Distribution and diversity of begomoviruses in tomato and sweet pepper plants in Costa Rica

Begomoviruses (genus Begomovirus, family Geminiviridae) have emerged as important plant pathogens in tropical and subtropical regions worldwide. Although these viruses were reported during the 1970s in Costa Rica, they are still poorly known. Therefore, the objective of this study was to analyse the diversity and distribution of begomoviruses in commercial tomato and sweet pepper fields from different agricultural production systems of the major growing regions of Costa Rica. A total of 651 plants were randomly sampled from greenhouses and open field crops during 2011 and 2012 in three different geographical locations. The bipartite begomoviruses Tomato yellow mottle virus, Tomato leaf curl Sinaloa virus and Pepper golden mosaic virus, and the monopartite begomovirus Tomato yellow leaf curl virus were detected in the collected samples. The complete genome of isolates from each species was cloned and sequenced. The frequency of detection of these four begomoviruses in the analysed samples ranged from 0 to 9%, the presence, and the prevalent virus varied largely according to the geographical location, the host (tomato and pepper), and the production system (greenhouses or open fields). An association between geographical region and begomovirus species was observed suggesting that in Costa Rica the heterogeneity on climate, topography and agricultural system might influence the distribution of begomovirus species in the country. A broader survey needs to be conducted to confirm it, although these preliminary results may contribute to the management of begomoviruses in Costa Rica.     

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

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

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

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

The circulative pathway of begomoviruses in the whitefly vector Bemisia tabaci— insights from studies with Tomato yellow leaf curl virus

Our current knowledge concerning the transmission of begomoviruses by the whitefly vector Bemisia tabaci is based mainly on research performed on the Tomato yellow leaf curl virus (TYLCV) complex and on a number of viruses originating from the Old World, such as Tomato leaf curl virus, and from the New World, including Abutilon mosaic virus, Tomato mottle virus, and Squash leaf curl virus. In this review we discuss the characteristics of acquisition, transmission and retention of begomoviruses by the whitefly vector, concentrating on the TYLCV complex, based on both published and recent unpublished data. We describe the cells and organs encountered by begomoviruses in B. tabaci. We show immunolocalisation of TYLCV to the B. tabaci stylet food canal and to the proximal part of the descending midgut, and TYLCV‐specific labelling was also associated with food in the lumen. The microvilli and electron‐dense material in the epithelial cells of the gut wall were also labelled by the anti TYLCV serum, pointing to a possible virus translocation route through the gut wall and to a putative site of long‐term virus storage. We describe the path of begomoviruses in their vector B. tabaci and in the non‐vector whitefly Trialeurodes vaporariorum, and we follow the rate of virus translocation in these insects. We discuss TYLCV transmission between B. tabaci during mating, probably by exchange of haemolymph. We show that following a short acquisition access to infected tomato plants, TYLCV remains associated with the B. tabaci vector for weeks, while the virus is undetectable after a few hours in the non‐vector T. vaporariorum. The implications of the long‐term association of TYLCV with B. tabaci in the light of interactions of the begomovirus with insect receptors are discussed.     

A rapid PCR method to discriminate between Tomato yellow leaf curl virus isolates

Tomato yellow leaf curl virus (TYLCV) was recently divided into two different species: Tomato yellow leaf curl virus‐Israel (TYLCV‐Is) and Tomato yellow leaf curl virus‐Sardinia (TYLCV‐Sar). There are no rapid methods by which TYLCV viruses may be assigned to either TYLCV‐Is or TYLCV‐Sar species. In the present work, using an extensive alignment of begomovirus sequences, TYLCV‐specific primers were designed and tested which allow the specific amplification of DNA fragments from any isolate of TYLCV. Also, a primer was designed and tested which allows the specific amplification of TYLCV‐Sar. Furthermore, a combination of these primers was selected to develop a duplex PCR method, which has the potential to detect either TYLCV‐Is or TYLCV‐Sar. The PCR methods were also highly effective with minimal sample preparation and allowed direct amplification of TYLCV from infected leaf extracts. This approach may be used in the laboratory as a tool for rapid, large‐scale diagnostics of TYLCV‐infected samples.     

Distribution of tomato-infecting begomoviruses and Bemisia tabaci biotypes in Morocco

Tomato yellow leaf curl virus (TYLCV) and tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae) as well as their whitefly vector Bemisia tabaci were reported from the south‐west and central regions of Morocco. To establish a more comprehensive view of tomato begomoviruses and B. tabaci biotypes throughout Morocco, 32 tomato fields were surveyed for tomato yellow leaf curl disease (TYLCD) symptoms in southern and northern regions, and 54 samples of leaves from cultivated plants or weeds and 35 B. tabaci individuals were collected and analysed by PCR, randomly amplified polymorphic DNA and sequencing. Only TYLCV or TYLCSV were detected. TYLCV was detected in 15 plant samples whereas TYLCSV only in 4. Sequence analyses revealed the presence of the ‘Spanish’ strain of TYLCSV and distinguished two genetically distinct strains of TYLCV. The begomovirus infections were unevenly distributed throughout Morocco. In the north‐west and north‐central regions where tomato plants exhibiting TYLCD symptoms were rarely observed, only 1 sample out of 13 tested positive for the presence of a begomovirus. In the north‐east region, the ratio of begomovirus‐positive samples was higher, 6/13, and in the south‐west region, it was the highest, 13/14. Consistently the frequency of plants exhibiting TYLCD‐like symptoms in the northern regions was lower than that in the south‐west region. B. tabaci biotype Q is present throughout the country and in Algeria, whereas biotype B, identified for the first time in Morocco, was detected only in the north‐east region.     

Evaluation of Tomato Hybrids Carrying Ty‐1 and Ty‐2 Loci to Japanese Monopartite Begomovirus Species

Tobacco leaf curl Japan virus, Honeysuckle yellow vein mosaic virus and Tomato yellow leaf curl virus are three begomoviruses that infect tomato crops in Japan. Tomato infection by begomoviruses has increased in Japan after the development of a high level of resistance to certain insecticides in some populations of the vector B. tabaci biotypes ‘B and Q’. Ty‐1 and Ty‐2 homozygous tomato hybrids were evaluated for reaction to monopartite begomovirus species in Japan by Agrobacterium‐mediated inoculation. Test plants were evaluated by a disease assessment scale (DAS), varying from 1 = no symptoms to 4 = severe symptoms, and systemic infection was evaluated by polymerase chain reaction (PCR), using specific begomovirus primers for each virus. Ty‐1 hybrids showed tolerance to HYVMV and with a large number of plants being neither virus‐free nor symptom‐free. The response of Ty‐1 hybrids was also resistant to moderately resistant against TbLCJV. The response of Ty‐2 hybrids was resistant to highly resistant against the three monopartite begomoviruses, when compared with susceptible plants.     

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Background

Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.

Results

All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.

Conclusions

Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.     

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

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

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

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

Horizontal transmission of begomoviruses between Bemisia tabaci biotypes

We have previously shown that the monopartite Tomato yellow leaf curl virus (TYLCV), a begomovirus (family Geminiviridae, genus Begomovirus) infecting tomato plants can be transmitted in a gender-dependent manner among its insect vector the whitefly Bemisia tabaci type B (Gennaduis) (Aleyrodidae: Hemiptera) during mating. Viruliferous females were able to transmit the virus to non-viruliferous males and vice versa, in the absence of any other virus source. The recipient insects were able to infect tomato plants. In this communication, we present evidence that two bipartite begomoviruses infecting cucurbits, Squash leaf curl virus (SLCV) and Watermelon chlorotic stunt virus (WmCSV) can be transmitted in a gender-dependent manner among whiteflies. In addition we show that TYLCV can be transmitted during mating among individuals from the same biotype (from B-males to B-females and vice versa; and from Q-males to Q-females and vice versa). However, viruliferous males of the B biotype are unable to transmit the virus to females of the Q biotype (and vice versa); similarly, viruliferous males of the Q biotype are unable to transmit the virus to females of the B biotype (and vice versa). These findings support the hypothesis that a pre-zygotic mating barrier between the Q and B biotypes is the cause for the absence of gene flow between the two biotypes, and that virus transmission can be used as a marker for inter-biotype mating. To be transmitted during mating, the virus needs to be present in the haemolymph of the donor insect. Abutilon mosaic virus (AbMV), a bipartite begomovirus that can be ingested but not transmitted by B. tabaci, is absent in the whitefly haemolymph, and cannot be transmitted during mating. Mating was a precondition for horizontal virus transfer from male to female, or female to male. Virus was not transmitted when viruliferous B. tabaci were caged with the non-vector non-viruliferous whitefly Trialeurodes vaporariorum (Westwood) (Aleyrodidae: Hemiptera) and vice versa.     

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

Background

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

Results

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

Conclusion

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

Pyramiding Ty-1/Ty-3 and Ty-2 in tomato hybrids dramatically inhibits symptom expression and accumulation of tomato yellow leaf curl disease inducing viruses

Tomato yellow leaf curl disease is a major constraint for tomato production worldwide and availability of new resistant materials is of great importance for breeding programmes. A phenotypic survey was undertaken to evaluate the level of resistance to the main tomato yellow leaf curl disease-inducing viruses Tomato yellow leaf curl virus and Tomato yellow leaf curl Sardinia virus, in several commercial tomato cultivars, never characterised before. Seven weeks post inoculation, two cultivars resulted in high resistant phenotypes to both begomoviruses, and four were tolerant to at least one of them. In the two highly resistant hybrids (SJ12, RFT112), symptoms were completely absent and viral DNA was from 10² to 10⁵ fold lower than in susceptible plants. Molecular marker analysis revealed that these genotypes harbour the resistant genes Ty-1/Ty-3 and Ty-2. Given their high resistance, they can be considered good candidates for cultivation and breeding in areas where incidence of TYLCD is very elevated.     

Multiple Introductions of the Old World Begomovirus Tomato yellow leaf curl virus into the New World

A phylogenetic analysis of three genomic regions revealed that Tomato yellow leaf curl virus (TYLCV) from western North America is distinct from TYLCV isolated in eastern North America and the Caribbean. This analysis supports a second introduction of this Old World begomovirus into the New World, most likely from Asia.     

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

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

Within-Host Dynamics of the Emergence of Tomato Yellow Leaf Curl Virus Recombinants

Tomato yellow leaf curl virus (TYLCV) is a highly damaging begomovirus native to the Middle East. TYLCV has recently spread worldwide, recombining with other begomoviruses. Recent analysis of mixed infections between TYLCV and Tomato leaf curl Comoros begomovirus (ToLCKMV) has shown that, although natural selection preserves certain co-evolved intra-genomic interactions, numerous and diverse recombinants are produced at 120 days post-inoculation (dpi), and recombinant populations from different tomato plants are very divergent. Here, we investigate the population dynamics that lead to such patterns in tomato plants co-infected with TYLCV and ToLCKMV either by agro-inoculation or using the natural whitefly vector Bemisia tabaci. We monitored the frequency of parental and recombinant genotypes independently in 35 plants between 18 and 330 dpi and identified 177 recombinants isolated at different times. Recombinants were detected from 18 dpi and their frequency increased over time to reach about 50% at 150 dpi regardless of the inoculation method. The distribution of breakpoints detected on 96 fully sequenced recombinants was consistent with a continuous generation of new recombinants as well as random and deterministic effects in their maintenance. A severe population bottleneck of around 10 genomes was estimated during early systemic infection–a phenomenon that could account partially for the heterogeneity in recombinant patterns observed among plants. The detection of the same recombinant genome in six of the thirteen plants analysed beyond 30 dpi supported the influence of selection on observed recombination patterns. Moreover, a highly virulent recombinant genotype dominating virus populations within one plant has, apparently, the potential to be maintained in the natural population according to its infectivity, within-host accumulation, and transmission efficiency - all of which were similar or intermediate to those of the parent genotypes. Our results anticipate the outcomes of natural encounters between TYLCV and ToLCKMV.     

Begomoviruses Infecting Tomato Crops in Panama

The key regions in Panama involved in open field‐ and greenhouse‐grown commercial tomato production, including the Chiriquí, Veraguas, Herrera, Los Santos, Coclé and Panama Oeste provinces, were surveyed for the incidence and distribution of begomoviruses in the growing seasons of 2011 and 2012. The surveys took place in 14 of the 51 districts of the above‐mentioned provinces and comprised all relevant tomato production areas of the provinces. A total of 28 tomato plots were surveyed. The exact location of each plot was geo‐referenced using a hand‐held Global Positioning System unit. In total, 319 individual tomato plants (181 in 2011 and 138 in 2012) were sampled. Plants displayed diverse combinations of virus‐like symptoms of different severity, including necrosis, yellowing, mosaic, mottling, rolling, curling, distortion and puckering of leaves, reduced leaf size, and stunted growth. DNA was extracted from each plant for a subsequent polymerase chain reaction (PCR) analysis, using two sets of degenerate primers able to detect members of the genus Begomovirus. The samples displaying a positive reaction were subsequently analysed with specific primer pairs to identify the affecting begomoviruses. A total of 42.3% of all collected samples showed a positive signal to PCRs. Three begomovirus species were detected with the species‐specific set of primers; in particular, in the samples obtained in 2011, Potato yellow mosaic Panama virus (PYMPV), Tomato leaf curl Sinaloa virus (ToLCSiV) and Tomato yellow mottle virus (TYMoV) were detected, while in the 2012 samples, only PYMPV and ToLCSiV were found. To our knowledge, this is the first reported incidence of ToLCSiV and TYMoV in Panamanian tomato crops.