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.     

Evidence of local evolution of tomato-infecting begomovirus species in West Africa: characterization of tomato leaf curl Mali virus and tomato yellow leaf crumple virus from Mali

Tomato yellow leaf curl (TYLC) and tomato leaf curl (ToLC) diseases are serious constraints to tomato production in Mali and other countries in West Africa. In 2003 and 2004, samples of tomato showing virus-like symptoms were collected during a survey of tomato virus diseases in Mali. Three predominant symptom phenotypes were observed: (1) TYLC/ToLC (stunted upright growth and upcurled leaves with interveinal yellowing and vein purpling), (2) yellow leaf crumple and (3) broccoli or bonsai (severe stunting and distorted growth). Squash blot (SB) hybridization with a general begomovirus probe and/or SB/PCR analyses revealed begomovirus infection in plants with each of these symptom phenotypes and no evidence of phytoplasma infection. Sequence analysis of PCR-amplified begomovirus fragments revealed two putative new begomovirus species associated with the TYLC/ToLC and yellow leaf crumple symptom phenotypes, respectively. Full-length clones of these begomoviruses were obtained using PCR and overlapping primers. When introduced into N. benthamiana and tomato plants, these clones induced upward leaf curling and crumpling (the TYLC/ToLC-associated begomovirus) or downward leaf curl/yellow mottle (yellow leaf crumple-associated begomovirus) symptoms. Thus, these begomoviruses were named tomato leaf curl Mali virus (ToLCMLV) and tomato yellow leaf crumple virus (ToYLCrV). The genome organization of both viruses was similar to those of other monopartite begomoviruses. ToLCMLV and ToYLCrV were most closely related to each other and to tobacco leaf curl Zimbabwe virus (TbLCZV-[ZW]) and tomato curly stunt virus from South Africa (ToCSV-ZA). Thus, these likely represent tomato-infecting begomoviruses that evolved from indigenous begomoviruses on the African continent. Mixed infections of ToLCMLV and ToYLCrV in N. benthamiana and tomato plants resulted in more severe symptoms than in plants infected with either virus alone, suggesting a synergistic interaction. Agroinoculation experiments indicated that both viruses induced symptomatic infections in tomato and tobacco, whereas neither virus induced disease symptoms in pepper, common bean, small sugar pumpkin, African eggplant, or Arabidopsis. Virus-specific PCR primers were developed for detection of ToLCMLV and ToYLCrV and will be used to further investigate the distribution and host range of these viruses.     

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.     

Founder effect, plant host, and recombination shape the emergent population of begomoviruses that cause the tomato yellow leaf curl disease in the Mediterranean basin

Tomato yellow leaf curl disease (TYLCD)-associated viruses present a highly structured population in the western Mediterranean basin, depending on host, geographical region and time. About 1,900 tomato and common bean samples were analyzed from which 111 isolates were characterized genetically based on a genome sequence that comprises coding and non-coding regions. Isolates of three distinct begomoviruses previously described were found (Tomato yellow leaf curl virus, TYLCV, Tomato yellow leaf curl Sardinia virus, TYLCSV, and Tomato yellow leaf curl Málaga virus, TYLCMalV), together with a novel recombinant virus. Mixed infections were detected in single plants, rationalizing the occurrence of recombinants. Except for TYLCV-type strain, single, undifferentiated subpopulations were present for each virus type, probably the result of founder effects. Limited genetic variation was observed in genomic regions, with selection against amino acid change in coding regions.     

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.     

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.     

Phylogenetic lineage of Tobacco leaf curl virus in Korea and estimation of recombination events implicated in their sequence variation

New strains of Tobacco leaf curl virus (TbLCV) were isolated from tomato plants in four different local communities of Korea, and hence were designated TbLCV-Kr. Phylogenetic analysis of the sequences of the whole genome and of individual ORFs of these viruses indicated that they are closely related to the Tobacco leaf curl Japan virus (TbLCJV) cluster, which includes Honeysuckle yellow vein virus (HYVV), Honeysuckle yellow vein mosaic virus (HYVMV), and TbLCJV isolates. Four putative recombination events were recognized within these virus sequences, suggesting that the sequence variations observed in these viruses may be attributable to intraspecific and interspecific recombination events involving some TbLCV-Kr isolates, Papaya leaf curl virus (PaLCV), and a local isolate of Tomato yellow leaf curl virus (TYLCV).     

A worldwide survey of tomato yellow leaf curl viruses

Summary.  The name tomato yellow leaf curl virus (TYLCV) has been given to several whitefly-transmitted geminiviruses affecting tomato cultures in many tropical and subtropical regions. Hybridization tests with two DNA probes derived from a cloned isolate of TYLCV from Israel (TYLCV-ISR) were used to assess the affinities of viruses in naturally infected tomato plants with yellow leaf curl or leaf curl symptoms from 25 countries. Probe A which included most of the intergenic region was expected to detect only isolates closely related to TYLCV-ISR, especially after high stringency washes. In contrast probe B, which included the full-length genome, was expected to detect a wide range of whitefly-transmitted geminiviruses. Tomato samples from six countries in the Middle East, from Cuba or the Dominican Republic proved to be closely related to TYLCV-ISR and probably were infected by strains of the same virus. Samples from Senegal and Cape Verde Islands were also related to the Middle Eastern virus. Samples from nine other countries in the western Mediterranean area, Africa, or South-East Asia were more distantly related and probably represent one or more additional geminivirus species. Samples from five countries in Africa, Central or South America gave hybridization signals with the full-length viral genome, only after low stringency wash, indicating that these samples were infected by remote viruses. These results were supported by DNA and protein sequence comparison, which indicate that tomato geminiviruses fall into three main clusters representing viruses from 1) the Mediterranean/Middle East/African region, 2) India, the Far East and Australia, and 3) the Americas. Within the first cluster, two sub-clusters of viruses from the western Mediterranean or from the Middle East/Caribbean Islands were distinguished. The incidence of tomato yellow leaf curl diseases has increased considerably between 1990 and 1996. Accepted January 28, 1997; Received April 19, 1996     

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.     

Sweet pepper confirmed as a reservoir host for tomato yellow leaf curl virus by both agro-inoculation and whitefly-mediated inoculation

Tomato yellow leaf curl virus (TYLCV), a member of the genus Begomovirus, has a single-stranded DNA genome. TYLCV can induce severe disease symptoms on tomato plants, but other hosts plants such as cucurbits and peppers are asymptomatic. A full-length DNA clone of a Korean TYLCV isolate was constructed by rolling-circle amplification from TYLCV-infected tomatoes in Korea. To assess relative susceptibility of sweet pepper varieties to TYLCV, 19 cultivars were inoculated with cloned TYLCV by agro-inoculation. All TYLCV-infected sweet peppers were asymptomatic, even though Southern hybridization and polymerase chain reaction analysis showed TYLCV genomic DNA accumulation in roots, stems, and newly produced shoots. Southern hybridization indicated that TYLCV replicated and moved systemically from agro-inoculated apical shoot tips to roots or newly produced shoots of sweet peppers. Whitefly-mediated inoculation experiments showed that TYLCV can be transmitted to tomatoes from TYLCV-infected sweet peppers. Taken together, these results indicate that sweet pepper can be a reservoir for TYLCV in nature.     

Whiteflies (<Emphasis Type="BoldItalic">Bemisia tabaci</Emphasis>) issued from eggs bombarded with infectious DNA clones of <Emphasis Type="BoldItalic">Tomato yellow leaf curl virus</Emphasis> from Israel (TYLCV) are able to infect tomato plants

Summary.  We have reported previously that Tomato yellow leaf curl virus from Israel (TYLCV) penetrates the reproductive system of its vector, the whitefly Bemisia tabaci biotype B, and may be transmitted to progeny [9]. In order to mimic this phenomenon and to understand how TYLCV accompanies the development of the insect, we have bombarded B. tabaci eggs with an infectious DNA clone of TYLCV. After a linear full-length genomic copy of TYLCV DNA was delivered to eggs, the DpnI-sensitive DNA became circular and DpnI resistant. When a dimeric copy of TYLCV DNA was delivered to eggs, the viral DNA was detected in all the whitefly developmental stages. Adult insects that developed from the treated eggs were able to infect tomato test plants with variable frequency. Viral DNA was detected in the progeny of whiteflies that developed from eggs bombarded with TYLCV. Similarly, when insect eggs were bombarded with a dimeric copy of an infectious clone of the genome of Tomato yellow leaf curl virus from Sardinia, Italy (TYLCSV), adults that eclosed from the treated eggs were able to infect tomato test plants. Received February 19, 2001 Accepted September 20, 2001     

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.     

PCR–RFLP-based typing for differentiation of Tomato yellow leaf curl virus (TYLCV) genotypes from infected host plants in Korea

A polymerase chain reaction (PCR) using two sets of primers designed from published Tomato yellow leaf curl virus (TYLCV) genomes was developed to distinguish from the TYLCV-IL groups. The specificity of the two sets of primers was proven by testing against control TYLCV genomes and the symptomatic leaves of 34 different tomato cultivars naturally infected with TYLCV in greenhouses. One set for TYLCV-IL strain-specific primers (TYLCV-UNI-F and TYLCV-UNI-R) amplified full-length genome fragments from all the 34 tomato cultivars. Another set for TYLCV-IL group-II strain-specific primers (TYLCV-GPII-F and TYLCV-GPII-R) amplified target DNA fragments from only 9 tomato cultivars. Digestion by BglII and EcoRV of the PCR amplicons produced restriction fragment length polymorphism pattern that distinguished the TYLCV-IL group-I with two fragments from the TYLCV-IL group-II with no digested fragment. PCR coupled with BglII and EcoRV digestion confirmed that the 9 tomato cultivars were infected with the TYLCV-IL group-II and the remained 25 tomato cultivars were infected with the TYLCV-IL group-I.     

TYLCSV DNA, but not infectivity, can be transovarially inherited by the progeny of the whitefly vector Bemisia tabaci (Gennadius)

The transovarial transmission of two species of begomovirus, Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV), through generations of Bemisia tabaci of the B and Q biotypes has been investigated. Different life stages of the progeny of viruliferous female whiteflies have been analysed by PCR detection of viral DNA and infectivity tests. Our results indicate that TYLCSV DNA can be detected in eggs and nymphs, and to a lesser extent adults, of the first-generation progeny. Infectivity tests using a large number of adult progeny of the first, second, and third generation indicate that even when viral DNA is inherited, infectivity is not. For TYLCV, neither viral DNA nor infectivity were associated with the progeny of viruliferous female whiteflies. Because the inherited viral DNA is unable to give rise to infections, the transovarial transmission of TYLCSV DNA appears to have no epidemiological relevance.     

Begomovirus genetic diversity in the native plant reservoir Solanum nigrum: Evidence for the presence of a new virus species of recombinant nature

We examined the native plant host Solanum nigrum as reservoir of genetic diversity of begomoviruses that cause the tomato yellow leaf curl disease (TYLCD) emerging in southern Spain. Presence of isolates of all the species and strains found associated with TYLCD in this area was demonstrated. Mixed infections were common, which is a prerequisite for recombination to occur. In fact, presence of a novel recombinant begomovirus was demonstrated. Analysis of an infectious clone showed that it resulted from a genetic exchange between isolates of the ES strain of Tomato yellow leaf curl Sardinia virus and of the type strain of Tomato yellow leaf curl virus. The novel biological properties suggested that it is a step forward in the ecological adaptation to the invaded area. This recombinant represents an isolate of a new begomovirus species for which the name Tomato yellow leaf curl Axarquia virus is proposed. Spread into commercial tomatoes is shown.     

A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus

Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A approximately 63-kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that tomato yellow leaf curl virus (TYLCV) particles displayed a specific affinity for the B. tabaci 63-kDa GroEL homologue. Feeding whiteflies anti-Buchnera GroEL antiserum before the acquisition of virions reduced TYLCV transmission to tomato test plants by >80%. In the haemolymph of these whiteflies, TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph.     

Development of a real-time PCR for Tomato yellow leaf curl Sardinia virus

Recently, tomato yellow leaf curl disease has become important for the tomato grown both in greenhouse and field conditions in Tunisia. Here, we describe a rapid, specific, reliable, and sensitive real-time PCR, based on TaqMan chemistry, for Tomato yellow leaf curl Sardinia virus (TYLCSV). This method proved suitable for the detection and quantification of this virus in tomato, pepper and bean plants. It detected the virus even in the samples that were negative by conventional assays.     

Lamium amplexicaule (Lamiaceae): a weed reservoir for tomato yellow leaf curl virus (TYLCV) in Korea

After the first identification of tomato yellow leaf curl virus (TYLCV) in the southern part of Korea in 2008, TYLCV has rapidly spread to tomato farms in most regions of Korea. From 2008 to 2010, a survey of natural weed hosts that could be reservoirs of TYLCV was performed in major tomato production areas of Korea. About 530 samples were collected and identified as belonging to 25 species from 11 families. PCR and Southern hybridization were used to detect TYLCV in samples, and replicating forms of TYLCV DNA were detected in three species (Achyranthes bidentata, Lamium amplexicaule, and Veronica persica) by Southern hybridization. TYLCV transmission mediated by Bemisia tabaci from TYLCV-infected tomato plants to L. amplexicaule was confirmed, and TYLCV-infected L. amplexicaule showed symptoms such as yellowing, stunting, and leaf curling. TYLCV from infected L. amplexicaule was also transmitted to healthy tomato and L. amplexicaule plants by B. tabaci. The rate of infection of L. amplexicaule by TYLCV was similar to that of tomato. This report is the first to show that L. amplexicaule is a reservoir weed host for TYLCV.