Delhi Virus Research Articles

Distribution of Mosaic Disease in Ridge Gourd (Luffa acutangula (L.) Roxb.), Characterization of Associated Virus and Screening for Virus Resistance

Aims: A survey was conducted in the ridge gourd growing areas of Kasaragod, Kannur and Kozhikkode district, India, to assess the prevalence of virus diseases. Molecular detection and characterization indicated the presence of mixed infection of Tomato leaf curl New Delhi virus (ToLCNDV) and Cucumber mosaic virus (CMV) in infected samples of ridge gourd collected from surveyed locations. Additionally, a screening of popular varieties/hybrids was undertaken to identify resistance sources against the Begomovirus in Ridge gourd.
 Study Design: CRD design for screening for resistance genotype.
 Place and Duration of Study: Department of Plant Pathology, College of Agriculture, Padannakkad. November 2022 - 2023.
 Methodology: Twenty-five ridge gourd plants were selected from a field and scored during survey. Major weeds and insects in and around the location were collected and identified. Screening for resistance was carried out in vitro by grafting infected scion on to healthy ridge gourd plants. Begomovirus infections were confirmed by polymerase chain reaction (PCR) using specific coat protein primers. Presence of CMV was detected using specific primers.
 Results: Disease incidence (DI) of surveyed locations ranged from 5.3-100 per cent and vulnerability index (VI) from 26.57-84.08. Kunjathur, Thalangady, and Hosabettu areas of Kasaragod district recorded the highest incidence (100%). The highest VI (84.08) was recorded in Thalangady (Kasaragod). Bemisia tabaci and Aphis gossypii were the associated insects and Synedrella sp., Ageratum conizoides and Alternanthera sp. were the associated weeds.
 Screening of varieties indicated moderately susceptible reactions of RG7 and RG10 after 60 days of grafting. PCR based molecular detection of ToLCNDV using coat protein specific primers yielded amplicons of approximately 575 bp. All the isolated sequences were found to be related to ToLCNDV on sequencing and blast analysis. Phylogenetic studies revealed that isolates from Thrikaripur, Mavichery, and Poolacode were closely related to each other. Cucumber mosaic virus, a RNA virus was also detected from isolated samples using specific primers of CMV with an amplicon size of 400 bp.
 Conclusion: The study revealed that the ridge gourd cultivated in areas of Kasaragod, Kannur and Kozhikode were infected with ToLCNDV and CMV; RG7 and RG10 were moderately susceptible among the varieties and hybrids screened. The presence of the begomovirus was detected in weeds indicating its role in perpetuation and spread of the virus disease in the field.

Molecular characterization of coat protein of tomato leaf curl New Delhi virus (Begomovirus, F: Geminiviridae) infecting ridge gourd and its expression studies in Escherichia coli

Molecular characterization of coat protein of tomato leaf curl New Delhi virus (Begomovirus, F: Geminiviridae) infecting ridge gourd and its expression studies in Escherichia coli

Molecular Insights into the Adaptability and Pathogenicity of Tomato Leaf Curl New Delhi Virus

Molecular Insights into the Adaptability and Pathogenicity of Tomato Leaf Curl New Delhi Virus

Cotton leaf curl Multan betasatellite impaired ToLCNDV ability to maintain cotton leaf curl Multan alphasatellite.

Alphasatellites (family Alphasatellitidae) are circular, single-stranded (ss) DNA molecules of ~1350 nucleotide in size that have been characterized in both the Old and New Worlds. Alphasatellites have inherent ability to self-replicate, which is accomplished by a single protein, replication-associated protein (Rep). Although the precise function of alphasatellite is yet unknown, and these consider dispensable for infectivity, however, their Rep protein functions as a suppressor of host defence. While alphasatellites are most frequently associated with begomoviruses, particularly with monopartite than bipartite begomoviruses, they have recently been found associated with mastreviruses. The in planta maintenance of alphasatellites by helper geminivirus is still an enigma, with no available study on the topic. This study aimed to investigate whether a widely distributed bipartite begomovirus, tomato leaf curl New Delhi virus (ToLCNDV), can maintain cotton leaf curl Multan alphasatellite (CLCuMuA) in the presence or absence of cotton leaf curl Multan betasatellite (CLCuMuB). The findings of this study demonstrated that ToLCNDV or its DNA A could maintain CLCuMuA in Nicotiana benthamiana plants. However, the presence of CLCuMuB interferes with the maintenance of CLCuMuA, and mutations in the CP of ToLCNDV further reduces it. Our study highlighted that the maintenance of alphasatellites is impaired in the presence of a betasatellite by ToLCNDV. Further investigation is needed to unravel all the interactions between a helper virus and an alphasatellites.

Cloning and Characterization of Rep Gene of the Strain of Tomato Leaf Curl New Delhi Virus (G: Begomovirus, F: Geminiviridae) Infecting Ridge Gourd in Karnataka and in silico Analysis of Rep Protein

Ridge gourd [Luffa acutangula (Roxb.) L.,] is one of the important vegetable crop affected by various diseases particularly viral disease tomato leaf curl New Delhi virus (ToLCNDV). The ridge gourd plants with leaf curling and yellowing symptoms were collected and used in for the amplification of the of the Rep gene. Cloning, sequencing and bioinformatics analysis of the Rep gene was carried out. The results revealed the association of this virus isolate with the different host of Cucurbitaceae family. Pairwise identity derived implemented by BLAST identified that the replicator initiator protein gene (Rep) of the bipartite virus showed maximum 96.90% sequence identity to the corresponding Rep gene of ridge gourd tomato leaf curl New Delhi virus isolate RG3 in ridge gourd - complete genome from Madikari, Karnataka (accession no KT426905). This also showed 97.90% sequence similarities with tomato leaf curl New Delhi virus strain CH7 of Chrysanthemum. Phylogenetic dendrogram of the Rep gene depicted that it grouped with ToLCNDV of different Indian isolates of ridge gourd and with ToLCNDV of potato from Pakistan (LN908936), and with tomato from Bangladesh (KM383738.1) showing 93.12% and 92.94% sequence similarity respectively. This study provides association of the Rep gene infected ridge gourd pants with Cucurbitaceae family crops and other related crops. This will helps in carrying further research in the management of ToLCNDV diseases using different biotechnological applications.

Accelerated Long-Fragment Circular PCR for Genetic Manipulation of Plant Viruses in Unveiling Functional Genomics.

Molecular cloning, a crucial prerequisite for engineering plasmid constructs intended for functional genomic studies, relies on successful restriction and ligation processes. However, the lack of unique restriction sites often hinders construct preparation, necessitating multiple modifications. Moreover, achieving the successful ligation of large plasmid constructs is frequently challenging. To address these limitations, we present a novel PCR strategy in this study, termed 'long-fragment circular-efficient PCR' (LC-PCR). This technique involves one or two rounds of PCR with an additional third-long primer that complements both ends of the newly synthesized strand of a plasmid construct. This results in self-circularization with a nick-gap in each newly formed strand. The LC-PCR technique was successfully employed to insert a partial sequence (210 nucleotides) of the phytoene desaturase gene from Nicotiana benthamiana and a full capsid protein gene (770 nucleotides) of a begomovirus (tomato leaf curl New Delhi virus) into a 16.4 kb infectious construct of a tobamovirus, cucumber green mottle mosaic virus (CGMMV), cloned in pCambia. This was done to develop the virus-induced gene silencing vector (VIGS) and an expression vector for a foreign protein in plants, respectively. Furthermore, the LC-PCR could be applied for the deletion of a large region (replicase enzyme) and the substitution of a single amino acid in the CGMMV genome. Various in planta assays of these constructs validate their biological functionality, highlighting the utility of the LC-PCR technique in deciphering plant-virus functional genomics. The LC-PCR is not only suitable for modifying plant viral genomes but also applicable to a wide range of plant, animal, and human gene engineering under in-vitro conditions. Additionally, the LC-PCR technique provides an alternative to expensive kits, enabling quick introduction of modifications in any part of the nucleotide within a couple of days. Thus, the LC-PCR proves to be a suitable 'all in one' technique for modifying large plasmid constructs through site-directed gene insertion, deletion, and mutation, eliminating the need for restriction and ligation.

Molecular characterization and pathogenicity of an infectious clone of tomato leaf curl New Delhi virus isolate infecting Cucumis melo

Tomato leaf curl New Delhi virus (ToLCNDV) is a member of the genus Begomovirus, and causes devastating disease in the world. In recent years, ToLCNDV was rapidly spreading in China and induces severe economic losses in agriculture. In this study, we sequenced and characterized the complete genome of ToLCNDV isolates from melon plants showing leaf curling and stunting symptoms in Jiangsu Province of China. We constructed a full-length infectious cDNA clone of ToLCNDV, which could induce systemic infection with typical symptoms in Nicotiana benthamiana, Citrullus melo, and Citrullus lanatus plants through agrobacterium-mediated inoculation. Further experimental evidence demonstrated that the virions produced in plants infected with the infectious clone of ToLCNDV are biologically active and sap-transmissible. We also evaluated the resistance of commercial melon cultivars to ToLCNDV and found all testing melon cultivars were susceptible to ToLCNDV. Collectively, the reverse genetic system developed herein will facilitate further research on biological functions of proteins encoded by ToLCNDV and plant-ToLCNDV interactions, which might provide new insights into breeding resistance germplasm in crops.

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 and molecular characterization of tomato leaf curl and its vector across the Himalayan foothills in Uttarakhand, India

AbstractTomato leaf curl disease is a serious constraint to tomato cultivation across the globe. The disease is characterized by leaf curling, crinkling, stunting and puckering with the drastic reduction in plant growth and yield. During two‐year survey in major tomato‐growing belt of Tarai region located in foothills of Himalayas in Uttarakhand, India, the mean incidence and per cent disease index of the disease were found to be 68.76% and 67.19%, respectively. In the morphological analysis using electron microscopy, pathogen contains twin geminate particles measuring 30 × 20 nm. The PCR amplification using RUGEMF1 and RUGEMR1 primers and sequencing of the amplicons, revealed more than 97% similarity to Tomato leaf curl New Delhi Virus (ToLCNDV). Thus, based on the sequence similarity, morphology and whitefly transmission, begomovirus isolate was recognized as a Tomato leaf curl New Delhi virus and designated as ToLCNDV_[Pant]. Amplification and sequencing of the Bemisia tabaci partial mtCOI gene from the Tarai region revealed the prevalence of Asia‐II‐1 cryptic species in the region. The overall mean pairwise distance across the 10 sequences studied was 0.0139, whereas the reference Asia‐II‐1 distance ranged from 0.0031 to 0.0332. The current investigation will aid in the development of an integrated strategy to ToLCD management.

A distinct strain of tomato leaf curl New Delhi virus that causes mosaic disease in ash gourd and other cucurbitaceous crops.

Ash gourd (Benincasa hispida) is a cucurbitaceous crop cultivated as an edible vegetable rich in vitamins, minerals, dietary fibers and antioxidants. In a field survey conducted in the Udumalpet region of Tamil Nadu during 2019, the incidence of mosaic disease on ash gourd crop was observed to be 75%. The DNA-A and DNA-B components of begomovirus genome have been identified as associated with this disease. Both the cloned DNA-A and DNA-B genomic components shared highest pairwise sequence identities with the isolates of tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus. Recombinant analysis showed that both the components are possibly evolved through intra-species recombination between ToLCNDV isolates. Tomato leaf curl Bangladesh betasatellite (ToLCBB) is not naturally associated with this sample. The results of infectivity studies on ash gourd and other cucurbitaceous crops demonstrates the Koch's postulates, when co-inoculation of DNA-A and DNA-B of ToLCNDV was undertaken. However, the inoculation of non-cognate ToLCBB along with DNA-A and DNA-B enhances the symptom expression and reduces the time taken for symptom development. Thus, Koch's postulates were proved for these virus complexes on cucurbitaceous crops. Furthermore, an enhanced accumulation of DNA-A component was detected in the cucurbits co-inoculated with ToLCNDV and ToLCBB. This report highlights the importance of investigating the spread of these disease complexes with other cucurbitaceous crops in India.

Identifying new sources of resistance to tomato leaf curl New Delhi virus from Indian melon germplasm by designing an improved method of field screening

Identifying new sources of resistance to tomato leaf curl New Delhi virus from Indian melon germplasm by designing an improved method of field screening

Molecular Identification of Tomato Leaf Curl New Delhi Virus Associated with Mosaic Disease of Pumpkin from Central India

The Tomato leaf curl New Delhi virus is an emerging disease of pumpkin associated with mosaic disease in central, India. Symptomatic pumpkin leaf samples from different locations in the Bhopal district were collected and confirmed for begomovirus disease by PCR using a begomovirus coat-protein gene-specific primer. All five samples were amplified, expecting ~800 bp of amplicons of associated begomovirus; these amplicons were purified and sequenced for identification of begomovirus species associated with mosaic disease on the pumpkin. The consensus nucleotide sequence data were submitted to the NCBI database under the accession numbers OQ320768, OQ320770, OQ320774, OQ116977, and OQ116978. The understudy begomovirus isolates showed the highest 95–97% nucleotide sequence identities and close phylogenetic relationships with several isolates of Tomato leaf curl New Delhi virus. To our knowledge, this was the first record of the Tomato leaf curl New Delhi virus associated with the mosaic disease of pumpkin in Central India.

Unraveling the devastating impact of Tomato Leaf Curl New Delhi Virus (ToLCNDV): understanding its characteristics, host range, and management strategies

Due to their stagnation, plants are frequently exposed to a variety of stresses, including both abiotic and biotic factors. Biotic stress in plants is due to many microbes that include viruses, fungi, bacteria, and parasitic organisms as plant pathogens. Plant viruses are one of those pathogens that frequently evolve and lower global agricultural productivity. Geminiviruses are the most damaging type of plant viruses. Tomato Leaf Curl New Delhi Virus (ToLCNDV) is a begomovirus belonging to the family Geminiviridae. This is a ssDNA virus having a bipartite genome with genomic length ranging between 2.6-2.7 kb. This virus was originally discovered in India in 1995 from tomato (Lycopersicon esculentum). It mostly infects solanaceous and cucurbitaceous crops and is a whitefly-transmitted, circulative and persistent virus. Its prominent symptoms are severe upward leaf curling, enations, stunting, puckering, yellow spots and yellow mosaic. This virus has devastating effects on the yield of different crops with yield losses ranging between 17.6-99.4 % depending upon the disease-causing factors. ToLCNDV-ES and ToLCNDV-OM are the most devastating strains of this virus. This comprehensive review will provide basic information on the characteristic features of ToLCNDV. The evolutionary mechanism of this virus is studied to know about the strain’s evolution with the passage of time on different host plants cultivated in several regions of the world. This review also covers the genome characteristics, geographical distribution, host range, transmission methods, genetic diversity, different molecular & serological assays, yield losses, and the management of this virus.

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.

Genetics of begomovirus resistance in Cucurbita moschata Duchesne: A novel resistant source PVR-1343

Whitefly transmitted begomoviruses are the most destructive virus(es) that causes quantitative and qualitative losses to pumpkin (Cucurbita moschata) crop worldwide. In present study we have molecularly characterized three types of viral infections, which were caused by squash leaf curl China virus (SLCCNV), tomato leaf curl New Delhi virus (ToLCNDV), and their mixed infection (MI-Sq/To). Screening of diverse germplasm through whitefly (Bemisia tabaci mitotype Asia II) mediated artificial inoculation and PCR confirmed the 'PVR-1343' genotype as a resistance source against identified begomoviruses. To understand the genetic control of begomovirus resistant gene(s), segregating populations derived from crosses of 'PVR-1343' and four susceptible genotypes (MVSR-6711, Punjab Samrat, MBN-6242 and P-278) inoculated with SLCCNV, ToLCNDV and MI-Sq/To by whitefly in closed environment for three consecutive years (2018–2020). Disease scoring at 75 days after sowing rendered all F1 progenies as susceptible indicating recessive nature of resistance with no maternal or cytoplasmic effects. Chi-square analysis indicated a good fit of 9(S):3(MS):3(MR):1(R) ratio in F2 progenies of all crosses. Backcross of the F1’s to the resistant parent segregated into 1:1:1:1 ratio, while all progeny were susceptible in backcross of the F1’s with susceptible parent. Phenotypic segregation ratio of F2:3 confirmed the F2 genotypic ratio of 1:2:2:4:1:2:1:2:1. This substantiated that multiple begomovirus resistance in 'PVR-1343' is controlled by digenic recessive genes. Moreover, F2:3 progenies resistant to SLCCNV were also resistant to ToLCNDV and MI-Sq/To and vice versa, suggesting that common or tightly linked gene(s) might be involved in resistance to both viruses.

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.

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

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 ~ 10nm (MSNPDEA), ~ 32nm (MSNPTEA) and ~ 66nm (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.

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 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.

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.

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 20days 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.