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Viruses of the genus Begomovirus (family Geminiviridae) are emergent pathogens of crops throughout the tropical and subtropical regions of the world. By virtue of having a small DNA genome that is easily cloned, and due to the recent innovations in cloning and low-cost sequencing, there has been a dramatic increase in the number of available begomovirus genome sequences. Even so, most of the available sequences have been obtained from cultivated plants and are likely a small and phylogenetically unrepresentative sample of begomovirus diversity, a factor constraining taxonomic decisions such as the establishment of operationally useful species demarcation criteria. In addition, problems in assigning new viruses to established species have highlighted shortcomings in the previously recommended mechanism of species demarcation. Based on the analysis of 3,123 full-length begomovirus genome (or DNA-A component) sequences available in public databases as of December 2012, a set of revised guidelines for the classification and nomenclature of begomoviruses are proposed. The guidelines primarily consider a) genus-level biological characteristics and b) results obtained using a standardized classification tool, Sequence Demarcation Tool, which performs pairwise sequence alignments and identity calculations. These guidelines are consistent with the recently published recommendations for the genera Mastrevirus and Curtovirus of the family Geminiviridae. Genome-wide pairwise identities of 91 % and 94 % are proposed as the demarcation threshold for begomoviruses belonging to different species and strains, respectively. Procedures and guidelines are outlined for resolving conflicts that may arise when assigning species and strains to categories wherever the pairwise identity falls on or very near the demarcation threshold value.

The begomoviral V2 protein is known to be multifunctional, including its interaction with and inhibition of CYP1, a papain‐like cysteine protease (PLCP). However, the effect of this interaction on viral pathogenicity remains unclear. Cotton leaf curl Multan virus (CLCuMuV), a typical monopartite begomovirus associated with a betasatellite, is one of the main pathogens responsible for cotton leaf curl disease. This study verifies the interaction between CLCuMuV V2 and NbCP15, a PLCP homologue in Nicotiana benthamiana. The results show that V2 can be cleaved by NbCP15 in vitro, with the N‐terminal cleavage site located between the second and third amino acids. Using an Agrobacterium‐mediated inoculation method, we investigated the influence of cleavage sites on viral pathogenicity. The findings indicate that mutation of the third amino acid in V2 (V2D3A) reduced the pathogenicity of both heterologous PVX and CLCuMuV. Additionally, the NbCP15 gene mutation in N. benthamiana (nbcp15) also resulted in reduced CLCuMuV pathogenicity. These results suggest that CLCuMuV V2 may promote viral infection through its interaction with plant PLCPs. Cotton leaf curl Multan virus uses its V2 protein to promote viral infection through its interaction with a plant papain‐like cysteine protease (NbCP15), which can cleave V2 after the first two amino acids at the N‐terminus.

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.

Yellow mosaic disease (YMD) is a major constraint for the low productivity of mungbean, mainly in South Asia. Addressing this issue requires a comprehensive approach, integrating field and challenge inoculation evaluations to identify effective solutions. In this study, an infectious clone of Begomovirus vignaradiataindiaense (MYMIV) was developed to obtain a pure culture of the virus and to confirm resistance in mungbean plants exhibiting resistance under natural field conditions. The infectivity and efficiency of three Agrobacterium tumefaciens strains (EHA105, LBA4404, and GV3101) were evaluated using the susceptible mungbean genotype PS16. Additionally, a recombinant inbred line (RIL) population comprising 175 lines derived from Pusa Baisakhi (MYMIV susceptible) and PMR-1 (MYMIV resistant) cross was developed and assessed for YMD response. Among the tested Agrobacterium tumefaciens strains, EHA105 exhibited the highest infectivity (84.7%), followed by LBA4404 (54.7%) and GV3101 (9.80%). Field resistance was evaluated using the coefficient of infection (CI) and area under disease progress curve (AUDPC), identifying seven RILs with consistent resistant reactions (CI≤9) and low AUDPC (≤190). Upon challenge inoculation, six RILs exhibited resistance, while RIL92 displayed a resistance response, with infection occurring in less than 10% of plants after 24 to 29 days post inoculation (dpi). Despite some plants remaining asymptomatic, MYMIV presence was confirmed through specific PCR amplification of the MYMIV coat protein (AV1) gene. Quantitative PCR revealed a very low relative viral load (0.1–5.1% relative fold change) in asymptomatic RILs and the MYMIV resistant parent (PMR1) compared to the susceptible parent (Pusa Baisakhi). These findings highlight the potential utility of the developed infectious clone and the identified MYMIV-resistant RILs in future mungbean breeding programs aimed at cultivating MYMIV-resistant varieties.

Tomato leaf curl New Delhi virus (ToLCNDV) is a whitefly-transmitted bipartite begomovirus (genus Begomovirus, family Geminiviridae) that causes damage to multiple cultivated plant species mainly belonging to the Solanaceae and Cucurbitaceae families. ToLCNDV was limited to Asian countries until 2012, when it was first reported in Spain, causing severe epidemics in cucurbit crops. Here, we show that a genetically-uniform ToLCNDV population is present in Spain, compatible with a recent introduction. Analyses of ToLCNDV isolates reported from other parts of the world indicated that this virus has a highly heterogeneous population genetically with no evident geographical, plant host or year-based phylogenetic groups observed. Isolates emerging in Spain belong to a strain that seems to have evolved by recombination. Isolates of this strain seem adapted to infecting cucurbits, but poorly infect tomatoes.

In this work, a begomovirus isolated from a bean plant coinfected with the potyviruses bean common mosaic virus and bean common mosaic necrosis virus was characterized. The three viruses were detected by high-throughput sequencing and assembly of total small RNAs, but the begomovirus-related contigs did not allow precise identification. Molecular analysis based on standard DNA amplification techniques revealed the presence of a single bipartite virus, which is a novel begomovirus according to the current taxonomic criteria. Infectious clones were generated and agroinoculated into Phaseolus vulgaris and Nicotiana benthamiana plants. In all cases, viral DNA-A and DNA-B were detected in new growths, but no symptoms were observed, thus indicating that this virus produces asymptomatic infections in both host species.

In India, plants from the non-cultivated, horticultural, and agricultural categories are commonly infected with various begomoviruses, most of which produce yellow mosaic, bright yellow mosaic, or curling symptoms on leaves. In this study, the complete genome of a new bipartite begomovirus causing yellow mosaic disease (YMD) in butterfly pea (Clitoria ternatea L.) was characterized using rolling-circle amplification followed by restriction digestion, cloning, and sequencing to obtain the full-length DNA-A (2727 nt) and DNA-B (2648 nt) sequences. The DNA-A and DNA-B components have a genome organization that is typical of the Old World bipartite begomoviruses, and the common regions (95 nt) of DNA-A and DNA-B share 91% nucleotide sequence identity, which is well above the threshold (>85%) for them to be considered cognate components. Both DNA molecules contain a begomovirus nonanucleotide motif, conserved iterons, and a putative stem-loop structure. The DNA-A of this virus was found to be most similar to that of Cajanus scarabaeoides yellow mosaic virus (CsYMV), with 82.61% nucleotide sequence identity, which is far below the species demarcation threshold (< 91%) for begomoviruses, whereas the DNA-B sequence showed the most similarity (77.16% identity) to that of Rhynchosia yellow mosaic virus (RhYMV). In phylogenetic analysis, both DNA-A and DNA-B were formed a clade with the corresponding genome segments of other YMD-causing begomoviruses. Based on these characteristics, this virus, for which the name \"butterfly pea yellow mosaic virus\" (BpYMV) is proposed, should be considered a member of a new species in the genus Begomovirus.

This is the first description of the complete genome sequence of a newly characterized monopartite begomovirus isolated from an asymptomatic uncultivated plant, Melochia tomentosa, collected in Burkina Faso. The sequence was obtained through rolling-circle amplification, cloning, and Sanger sequencing. The provisional species name “Begomovirus melochiae” and common virus name “melochia associated virus” (MeAV) are proposed. The MeAV genome was found to share the most nucleotide sequence similarity with three African monopartite begomoviruses: tomato curly stunt virus (74%), pepper yellow vein Mali virus (73%), and tomato leaf curl Cameroon virus (73%). Phylogenetic analysis confirmed its relationship to Old World monopartite begomoviruses. The discovery of MeAV in an uncultivated and asymptomatic plant provides a further example of the high diversity of begomoviruses in sub-Saharan African ecosystems.

Phytopathogenic viruses severely impact major crops, leading to significant social and economic losses. Among them, begomoviruses pose a serious threat to key cultivars in subtropical and tropical regions despite ongoing efforts to limit their spread. Early detection of these pathogens in field crops and associated weeds is essential for the timely implementation of management strategies to mitigate viral disease outbreaks. In this study, we applied a sensitive loop-mediated isothermal amplification (LAMP) assay for the detection of tomato yellow leaf curl virus (TYLCV), tomato latent virus (TLV), and tomato mottle Taino virus (ToMoTV) in agro-inoculated Nicotiana benthamiana and Solanum lycopersicum. Importantly, LAMP assays also enabled the identification of these viruses in both symptomatic and asymptomatic field-grown tomato plants, detecting a higher number of infected plants than dot blot hybridization and PCR. Field surveys further revealed mixed infections of TYLCV, TLV, and ToMoTV within individual tomato plants, uncovering a complex epidemiological scenario.

Tomato yellow leaf curl Guangdong virus (TYLCGdV), a monopartite begomovirus first identified in 2004, remains poorly characterised. In this study, we demonstrate that TYLCGdV associates with a betasatellite, TYLCGdB, and the βC1 protein encoded by TYLCGdB is essential for symptom development. We also explore the role of TYLCGdV C4 protein by generating a C4‐deficient infectious clone (TYLCGdVmC4), revealing a dynamic role for TYLCGdV C4. Specifically, viral accumulation in TYLCGdVmC4/TYLCGdB‐inoculated plants was significantly lower than that in TYLCGdV/TYLCGdB‐inoculated plants at 7 and 14 days post‐inoculation (dpi), but surpassed that of TYLCGdV/TYLCGdB‐inoculated plants by 25 dpi. Furthermore, although C4 proteins in other begomoviruses typically exhibit one or more of the following properties: (i) suppression of post‐transcriptional gene silencing (PTGS), (ii) suppression of transcriptional gene silencing (TGS), (iii) enhancement of pathogenicity in potato virus X (PVX) and (iv) symptom induction when transgenically expressed, TYLCGdV C4 did not exhibit any of these properties. However, the dynamic role of TYLCGdV C4 in viral infection appears to result from its effects on viral DNA methylation. At 7 dpi, the cytosine methylation level in the TYLCGdVmC4 genome was notably elevated compared to that of the wild‐type virus. However, this trend reversed by 14 dpi, with the wild‐type virus exhibiting a higher methylation level. By 25 dpi, the cytosine methylation levels of both TYLCGdVmC4 and TYLCGdV were comparable. These results indicate that TYLCGdV C4 modulates viral infection via an unconventional mechanism. This novel observation highlights the need for further investigation into the diverse roles of C4 proteins in begomoviruses. The C4 protein, encoded by tomato yellow leaf curl Guangdong virus (TYLCGdV), does not inhibit PTGS or TGS; the C4 protein modulates TYLCGdV infection by regulating the cytosine methylation level of viral genome at different infection stages.