Evidence for Transovarial Transmission of Tomato Yellow Leaf Curl Virus by Its Vector, the WhiteflyBemisia tabaci

The whiteflyBemisia tabaciis the only vector of the tomato yellow leaf curl geminivirus (TYLCV). The insect transmits the virus in a persistent-circulative manner. TYLCV DNA was detected by polymerase chain reaction and by Southern blot hybridization in progeny (eggs, first and second instars, adults) of single viruliferous whiteflies that developed on eggplant or on cotton (two TYLCV nonhost plants). Furthermore, TYLCV DNA was present in the progeny of insects that had acquired the virus through the egg. The adult progeny of the viruliferous insects and their own progeny were able to infect tomato test plants, producing typical disease symptoms. Ovaries and maturing eggs of viruliferous insects contained viral DNA, as did eggs laid by viruliferous insects maintained on an artificial diet. Eggs laid by nonviruliferous whiteflies on cotton plants previously caged with viruliferous insects did not acquire viral DNA from the plant. Hence, TYLCV can be transmitted through the egg for at least two generations. In the absence of an available plant host, the whitefly may serve as a reservoir of the virus between growing seasons.     

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     

Tomato yellow leaf curl virus: a whitefly-transmitted geminivirus with a single genomic component

The genome of the tomato yellow leaf curl virus (TYLCV), a Bemisia tabaci-transmitted geminivirus, was cloned. All clones obtained were of one genomic molecule, analogous to DNA A of African cassava mosaic virus. Nucleotide sequence analysis of the TYLCV genome showed that it comprises 2787 nucleotides, encoding six open reading frames, two on the virion strand and four on the complementary strand. All of them have counterparts in other geminiviruses. Dimeric copies of the cloned viral genome were introduced into tomato plants by agroinoculation. Severe yellow leaf curl disease symptoms developed in all of them. Effective whitefly-mediated transmission of the virus from agroinoculated plants to test plants demonstrated that the cloned molecule carries all the information needed for virus replication, systemic infection, and transfer by whiteflies. Restriction and hybridization analyses of viral DNA forms in infected plants and viruliferous whiteflies did not support the presupposed existence of a second genomic component. This is the first report of a whitefly-transmitted geminivirus that possesses a single genomic molecule.     

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.     

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.     

Real-time PCR for the quantitation of Tomato yellow leaf curl Sardinia virus in tomato plants and in Bemisia tabaci

Tomato yellow leaf curl Sardinia virus (TYLCSV) (Geminiviridae) is an important pathogen severely affecting tomato production in the Mediterranean basin. Although diagnostic protocols are available for its detection in plants and its vector Bemisia tabaci (Gennadius), suitable tools for estimating and comparing viral loads in plant and insect tissues are needed. In this paper, real-time PCR methods are described for quantitation of TYLCSV in both tomato plant and whitefly extracts. The DNA extraction method was optimised on TYLCSV-infected tomato tissue. The amount of virus was determined using specific primers and probe and standardised to the amount of DNA present in each sample, using selected endogenous tomato or Bemisia genes as internal references. The distribution of TYLCSV was relatively quantified within the four uppermost leaves of plants. An absolute estimation of the amount of TYLCSV in the first leaf below the apex was obtained. The kinetics of virus retention within different batches of viruliferous whiteflies was also analysed. The real-time PCR was 2200-fold more sensitive than membrane hybridisation, allowing detection of as few as 10 viral copies in a sample. These methods are potentially suitable for several applications, such as plant breeding for resistance, analysis of virus replication, and virus-vector interaction studies.     

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.     

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.     

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.     

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.     

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.     

Single-stranded DNA of Tomato leaf curl virus accumulates in the cytoplasm of phloem cells

Geminiviruses have been reported to replicate in, and localize to, the nuclei of host plant cells. We have investigated the tissue and intracellular distribution of the monopartite Tomato leaf curl virus (TLCV) by in situ hybridization. Contrary to the current understanding of geminiviral localization, single-stranded (ss) DNA of TLCV accumulated in the cytoplasm. TLCV ssDNA was also found in the nucleus, as was lower levels of replicative form double-stranded (ds) DNA. Under the same conditions, Tomato golden mosaic virus (TGMV) ssDNA and dsDNA were found in nuclei. ssDNA of TLCV, TGMV, and Tomato yellow leaf curl Sardinia virus (TYLCSV) was detected in some xylem vessels under specific hybridization conditions. Tissue specificity of TLCV was partially released by co-infection with TGMV. Our observations suggest that the mechanism of TLCV movement may differ from that of bipartite begomoviruses.     

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 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     

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.     

Whitefly-mediated transmission of cotton leaf curl Multan betasatellite: evidence for betasatellite encapsidation in coat protein of helper begomoviruses

Cotton leaf curl Multan betasatellite (CLCuMB) is responsible for symptom expression of a devastating disease of cotton in the Indian subcontinent. CLCuMB depends on helper virus replication-associated protein for its replication and on viral coat protein (CP) for its encapsidation. However, no direct evidence of encapsidation of CLCuMB in viral CP has been available. In the present study, non-viruliferous whiteflies were placed on tomato plants that had been agroinoculated with infectious clones of an Iranian isolate of tomato yellow leaf curl virus (TYLCV-[Ab]) and CLCuMB for an acquisition access period of 72 h and then transferred to healthy tomato seedlings at the 3- to 4-leaf stage. Typical symptoms of TYLCV-[Ab] appeared on inoculated seedlings 30-45 days post-inoculation. The presence of TYLCV-[Ab] and CLCuMB DNAs in symptomatic test plants and viruliferous whiteflies was confirmed by PCR analysis using specific primers and DIG Southern blotting. Furthermore, the possibility of CLCuMB DNA encapsidation in TYLCV-[Ab] CP within infected plants was examined by immunocapture PCR. The results showed that CLCuMB DNA was encapsidated in TYLCV-[Ab] CP. Whitefly-mediated transmission of CLCuMB in the presence of helper virus is additional evidence for encapsidation of CLCuMB by TYLCV-[Ab] CP.     

Trapping of Tomato yellow leaf curl virus (TYLCV) and other plant viruses with a GroEL homologue from the whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Summary. To avoid destruction in the haemolymph of their vector, many plant circulative viruses interact with GroEL homologues produced by insect endosymbiotic bacteria. We have exploited this phenomenon to devise tools allowing trapping of plant viruses by either GroEL purified from the whitefly Bemisia tabaci or by whitefly GroEL over-expressed in E. coli. PCR tubes or 96-well plates coated with a GroEL preparation were incubated with cleared sap of virus infected plant leaves or insect vectors. GroEL-bound viruses were then identified by PCR or RT-PCR using virus-specific primers or by ELISA with virus specific antibodies. In this way Tomato yellow leaf curl virus (TYLCV) – a whitefly-transmitted geminivirus – was detected in plant sap, in extracts of leaf squashes and in homogenates of individual viruliferous whiteflies. Anti-GroEL antibody prevented TYLCV binding to GroEL. GroEL-bound virus was also detected by ELISA. GroEL was much more potent in binding TYLCV than commercial anti-TYLCV antibodies. In addition to several other geminiviruses, these procedures allowed detecting a variety of RNA viruses such as Cucumber mosaic virus (CMV), Prune dwarf virus (PDV) and Tomato spotted wilt (TSWV), but not Potato virus X and Potato virus Y (PVX and PVY), Grapevine leafroll-associated viruses (GLRV) and Tobacco mosaic virus (TMV). Predictions pertaining to viruses that do, or do not bind to GroEL, and applications in plant virus diagnosis, are presented.     

Dual interaction of plant PCNA with geminivirus replication accessory protein (Ren) and viral replication protein (Rep)

Geminiviruses replicate their small, single-stranded DNA genomes in plant nuclei using host replication machinery. Similar to most dicotyledonous plant-infecting geminiviruses, Tomato yellow leaf curl Sardinia virus (TYLCSV) encodes a protein, REn, that enhances viral DNA accumulation through an unknown mechanism. Earlier studies showed that REn protein from another geminivirus, Tomato golden mosaic virus (TGMV), forms oligomers and interacts with Rep protein, the only viral protein essential for replication. It has been shown that both proteins from TGMV also interact with a plant homolog of the mammalian tumor suppressor retinoblastoma protein (RBR). By using yeast two-hybrid technology and the TYLCSV REn protein as bait, we have isolated three clones of the proliferating cell nuclear antigen (PCNA) of Arabidopsis thaliana, a ring-shaped protein that encircles DNA and plays an essential role in eukaryotic chromosomal DNA replication. We also demonstrate by the two-hybrid system and a pull-down assay that REn interacts with tomato PCNA (LePCNA). Analysis of truncated proteins has located the REn-binding domain of LePCNA between amino acids 132 and 187, whereas all REn deletions used abolished or decreased dramatically its ability to interact with PCNA. Tomato PCNA also interacts with TYLCSV Rep. We propose that the interaction between PCNA and REn/Rep takes place during virus infection, inducing the assembly of the plant replication complex (replisome) close to the virus origin of replication.     

The complete nucleotide sequence of an isolate of Tomato yellow leaf curl Sardinia virus found in Sicily

Partial sequences of Tomato yellow leaf curl Sardinia virus (TYLCSV) derived from tomato samples collected in Sicily in 1999, 2002 and 2004 indicated the presence of a TYLCSV different from the one previously described as the Sic strain. Here, we report a complete DNA sequence that is classified as belonging to the TYLCSV type strain (Sar strain), confirming the co-existence in Sicily of virus populations of both strains. Moreover, comparisons between this new sequence and those of the two recombinants recently described in Sicily revealed unequivocally (99% identity) that their TYLCSV-derived portion originated from the Sar strain.