Tomato yellow leaf curl viruses: ménage à trois between the virus complex,the plant and the whitefly vector

Tomato yellow leaf curl disease (TYLCD) is one of the most devastating viral diseases affecting tomato crops in tropical, subtropical and temperate regions of the world. Here, we focus on the interactions through recombination between the different begomovirus species causing TYLCD, provide an overview of the interactions with the cellular genes involved in viral replication, and highlight recent progress on the relationships between these viruses and their vector, the whitefly Bemisia tabaci. Taxonomy: The tomato yellow leaf curl virus‐like viruses (TYLCVs) are a complex of begomoviruses (family Geminiviridae, genus Begomovirus) including 10 accepted species: Tomato yellow leaf curl Axarquia virus (TYLCAxV), Tomato yellow leaf curl China virus (TYLCCNV), Tomato yellow leaf curl Guangdong virus (TYLCGuV), Tomato yellow leaf curl Indonesia virus (TYLCIDV), Tomato yellow leaf curl Kanchanaburi virus (TYLVKaV), Tomato yellow leaf curl Malaga virus (TYLCMalV), Tomato yellow leaf curl Mali virus (TYLCMLV), Tomato yellow leaf curl Sardinia virus (TYLCSV), Tomato yellow leaf curl Thailand virus (TYLCTHV), Tomato yellow leaf curl Vietnam virus (TYLCVNV) and Tomato yellow leaf curl virus(TYLCV). We follow the species demarcation criteria of the International Committee on Taxonomy of Viruses (ICTV), the most important of which is an 89% nucleotide identity threshold between full‐length DNA‐A component nucleotide sequences for begomovirus species. Strains of a species are defined by a 93% nucleotide identity threshold. Host range: The primary host of TYLCVs is tomato (Solanum lycopersicum), but they can also naturally infect other crops [common bean (Phaseolus vulgaris), sweet pepper (Capsicum annuum), chilli pepper (C. chinense) and tobacco (Nicotiana tabacum)], a number of ornamentals [petunia (Petunia×hybrida) and lisianthus (Eustoma grandiflora)], as well as common weeds (Solanum nigrum and Datura stramonium). TYLCVs also infect the experimental host Nicotiana benthamiana. Disease symptoms: Infected tomato plants are stunted or dwarfed, with leaflets rolled upwards and inwards; young leaves are slightly chlorotic; in recently infected plants, fruits might not be produced or, if produced, are small and unmarketable. In common bean, some TYLCVs produce the bean leaf crumple disease, with thickening, epinasty, crumpling, blade reduction and upward curling of leaves, as well as abnormal shoot proliferation and internode reduction; the very small leaves result in a bushy appearance.     

Begomoviruses Associated with Leaf Curl Disease of Tomato in Java,Indonesia*

We report that several begomoviruses are associated with tomato leaf curl disease in Java, Indonesia. Tomato plants with leaf curl symptoms were collected from Bandung (west Java), Purwokerto (central Java), Magelang (central Java) and Malang (east Java) of Indonesia, the major tomato‐growing areas of the country. Viruses were detected using the polymerase chain reaction (PCR), with universal primers for the genus Begomovirus. PCR‐amplified fragments were cloned and sequenced. Based on sequence comparisons and phylogenetic analyses, the viruses were divided into three groups. With respect to amino acid (aa) identities of the N‐terminal halves of the coat proteins compared in this study, group I was most closely related to Ageratum yellow vein virus (AYVV) (97%), Ageratum yellow vein China virus‐[Hn2] (AYVCNV‐[Hn2]) (96%) and Ageratum yellow vein virus‐[Taiwan] (AYVV‐[Tai]) (95%), and ageratum‐infecting begomovirus from Java (99%). Group II had high sequence identity with a tentative species of tomato leaf curl Java virus (ToLCJAV) (96% aa) for the CP. Group III was most closely related to a proposed species of Pepper yellow leaf curl Indonesia virus (PepYLCIDV) (90% aa identity) by its partial CP sequence.     

Begomovirus and Associated Satellite Components Infecting Cluster Bean (Cyamopsis tetragonoloba) in Pakistan

Cluster bean (Cyamopsis tetragonoloba) is a legume that is grown widely on the Indian subcontinent. Leaf curl symptoms of cluster bean plants collected in the Punjab, Pakistan, were shown to be associated with the begomovirus Papaya leaf curl virus; the first time this virus has been identified infecting cluster bean in Pakistan. The virus was shown to be associated with Tomato leaf curl betasatellite. Additionally, some cluster bean plants were shown to also harbour Cotton leaf curl Multan alphasatellite. The significance of these findings is discussed.     

Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus Invade South-east Coast of China

An epidemic outbreak of severe yellow leaf curl disease was reported in field grown tomato within Zhejiang Province of China in the autumn–winter cropping season of 2006. A molecular diagnostic survey was carried out based on comparisons of partial and complete viral DNA sequences. Comparison of partial DNA‐A sequences amplified with degenerate primers specific for begomoviruses confirmed the presence of two types of begomoviruses. The complete DNA sequences of five isolates, corresponding to the two types, were determined. Sequence comparisons and phylogenetic analysis revealed that they correspond to two previously identified begomoviruses, Tomato yellow leaf curl virus and Tomato leaf curl Taiwan virus. The satellite DNAβ molecule was not detected in these samples by either PCR or Southern blot hybridization analysis. There has been no previous report of geminivirus disease incidence in Zhejiang Province, indicating that the introduction of these two tomato infecting geminiviruses into the agro‐ecological zone of South‐eastern China is a fairly recent event. The implications for disease control are discussed.     

Molecular Characterization of a Strain of Squash Leaf Curl China Virus from the Philippines

The complete nucleotide sequence of infectious cloned DNA components (A and B) of the causal agent of squash leaf curl disease in the Philippines was determined. DNA‐A and DNA‐B comprise 2739 and 2705 nucleotides, respectively; the common region is 174 bases in length. Five ORFs were found in DNA‐A and two in DNA‐B. Partial dimeric clones containing DNA‐A and DNA‐B, constructed in a binary vector and transformed into Agrobacterium tumefaciens, induced systemic infection in agro‐inoculated pumpkin plants (Cucurbita moschata). The total DNA‐A sequence was most closely related to that of Squash leaf curl China virus (SLCCNV) (88% identity), although the existence of B component of SLCCNV has not been reported. The deduced coat protein was like that of SLCCNV (98% amino acid sequence identity) and the Philippines virus has low sequence identity to Squash leaf curl virus (SLCV) and Squash mild leaf curl virus (SMLCV) (63 and 64% total nucleotide sequence identities, respectively). From these results, we propose that the Philippines virus be designated Squash leaf curl China virus‐[Philippines] (SLCCNV‐[PH]).     

Incidences and progression of tomato chlorosis virus disease and tomato yellow leaf curl virus disease in tomato under different greenhouse covers in southeast Spain

Epidemics of whitefly‐transmitted Tomato chlorosis virus, Tomato yellow leaf curl Sardinia virus and Tomato yellow leaf curl virus have been present in the south east of Spain since the 1990s. A survey was performed in 40 greenhouses and nethouses during 2003 to establish the relationship between the disease incidence and the quality of greenhouse or nethouse coverings, providing a physical protection of crops against whiteflies. For tomato chlorosis virus disease (ToCD), the incidence correlated with the type of greenhouse cover and was most reduced under higher quality covers. Control of tomato yellow leaf curl disease (TYLCD) was achieved only for crops grown in the highest quality greenhouses. TYLCD incidence in tolerant tomatoes remained below 100% within the 5 months of sampling, despite the disease progress rate at the initial stage of the cultivation being higher than that of ToCD, which did reach 100% incidence in many greenhouses. Linear regression analysis showed that the development of ToCD and TYLCD in most of the greenhouses was best described by the monomolecular model and the Gompertz model, respectively. Tomato infectious chlorosis virus was not detected in parallel surveys carried out during this study, although it has been described previously in the area studied.     

Rapid Detection and Identification of Six Tomato yellow leaf curl virus Isolates from Different Regions Using Polymerase Chain Reaction and Restriction Enzyme Analysis

The combinational analysis of polymerase chain reaction and restriction enzyme analysis (PCR‐RE) to distinguish six Tomato yellow leaf curl virus (TYLCV) isolates from five countries was developed. Tomato yellow leaf curl virus has spread from the Middle East to Western Europe, Central America and Eastern Asia, and occurs on infected crops such as tomatoes, peppers, cucurbits and beans. Tomato yellow leaf curl virus isolates from Jordan (TYLCV‐Mld[Jo:Cuc] and TYLCV‐IL[Jo:Cuc]), Israel (TYLCV‐IL[IL:Reo:86]), Spain (TYLCV‐Mld[ES72/97]), USA (TYLCV‐IL[US:F10:04]) and Korea (TYLCV‐KR) were collected, and the sequences of the six isolates were analysed to distinguish them by PCR‐RE combination analysis. Oligonucleotide primers for the six TYLCV isolates were designed to amplify approximately 740 base pairs including the intergenic region (IR) and parts of V1 and V2 ORF. Unique restriction enzyme sites were analysed to identify isolate‐specific restriction enzyme sites on the PCR products of each isolate. Three enzymes (DdeI, FauI and BssSI) were selected by in silico analysis, and then, the PCR products following the serial digestion of each restriction enzyme were separated by agarose gel electrophoresis to distinguish the TYLCV isolates. Taken together, the PCR‐RE combination analysis by serial digestion with three restriction enzymes could be a useful method for distinguishing the six isolates.     

Identification of a distinct strain of Cotton leaf curl Gezira virus infecting tomato in Oman

Tomato (Solanum lycopersicum) plants exhibiting yellowing, curling and stunting symptoms were identified in fields of the Tawoos Agricultural Systems, in Al‐Batinah in Oman. Cloning and sequencing of restriction endonuclease digested rolling circle amplified viral DNA identified a cotton begomovirus (family Geminiviridae) associated with the symptomatic tomato plants. Detailed analysis of complete sequences showed the virus to be a previously unknown strain of Cotton leaf curl Gezira virus (CLCuGeV) in association with the betasatellite Tomato leaf curl betasatellite (ToLCB). The new CLCuGeV strain, for which the name “Al Batinah” strain is suggested, has the greatest levels of sequence identity (91.9%) to an isolate of CLCuGeV recently reported from the neighbouring United Arab Emirates. Additionally, CLCuGeV‐Al Batinah was shown to have a recombinant origin with sequences donated by an African cassava mosaic virus‐like parent. This is the first identification of this Malvaceae‐adapted begomovirus in tomato. Although ToLCB is common in Oman, being one of only two betasatellites identified there so far, this is the first identification of this betasatellite with CLCuGeV. The significance of these findings is discussed.     

Detection and Molecular Characterization of Squash leaf curl virus (SLCV) in Jordan

Squash leaf curl virus (SLCV) was detected for the first time in Jordan using degenerated oligonucleotide primers. Two isolates of the virus, SLCV‐E and SLCV‐R, were detected using specific oligonucleotide primers in symptomatic Cucurbita pepo. SLCV was also found to occur naturally in Malva parviflora, which showed severe leaf curling, yellowing and stunting of the whole plants. The full‐length genomes of Squash leaf curl virus‐Malva (SLCV‐Malva) isolate were amplified using the bacteriophage Φ DNA polymerase enzyme. Nucleotide sequence analysis showed that SLCV‐Malva shared high nucleotide identity (98% and 97%) with SLCV‐EG and SLCV‐E from Egypt and USA, respectively. A survey using dot‐blot hybridization indicated that squash leaf curl disease occurred in all surveyed areas. The highest disease incidence (95%) was recorded in Dir Alla area, whereas disease incidence did not exceed 69% in squash samples collected from North Ghor.     

Engineering tomato for resistance to tomato leaf curl disease using viral <Emphasis Type="Italic">rep</Emphasis> gene sequences

Transgenic tomato resistant to tomato leaf curl disease (ToLCD) using replicase (rep) gene sequences of Tomato leaf curl virus in antisense orientation were developed via Agrobacterium-mediated transformation. A binary vector carrying the antisense rep gene (untranslatable full length sequence, 1086 bp) along with the npt II gene was used for transformation. High level of resistance and inheritability of the transgene was observed up to T₂ stage following challenge inoculation with the virus. The mechanism of resistance appears RNA-mediated, since the plants carried the untranslatable antisense rep gene. Progeny analysis of these plants showed classical Mendelian pattern of inheritance in two of the six transgenic lines having single transgene insertion.     

Biology and interactions of two distinct monopartite begomoviruses and betasatellites associated with radish leaf curl disease in India

Background

Emerging whitefly transmitted begomoviruses are major pathogens of vegetable and fibre crops throughout the world, particularly in tropical and sub-tropical regions. Mutation, pseudorecombination and recombination are driving forces for the emergence and evolution of new crop-infecting begomoviruses. Leaf curl disease of field grown radish plants was noticed in Varanasi and Pataudi region of northern India. We have identified and characterized two distinct monopartite begomoviruses and associated beta satellite DNA causing leaf curl disease of radish (Raphanus sativus) in India.

Results

We demonstrate that RaLCD is caused by a complex of two Old World begomoviruses and their associated betasatellites. Radish leaf curl virus-Varanasi is identified as a new recombinant species, Radish leaf curl virus (RaLCV) sharing maximum nucleotide identity of 87.7% with Tomato leaf curl Bangladesh virus-[Bangladesh:2] (Accession number AF188481) while the virus causing radish leaf curl disease-Pataudi is an isolate of Croton yellow vein mosaic virus-[India] (CYVMV-IN) (Accession number AJ507777) sharing 95.8% nucleotide identity. Further, RDP analysis revealed that the RaLCV has a hybrid genome, a putative recombinant between Euphorbia leaf curl virus and Papaya leaf curl virus. Cloned DNA of either RaLCV or CYVMV induced mild leaf curl symptoms in radish plants. However, when these clones (RaLCV or CYVMV) were individually co-inoculated with their associated cloned DNA betasatellite, symptom severity and viral DNA levels were increased in radish plants and induced typical RaLCD symptoms. To further extend these studies, we carried out an investigation of the interaction of these radish-infecting begomoviruses and their associated satellite, with two tomato infecting begomoviruses (Tomato leaf curl Gujarat virus and Tomato leaf curl New Delhi virus). Both of the tomato-infecting begomoviruses showed a contrasting and differential interaction with DNA satellites, not only in the capacity to interact with these molecules but also in the modulation of symptom phenotypes by the satellites.

Conclusion

This is the first report and experimental demonstration of Koch's postulate for begomoviruses associated with radish leaf curl disease. Further observations also provide direct evidence of lateral movement of weed infecting begomovirus in the cultivated crops and the present study also suggests that the exchange of betasatellites with other begomoviruses would create a new disease complex posing a serious threat to crop production.     

Identification and Molecular Characterization of a New Geminivirus Betasatellite Infecting Malvastrum coromandelianum in China

The complete nucleotide sequence of a satellite molecule associated with Malvastrum leaf curl Guangdong virus (MLCuGdV) infecting M. coromandelianum plants exhibiting leaf curl symptoms in a suburb of Guangzhou, Guangdong Province of China, is described and analysed. The molecule has typical features of betasatellites, containing a single ORF in the complementary‐sense strand, an A‐rich region, the satellite‐conserved region and a stem–loop structure. Compared with the geminivirus betasatellites in GenBank database, this molecule shows the highest nucleotide sequence identity of 71.9% with Tomato leaf curl Philippine betasatellite isolate Laguna1 (ToLCPB, AB307732). Phylogenetic analysis indicates that it is more related to isolate Laguna 1 and Laguna 2 of ToLCPB. According to the proposed species demarcation threshold of betasatellites (78% nucleotide identity), it is a novel betasatellite species, for which we propose the name Malvastrum leaf curl Guangdong betasatellite (MLCuGdB).     

First Report of Tomato Yellow Leaf Curl Virus-Israel Species Infecting Tomato, Pepper and Bean in Tunisia

Tomato yellow leaf curl virus disease (TYLCVD) has been observed in Tunisia for more than 20 years. Until year 2004, only the Tomato yellow leaf curl Sardinia virus‐Sicily (TYLCSV‐[Sic]) was detected in tomato, pepper and bean crops. In the Sahel region, some tomato samples showing severe TYLCVD symptoms were collected from greenhouses in 2004 and 2005. Typing of these isolates revealed for the first time the presence of the TYLCV Israel in Tunisia. This result was confirmed by using several sets of specific primers and by sequencing. This species has also been detected on pepper and bean collected from fields in the same region. The sequencing of a tomato and a bean isolate showed that they both share more than 97% of sequence identity with the TYLCV from Dominican Republic ( AF024715 ). The TYLCV has been found in single and mixed infection with the TYLCSV‐[Sic].     

Identification of Mungbean yellow mosaic Indian virus Associated with Tomato Leaf Curl Betasatellite Infecting Phaseolus vulgaris in Oman

Kidney bean (Phaseolus vulgaris) plants exhibiting foliar yellow mosaic symptoms and some leaf crumpling were identified in the Al Batinah region of Oman. Rolling circle amplification and polymerase chain reaction identified a bipartite begomovirus (family Geminiviridae) and a betasatellite in association with the symptomatic plants. Analysis of full‐length sequences showed the virus to be Mungbean yellow mosaic Indian virus (MYMIV) and the betasatellite Tomato leaf curl betasatellite (ToLCB). This is the first identification of a legume‐adapted begomovirus in Oman and the first identification of MYMIV in association with the betasatellite ToLCB. The isolate of MYMIV from Oman shows the greatest levels of sequence identity to isolates occurring in South Asia and South‐East Asia, suggesting that the virus has only recently been introduced. The significance of these findings is discussed.     

Different transmission efficiencies may drive displacement of tomato begomoviruses in the fields in Taiwan

A progressive displacement of Tomato leaf curl Taiwan virus (ToLCTWV) by Tomato yellow leaf curl Thailand virus (TYLCTHV) from 2005 to 2009 has been recorded in tomato fields in Taiwan. Begomoviruses are exclusively transmitted by Bemisia tabaci complex, so we hypothesised that the displacement of tomato begomoviruses in the fields may be due to the invasion of a new virus/vector and the different transmission efficiencies of the viruses by the vectors. The objective of this research was to compare the transmission efficiency of TYLCTHV and ToLCTWV by the B and Q biotypes of B. tabaci complex. When transmission efficiency, virus retention in vector, and latent period for vector transmission were compared, the B biotype transmitted TYLCTHV and ToLCTWV more efficiently than did the Q biotype, and transmitted TYLCTHV more efficiently than ToLCTWV. The B biotype retained both viruses and remained infective throughout adulthood, but the Q biotype did not keep its infectivity, although it did retain both viruses lifelong. The B biotype transmitted TYLCTHV and ToLCTWV with the shortest latent period. In summary, B. tabaci B biotype and TYLCTHV is the best alliance for disease transmission, so we conclude that this may be one of drivers responsible for the displacement of ToLCTWV by TYLCTHV in tomato fields in Taiwan.     

Molecular characterization of tomato leaf curl China virus, infecting tomato plants in China, and functional analyses of its associated betasatellite

A novel tomato-infecting begomovirus from Guangxi province, China, was identified and characterized, for which the name Tomato leaf curl China virus (ToLCCNV) was proposed. Phylogenetic and recombination analyses of the virus genomic sequences suggested that ToLCCNV may have arisen by recombination among Tomato leaf curl Vietnam virus (ToLCVV), Tomato leaf curl Gujarat virus (ToLCGV), and an unknown virus. A betasatellite molecule was found to be associated with ToLCCNV (ToLCCNB), and its complete nucleotide sequences were determined. Infectious clones of ToLCCNV and ToLCCNB were constructed and then used for agro-inoculation of plants; ToLCCNV alone infected Nicotiana benthamiana, Nicotiana glutinosa, Petunia hybrida, and Solanum lycopersicum plants, but no symptoms were induced. ToLCCNB was required for induction of leaf curl disease in these hosts. The βC1 protein of ToLCCNB was identified as a suppressor of RNA silencing and accumulated primarily in the nucleus. Deletion mutagenesis of βC1 showed that the central part of βC1 (amino acids 44 to 74) was responsible for both the suppressor activity and nuclear localization.     

A survey of mixed Begomovirus infection in solanaceae and fabaceae at different altitudes in East Java,Indonesia

A multiplex primer set was developed to detect four Begomoviruses in East Java, Indonesia, i.e. Tomato leaf curl New Delhi virus (ToLCNDV), Tomato yellow leaf curl Kanchanaburi virus (TYLCKaV), Pepper yellow leaf curl Indonesia virus (PepYLCIV) and Mungbean yellow mosaic India virus (MYMIV). Survey at different altitudes found that begomoviruses infecting pepper, tomato and long bean were more variable, while in eggplant and string bean were more uniform. As a single virus, TYLCKaV infected eggplant, and sometimes tomato and pepper; PepYLCIV infected pepper, tomato and long bean; ToLCNDV only infected long bean and tomato at low frequency; and MYMIV infected beans. Mixed infection occurred more frequently in the low altitude areas. Subsequent examination indicated that Cucumber mosaic virus (CMV) and potyviruses were also responsible for diseased fabaceous. Our data suggest a relationship between altitudes and virus species occurrence. However, which viral species infects a crop is mainly influenced by the crop rather than by altitude.     

Molecular Characterization of a Distinct Begomovirus Infecting Euphorbia pulcherrima in China

Virus isolate G35 was obtained from Euphorbia pulcherrima showing leaf curl and vein thickening symptoms in Tianyang, Guangxi Province, China. The virus was transmitted by whiteflies to Nicotiana tabacum, Lycopersicon esculentum, Datura stramonium and E. pulcherrima. DNA‐A contains 2746 nucleotides, with two open reading frames (ORFs) in the virion‐sense DNA and four ORFs in the complementary‐sense DNA. When compared with the DNA‐A sequence of other begomoviruses, the total DNA‐A of isolate G35 was most closely related to that of Ageratum enation virus (79.9% sequence identity). However, the deduced coat protein of G35 is most like that of Pepper leaf curl virus from Bangladesh (94.9% amino acid sequence identity), and the AC1 of G35 is most like that of Cotton leaf curl Multan virus‐Okra (87.2% amino acid sequence identity). The molecular data showed that G35 is a distinct Begomovirus species, for which the name Euphorbia leaf curl virus (ELCV) is proposed.     

Occurrence of Six Begomoviruses Infecting Tomato Fields in Venezuela and Genetic Characterization of Potato Yellow Mosaic Virus Isolates

Begomoviruses are one of the major pathogens in tomato crops worldwide. In Venezuela, six begomovirus species have been described infecting tomato: Potato yellow mosaic virus (PYMV), Euphorbia mosaic Venezuela virus (EuMVV), Merremia mosaic virus (MeMV), Tomato chlorotic leaf distortion virus (ToCLDV), Tomato yellow margin leaf curl virus (TYMLCV) and Tomato yellow leaf curl virus (TYLCV). In this study, the occurrence of these viruses was analysed by PCR in 338 tomato plants exhibiting virus‐like symptoms. Sixty‐three per cent of the plants were positive at least to one of the begomoviruses tested. PYMV and TYLCV were the most frequent viruses showing 39.6 and 23.7% occurrence, respectively. Phylogenetic analyses revealed two groups of PYMV isolates from several Caribbean Basin countries. The first group clustered isolates from several countries, including Venezuela, and the second group clustered only Colombian isolates. Due to the high prevalence of PYMV and TYLCV in Venezuela, it is suggested that the surveillance and control strategies currently applied in the country should be focused on these two begomoviruses.