A new strain of tomato severe leaf curl virus and a unique variant of tomato yellow leaf curl virus from Mexico

The complete genome sequence of a distinct variant of tomato yellow leaf curl virus-Israel (TYLCV-IL) and the DNA-A sequence of a new strain of tomato severe leaf curl virus (ToSLCV) isolated in San Luis Potosi, Mexico, are described and analyzed. The TYLCV-IL[MX:SLP:11] variant differs from all TYLCV-IL isolates described so far by a unique 42-nt duplicated sequence comprising a part of the conserved stem-loop element of the virion-strand replication origin and adjacent regulatory sequences. TYLCV-IL[MX:SLP:11] was associated with tomato chino La Paz virus (ToChLPV-B[MX:SLP:11]) in a Solanum pimpinellifolium plant, and with pepper huasteco yellow vein virus (PHYVV-[MX:SLP:11]) and ToSLCV-GT[MX:SLP:11] in a Solanum lycopersicum plant. In addition, a distinct ToSLCV exhibiting low sequence identity (<89?%) to other ToSLCV isolates from Mexico was found in a tomato plant collected in the same field. Sequence analysis of this new ToSLCV strain indicates that it is a recombinant of close relatives of ToSLCV-GT[MX:SLP:11] and ToChLPV-B[MX:SLP:11] found in mixed infections with TYLCV-IL[MX:SLP:11].     

Predominance of tomato leaf curl Gujarat virus as a monopartite begomovirus: association with tomato yellow leaf curl Thailand betasatellite

Tomato leaf curl is a serious malady in the state of Maharashtra, India, causing nearly 100 % yield loss. An extensive survey was done in the affected fields of tomato in the year 2008, and members of three species of begomoviruses were identified as causing the disease. More than 60 % of the samples from diseased plants were infected with tomato leaf curl Gujarat virus (ToLCGuV). Isolates collected from these fields differed from the Varanasi isolate of ToLCGuV in not having a DNA B component. Instead, they were like typical Old World monopartite begomoviruses in that they were associated with only one betasatellite, tomato yellow leaf curl Thailand betasatellite (TYLCTHB). ToLCGuV alone is readily infectious, expressing systemic symptoms in Nicotiana benthamiana and tomato. Co-inoculation of ToLCGuV with TYLCTHB, increased symptom severity and reduced the incubation time required for symptom expression. ToLCGuV successfully interacted with heterologous DNA B component of ToLCNDV [IN:Pun:JID:08], and co-inoculation of these two resulted in yellow mottling symptoms that were typical of DNA B.     

Biolistic inoculation of plants with tomato yellow leaf curl virus DNA

Tomato yellow leaf curl virus (TYLCV) full-length DNA was amplified by PCR and cloned into a bacterial plasmid. The cloned TYLCV DNA was excised from the plasmid, ligated and the resulting monomeric circular double-stranded TYLCV DNA was used to inoculate tomato (Solanum lycopersicom) and datura (Datura stramonium) plants by particle bombardment. The bombarded plants produced typical disease symptoms, similar to those produced following whitefly-mediated inoculation, albeit 5-7 days later than whitefly-inoculated plants. The success rate of inoculating tomato plants by particle bombardment averaged 37%, whereas with datura plants, it averaged 85%. With whitefly-mediated inoculation of TYLCV, the success rate of inoculation was also higher in datura plants than in tomato plants. Bombardment of datura plants with a linear form of TYLCV DNA also resulted in viral infection, with an inoculation success rate similar to that with the closed-circular TYLCV DNA. Bombarding datura plants with the bacterial plasmid containing the cloned TYLCV DNA did not result in viral infection, but bombardment with a bacterial plasmid containing a cloned dimer of TYLCV DNA yielded an infection rate of 50-100%. This is the first report of TYLCV inoculation of plants using particle bombardment of a cloned monomeric linear or closed-circular form of TYLCV double-stranded DNA.     

Genetic structure and evolution of natural populations of viruses causing the tomato yellow leaf curl disease in Spain

The population structure and genetic variation of two begomoviruses: tomato yellow leaf curl Sardinia virus (TYLCSV) and tomato yellow leaf curl virus (TYLCV) in tomato crops of Spain were studied from 1997 until 2001. Restriction digestion of a genomic region comprised of the CP coat protein gene (CPR) of 358 TYLC virus isolates enabled us to classify them into 14 haplotypes. Nucleotide sequences of two genomic regions: CPR, and the surrounding intergenic region (SIR) were determined for at least two isolates per haplotype. SIR was more variable than CPR and showed multiple recombination events whereas no recombination was detected within CPR. In all geographic regions except Murcia, the population was, or evolved to be composed of one predominant haplotype with a low genetic diversity (<0.0180). In Murcia, two successive changes of the predominant haplotype were observed in the best studied population. Phylogenetic analysis showed that the TYLCSV sequences determined clustered with sequences obtained from the GenBank of other TYLCSV Spanish isolates which were clearly separated from TYLCSV Italian isolates. Most of our TYLCV sequences were similar to those of isolates from Japan and Portugal, and the sequences obtained from TYLCV isolates from the Canary island of Lanzarote were similar to those of Caribbean TYLCV isolates.     

Molecular characterization and pathogenicity of tomato yellow leaf curl virus in China

Several tomato production regions in China were surveyed for tomato yellow leaf curl disease (TYLCD), and 31 tomato leaf samples showing TYLCD-like symptoms were collected. The partial or full-length genomes of these isolates were sequenced and tomato yellow leaf curl virus (TYLCV) was detected in Shanghai, Zhejiang, Jiangsu Shandong and Hebei provinces of China. The TYLCV isolates found in China share high sequence identity (>98%) and have more than 97% sequence identity with TYLCV-IL[IL:Reo] (X15656). Phylogenetic relationship analysis reveals that although with little genetic variability, they can form two groups and all the TYLCV isolates in China belong to the group I. An infectious clone of TYLCV-[CN:SH2] (AM282874) was constructed and agro-inoculated into Nicotiana benthamiana, N. tabacum Samsun, N. glutinosa, Solanum lycopersicum, Petunia hybrida, Cucumis sativus, Gossypium hirsutum, S. melongena, and Capsicum annuum. TYLCV-[CN:SH2] can induce severe leaf curling and stunting symptoms in these plants except C. sativus, G. hirsutum, S. melongena and C. annuum. We verified that TYLCV can trans-replicate tomato yellow leaf curl China virus DNA-β in N. benthamiana and S. lycopersicum and induced more severe symptoms with distortion and yellow vein.     

Detection of tomato yellow leaf curl virus by loop-mediated isothermal amplification reaction

The genomic DNA molecule of tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted geminivirus, was amplified from total DNA extracts of TYLCV-infected tomato (Lycopersicon esculentum) by the use of loop-mediated isothermal amplification (LAMP). The procedure was also used to amplify TYLCV DNA from total DNA extracts of individual whiteflies (Bemisia tabaci) that had fed on TYLCV-infected plants. One of the characteristics of the LAMP method is its ability to synthesize an extremely large amount of DNA. Accordingly, a large amount of by-product, pyrophosphate ion, is produced yielding a white precipitate of magnesium pyrophosphate in the reaction mixture. The presence or absence of this white precipitate allows easy detection of amplification of TYLCV genomic DNA without gel electrophoresis.     

Tomato yellow leaf curl virus, an emerging virus complex causing epidemics worldwide

Tomato yellow leaf curl (TYLC) is one of the most devastating viral diseases of cultivated tomato (Lycopersicon esculentum) in tropical and subtropical regions worldwide, and losses of up to 100% are frequent. In many regions, TYLC is the main limiting factor in tomato production. The causal agents are a group of geminivirus species belonging to the genus Begomovirus of the family Geminiviridae, all of them named Tomato yellow leaf curl virus (TYLCV) (sensu lato). There has been almost 40 years of research on TYLCV epidemics and intensive research programmes have been conducted to find solutions to the severe problem caused by these viruses. This paper provides an overview of the most outstanding achievements in the research on the TYLCV complex that could lead to more effective control strategies.     

A single-tube PCR assay for detecting viruses and their recombinants that cause tomato yellow leaf curl disease in the Mediterranean basin

Tomato yellow leaf curl disease (TYLCD) is well known in Mediterranean countries, where it has been causing severe losses in tomato crops for decades. Until recently, two viruses (with several isolates) in the genus Begomovirus, family Geminiviridae, have been associated with the epidemics: Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV). However, recombinants between these, such as Tomato yellow leaf curl Malaga virus (TYLCMalV), are spreading, and new methods for detecting all viruses present in the region are needed. By considering all DNA sequences available of viruses causing TYLCD in the Mediterranean basin, a PCR/RFLP protocol was developed that amplifies the intergenic region in a multiplex reaction, followed by digestion with AclI (=Psp1406I) restriction enzyme. This procedure generates an easily recognizable pattern on gels, with DNA fragments of specific size for each virus species and each recombinant: 800 bp for TYLCSV, 410 bp for TYLCV, 570 bp for TYLCMalV and the other detected recombinants, 640 bp for hypothetical recombinants of different type. This new method gives, with a single reaction, an overview of the species present in the sample and will be useful for screening the causal agents of TYLCD, as well as in breeding programs for resistance.     

Interaction of tomato yellow leaf curl virus with diverse betasatellites enhances symptom severity

The complete nucleotide sequence was determined for a begomovirus isolated from tomato exhibiting leaf curling and yellowing symptoms in Tochigi Prefecture in Japan. The genome organization of this virus was similar to those of other Old World monopartite begomoviruses. Neither a DNA betasatellite nor a DNA-B component was detected. It had the highest total nucleotide sequence identity (99%) with tomato yellow leaf curl virus-Israel[Japan:Tosa:2005] (TYLCV-IL[JR:Tos:05]) and TYLCV-Israel[Japan:Haruno:2005] (TYLCV-IL[JR:Han:05]). Its coat protein V1 also showed an identical amino acid sequence with those of TYLCV-IL[JR:Tos:05] and TYLCV-IL[JR:Han:05]. Thus, the begomovirus was determined to be an isolate of TYLCV-IL designated as TYLCV-Israel[Japan:Tochigi:2007] (TYLCV-IL[JR:Toc:07]). We investigated the interaction of TYLCV-IL[JR:Toc:07] with two known satellites associated with tomato yellow dwarf disease in Japan, tobacco leaf curl Japan betasatellite [Japan:Ibaraki:2006] and honeysuckle yellow vein mosaic betasatellite [Japan:Nara:2006], as well as with tomato leaf curl Philippines betasatellite [Philippines:Laguna1:2008], in tomato and Nicotiana benthamiana plants. TYLCV-IL[JR:Toc:07] trans-replicated these betasatellites, inducing more severe tomato yellow leaf curl disease-related symptoms than TYLCV-IL[JR:Toc:07] alone.     

A multiplex PCR method discriminating between the TYLCV and TYLCV-Mld clades of tomato yellow leaf curl virus

Tomato yellow leaf curl virus (TYLCV) is one of the causal agents of tomato yellow leaf curl disease (TYLCD) and can cause up to 100% yield losses in tomato fields. As TYLCV continues to spread, many isolates have been described in different parts of the world. Recently two closely related but distinct TYLCV clades, called TYLCV and TYLCV-Mld, have been identified. Isolates from those two clades differ mainly in the nucleotide sequences of their replication associated protein genes but do not display significantly different symptomatology. In order to improve monitoring of the rapidly expanding worldwide TYLCD epidemic, a multiplex polymerase chain reaction assay (mPCR) was developed. A set of three primers were designed to detect and characterize the TYLCV and TYLCV-Mld clade isolates. The specificity and sensitivity of the mPCR were validated on TYLCV infected tomato plants and Bemisia tabaci whiteflies. Being cheap, fast and highly sensitive this new diagnostic tool should greatly simplify efforts to trace the global spread of TYLCV.     

Characterization and distribution of tomato yellow margin leaf curl virus, a begomovirus from Venezuela

A begomovirus causing mottling and leaf deformation in tomato from the State of Mérida was cloned and sequenced. The virus has a bipartite genome comprised of a DNA-A (2,572 nucleotides) and a DNA-B (2,543 nucleotides) with a genome organization typical of New World begomoviruses. Both components share a common region of 115 nucleotides with 98 % sequence identity. Phylogenetic analysis indicated that while no virus sequences were closely related, the A component was distantly related to those of two other tomato-infecting viruses, tomato leaf deformation virus and Merremia mosaic virus; and the DNA-B, to those of pepper huasteco yellow vein virus and Rhynchosia golden mosaic Yucatan virus. The DNA-A and DNA-B sequences were submitted to GenBank (accession no. AY508993 and AY508994, respectively) and later accepted by the International Committee on Taxonomy of Viruses as the genome of a member of a unique virus species with the name Tomato yellow margin leaf curl virus (TYMLCV). Tomato (Solanum lycopersicum L. ‘Fl. Lanai’) plants inoculated with cloned TYMLCV DNA-A and DNA-B became systemically infected and showed chlorotic margins and leaf curling. The distribution of TYMLCV in tomato-producing states in Venezuela was determined by nucleic acid spot hybridization analysis of 334 tomato leaf samples collected from ten states using a TYMLCV-specific probe and confirmed by PCR and sequencing of the PCR fragment. TYMLCV was detected in samples from the states of Aragua, Guárico, and Mérida, suggesting that TYMLCV is widely distributed in Venezuela.     

Pathogenicity and insect transmission of a begomovirus complex between tomato yellow leaf curl virus and Ageratum yellow vein betasatellite

Tomato yellow leaf curl virus (TYLCV) and Ageratum yellow vein betasatellite (AYVB) are members of the genus Begomovirus (family Geminiviridae). TYLCV and AYVB have been found in Japan over the last 15 years, and are associated with tomato leaf curl and the tomato yellow leaf curl diseases (TYLCD). AYVB is also associated with some monopartite begomoviruses. We have cloned both TYLCV and AYVB and demonstrated that TYLCV can trans-replicate with AYVB in Nicotiana benthamiana and tomato plants. A mixed infection of TYLCV and AYVB induced more severe symptoms of upward leaf curl, stunting, vein thickening, and swelling compared with TYLCV infection alone. The symptoms induced by infection of AYVB included a rise in abnormal cell proliferation, and pigmentation around leaf vein tissues. This is the first study to show that a complex of TYLCV and AYVB can be transmitted by vector insects among tomato plants. These results indicate that TYLCV possesses the potential to induce severe TYLCD by associating with AYVB.     

Infectivity of Euphorbia leaf curl virus and interaction with Tomato yellow leaf curl China betasatellite

To investigate the infectivity of Euphorbia leaf curl virus (EuLCV), an infectious clone was constructed and tested by agroinoculation and whitefly inoculation. EuLCV infected Nicotiana benthamiana, N. glutinosa, Solanum lycopersicum, Petunia hybrida efficiently upon agroinoculation and induced leaf curling, vein swelling and stunting in these plants but no symptoms in N. tabacum. Co-inoculation of EuLCV with a betasatellite DNA from an unrelated begomovirus enhanced symptoms in N. benthamiana, N. glutinosa, N. tabacum, S. lycopersicum and P. hybrida plants but had no effect on the accumulation of EuLCV DNA. Euphorbia pulcherrima plants were only infectable by insect transmission from agro-infected P. hybrida as a source. This is the first report about a monopartite begomovirus that has been reintroduced into a plant of the genus Euphorbia.     

Application of Phi29 DNA polymerase in identification and full-length clone inoculation of tomato yellow leaf curl Thailand virus and tobacco leaf curl Thailand virus

Summary. Tomato plants grown in greenhouses in Thailand developed typical symptoms of a tomato yellow leaf curl Thailand virus (TYLCTHV) infection. After confirmation by ELISA, a Phi29 DNA polymerase approach was chosen for further molecular analysis of TYLCTHV. Total DNA purified from infected tomato leaves was subjected to rolling-circle amplification (RCA) of DNA-A and DNA-B of TYLCVTHV. In addition, a new monopartite geminivirus with a putative recombinant background was identified by RCA and tentatively named tobacco leaf curl Thailand virus (TbLCTHV). To confirm the composition of both geminiviruses, full-length clones were established and used for inoculation of Nicotiana benthamiana by particle bombardment or agroinfection. When TYLCTHV DNA-A and DNA-B were applied together by particle bombardment or agroinfection, severe stunting, yellowing, and leaf curling were observed. Whereas TYLCTHV DNA-A and TbLCTHV revealed no infection after'particle bombardment, similar symptoms in N. benthamiana, like leaf upward curling and yellowing were observed following agroinfection. DNA components of TYLCTHV DNA-A and DNA-B were excised from their respective plasmids, ligated, and amplified by Phi29 DNA polymerase. The ability of viral concatamere inoculation was evaluated in particle co-bombardment experiments on N. benthamiana. Thus, particle bombardment of RCA-derived multimeric products proved to be at least as effective as inoculation with a partial repeat construct and tenfold as effective as inoculation with excised unit-lengths of DNA-A and DNA-B of TYLCVTHV when using each DNA component in an amount of 5 ng.     

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.     

Effect of plant age at inoculation on expression of genetic resistance to tomato yellow leaf curl virus

Summary To determine the effects of plant age on the expression of genetic resistance to tomato yellow leaf curl virus (TYLCV), six TYLCV-resistant and two susceptible tomato varieties were inoculated at 14, 28 or 45 days after sowing (DAS). Inoculation at 14 and 28 DAS was performed in the greenhouse, and the plants were transplanted to the field at 30 DAS. Inoculation at 45 DAS was performed in the field, by covering the target plants with polypropylene (“Agril”) sheets and releasing viruliferous whiteflies under them. Resistance was assayed mainly by comparing yield components of inoculated plants to those of control, non-inoculated plants of the same variety. Symptom severity and plant height were also followed. Plant age at inoculation had no effect on disease-severity scores of the susceptible varieties, and little or no effect on those of the resistant varieties. In contrast, plant age at inoculation had a significant effect on the yield of all varieties tested. All varieties suffered a significant yield reduction due to inoculation with TYLCV; the lowest yield was produced by plants inoculated at 14 DAS. A smaller TYLCV-induced yield reduction (yield increase of 50 to 100%, depending on the variety’s resistance level), was achieved following inoculation at 28 DAS. A further reduction in yield loss (yield increase of 30 to 40%) was achieved following inoculation at 45 DAS. Our results clearly demonstrate the occurrence of age-related (or mature-plant) resistance in tomato plants to TYLCV. Correspondence: Moshe Lapidot, Volcani Center, Department of Vegetable Research, Institute of Plant Sciences, Agricultural Research Organization, Bet Dagan 50250, Israel