INTRODUCTION
Raphanus sativus L. is a widely cultivated root vegetable belonging to the family Brassicaceae and is valued for its nutritional importance and economic significance. Its short growth cycle and adaptability to various agroclimatic environments, make radish an important vegetable crop cultivated across a wide range of agricultural regions (Manivannan et al., 2019; Nowak et al., 2024).
Despite its global adaptability, the radish cultivation is affected by several viral diseases, among which begomoviruses represent an important group of pathogens. They are circular single stranded DNA viruses belonging to family Geminiviridae transmitted by whiteflies (Bemisia tabaci). This family comprises taxonomically 15 different genera (Fiallo-Olive et al., 2021), among which Begomovirus is the most destructive and diverse genus in the world. These viruses are responsible for substantial yield losses in many economically important crops, including cassava, cotton and tomato (Briddon and Markham, 2000; Chakraborty et al., 2003; Varma and Malathi, 2003). Betasatellites consist of compact ssDNA genomic elements of nearly ~1.3kb that commonly occur in association with Old World begomoviruses. The betasatellite molecules depend on DNA-A or helper virus for systemic movement and replication. They are considered as major factors in contributing symptom development of the disease (Briddon and Stanley, 2006; Kumar et al., 2014). Betasatellites code a very conserved protein named as βC1, which functions as symptoms determinant and suppressor of host RNA silencing defense mechanisms (Briddon and Stanley, 2006).
Southeast Asia represents a geographically important hot spot of Old World begomoviruses diversification. This region is associated with exceptionally high levels of genetic heterogeneity considerably through inter-species recombination and pervasive interactions with virus and associated molecules (Nawaz-ul-Rehman et al., 2012). Such evolutionary dynamism helps to achieve a quick adaptation of the host, and thus far, these viruses have been able to infect a vast array of economically important vegetable crops such as Capsicum spp., Ipomoea batatas, Solanum melongena, and Solanum lycopersicum (More et al., 2022; Shingote et al., 2022; Venkataravanappa et al., 2023).
Begomovirus infection of radish was first documented in Pakistan by Mansoor et al. (2000). The radish plants exhibiting typical symptoms of leaf enations and curling were harboring Cotton leaf curl virus, suggesting expansion of the virus host range beyond the family Malvaceae (Mansoor et al., 2000). Infection severity levels ranging from 10-90% were observed in the kitchen gardens and commercial fields in Punjab. PCR and nucleic acids-based hybridization methods confirmed occurrence of DNA-A and the betasatellite molecules in symptomatic radish plants. Later on, Singh et al. (2007) documented significant disease incidence in radish crops in India-Varanasi district, where infected plants were exhibiting similar symptoms associated with begomoviruses. Molecular analyses of early isolates indicated phylogenetic relationships with Tomato leaf curl Bangladesh virus, suggesting possible host-shift events. The begomoviruses in the Indian sub-continent, are considered as important pathogens of vegetables crops, causing leaf curling, stunting and significant yield losses. For examples, Kumar et al. (2012), reported that okra plants exhibiting sever leaf curling and stunted plant growth were found to harbor a monopartite begomovirus along with alphasatellite and betasatellite molecules. Sequence analysis of the begomoviruses showed high nucleotide identity with radish leaf curl virus and associated satellites.
Although begomoviruses-associated diseases in radish have been reported from several regions, information regarding the molecular diversity of these viruses and their satellite molecules remains limited. This lack of information restricts effective surveillance and management strategies. Therefore, the present study aimed to characterize the begomovirus complex associated with symptomatic radish plants in Pakistan using coat-protein-specific and universal betasatellite primers to identify the viral DNA-A and betasatellite molecules.
MATERIALS AND METHODS
Sample Collection and DNA Extraction: During 2020-2021, field surveys were conducted in radish growing areas of Punjab Province, Pakistan including Faisalabad, Okara, Toba Tek Singh, Multan, Lahore, and Layyah. A total of 120 sites of these six districts along with their vegetable markets were surveyed for the presence of virus-like symptoms. These locations were selected based on the availability of radish crop and accessibility to the fields. Symptomatic plants with typical symptoms of virus infection were observed in the Thal Desert region near Layyah (31.08 N, 71.12 E). Infected plants exhibited severe stunting, reduced leaf size and pronounced vein thickening (Figure-1B). Leaf tissues from six symptomatic plants were collected, properly labeled and immediately-preserved on dry ice during transportation. The samples were subsequently transferred to the molecular virology lab, University of Agriculture Faisalabad, for genomic analysis. Total genomic DNA was extracted from ~100mg leaf tissues, using modified cetyltrimethyl-ammonium bromide (CTAB) method based on protocol of Doyle (1991). Briefly, frozen leaf tissues were ground into a fine powder using liquid nitrogen. Cellular lysate of the homogenate was then incubated in 2% CTAB buffer at 65°C for a period of 45 minutes following chloroform: isoamyl alcohol (24:1 v/v) and ethanol precipitation. The quality and quantity of extracted DNA were assessed using a Bio-Rad SmartSpecTM Plus spectro-photometer. For downstream PCR applications, 100 ng of DNA was standardized in a final reaction volume of 25 µL.
PCR Amplification of Viral Components: Viral detection was targeted based on begomovirus(es) and satellite molecules. The CP gene of DNA-A component was amplified with previously reported degenerate primers AV1F and AV1R (Khan et al., 2014; Brown et al., 2001). Universal betasatellite primers were simultaneously used for the amplification of betasatellites using the following reaction mixture: 100 ng genomic DNA, 0.2 µM primer concentration, 200 µM dNTP mixture, 1.5 mM MgCl2, 1x manufacturer-supplied reaction buffer, and Phusion™ High-Fidelity DNA Polymerase (Thermo scientific) at 0.05U/reaction. The amplification profile included an initial heating step at 95°C for 3 min, followed by 25 repeated cycles of denaturation (95°C for 30 seconds), primers hybridization (55°C for 30 seconds), and DNA synthesis at 72°C for 60 seconds. The reaction was completed with a final elongation step at 72°C for 10 minutes. The amplification was confirmed by electrophoresing of PCR products on ethidium stained 1% agarose gels. Electrophoresis was performed in in the Tris-acetate-EDTA (TAE) buffer at 80V for 60 min, and the amplified products were visualized under ultraviolet transillumination.
Amplicon Purification and Sequencing: PCR products corresponding to the coat protein region and associated betasatellite DNA were recovered from agarose gels and purified using the QIAquick PCR Purification Kit (Qiagen, Cat. No.28104) following the manufacturer's recommended procedure. Approximately 3 μg of each purified PCR product was forwarded to Macrogen Inc. for bidirectional Sanger sequencing analysis. Raw sequence chromatograms were quality-checked, base-called, and assembled into consensus sequences using the SeqMan module within the DNASTAR Lasergene software suite (Version 6, Madison, WI, USA). Finally, the high-quality consensus sequences were assembled for both viral/satellite components and deposited in the National Center for Biotechnology Information.
Phylogenetic and Evolutionary Analysis: The PCR amplified CP and betasatellite nucleotide sequences were comparatively analyzed against reference sequences present in the National Center for Biotechnology Information (NCBI-GenBank). The Sequence Demarcation Tool (SDT v1.3) was used to calculate sequence similarity matrices and pairwise-identity percentages through Muscle alignment module (Muhire et al., 2014). For phylogenetic tree construction, the sequence datasets comprising 15 representative molecules per viral component were aligned using Clustal-W module integrated in the MEGA program (MEGA-version 12, Kumar et al., 2024). Following sequence alignment, phylogenetic relationships were inferred using Maximum Likelihood approach. The Kimura 2-parameter with Gamma distributions and invariant sites substitution model was employed for the coat protein dataset, whereas. for the betasatellite sequences, Tamura 3-parameter model with Invariant sites substitution model was employed for phylogenetic tree construction. The tree topologies were validated through 1,000 bootstrap replications, with nodes retaining >70% bootstrap support were considered phylogenetically significant. The resulting phylogenetic trees were annotated to visualize clustering patterns and their taxonomic affiliations.
RESULTS
Molecular Detection of Begomovirus and associated satellite molecule: During field surveys for radish crop in 6 different districts of Punjab, only six plants exhibited discernable disease symptoms. The infected plants revealed prominent phenotypic abnormalities including severe leaf size reduction and overall stunted growth, resulting in considerably reduced biomass accumulation compared to healthy plants (Figure 1B). The presence of large whitefly populations on diseased plants provided initial evidence of begomovirus infection.
Molecular diagnosis through PCR confirmed the field observation of begomoviruses infection. The extracted DNA was used as a template for begomovirus detection using two primer sets: degenerate primers AV1F/AV1R targeting the coat protein gene of DNA-A component, and Universal primers Beta-01/Beta-02 specific to betasatellite molecules. The analysis of PCR products through agarose gel electrophoresis revealed amplification of a distinct ~1361 bp fragment representing betasatellites (Figure 1C, lanes 1-2). While, coat protein gene of approximately 771 base pair (bp) (Figure 1C, lanes 3-7) was observed, representing the DNA-A component.
The purified PCR products were subjected to bidirectional Sanger sequencing. The DNA contigs assembly for both the components generated a complete coding sequence of coat protein gene (771-nucleotide) and a 1361-nt betasatellite genome. BLAST analysis was used to confirm the molecular identity of each component. The complete annotated sequences have been submitted in GenBank under the accession numbers PV872843 (CP gene) and PV872842 (betasatellite).
Sequence analysis of viral and satellite molecules: To identify the begomovirus infecting radish plants, the coat protein (CP) sequence was analyzed using the BLASTn algorithm with a 100% query coverage threshold to ensure accurate and high-confidence matches. The BLAST results revealed a high level of sequence identity between the radish-derived CP sequence and those of previously known begomoviruses from Indian sub-continent. The sequence showed strong similarity with Pedilanthus leaf curl virus (PedLCV), with a percent identity ranging from 95.07% to 99.48% (accession numbers OM993557 to AM948961 table-1). Other closely related viruses included Papaya leaf curl virus (PaLCuV) with 94.99% to 99.35% identity (MF278787 to FM955602), Cotton leaf curl Kokhran virus (CLCuKoV) with 94.94% to 97.02% identity (OL436149 to KR816000), Cotton leaf curl Burewala virus (CLCuBuV) with 94.94% to 95.07% identity (LN713477 to HG937518), and Tomato leaf curl Pakistan virus (ToLCPKV) with 95.33% identity (GU732204). Based on the highest identity score of 99.48% with AM948961, a PedLCV isolate, it is hypothesized that the virus infecting the radish crop is most likely a newly introduced strain of PedLCV. To further confirm this hypothesis, pairwise sequence comparisons were performed via MUSCLE alignment in SDT, which included the aforementioned accessions as well as other begomoviruses previously reported from radish (LT600729, EF175733, KY026597, KY026598, FJ593629, and MW588387). The SDT analysis revealed a wide range of sequence identity from 72% to 100% among the coat protein sequences, with the highest identity observed between the radish isolate and PedLCV (AM948961), further supporting the initial BLAST-based identification (Table-1).
The associated betasatellite component was also analyzed to understand its relationship with known betasatellites associated with begomoviruses. Structural examination of the betasatellite revealed conserved features characteristic of typical betasatellites, including a nonanucleotide motif at the origin of replication, a betaC1 gene of 357 nucleotides located between genome positions 196 and 552, and an adenine-rich region (A-rich region). BLAST analysis of the betasatellite sequence, using the same parameters applied for the coat protein, revealed the closest matches to several known betasatellites. The top nucleotides BLAST hits included Papaya leaf curl betasatellite (78.98–85% identity; MF374787 to MZ172978), Tomato leaf curl New Delhi betasatellite (82.40–82.88% identity; JX679002 to MK040727), Tomato leaf curl Karnataka betasatellite (82.52–82.73% identity; KF964659 to KF964648), Sunflower leaf curl virus betasatellite (83.36–83.44% identity; KX219745 to JX678964), and Potato apical leaf curl disease-associated betasatellite (83.14% identity; EF043234/NC_008605). The highest nucleotides identity value was 83.56% with MZ172978 (Papaya leaf curl betasatellite, Indian isolate). This value is below the 91% species demarcation threshold level for betasatellites as established by ICTV (Briddon et al., 2008; Brown et al., 2015; Adams et al., 2017; Fiallo-Olivé et al., 2021), indicating that the radish-associated betasatellite may represent a novel recombinant molecule. To further confirm this, pairwise sequence identity analysis using SDT was conducted with both the top BLAST matches and other betasatellites previously reported in radish leaf curl disease (see Table 2). The analysis showed 72% to 85% sequence similarity, with the highest identity (85%) observed with MZ172978, JX678964, and KX219745, all of which are Indian isolates. These findings support the hypothesis that the identified betasatellite is a distinct and possibly new variant of papaya infecting betasatellite, potentially contributing to the pathogenicity of the associated begomovirus in radish.Based on our findings, we propose its name as Radish leaf curl Layyah betasatellite (RaLCLyB).
Figure 1. Disease symptoms and molecular detection of begomovirus infection in radish (Raphanus sativus) (A) Healthy radish plant displaying normal leaf development. (B) Symptomatic plant exhibiting characteristic begomovirus infection features: severe leaf shortening (lamina reduction) and stunted growth, resulting in significantly reduced biomass. (C) Electrophoresis of PCR amplified products: Lanes 1-2 show ~1360 bp fragments amplified with betasatellite-specific primers (Beta-01/Beta-02); Lanes 3-7 show ~771 bp fragments corresponding to the coat protein gene amplified with degenerate primers (AV1F/AV1R). M: 1 kb DNA ladder. Negative control (indicated) shows no amplification.
Phylogenetic Analysis of DNA-A and Betasatellite Sequences: Phylogenetic analysis of the coat protein, using the same dataset as employed in the sequence demarcation tool (SDT) analysis, further supported the preliminary findings. The resulting phylogenetic tree (Figure 2A) revealed that the coat protein sequence clustered closely with isolates of PedLCV and Cotton leaf curl Kokhran virus (CLCuKoV) previously reported from Pakistan. These results, along with the SDT-based sequence identity, strongly suggest that the observed infection in radish is caused by PedLCV.
Similarly, the phylogenetic tree of the associated betasatellite was constructed using the same dataset used for the SDT analysis. A Sida yellow vein alphasatellite isolate from Vietnam (DQ641718) was included as an outgroup (Figure 2B). The resulting dendrogram showed that the isolated betasatellite sequence did not closely cluster with Papaya leaf curl betasatellite (MZ172978), however, it makes a separate branch within the cluster. The clustering pattern and high sequence identity indicate that the detected betasatellite likely belongs to a different species, or a closely related variant, as Papaya leaf curl betasatellite.
Figure-2. Phylogenetic analysis of DNA-A and betasatellites isolated from radish plant. Panel-A describes the phylogenetic tree for coat protein sequences while panel-B describes the phylogenetic tree of betasatellites. The acronyms for DNA-A are Cotton leaf curl Burewala virus (CLCuBuV), Papaya leaf curl virus (PaLCV), Cotton leaf curl Kokhran virus (CLCuKoV), PedLCV, Tomato leaf curl Pakistan virus (ToLCPKV), Radish leaf curl virus (RaLCV), and Squash leaf curl virus (SLCV) and acronyms for betasatellites are Sunflower leaf curl betasatellite (SfLCB), Tomato leaf curl New Delhi betasatellite (ToLCNDB), Potato apical leaf curl disease associated betasatellite (PoALCDAB), Tomato leaf curl Karnataka betasatellite (ToLCKB), Papaya leaf curl betasatellite (PaLCB), Croton yellow vein mosaic betasatellite (CroYVMB), Radish leaf curl betasatellite (RaLCB), and Sida yellow vein Vietnam alphasatellite (SiYVVA). The vertical branches are not scaled to any measures, whereas horizontal branches reflect the mutation distances. Node values represent bootstrap support percentages. The coat protein tree was rooted at squash leaf curl virus coat protein sequence, as it is a new world begomovirus. The betasatellite tree was arbitrarily rooted on Sida yellow vein Vietnam alphasatellite, which is an unrelated sequence of the same size. The sequences isolated in this study are highlighted in each case.
DISCUSSION
Unique disease symptoms on Raphanus sativus plants collected from Thal region of Layyah, Pakistan, included conspicuous stunting, lamina shortening and reduced biomass accumulation on leaves and taproot. These systemic phenotypical expressions were consistently observed in all diseased plants. Such symptom profiles are characteristics of viral infections, particularly those associated with phloem-limited geminiviruses belonging to the genus Begomovirus (Brown et al., 2015).
The detection of begomoviruses in radish is epidemiologically significant because it broadens the recognized host range of begomoviruses. Although radish is not generally considered as a major host of begomoviruses, several begomoviruses have previously been reported infecting this crop, including, PaLCV (KY026597), RaLCV (EF175733), CroYVMV (FJ593629), and SLCV (MW588387), the latter being the only New World begomovirus reported to infect radish (Singh et al., 2012; Ismail et al., 2017; Kumar et al., 2017; Fontenele et al., 2021; Kumar et al., 2021). Despite these reports, the interaction between radish and begomoviruses remains poorly understood. This study was therefore conducted to understand the radish-begomovirus interaction within agroecosystem.
The nucleotide sequence identity and phylogenetic analysis identified, PedLCV as the putative causal agent of radish leaf curl disease. The literature suggested that PedLCV can potentially infects a wide range of important crop hosts, including Brassica rapa, capsicum spp., Glycine max, Solanum lycopersicum, and Raphanus sativus, as well as several ornamental and weed species (Ismail et al., 2017, Shakir et al., 2018). The broad host range of PedLCV suggests its better adaptation to diverse agro-ecosystems and may readily establish infections in multiple plant species (Ismail et al., 2017; Shakir et al., 2018). The spread of PedLCV in the region may therefore, be facilitated by its better host interaction at molecular level and ecological adaptability. Recent experiments also highlighted the biological importance of βV1 protein encoded by radish leaf curl betasatellite (RaLCB). The βV1 protein was shown to function as a virulence factor and an inducer of hypersensitive responses in plants (Sattar, 2025; Gupta et al., 2022). These findings suggest that betasatellites-associated pathogenicity proteins can contribute to symptoms development through interactions with replication proteins encoded by DNA-A component, underscoring the epidemiological importance of radish associated begomovirus complex. In the present study identification of a distinct betasatellite associated with PedLCV supports the hypothesis that betasatellites play an important role in evolution of radish-infecting begomovirus complexes under field conditions.
The identification of Radish leaf curl Layyah betasatellite (RaLCLyB) demonstrates that satellite molecules are continually emerging in association with begomoviruses. The RaLCLyB was structurally and functionally conserved but showed moderate sequence identity compared to other known betasatellites and was well under the 91% threshold for species demarcation. This indicates the potential for recombination, or the emergence of a divergent lineage, which could be attributed to selection pressure from the host, or ecological settings, or in connection with several, yet unreported, satellite or helper viruses. Earlier reports have documented several betasatellites infecting radishes, including ToLCPKB-LT600728, PaLCB-MF374786, RaLCB-EF175734, and CroYVMB-FJ593630 (Singh et al., 2012; Ismail et al., 2017; Kumar et al., 2017; Fontenele et al., 2021; Kumar et al., 2021), confirming that radish can harbor genetically diverse satellite molecules. Further, the pronounced genetic divergence of RaLCLyB suggests that the newly emerging satellite variants are the result of novel combinations of multiple satellite and/or helper viruses, a phenomenon which can be a function of changing agro-ecosystems. The presence of both distinct PedLCV and betasatellite also suggests the co-existence of multiple viruses, which can lead to increased recombination potential and expression of virulent symptomology. As a reservoir, or spillover host, radish may also contribute to the sustaining, and shuffling of these viral elements in the agro-ecosystem.
The epidemiological significance of these findings may be further amplified by local agricultural and postharvest practices. In many production areas, radish is harvested and sold as a whole plant, increasing the likelihood that infected tissues may be transported into non-cultivated environment. Because begomovirus infections are generally systemic, infected but asymptomatic planting materials could contribute to unnoticed virus dissemination across production regions. Similar patterns of disease spread have previously been reported for TYLCV where infected tomato seedlings facilitated rapid regional dissemination of the virus. These observations show the importance of monitoring of infected planting materials and understanding their role in the persistence and spread of begomoviruses (Polston et al., 1999; Kil et al., 2016; Pérez-Padilla et al., 2020).
Overall, the present findings broaden the known host range of PedLCV and reinforce the importance of continuous molecular surveillance of begomoviruses in both conventional and non-traditional hosts. Early detection and understanding of emerging virus-satellite complexes will be essential for minimizing potential risks to vegetable production systems and regional agriculture.
Conclusions: This study documents the first report of a novel betasatellite in association with Pedilanthus leaf curl virus (PedLCV) infecting radish crops in Thal region of district Layyah, Pakistan. The identification of novel betasatellite species suggests ever increasing diversity of begomovirus complexes. The presence of PedLCV in radish indicates that this crop may serve as a reservoir host in mixed cropping systems. Therefore, continuous molecular surveillance of these viruses in vegetable crops, together with effective whitefly management through insecticides and the development of virus-resistant cultivars, will be the important strategies to limit the emergence and dissemination of begomovirus infection.
Conflict of interest: The authors declare no conflict of interest. All authors have read the final version of the manuscript for publication.
Authors’ contribution: MH performed all the experiments. MM conducted the sequence analysis and contributed to the interpretation of the bioinformatics results. AJ drafted and wrote the manuscript and participated in data interpretation. MSN provided overall supervision of the study, critically edited and finalized the manuscript.
Acknowledgements: The authors are grateful to virology lab members who helped in molecular analysis of this study. The current research was funded by Higher Education Commission of Pakistan, grant number 6427 to the corresponding author. This research is part of dissertation of first author.
REFERENCES
Adams, M.J., E.J. Lefkowitz, A.M. King, B. Harrach, R.L. Harrison, N.J. Knowles, A.M. Kropinski, M. Krupovic, J.H. Kuhn and A.R. Mushegian (2017). Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2017). Arch. Virol. 162(8): 2505-2538. https://doi.org/10.1007/s00705-017-3358-5
Briddon, R.W., J.K. Brown, E. Moriones, J. Stanley, M. Zerbini, X. Zhou and C.M. Fauquet (2008). Recommendations for the classification and nomenclature of the DNA-beta satellites of begomoviruses. Arch. Virol. 153(4): 763-81. https://doi.org/10.1007/s00705-007-0013-6
Briddon, R.W. and P.G. Markham (2000). Cotton leaf curl virus disease. Virus Res. 71(1-2): 151-159. https://doi.org/10.1016/S0168-1702(00)00195-7
Briddon, R.W. and J. Stanley (2006). Subviral agents associated with plant single-stranded DNA viruses. Virology 344(1): 198-210. https://doi.org/10.1016/j.virol.2005.09.042
Brown, J.K., F.M. Zerbini, J. Navas-Castillo, E. Moriones, R. Ramos-Sobrinho, J.C. Silva, E. Fiallo-Olivé, R.W. Briddon, C. Hernández-Zepeda, A. Idris and V.G. Malathi (2015). Revision of Begomovirus taxonomy based on pairwise sequence comparisons. Arch. Virol. 160: 1593–1619. https://doi.org/10.1007/s00705-015-2398-y
Brown, J.K., A.M. Idris, I. Torres-Jerez, G.K. Banks and S.D. Wyatt (2001). The core region of the coat protein gene is highly useful for establishing the provisional identification and classification of begomoviruses. Arch. Virol. 146(8): 1581-1598. https://doi.org/10.1007/s007050170080
Chakraborty, S., P.K. Pandey, M.K. Banerjee, G. Kalloo and C.M. Fauquet (2003). A new begomovirus species causing tomato leaf curl disease in Varanasi, India. Plant Dis. 87(3): 313-313. https://doi.org/10.1094/PDIS.2003.87.3.313A
Doyle, J. (1991). DNA protocols for plants. Molecular techniques in taxonomy. Springer (Berlin). 283-293 p https://doi.org/10.1007/978-3-642-83962-7_18
Fiallo-Olivé, E., J.M. Lett, D.P. Martin, P. Roumagnac, A. Varsani, F.M. Zerbini and J. Navas-Castillo (2021). ICTV virus taxonomy profile: Geminiviridae 2021. J. Gen. Virol. 102(12): 001696. https://doi.org/10.1099/jgv.0.001696
Fontenele, R.S., A. Bhaskara, I.N. Cobb, L.C. Majure, A.M. Salywon, J.A. Avalos-Calleros, G. R. Argüello-Astorga, K. Schmidlin, P. Roumagnac and S.G. Ribeiro (2021). Identification of the begomoviruses Squash leaf curl virus and Watermelon chlorotic stunt virus in various plant samples in North America. Viruses 13(5): 810. https://doi.org/10.3390/v13050810
Gupta, N., K. Reddy, P. Gnanasekaran, Y. Zhai, S. Chakraborty and H.R. Pappu (2022). Functional characterization of a new ORF βV1 encoded by Radish leaf curl betasatellite. Front. Plant Sci. 13: 972386. https://doi.org/10.3389/fpls.2022.972386
Ismail, H., I. Hassan, M. Zubair, M. Khan, S. Serfraz, N. Jamil, S. Mansoor, S. Asad and I. Amin (2017). First report of Pedilanthus leaf curl virus, Tobacco leaf curl betasatellite, and Guar leaf curl alphasatellite infecting radish (Raphanus sativus) in Pakistan. Plant Dis. 101(5): 845-845. https://doi.org/10.1094/PDIS-09-16-1265-PDN
Khan, M.S., A.K. Tiwari, S.K. Raj, A. Srivastava, S.H. Ji and S.C. Chun (2014). Molecular epidemiology of begomoviruses occurring on some vegetables, grain legume and weed species in the Terai belt of north India. J. Plant Dis. Prot. 121(2): 53-57. https://doi.org/10.1007/BF03356491
Kil, E.J., S. Kim, Y.J. Lee, H.S. Byun, J. Park, H. Seo, C.S. Kim, J.K. Shim, J.H. Lee, J.K. Kim, K.Y. Lee, H.S. Choi and S. Lee (2016). Tomato yellow leaf curl virus (TYLCV-IL): a seed-transmissible geminivirus in tomatoes. Sci. Rep. 6(1): 19013. https://doi.org/10.1038/srep19013
Kumar, J., A. Kumar, S.P. Singh, J.K. Roy, A. Lalit, D. Parmar, N.C. Sharma, and R. Tuli (2012). First report of Radish leaf curl virus infecting okra in India. New Dis. Rep. 25(1): 9-9. https://doi.org/10.5197/j.2044-0588.2012.025.009
Kumar, J., J. Kumar, S.P. Singh and R. Tuli (2014). βC1 is a pathogenicity determinant: not only for begomoviruses but also for a mastrevirus. Arch. Virol. 159(11): 3071-3076. https://doi.org/10.1007/s00705-014-2149-5
Kumar, R.V., D. Singh, A.K. Singh and S. Chakraborty (2017). Molecular diversity, recombination and population structure of alphasatellites associated with begomovirus disease complexes. Infect. Genet. Evol. 49: 39-47. https://doi.org/10.1016/j.meegid.2017.01.001
Kumar, S., R. Raj, L. Agrawal, T. Gupta, S.K. Raj, M. Jaidi and A. Srivastava (2021). Association of an isolate of Papaya leaf curl virus and Papaya leaf curl betasatellite with leaf curl disease of radish in India. J. Plant Pathol. 103(2): 629-634. https://doi.org/10.1007/s42161-021-00764-x
Kumar, S., G. Stecher, M. Suleski, M. Sanderford, S. Sharma and K. Tamura (2024). MEGA12: Molecular Evolutionary Genetic Analysis Version 12 for Adaptive and Green Computing. Mol. Biol. Evol. 41(12): msae263. https://doi.org/10.1093/molbev/msae263
Manivannan, A., J.H. Kim, D.S. Kim, E.S. Lee and H.E. Lee (2019). Deciphering the nutraceutical potential of Raphanus sativus-a comprehensive overview. Nutrients 11(2): 402. https://doi.org/10.3390/nu11020402
Mansoor, S., S. Mukhtar, M. Hussain, I. Amin, Y. Zafar, K.A. Malik and P.G. Markham (2000). Widespread Occurrence of Cotton leaf curl virus on Radish in Pakistan. Plant Dis. 84(7): 809-809. https://doi.org/10.1094/PDIS.2000.84.7.809B
More, P., B.M. Baraiya, P.K. Agarwal and P. Agarwal (2022). Geminiviruses infecting tomato: genetic diversity, distribution, natural and genetically engineered resistance. In Geminivirus: Detection, diagnosis and management. Academic Press (US). 611-627 p https://doi.org/10.1016/B978-0-323-90587-9.00018-3
Muhire, B.M., A. Varsani and D.P. Martin (2014). SDT: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One 9(9): e108277. https://doi.org/10.1371/journal.pone.0108277
Nawaz-ul-Rehman, M.S., R.W. Briddon and C.M. Fauquet (2012). A melting pot of Old World begomoviruses and their satellites infecting a collection of Gossypium species in Pakistan. PLoS One 7(8): e40050. https://doi.org/10.1371/journal.pone.0040050
Nowak, A., M. Majewska, A. Marzec-Grządziel, E. Ozimek, M. Przybyś, A. Słomka, N. Kutyrieva-Nowak, A. Gałązka and J. Jaroszuk-Ściseł (2024). Effect of long-term radish (Raphanus sativus var. sativus) monoculture practice on physiological variability of microorganisms in cultivated soil. J. Environ. Manage. 367: 122007. https://doi.org/10.1016/j.jenvman.2024.122007
Pérez-Padilla, V., I.M. Fortes, B. Romero-Rodríguez, M. Arroyo-Mateos, A.G. Castillo, C. Moyano, L. De León and E. Moriones (2020). Revisiting Seed Transmission of the Type Strain of Tomato yellow leaf curl virus in Tomato Plants. Phytopathol. 110(1): 121-129. https://doi.org/10.1094/PHYTO-07-19-0232-FI
Polston, J.E., R.J. McGovern and L.G. Brown (1999). Introduction of Tomato yellow leaf curl virus in Florida and implications for the spread of this and other geminiviruses of tomato. Plant Dis. 83(11): 984-988. https://doi.org/10.1094/PDIS.1999.83.11.984
Sattar, M.N. (2025). Interactive dynamics of geminiviruses with host plants. Front. Plant Sci. 16: 1616024. https://doi.org/10.3389/fpls.2025.1616024
Shakir, S., M.S. Nawaz-ul-Rehman, M. Mubin and Z. Ali (2018). Characterization, phylogeny and recombination analysis of Pedilanthus leaf curl virus-Petunia isolate and its associated betasatellite. Virol. J. 15(1):1-11. https://doi.org/10.1186/s12985-018-1047-y
Shingote, P.R., D.L. Wasule, V.S. Parma, S.K. Holkar, S.G. Karkute, N.D. Parlawar and D. Senanayake (2022). An Overview of Chili Leaf Curl Disease: Molecular Mechanisms, Impact, Challenges, and Disease Management Strategies in Indian Subcontinent. Front. Microbiol. 13: 899512. https://doi.org/10.3389/fmicb.2022.899512
Singh, A., B. Chattopadhyay and S. Chakraborty (2012). Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India. Virol. J. 9(1): 43. https://doi.org/10.1186/1743-422X-9-43
Singh, A.K., B. Chattopadhyay, P.K. Pandey, A.K. Singh and S. Chakraborty (2007). A new begomovirus species causing leaf curl disease of radish in India. Plant Dis. 91(8): 1053. https://doi.org/10.1094/PDIS-91-8-1053B
Varma, A. and V. Malathi (2003). Emerging geminivirus problems: a serious threat to crop production. Ann. Appl. Biol. 142(2): 145-164. https://doi.org/10.1111/j.1744-7348.2003.tb00240.x
Venkataravanappa, V., K.V. Ashwathappa, P. Kallingappa, H. Shridhar, P.H. Reddy, M.K. Reddy and C.N.L. Reddy (2023). Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant. Microb. Pathogen. 180: 106127. https://doi.org/10.1016/j.micpath.2023.106127