Sequence characterization of cotton leaf curl virus from Rajasthan: phylogenetic relationship with other members of geminiviruses and detection of recombination

Diseased cotton plants showing typical leaf curl symptoms were collected from experimental plot of Agriculture Research Station—Sriganganagar, Rajasthan. Complete DNA-A component from samples taken from two areas were amplified through rolling circle amplification (RCA) using templiphi kit (GE Healthcare) and characterized. DNA-A of one isolate consists of 2751 nucleotides and second isolate of 2759 nucleotide. Both sequences comprised six ORF’s. Genome organization of DNA-A of one isolate shows high sequence similarity with other characterized local begomovirus isolates of Rajasthan, while other isolate shows high sequence similarity with CLCuV reported from Pakistan. The maximum similarity of first isolate, CLCuV-SG01, shows highest sequence identity with Cotton leaf curl Abohar (Rajasthan) virus, and second isolate, CLCuV-SG02, shows highest sequence identity with cotton leaf curl virus from Pakistan. Both isolates showed 85% similarities with each other. The sequence data revealed probable infiltration of some strains of Cotton leaf curl virus from Pakistan to India, or co-existence of different isolates under similar geographical conditions. While CLCuV-SG01 shows highest nt sequence similarity with CLCuV Rajasthan (Abohar), nt identity of V1 ORF (encoding coat protein) of SG01 shows the highest nt identity (100%) with CLCuV Multan (Bhatinda) and Abohar virus while AC1 region also showed difference. Complete nucleotide sequence of SG01 shows only 86% similarity with CLCuV Multan virus. Similarity search revealed significant difference in AV1 and AC1 regions with respect to DNA-A suggesting an evolutionary history of recombination. Computer based analysis, recombination detection Program (RDP) supports the recombination hypothesis, indicated that recombination with other begomoviruses had taken place within V1 ORF and AC1 ORF of CLCuV-SG01 and AC1 ORF of CLCuV-SG02 and also in noncoding intergenic region (IR).     

A novel recombinant tomato-infecting begomovirus capable of transcomplementing heterologous DNA-B components

The genome of a tomato-infecting begomovirus from Ranchi, India, was cloned, sequenced and analysed. The viral genome shared 88.3% sequence identity with an isolate belonging to the species Tobacco curly shoot virus (TbCSV), and this virus should therefore be considered a member of a new species, tentatively named Tomato leaf curl Ranchi virus (ToLCRnV). The DNA-?? molecule, which had 74.5% sequence identity with tomato leaf curl Bangladesh betasatellite (ToLCBDB), is named tomato leaf curl Ranchi betasatellite (ToLCRnB). Phylogenetic analysis revealed that ToLCRnV is related to tomato leaf curl Bangladesh virus (ToLCBDV), tobacco curly shoot virus (TbCSV) and tomato leaf curl Gujarat virus (ToLCGV). An infectivity study with ToLCRnV established the monopartite nature of the viral genome, whereas inoculation with ToLCRnB resulted in increased symptom severity. ToLCRnV could transreplicate DNA-B of tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl New Delhi virus (ToLCNDV), both in N. benthamiana and tomato, although DNA-B accumulation of was less than with the wild-type combinations. ToLCRnB could be efficiently replicated by DNA-A of both ToLCNDV and ToLCGV. A leaf disk assay suggests that DNA-A could transreplicate the homologous DNA-B and DNA-?? more efficiently than the heterologous one.     

Further characterization of tomato-infecting begomoviruses in Brazil

Tomato cultivation in Brazil is threatened by a number of tomato-infecting viruses belonging to the genus Begomovirus of the family Geminiviridae. Here, we report the full DNA-A sequences of three Brazilian begomoviruses: a potentially new tomato-infecting viruses, tomato interveinal chlorosis virus (ToICV), and two previously proposed begomoviruses for which only partial DNA-A sequences are available in the databases: tomato mottle leaf curl virus (TMoLCV) and tomato golden vein virus (TGVV). The complete sequences of the DNA-B components of TMoLCV and TGVV and the DNA-A components of a number of tomato severe rugose virus variants are also presented. Collectively, all of the analyzed sequences were phylogenetically clustered within the two major groups of Brazilian tomato-infecting begomoviruses.     

Cotton leaf curl disease is associated with multiple monopartite begomoviruses supported by single DNA β

Summary For bipartite begomoviruses (family Geminiviridae) trans-replication of the DNA B component by the DNA A-encoded replication-associated protein (Rep) is achieved by virtue of a shared sequence, the common region, which contains repeated motifs (iterons) which are sequence-specific Rep binding sites and form part of the origin of replication. Recently cotton leaf curl disease (CLCuD), a major constraint to cotton production on the Indian subcontinent, has been shown to be caused by a monopartite begomovirus (Cotton leaf curl Multan virus [CLCuMV]) and a novel single-stranded DNA satellite molecule termed CLCuD DNA . The satellite molecule is trans-replicated by CLCuMV but does not possess the iteron sequences of this virus. We have investigated the ability of CLCuD DNA to interact with three further clones of monopartite begomoviruses, isolated from cotton, that have distinct Rep binding specificities. All three cloned viruses were capable of trans-replicating the satellite molecule and inducing CLCuD symptoms in cotton, indicating that the interaction between begomovirus and DNA is relaxed in comparison to the interaction between DNA A and DNA B components. Field surveys across all the cotton growing regions of Pakistan indicate that dual and multiple infections are the norm for CLCuD with no evidence of synergism. Despite the diversity of begomoviruses associated with CLCuD, only a single class of DNA has been detected, suggesting that this satellite has the capacity to be recruited by unrelated begomoviruses.Received January 13, 2003; accepted May 8, 2003     

Molecular characterization of tomato-infecting begomoviruses in Thailand

Bipartite geminiviruses infecting tomatoes in Thailand were detected by polymerase chain reaction (PCR) using CPA5/CPA2 primers. Products derived from PCR-amplified full-length DNA-A and DNA-B of TYLCV collected from Chiang Mai, Nong Khai, and Sakon Nakhon were cloned and sequenced. DNA-A from Chiang Mai was 2747 nts long; Nong Khai, 2744 nts; and Sakon Nakhon, 2747 nts, and those of DNA-B from Chiang Mai were 2750 nts long; Nong Khai, 2749 nts; and Sakon Nakhon, 2749 nts. The genomes of these virus isolates were organized like those of other begomoviruses. The DNA-A had two ORFs in the virion sense and four ORFs in the complementary sense. The DNA-B had two ORFs in the virion sense and one ORF in the complementary sense. Nucleotide sequences of DNA-A of TYLCV from Chiang Mai, Nong Khai, and Sakon Nakhon were closely related to those of Tomato yellow leaf curl Thailand virus (TYLCTHV) and Tomato yellow leaf curl Thailand virus-[Myanmar] (TYLCTHV-[MM]) with nucleotide sequence identity ranging from 89% to 95%. Based on sequence comparisons and phylogenetic analyses, these three virus isolates studied were identified as new strains of TYLCTHV and named Tomato yellow leaf curl Thailand virus-Chiang Mai (TYLCTHV-[CM]The GenBank accession codes for DNA-A of TYLCTHV-[CM], -[NK], and -[SK] are , and , respectively. The GenBank accession codes for DNA-B of TYLCTHV-[CM], -[NK], and -[SK] are , , and , respectively.), Nong Khai (TYLCTHV-[NK] and Sakon Nakhon (TYLCTHV-[SK]).     

Molecular characterization of a new begomovirus infecting Senecio scandens in China

Three begomovirus isolates, G46, G83 and G84 from Senecio scandens showing yellow mosaic symptoms were collected from Guangxi Province, P.R. China. The isolates were detected by PCR using universal primers for begomoviruses. Comparison of partial DNA-A sequences (approximately 500 bp) of the isolates revealed their 98.7-99.1% identity. The isolate G46, chosen for complete DNA-A sequencing, consisted of 2746 nt and had a typical genomic organization of begomoviruses. The G46 DNA-A had the highest sequence identity (72.4%) with that of Ageratum leaf curl virus among begomoviruses. The molecular data suggest that the isolate G46 is a new begomovirus (species), for which the name Senecio yellow mosaic virus (Senecio yellow mosaic virus) is proposed.     

Host-specific adaptation of diverse betasatellites associated with distinct Indian tomato-infecting begomoviruses

Tomato leaf curl viruses cause major crop loss hindering tomato cultivation worldwide. The ‘Old World’ begomoviruses are often associated with circular ssDNA satellite molecules called betasatellites. In the present study, replication compatibility of five different betasatellites with three distinct Indian tomato-infecting begomoviruses representing each of a monopartite, a mono-bipartite and a bipartite begomoviruses was studied. All the betasatellites could be trans-replicated by the begomoviruses in Nicotiana benthamiana plants, however, not uniformly in tomato. Tomato leaf curl Joydebpur betasatellite—Magrahat could not induce symptom with any of these begomoviruses in tomato, whereas only Tomato leaf curl Gujarat virus could trans-replicate Radish leaf curl betasatellite in this plant species. However, none of the betasatellites were found to complement the movement function of a bipartite begomovirus in tomato. Unlike tomato, the trans-replication/maintenance of betasatellites by these begomoviruses in N. benthamiana could be due to its compromised host defence machinery. Co-infection of betasatellites with these viruses did not enhance the helper virus accumulation, but the incubation period was reduced. The possible factors involved in this host-driven adaptability of betasatellites were also discussed.     

Sequence characterization of Tomato leaf curl Sinaloa virus and <Emphasis Type="Italic">Tomato severe leaf curl virus</Emphasis>: Phylogeny of New World begomoviruses and detection of recombination

Summary. Diseases caused by begomoviruses (family Geminiviridae, genus Begomovirus) constitute a serious constraint to tomato production in Nicaragua. In this study, the complete nucleotide (nt) sequences of the DNA-A and DNA-B components were determined for the first time for Tomato leaf curl Sinaloa virus (ToLCSinV). In addition, the complete nt sequence was determined for the DNA-A component of two isolates of Tomato severe leaf curl virus (ToSLCV). The genome organization of ToLCSinV and ToSLCV was identical to the bipartite genomes of other begomoviruses described from the Americas. A phylogenetic analysis of DNA-A including 45 begomovirus species showed that the indigenous begomoviruses of the New World can be divided into three major clades and an intermediate group: AbMV clade, SLCV clade, “Brazil clade”, and BGYMV group. Phylogenetic analyses of the DNA-A and DNA-B components and their open reading frames indicated that ToLCSinV and ToSLCV belong to different clades: ToLCSinV to the AbMV clade, and ToSLCV to the SLCV clade. The two Nicaraguan isolates of ToSLCV showed a close relationship with ToSLCV from Guatemala (ToSLCV-[GT96-1]) and Tomato chino La Paz virus (ToChLPV), but differed significantly in the AV1 and AC1 regions, respectively. Computer-based predictions indicated that recombination with another begomovirus had taken place within AV1 of ToSLCV dividing this species into two strains. A high probability was also found that ToChLPV is involved in the evolution of ToSLCV.     

Cotton leaf curl Gezira alphasatellite associated with tomato leaf curl Sudan virus approaches the expected upper size limit in the evolution of alphasatellites

Begomoviruses (family Geminiviridae) cause severe damage to tomato crops worldwide. Among them, tomato leaf curl disease (ToLCD)-associated begomoviruses are a major concern for tomato production in Sudan. Here, we report the detection of unexpectedly large cotton leaf curl Gezira alphasatellite molecules (up to 1467 nt) associated with an isolate of a novel strain of tomato leaf curl Sudan virus (ToLCSDV) in tomato plants affected by ToLCD. A recombinant nature is suggested for this ToLCSDV isolate.     

Differential pathogenicity among Tomato leaf curl Gujarat virus isolates from India

Tomato leaf curl Gujarat virus (ToLCGV) has been identified as one of the most destructive pathogens causing tomato leaf curl disease (ToLCD) in India. In the tomato growing regions of Dhanbad and Ramgarh, plants bearing severe symptoms of ToLCD such as leaf curling, leaf crinkling, yellowing and leaf rolling was observed in the farmer fields. The association of begomovirus in these samples was confirmed by PCR and the causal viruses were identified as the isolates of ToLCGV. However, association of cognate DNA B component could not be ascertained from these samples. Indeed, like other Old World begomoviruses, the present ToLCGV isolates were found to be associated with a particular betasatellite, Tomato yellow leaf curl Thailand betasatellite (TYLCTHB). Although DNA A of both ToLCGV isolates could alone infect tomato inducing systemic symptoms, the difference in virulence was observed. Co-inoculation of TYLCTHB reduced the incubation period without influencing the accumulation of helper virus DNA and hence, differential pathogenesis among ToLCGV isolates was governed by the helper component rather than betasatellite. ToLCGV infection with DNA B increases the accumulation of DNA A component of Dhanbad isolate but not of Ramgarh isolate. Results indicated that the begomovirus identified from Ramgarh sample was a mild strain of ToLCGV.     

Strains of a new bipartite begomovirus, pepper yellow leaf curl Indonesia virus, in leaf-curl-diseased tomato and yellow-vein-diseased ageratum in Indonesia

The complete nucleotide sequences of begomoviruses from pepper with leaf curl and yellowing symptoms, tomato with leaf curl symptoms, and ageratum with yellow vein in Indonesia were determined. On the basis of genome organization and sequence homology, they were proposed to belong to a new species, Pepper yellow leaf curl Indonesia virus (PepYLCIV), which includes the new strains PepYLCIV-Tomato and PepYLCIV-Ageratum. These viruses had bipartite genomes. Pepper virus DNAs from Indonesia (PepYLCIV, PepYLCIV-Tomato and PepYLCIV-Ageratum DNA-As) were noticeably distinct, forming a separate branch from the viruses infecting pepper. Considerable divergence was observed in the common region (CR) of the genomic components of PepYLCIV (77%), PepYLCIV-Tomato (82%) and PeYLCIV-Ageratum (75%). A stem-loop-forming region and a Rep-binding motif were identical in the CR of the three viruses. The CRs of PepYLCIV-Ageratum DNA-A was approximately 10 nucleotides longer than that of PepYLCIV DNA-A and PepYLCIV-Tomato DNA-A. A similar insertion was also found in the CR of PepYLCIV-Ageratum DNA-B. PepYLCIV DNA-A alone was infectious in pepper and Nicotiana benthamiana plants, and association with DNA-B increased symptom severity.     

Molecular characterization of a new begomovirus infecting Sida cordifolia and its associated satellite DNA molecules

Two virus isolates Hn57 and Hn60 were obtained from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province of China. Comparison of partial sequences of DNA-A like molecule confirmed the existence of a single type of begomovirus. The complete nucleotide sequence of DNA-A of Hn57 was determined to be 2757 nucleotides, with a genomic organization typical of begomoviruses. Complete sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAβ and DNA1 were found to be associated with SiLCV isolates. Both DNAβ and DNA1 were approximately half the size of their cognate genomic DNA. Sequence analysis shows that DNAβ of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and they have the highest sequence identity (58.5%) with DNAβ associated with Ageratum leaf curl disease (AJ316027). The nucleotide sequence identity between DNA1 of Hn57 and that of Hn60 was 83.8%, they share 58.2–79.3% nucleotide sequence identities in comparison with other previously reported DNAl. The GenBank accession numbers of the sequences reported in this paper are AM050730-35.     

Molecular characterization of a distinct bipartite begomovirus species infecting tomato in India

A distinct bipartite begomovirus was found associated with tomato plants showing yellowing, curling, and crumpling of the leaves, in a sub-temperate region in India. The complete DNA-A and DNA-B components were amplified through rolling circle amplification (RCA) using Φ-29 DNA polymerase and characterized. The DNA-A of the isolate was comprised of 2,756 nucleotides, encoding six open reading frames (ORFs) and DNA-B that of 2,725 nucleotides, encoding two ORFs. Genome organization of the isolate was typical of an old world bipartite begomovirus. Comparisons showed that DNA-A and its intergenic region (IR) have the highest sequence identity (86% and 84%, respectively) with the Tomato leaf curl New Delhi virus (ToLCNDV; DQ116885) and some other begomoviruses (>84%) reported from cucurbits and tomato. This data suggested that the isolate is a distinct begomovirus species for which a name Tomato leaf curl Palampur virus (ToLCPMV) is proposed. DNA-B showed the maximum sequence identity (73%) with Tomato leaf curl New Delhi virus-India-[Pakistan:Dargai:T5/6:2001] (AY150305). The common region (CR) of DNA-A and DNA-B showed 94% sequence similarity with each other. In the present study, phylogenetic relationship of this new species was also established with different begomoviruses reported from tomato and other begomoviruses showing highest homologies with complete DNA-A and DNA-B sequences. ToLCPMV is being reported from a sub-temperate region in India which was previously unaffected by begomoviruses and its whitefly vector. An erratum to this article can be found at     

Recent evolution of a novel begomovirus causing tomato leaf curl disease in the Al-Batinah region of Oman

For last two decades, begomoviruses (family Geminiviridae) have been a major constraint for tomato production in Oman, particularly in the Al-Batinah region, the major agricultural area of Oman. Farms in the Al-Batinah region were surveyed during January-March and November-December in 2012 and January-February in 2013. Leaf samples of tomato plants showing typical leaf curl disease symptoms were collected and analyzed for begomoviruses. Out of fifteen begomovirus clones sequenced, seven were shown to be tomato yellow leaf curl virus strain Oman (TYLCV-OM); three, chili leaf curl virus strain Oman (ChLCV-OM); and one, tomato leaf curl Oman virus (ToLCOMV) – viruses that have previously been shown to occur in Oman. Four sequences were shown to have relatively low percent identity values to known begomoviruses, with the highest (86 %) to isolates of pepper leaf curl Lahore virus, indicating that these should be included in a new species, for which the name “Tomato leaf curl Al Batinah virus” (ToLCABV) is proposed. Although the betasatellite tomato leaf curl betasatellite (ToLCB; 7 full-length sequences isolated) was identified with some isolates of ChLCV-OM, TYLCV-OM and ToLCOMV, it was not identified in association with any of the ToLCABV isolates. Analysis of the sequences of the TYLCV-OM and ToLCOMV isolates characterized here did not show them to differ significantly from previously characterized isolates of these viruses. The three isolates of ChLCV-OM characterized were shown to have a recombination pattern distinct from earlier characterized isolates. ToLCABV was shown to have resulted from recombination between ChLCV-OM and ToLCOMV. A clone of ToLCABV was infectious by Agrobacterium-mediated inoculation to Nicotiana benthamiana and tomato, inducing symptoms typical of those seen in tomato in the field. Additionally, ToLCABV was shown to be able to interact in planta with ToLCB, resulting in a change in symptom phenotype, although the betasatellite did not appear to affect viral DNA levels.     

Transmission of cotton leaf curl disease: answer to a long-standing question

Cotton leaf curl disease (CLCuD) has been a major constraint to cotton production across Pakistan and northwestern India since the early 1990s. The disease is caused by a number of begomoviruses, including Cotton leaf curl Multan virus (CLCuMuV), which associate with a specific host range and symptom determining betasatellite known as Cotton leaf curl Multan betasatellite (CLCuMuB). Bemisia tabaci is a complex of cryptic species that consists of numerous (>?44) morphologically indistinguishable and, at least partially, reproductively isolated species. CLCuD has recently been introduced into parts of China but has not, at least so far, become a problem in the major cotton regions. The disease in China has been shown to be caused by CLCuMuV with CLCuMuB, which is believed to have been introduced from South Asia in ornamental plants. To understand the basis for this lack of spread of CLCuD into the cotton-growing areas of China, Pan et al. (Phytopathology 108:1172–1183, 2018) investigated the transmission of CLCuMuV/CLCuMuB by B. tabaci. The study showed that, of the four cryptic B. tabaci species investigated, only the cryptic species Asia II 1 was able to efficiently transmit CLCuMuV/CLCuMuB. Significantly, Asia II 1 is not present in the major cotton-growing regions of China. The results of Pan et al. (Phytopathology 108:1172–1183, 2018) are discussed with particular emphasis on the situation of CLCuD in Pakistan and northwestern India, which differs significantly from the situation in China.     

Diverse population of a new bipartite begomovirus infecting tomato crops in Uruguay

Several isolates of a novel begomovirus were characterized from tomato samples collected in northern Uruguay exhibiting disease symptoms associated with Bemisia tabaci infestations. Analysis of full-length sequences of DNA-A and DNA-B components revealed the presence of a new begomovirus with the typical genome organization of a New World begomovirus, for which the name tomato rugose yellow leaf curl virus (ToRYLCV) is proposed. A high degree of nucleotide sequence diversity was found for both components, suggesting the presence of a diverse virus population. Recombination analysis suggested relationships of ToRYLCV to begomoviruses reported from the New World. Although common regions from DNA-As and DNA-Bs were surprisingly divergent for a cognate pair, a DNA-A and DNA-B pair cloned from one sample were infectious in Nicotiana benthamiana and tomato and reproduced symptoms observed in field-infected tomato plants, suggesting that ToRYLCV is the causal agent of the disease observed. This is the first report of a begomovirus infecting tomato crops in Uruguay and of the presence of begomovirus in this country.     

Transreplication of a Tomato yellow leaf curl Thailand virus DNA-B and replication of a DNAbeta component by Tomato leaf curl Vietnam virus and Tomato yellow leaf curl Vietnam virus

The genomes of two tomato-infecting begomoviruses from Vietnam were cloned and sequenced. A new variant of Tomato leaf curl Vietnam virus (ToLCVV) consisting of a DNA-A component and associated with a DNAbeta molecule as well as an additional begomovirus tentatively named Tomato yellow leaf curl Vietnam virus (TYLCVV) consisting also of a DNA-A component were identified. To verify if monopartite viruses occurring in Vietnam and Thailand are able to transreplicate the DNA-B component of Tomato yellow leaf curl Thailand virus-[Asian Institute of Technology] (TYLCTHV-[AIT]) infectivity assays were performed via agroinoculation and mechanically. As result, the DNA-B component of TYLCTHV-[AIT] was transreplicated by different DNA-A components of viruses from Vietnam and Thailand in Nicotiana benthamiana and Solanum lycopersicum. Moreover, the TYLCTHV-[AIT] DNA-B component facilitated the mechanical transmission of monopartite viruses by rub-inoculation as well as by particle bombardment in N. benthamiana and tomato plants. Finally, defective DNAs ranging from 735 to 1457 nucleotides were generated in N. benthamiana from those combinations containing TYLCTHV-[AIT] DNA-B component.     

Two genetically related begomoviruses causing tomato leaf curl disease in Togo and Nigeria differ in virulence and host range but do not require a betasatellite for induction of disease symptoms

Tomato leaf curl disease (ToLCD) has emerged as a major constraint on tomato production in some parts of West Africa. In this study, begomoviruses associated with ToLCD in Togo and Nigeria were characterized, as well as a betasatellite associated with the disease in Togo. The genome organization of both viruses is typical of Old World monopartite begomoviruses. Sequence analysis revealed that the begomovirus from Togo is a variant of tomato leaf curl Kumasi virus (ToLCKuV) from Ghana, and it is designated ToLCKuV-[Togo:Pagouda:2006] (ToLCKuV-[TG:Pag:06]). The begomovirus from Nigeria has a recombinant genome, composed of sequences of ToLCKuV (major parent) and a cotton leaf curl Gezira virus (CLCuGV)-like virus, and possesses an unusual non-reiterated replication-associated protein (Rep) binding site. Moreover, because the sequence has <89% identity with those of previously characterized begomoviruses, it is a new species and is designated tomato leaf curl Nigeria virus-[Nigeria:Odogbo:2006] (ToLCNGV-[NG:Odo:06]). The cloned DNAs of ToLCKuV-TG and ToLCNGV were infectious and induced leaf curl symptoms in tomato plants, but ToLCNGV was comparatively more virulent. Both viruses also induced stunted growth and leaf curl symptoms in other solanaceous species (various Nicotiana spp. and Datura stramonium), whereas ToLCNGV but not ToLCKuV-TG induced symptoms in common bean plants. The betasatellite associated with ToLCD in Togo is genetically distinct (i.e., <78% nucleotide sequence identity with previously identified betasatellites) and is designated tomato leaf curl Togo betasatellite-[Togo:Pagouda:2006] (ToLCTGB-[TG:Pag:06]). Replication and systemic spread of ToLCTGB in tomato was mediated by ToLCKuV-TG and ToLCNGV; however, the betasatellite had no effect on disease symptoms induced by either begomovirus. In contrast, ToLCTGB increased symptom severity induced by both viruses in Nicotiana spp. and D. stramonium. Thus, although ToLCTGB increased symptom severity in a host-dependent manner, it does not appear to play a role in ToLCD and may have been present with ToLCKuV-TG as a reassortant.