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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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).     

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     

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.     

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.     

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.     

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.     

Complete sequences of tomato leaf curl Palampur virus isolates infecting cucurbits in Iran

Tomato leaf curl disease (TLCD) and and tomato yellow leaf curl (TYLCD) is caused by a number of begomovirus species that collectively threaten tomato production worldwide. We report here that an ongoing TLCD and TYLCD epidemic in Iran is caused by variants of tomato leaf curl Palampur virus (ToLCPMV), a newly proposed begomovirus species previously only detected in India. Besides infecting tomatoes, we identified ToLCPMV as the causal agent of a cucurbit disease that has devastated greenhouse cucumber and melon farms in Jiroft, southeastern Iran. We found no convincing evidence that the ToLCPMV DNA-B sequences have been derived through inter-species recombination, however, all of the currently sampled ToLCPMV DNA-A sequences are descendents of a sequence that probably arose through recombination between a ToLCNDV isolate and a currently unsampled geminivirus species that falls outside the ToLCNDV-ToLCPMV cluster. The increasing incidence of ToLCPMV in different cultivated species throughout Iran may signal the emergence of a serious new threat to agricultural production throughout the Middle East. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular characterization of tomato-infecting begomoviruses in Thailand

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

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.