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Garden croton ( Codiaeum variegatum L.) plants showing typical begomovirus symptoms of vein twisting, enation and curling were collected from different gardens at Varanasi, Uttar Pradesh state of India ranged from 20 to 30%. All the 10 ten (CR1-CR10) infected samples of garden croton resulted in expected amplicon of 1.2 Kb in PCR specific to begomoviruses. No amplification was obtained for betasatellite and alphasatellite specific primers. The complete genome sequence of DNA-A and DNA-B for two isolates (CR1 and CR2) was obtained through rolling cycle amplification (RCA) and comparisons were made with other begomoviruses using Sequence Demarcation Tool (SDT) which revealed that, DNA-A of two isolates, CR1 (Acc.No.: MW816855) and CR2 (Acc.No.: MW816856) showed maximum nucleotide (nt) identity of 85.7–85.9% with Tomato leaf curl Karnataka virus, which is below the threshold percentage of begomovirus species demarcation, hence considered as novel begomovirus and proposed the name Garden croton enation leaf curl virus (CroELCuV) [IN: Varanasi: Croton: 18]. Further, DNA-B these isolates shared maximum nt identity of 91.0–92.2% (DNA-B) with Tomato leaf curl New Delhi virus . Recombination and GC plot analysis showed that the recombination occured at in low GC content regions of DNA-A and DNA-B of the CroELCuV and are derived from the previously reported several begomoviruses. This is the first record of novel bipartite begomovirus associated with vein twisting, enation and leaf curling of disease of garden garden croton in India and world.

Chilli, an important vegetable cum spice crop in India, is affected by various biotic and abiotic factors, which leads to a significant reduction in the growth and yield of the chilli crop. One of the most prominent biotic factors posing threat to chilli production in southern parts of India especially, in Tamil Nadu is begomoviruses (Family Geminiviridae ). Begomoviruses are transmitted by the insect vector, whitefly ( Bemisia tabaci ) and they cause curling, yellowing, puckering, and reduction in the size of the leaf in the infected chilli plants. In this study, molecular (PCR ) diagnostics were used to detect the presence of begomovirus, betasatellite, and six begomovirus species viz tomato leaf curl New Delhi virus (ToLCNDV), tomato leaf curl Bangalore virus (ToLCBV), tomato leaf curl Palampur virus (ToLCPalV), tomato leaf curl Gujarat virus (ToLCGV), tomato leaf curl Joydebpur virus (ToLCJoV) and chilli leaf curl virus (ChiLCV), in the chilli samples collected from the major chilli growing areas of all the five agro-climatic zones of Tamil Nadu state of India. A total number of 833 samples collected from different locations in Tamil Nadu during the period of 2018-2022 were analysed by generic as well as species-specific PCR. The PCR results of 833 samples showed positive amplification of 20.5% for generic, 13.4% for beta satellite, 42.1% for ChiLCV, 17.8% (ToLCNDV), 16.6% (ToLCGV), 6.7% (ToLCBV), 2.2% (ToLCPalV), and 0.7% for ToLCJoV specific primers respectively. The percentage of mixed infection of two or more than two begomoviruses among the total samples is 39.2. Our study has shown that the ChiLCV and multiple tomato-infecting begomoviruses were prevalent in the major chilli-growing areas of Tamil Nadu. The present study also showed that species of begomovirus infecting chilli plants in Northern and Southern India have a differential distribution.

Tomato, worldwide, is infected by several viral diseases which cause stunting, leaf curl, yellowing, mosaic, mottling, necrosis and shoe-string symptoms on plants, leaves or fruits. Among them, bud necrosis disease caused by an orthotospovirus is emerging as a major constraint to the cultivation of tomato for resource-poor farmers. In the IndoGangetic eastern plains, bud necrosis disease incidence on tomato ranged from 0 to 45% under field conditions during 2015 and 2016, along with other diseases such as leaf curl and mosaic caused by begomoviruses (0–35%) and tobamoviruses (0–18%) respectively. Thirty four viral infected samples collected from 11 different villages were screened for different viruses using serological and PCR-based methods. The result revealed that most samples were positive for RNA (peanut bud necrosis virus, tomato mosaic virus) and DNA (tomato leaf curl New Delhi virus, tomato leaf curl Gujarat virus, tomato leaf curl Palampur virus, tomato leaf curl Bangalore virus, tomato leaf curl Karnataka virus) viruses along with their satellites (DNA-α and DNA-ß) respectively. Further, the incidence of bud necrosis correlates to the weather parameters, which demonstrate that a long dry spell leads to higher incidence of viral disease, whereas lower incidence was observed during the rainy period considering a reduced vector population.

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.

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.

One important factor for successful disease management is the ability to rapidly and accurately identify the causal agent. Plant viruses cause severe economic losses and pose a serious threat to sustainable agriculture. Therefore, optimization of the speed, sensitivity, feasibility, portability, and accuracy of virus detection is urgently needed. Here, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based nucleic acid diagnostic method utilizing the CRISPR–Cas12a system for detecting two geminiviruses, tomato yellow leaf curl virus (TYLCV) and tomato leaf curl New Delhi virus (ToLCNDV), which have single-stranded DNA genomes. Our assay detected TYLCV and ToLCNDV in infected plants with high sensitivity and specificity. Our newly developed assay can be performed in ~1 h and provides easy-to-interpret visual readouts using a simple, low-cost fluorescence visualizer, making it suitable for point-of-use applications.

The ongoing global spread of Tomato yellow leaf curl virus (TYLCV) poses an endemic threat to tomato production in all tropical and sub-tropical regions of the world. In Oman, TYLCV evolved into the new species Tomato leaf curl Oman virus (ToLCOMV) by recombination with Papaya leaf curl virus (PaLCuV). In March 2017, during a field survey of Sindh province of Pakistan, tomato (Solanum lycopersicum L.) plants showing typical symptoms of Tomato leaf curl disease (ToLCD); such as leaf curling, yellowing and stunted plant growth were observed. For identification and characterization of the causative agent, leaf samples were collected and tested for the presence of begomoviruses and associated DNA satellites. We have recently reported the presence of ToLCOMV-PK (previously known as TYLCV-PK strain) on Cyamopsis tetragonoloba in the same province of Pakistan. The tomato samples were analyzed for the presence of ToLCOMV-PK using specific primers. Approx. 2.8 kb and 1.4 kb PCR products were obtained which were cloned and sequenced; the results indicated the presence of ToLCOMV-PK and Tomato leaf curl betasatellite (ToLCB). We were also able to identify defective molecules of ToLCOMV-PK which were about half the size of the complete virus. Phylogenetic analysis and the sequence demarcation tool (SDT) showed that these are new isolates of ToLCOMV-PK (ToLCOMV-Pakistan strain) associated with ToLCB. Recombination analysis detected a strong recombination event in ToLCOMV-PK in which TYLCV (JQ928346) and PaLCuV (LT009400) were major and minor parents, respectively. We also corrected the classification of the previously identified distinct strain i.e. TYLCV-PK (TYLCV-Pakistan strain) into ToLCOMV-PK strain, based on our current analysis and classification. Biolistic inoculation of ToLCOMV-PK and associated ToLCB in Nicotiana benthamiana plants showed that coinfection with ToLCB resulted in enhanced symptoms and virus accumulation. This report of ToLCOMV-PK associated with ToLCB infecting tomato in Pakistan represents the widespread and vast host range of ToLCOMV and which poses serious threat to tomato production in Pakistan.

Cotton leaf curl disease (CLCuD) is the major biotic constraint to cotton production on the Indian subcontinent, and is caused by monopartite begomoviruses accompanied by a specific DNA satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Since the breakdown of resistance against CLCuD in 2001/2002, only one virus, the “Burewala” strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bur), and a recombinant form of CLCuMB have consistently been identified in cotton across the major cotton growing areas of Pakistan. Unusually a bipartite isolate of the begomovirus Tomato leaf curl virus was identified in CLCuD-affected cotton recently. In the study described here we isolated the bipartite begomovirus Tomato leaf curl New Delhi virus (ToLCNDV) from CLCuD-affected cotton. To assess the frequency and geographic occurrence of ToLCNDV in cotton, CLCuD-symptomatic cotton plants were collected from across the Punjab and Sindh provinces between 2013 and 2015. Analysis of the plants by diagnostic PCR showed the presence of CLCuKoV-Bur in all 31 plants examined and ToLCNDV in 20 of the samples. Additionally, a quantitative real-time PCR analysis of the levels of the two viruses in co-infected plants suggests that coinfection of ToLCNDV with the CLCuKoV-Bur/CLCuMB complex leads to an increase in the levels of CLCuMB, which encodes the major pathogenicity (symptom) determinant of the complex. The significance of these results are discussed.

Key messageA begomovirus resistance gene pepy-1, which encodes the messenger RNA surveillance factor Pelota, was identified in pepper (C. annuum) through map-based cloning and functional characterization.Pepper yellow leaf curl disease caused by begomoviruses seriously affects pepper (Capsicum spp.) production in a number of regions around the world. Ty genes of tomato, which confer resistance to the tomato yellow leaf curl virus, are the only begomovirus resistance genes cloned to date. In this study, we focused on the identification of begomovirus resistance genes in Capsicum annuum. BaPep-5 was identified as a novel source of resistance against pepper yellow leaf curl Indonesia virus (PepYLCIV) and pepper yellow leaf curl Aceh virus (PepYLCAV). A single recessive locus, which we named as pepper yellow leaf curl disease virus resistance 1 (pepy-1), responsible for PepYLCAV resistance in BaPep-5 was identified on chromosome 5 in an F2 population derived from a cross between BaPep-5 and the begomovirus susceptible accession BaPep-4. In the target region spanning 34 kb, a single candidate gene, the messenger RNA surveillance factor Pelota, was identified. Whole-genome resequencing of BaPep-4 and BaPep-5 and comparison of their genomic DNA sequences revealed a single nucleotide polymorphism (A to G) located at the splice site of the 9th intron of CaPelota in BaPep-5, which caused the insertion of the 9th intron into the transcript, resulting in the addition of 28 amino acids to CaPelota protein without causing a frameshift. Virus-induced gene silencing of CaPelota in the begomovirus susceptible pepper No.218 resulted in the gain of resistance against PepYLCIV, a phenotype consistent with BaPep-5. The DNA marker developed in this study will greatly facilitate marker-assisted breeding of begomovirus resistance in peppers.

Abstract 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. Resumo Alfassatélites (família Alphasatellitidae) são moléculas de DNA circulares de fita simples (ss) de ~1350 nucleotídeos de tamanho, que foram caracterizadas tanto no Velho como no Novo Mundo. Os alfassatélites têm capacidade inerente de autorreplicação, o que é realizado por uma única proteína, a proteína associada à replicação (Rep). Embora a função precisa dos alfassatélites ainda seja desconhecida, e estes sejam considerados dispensáveis para infectividade, entretanto, sua proteína Rep funciona como supressora da defesa do hospedeiro. Embora os alfassatélites sejam mais frequentemente associados a begomovírus, particularmente com begomovírus monopartidos do que bipartidos, eles foram encontrados recentemente associados a mastrevírus. A manutenção in planta de alfassatélites por helper geminivirus ainda é um enigma, sem estudos disponíveis sobre o tema. Este estudo teve como objetivo investigar se um begomovírus bipartido amplamente distribuído, o tomate leaf curl New Delhi virus (ToLCNDV), pode manter o alfassatélite Multan do enrolamento das folhas de algodão (CLCuMuA) na presença ou ausência do betassatélite Multan do enrolamento das folhas de algodão (CLCuMuB). Os achados deste estudo demonstraram que ToLCNDV ou seu DNA A poderia manter CL CuMuA em plantas de Nicotiana benthamiana. No entanto, a presença de CLCuMuB interfere na manutenção de CLCuMuA, e mutações no CP de ToLCNDV a reduzem ainda mais. Nosso estudo destacou que a manutenção de alfassatélites é prejudicada na presença de um betassatélite por ToLCNDV. Mais investigações são necessárias para desvendar todas as interações entre um vírus auxiliar e um alfassatélite.