA Primase Large subunit (PRiL) of all accessions that exhibited resistance upon a challenge with ToLCNDV. Silencing of (native) Nicotiana benthamiana PriL and subsequent challenging with three different geminiviruses showed a severe reduction in titers of all three viruses, altogether emphasizing an important role of PRiL in geminiviral replication. A model is presented explaining the role of PriL during initiation of geminiviral DNA replication, i.e. as a regulatory subunit of primase that generates an RNA primer at the onset of DNA replication in analogy to DNA Primase-mediated initiation of DNA replication in all living organisms.

Seed Transmission of Begomoviruses: A Potential Threat for Bitter Gourd Cultivation.

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.

Occurrence of Tomato Leaf Curl New Delhi Virus in Cucurbit Plants in China.

In August 2022, melon (Cucumis melo), cucumber (Cucumis sativus) and luffa (Luffa aegyptiaca) plants showed virus-like symptoms characteristic of geminiviruses (yellowish green, mosaic patterns and severe curling of leaves, short internodes, and stunting) in 10 greenhouses in Fengxian district and 20 greenhoues in Jiading district of Shanghai, China. Fifty symptomatic leaf samples were randomly collected: 28 from melon, 17 from cucumber, and 5 from luffa. To investigate the etiology of the observed disease, total DNA and RNA was extracted via a DNA extraction kit (Tiangen, Not: DP350) and TRIzol reagent (Sigma-Aldrich, Not: T9424), respectively. Healthy melon plants grown in a growth chamber served as negative control. The DNA and RNA samples were screened for the presence of geminiviruses, Cucurbit chlorotic yellow virus (CCYV), Melon yellow spot virus (MYSV), Cucumber mosaic virus (CMV), Zucchini yellow mosaic virus (ZYMV) and Cucumber green mottle mosaic virus (CGMMV) through PCR or RT‒PCR with geminiviruses (Deng et al. 1994), CCYV, MYSV, CMV, ZYMV and CGMMV (Zeng et al. 2011, 2019) primers. The PCR results showed that 28 melon leaves and 17 cucumber leaves were positive for geminivirus and CCYV, respectively, 5 luffa samples were infected with only geminivirus, and virus was not detected from the healthy plants. These results indicate that these two viruses are widely distributed throughout cucurbit crops in Shanghai, China. All the geminiviruses sequences (approximately 510 bp) were quite similar to each other and were most similar (99.4%) to the Tomato leaf curl New Delhi virus (ToLCNDV) sequence (GenBank Accession No. OP356207) (Li et al. 2022). To confirm the presence of geminiviruses, the segments of DNA-A and DNA-B were amplified by PCR with 4 ToLCNDV-specific primer sets (Mizutani et al. 2011) and sequenced from 10 samples (4 melon, 4 cucumber and 2 luffa). Both DNA-A and DNA-B of the ToLCNDV sequences and features were deposited in GenBank under the accession numbers OQ190939-OQ190948 (DNA-A, 2739 nt) and OQ190949-OQ190958 (DNA-B, 2693 nt). BLASTn analysis of Shanghai isolates of ToLCNDV (DNA-A and DNA-B) showed that the sequences shared nucleotide identities ranged from 99.3% to 100% among them and with values of more than 99.4% nucleotide identity with ToLCNDV isolates from tomato in China (OP356207 and OP356208) (Li et al. 2022). To confirm the virus infection, we have successfully constructed an infectious clone for 0823-1 isolate in the binary plasmid and inoculated melon with and without an infectious clone. The melon plants inoculated with ToLCNDV 15 dpi showed the high accumulation of the virus and displayed symptoms similar to viruses in greenhouse. Based on the complete sequences, results of the molecular phylogenetic analysis (Fig. 2) and infectious clone, these geminiviruses were identified as ToLCNDV. ToLCNDV has been reported to occur and spread by the whitefly (Bemisia tabaci) in many Asian countries (Sohrab et al. 2003; Sohrab et al. 2011; Aamir et al. 2020) and Europe (Juárez et al. 2014; Ruiz et al. 2015; Luigi et al. 2019). Large populations of whiteflies were also present in all our surveyed areas. However, to our knowledge, this is the first report of the occurrence of ToLCNDV in cucurbit plants in China. The presence of ToLCNDV and CCYV can cause severe losses in crop yields or even crop failure. In addition to TYLCV, ToLCNDV is another major geminivirus-induced disease threatening cucurbit and other vegetable production in China.

Detecting Tomato Leaf Curl New Delhi Virus Causing Ridge Gourd Yellow Mosaic Disease, and Other Begomoviruses by Antibody-Based Methods.

The incidence and severity of begomovirus diseases have been increasing around the world recently, and the ridge gourd [Luffa acutangula (Roxb.) L.] is the latest example of a crop that has become highly susceptible to the outbreak of the tomato leaf curl New Delhi virus (ToLCNDV, genus Begomovirus) in India. Accurate diagnosis of causal agents is important in designing disease management strategies. In this study the coat protein (CP) gene from a ToLCNDV-Rg ridge gourd isolate was used to produce polyclonal antibodies (ToLCNDV-Rg-CP-PAb) in a rabbit. The antibodies successfully detected a 30.5 kDa ToLCNDV-Rg-CP in extracts of symptomatic ridge gourd leaf samples by several assays, such as Western Blotting (WB), Dot Immuno Binding Assay (DIBA), Direct Antigen Coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA), Immuno Capture Polymerase Chain Reaction (IC-PCR), and Immuno Capture Loop-Mediated Isothermal Amplification (IC-LAMP) assays. However, none of the negative samples tested positive in either of the detection methods. Among all the methods tested, the immunocapture assay, IC-LAMP, was the most sensitive in detecting ToLCNDV-Rg. Furthermore, antibodies generated in this study also detected other commonly occurring begomoviruses in South India, such as tomato leaf curl Palampur virus and squash leaf curl China virus in cucurbits. Together, ToLCNDV-Rg-CP-PAb can be used for detecting at least three species of begomoviruses infecting cucurbits. The obtained antibodies will contribute to monitoring disease outbreaks in multiple crops.

Characterization of a recombinant tomato leaf curl New Delhi Virus (ToLCNDV) in a perennial medicinal climber host (Ipomoea cairica (L.) Sweet).

During the year 2020-2021, a disease syndrome very commonly observed in railway creepers (Ipomoea cairica (L.) Sweet) was taken into consideration from Gorakhpur Province (UP East region). Whitefly, a common vector for plant-related viral diseases was observed for wide transmission of disease. DNA from 17 infected leaf samples was isolated and analyzed through PCR using specific primers designed for the detection of Begomoviruses. Further amplification of isolated DNA fragments supporting a begomovirus infection with an estimated size of (2.7 kb). RCA of the isolated DNA sample was carried out using ϕ29 DNA polymerase by digesting it through a set of restriction endonucleases (such as BamHI, Kpn1, HindIII, EcoRI) obtaining the best result through KpnI. The amplified segment was cloned into pUC 18vectors. The obtained sequences were matched using the NCBI BLAST database showing the highest sequence similarity of 98.7% with ToLCNDV of snake gourd (Accession no. KY780199), isolates of CP genes sequence in Varanasi. ToLCNDV, a begomovirus of bipartite nature was first reported to be from Tomato (Solanaceae), later affecting certain members of the Cucurbitaceae family in India and adjacent countries. The obtained DNA sequence was submitted at NCBI with the name ToLCNDV-IP (GenBank Accession no. OM777194). The phylogenetic analysis clubbed the current isolate ToLCNDV-IP with Asian isolates. All European isolates were clubbed in separate clades indicating two distinct groups of ToLCNDV isolates. ToLCNDV-IP isolate was found to be an intralineage recombinant between two Asian isolates viz ToLCNDV isolate from Papaya (India) and ToLCNDV isolate from Tomato (Pakistan). This study shows the association of recombinant ToLCNDV infection in a new host Ipomoea cairica for the first time in India.

Disruption of tomato TGS machinery by ToLCNDV causes reprogramming of vascular tissue-specific TORNADO1 gene expression.

MAIN CONCLUSION: Vascular development-related TRN1 transcription is suppressed by cytosine methylation in fully developed leaves of tomato. ToLCNDV infection disrupts methylation machinery and reactivates TRN1 expression - likely causing abnormal leaf growth pattern. Leaf curl disease of tomato caused by tomato leaf curl New Delhi virus (ToLCNDV) inflicts huge economical loss. Disease symptoms resemble leaf developmental defects including abnormal vein architecture. Leaf vein patterning-related TORNADO1 gene's (SlTRN1) transcript level is augmented in virus-infected leaves. To elucidate the molecular mechanism of the upregulation of SlTRN1 in vivo, we have deployed SlTRN1 promoter-reporter transgenic tomato plants and investigated the gene's dynamic expression pattern in leaf growth stages and infection. Expression of the gene was delimited in the vascular tissues and suppressed in fully developed leaves. WRKY16 transcription factor readily activated SlTRN1 promoter in varied sized leaves and upon virus infection, while silencing of WRKY16 gene resulted in dampened promoter activity. Methylation-sensitive PCR analyses confirmed the accumulation of CHH methylation at multiple locations in the SlTRN1 promoter in older leaves. However, ToLCNDV infection reverses the methylation status and restores expression level in the leaf vascular bundle. The virus dampens the level of key maintenance and de novo DNA methyltransferases SlDRM5, SlMET1, SlCMT2 with concomitant augmentation of two DNA demethylases, SlDML1 and SlDML2 levels in SlTRN1 promoter-reporter transgenics. Transient overexpression of SlDML2 mimics the virus-induced hypomethylation state of the SlTRN1 promoter in mature leaves, while silencing of SlDML2 lessens promoter activity. Furthermore, in line with the previous studies, we confirm the crucial role of viral suppressors of RNA silencing AC2 and AC4 proteins in promoting DNA demethylation and directing it to restore activated transcription of SlTRN1. Unusually elevated expression of SlTRN1 may negatively impact normal growth of leaves.