Tomato Yellow Leaf Curl virus-Israel Research Articles

Behavior of new entries and developed tomato hybrids carrying Ty-2 genes

Tomato yellow leaf curl disease (TYLCD) is a serious problem hampering tomato production worldwide. In the Mediterranean Basin, disease incidence and severity are higher in the dry season increasing whitefly (Bemisia tabaci) populations. Effectiveness of resistance to Tomato yellow leaf curl virus (TYLCV) depends on both tomato host resistance and TYLCV complex species. So far, six different Ty tomato resistance genes have been identified. Two main TYLCV complex species, Tomato yellow leaf curl virus-Israel (TYLCV-Is) and Tomato yellow leaf curl Sardinia virus (TYLCSV), have been identified in Tunisia. The present work aimed to evaluate entries heterozygous for Ty-2 gene to help predict hybrid performance. Two tomato entries homozygous for the Ty-2 TYLCV resistance gene, one tomato hybrid homozygous for Ty-2 and two heterozygous hybrids were included, besides two susceptible tomato entries. Resistance response to TYLCD was recorded based on disease incidence and severity levels. Data analysis was performed according to presence/absence of Ty-2 gene and taking into account homozygosity and heterozygosity of Ty-2. Generalized linear model analysis was applied to check significance of individual factors' effects (i.e. effect of tomato entries or tomato groups of entries based on presence or absence of homozygous/heterozygous Ty-2 gene, block unit within the field trial and the year of the trial) on the dependent variables (disease incidence and severity). Further multicomparison tests gave evidence on significant effect of Ty-2 homozygous gene tomato entries on TYLCD incidence and severity levels. The results were discussed with special focus on the relevance use of heterozygous hybrid tomato varieties.

First Evidence of Tomato Yellow Leaf Curl Virus-Israel IS76 Recombinant Isolates Associated with Severe Yellow Leaf Curl Epidemics in Resistant Tomatoes in Spain

First Evidence of Tomato Yellow Leaf Curl Virus-Israel IS76 Recombinant Isolates Associated with Severe Yellow Leaf Curl Epidemics in Resistant Tomatoes in Spain

First Report of Papaya (Carica papaya) Naturally Infected With the Introduced Tomato yellow leaf curl virus-Israel

First Report of Papaya (<i>Carica papaya</i>) Naturally Infected With the Introduced <i>Tomato yellow leaf curl virus</i>-Israel

Recessive Resistance Derived from Tomato cv. Tyking-Limits Drastically the Spread of Tomato Yellow Leaf Curl Virus.

The tomato yellow leaf curl disease (TYLCD) causes severe damage to tomato (Solanum lycopersicum L.) crops throughout tropical and subtropical regions of the world. TYLCD is associated with a complex of single-stranded circular DNA plant viruses of the genus Begomovirus (family Geminiviridae) transmitted by the whitefy Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae). The tomato inbred line TX 468-RG is a source of monogenic recessive resistance to begomoviruses derived from the hybrid cv. Tyking F1. A detailed analysis of this germplasm source against tomato yellow leaf curl virus-Israel (TYLCV-IL), a widespread TYLCD-associated virus, showed a significant restriction to systemic virus accumulation even under continuous virus supply. The resistance was effective in limiting the onset of TYLCV-IL in tomato, as significantly lower primary spread of the virus occurred in resistant plants. Also, even if a limited number of resistant plants could result infected, they were less efficient virus sources for secondary spread owing to the impaired TYLCV-IL accumulation. Therefore, the incorporation of this resistance into breeding programs might help TYLCD management by drastically limiting TYLCV-IL spread.

First Report of Tomato yellow leaf curl virus in Tomato in Costa Rica.

One of the most important invasive and harmful members of the genus Begomovirus (family Geminiviridae) is the monopartite Tomato yellow leaf curl virus (TYLCV), which is widespread over the world associated with tomato yellow leaf curl disease (TYLCD). Tomato (Solanum lycopersicum) plants infected with TYLCV show upward leaf curling and yellowing. In Latin America, isolates of TYLCV have been reported from Cuba, the Dominican Republic, Mexico and Puerto Rico (1), Guatemala (GenBank Accession No. GU355941), and Venezuela (partial genome sequence DQ302033). In Costa Rica, only isolates of the bipartite begomoviruses Tomato leaf curl Sinaloa virus (TLCSiV) (3) and Tomato yellow mottle virus (KC176780, KC176781) have been reported infecting tomatoes. During a survey conducted in 2012, similar begomovirus-like symptoms (leaf yellowing and upward leaf curling) were observed in tomato plants of five commercial growing areas in the Central Valley (Grecia region) of Costa Rica. In total, 65 tomato samples were randomly collected, 14 from greenhouses and 41 from open fields. Symptoms of upward leaf curling and yellowing were observed in three samples. Total DNA was extracted from collected samples and tested by dot blot hybridization using a probe to the coat protein (CP) gene of a Guatemalan isolate of Bean golden yellow mosaic virus (3). Only the three symptomatic samples tested positive, which represents an incidence of 14% (2 samples) in greenhouses and 2.4% (1 sample) in open field crops. These samples were subjected to rolling circle amplification (RCA) for viral circular genome amplification (2). The amplified products were then digested with MspI restriction endonuclease, which resulted into DNA fragments of 2,320 and 458 bp for all three samples. This suggested infection with a monopartite begomovirus. In order to obtain the full-length clone, the RCA product of two samples (5240 and 5241) was digested with BamHI, and the ~2.8 kb DNA fragment was cloned into pBluescript II SK(+) (Stratagene, La Jolla, CA) vector. After transformation of Escherichia coli DH5α, recombinant plasmids with inserts of expected size were selected and the insert was sequenced by primer walking (Macrogen Inc., Korea). The inserts of three clones from the two samples (CR:5240-16:2012, CR:5240-17:2012, and CR:5241-14:2012) were sequenced (deposited in GenBank as KF533855, KF533856, and KF533857, respectively). Sequences were all 2,781 nt long and shared 100% identity between themselves (1-nt mismatch between CR:5240-16:2012 and CR:5240-17:2012, and CR:5240-16:2012 and CR:5241-14:2012; and 2-nt mismatches between CR:5240-17:2012 and CR:5241-14:2012) and 99% with the sequence of Tomato yellow leaf curl virus-Israel[Japan:Haruno:2005] (TYLCV-IL[JR:Har:05]) (AB192966). These sequences represented full length genomes of isolates of the monopartite begomovirus TYLCV-IL and grouped in a phylogenetic clade (4) that comprised TYLCV-IL isolates reported from Asia (China and Japan) and from Mexico, while more distantly related to the clade comprising TYLCV-IL isolates reported from Central America (Cuba, Guatemala, Puerto Rico) and the United States, suggesting a distinct introduction event in Costa Rica. This is the first report of the presence of TYLCV in Costa Rica, therefore it is imperative to study the incidence and geographical spread of this virus in the country as well as its genetic diversity, since TYLCV infections might lead to significant yield losses, as reported in other countries.

A bead-based suspension array for the multiplexed detection of begomoviruses and their whitefly vectors

Bead-based suspension array systems enable simultaneous fluorescence-based identification of multiple nucleic acid targets in a single reaction. This study describes the development of a novel approach to plant virus and vector diagnostics, a multiplexed 7-plex array that comprises a hierarchical set of assays for the simultaneous detection of begomoviruses and Bemisia tabaci, from both plant and whitefly samples. The multiplexed array incorporates genus, species and strain-specific assays, offering a unique approach for identifying both known and unknown viruses and B. tabaci species. When tested against a large panel of sequence-characterized begomovirus and whitefly samples, the array was shown to be 100% specific to the homologous target. Additionally, the multiplexed array was highly sensitive, efficiently and concurrently determining both virus and whitefly identity from single viruliferous whitefly samples. The detection limit for one assay within the multiplexed array that specifically detects Tomato yellow leaf curl virus-Israel (TYLCV-IL) was quantified as 200fg of TYLCV-IL DNA, directly equivalent to that of TYLCV-specific qPCR. Highly reproducible results were obtained over multiple tests. The flexible multiplexed array described in this study has great potential for use in plant quarantine, biosecurity and disease management programs worldwide.

Panel of real‐time PCRs for the multiplexed detection of two tomato‐infecting begomoviruses and their cognate whitefly vector species

A new approach for the simultaneous identification of the viruses and vectors responsible for tomato yellow leaf curl disease (TYLCD) epidemics is presented. A panel of quantitative multiplexed real‐time PCR assays was developed for the sensitive and reliable detection of Tomato yellow leaf curl virus‐Israel (TYLCV‐IL), Tomato leaf curl virus (ToLCV), Bemisia tabaci Middle East Asia Minor 1 species (MEAM1, B biotype) and B. tabaci Mediterranean species (MED, Q biotype) from either plant or whitefly samples. For quality‐assurance purposes, two internal control assays were included in the assay panel for the co‐amplification of solanaceous plant DNA or B. tabaci DNA. All assays were shown to be specific and reproducible. The multiplexed assays were able to reliably detect as few as 10 plasmid copies of TYLCV‐IL, 100 plasmid copies of ToLCV, 500 fg B. tabaci MEAM1 and 300 fg B. tabaci MED DNA. Evaluated methods for routine testing of field‐collected whiteflies are presented, including protocols for processing B. tabaci captured on yellow sticky traps and for bulking of multiple B. tabaci individuals prior to DNA extraction. This work assembles all of the essential features of a validated and quality‐assured diagnostic method for the identification and discrimination of tomato‐infecting begomovirus and B. tabaci vector species in Australia. This flexible panel of assays will facilitate improved quarantine, biosecurity and disease‐management programmes both in Australia and worldwide.

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

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

Effect of a single amino acid substitution in the NLS domain of Tomato yellow leaf curl virus-Israel (TYLCV-IL) capsid protein (CP) on its activity and on the virus life cycle

The capsid protein (CP) of Tomato yellow leaf curl virus-Israel (TYLCV-IL), encoded by the v1 gene, is the only known component of the viral capsid. Three point mutations introduced into the conserved NLS region of the CP were investigated. One mutant, in which the Arg at position 19 was converted to Leu, had the most significant effect on the CP–CP homotypic interaction as well as on CP's interaction with its nuclear receptor karyopherin α1 and with the protein GroEL. The latter has been suggested to protect the virions in the insect vector hemolymph. These effects were first observed by yeast two-hybrid assay and then confirmed in tobacco protoplasts by measuring fluorescence resonance energy transfer (FRET) between YFP- and CFP-tagged proteins. Most importantly, when the point mutation converting Arg 19 to Leu was introduced into the full-length TYLCV genome, it disrupted its ability to cause symptoms.

Tomato yellow leaf curl virusin Australia: distribution, detection and discovery of naturally occurring defective DNA molecules

Tomato yellow leaf curl virus (TYLCV) was detected for the first time in Australia in March 2006 in field-grown tomatoes in Brisbane, Queensland. Surveys showed that the virus was confined to south-east Queensland. Virus transmission studies carried out using Bemisia tabaci (B biotype) verified that resistant tomato lines containing the Ty-1 or Ty-5 genes displayed tolerance to infection by TYLCV isolates from Australia. A PCR assay specific for TYLCV was designed and optimised to confirm the presence of the virus in samples that tested positive in begomovirus-specific double-antibody sandwich enzyme-linked immunosorbent assay. Eight isolates of TYLCV from various sites were cloned and sequenced, and were shown to have near-identical sequences and a high nucleotide sequence similarity (98%) to the monopartite Tomato yellow leaf curl virus-Israel (TYLCV-IL). No DNA-B, DNA-1 nor DNA-b satellite molecules were detected using degenerate PCR assays. Phylogenetic analysis revealed that Australian isolates of TYLCV separated into two sequence groups, TYLCV-IL[Au:Bri:06] and TYLCV-IL[Au:Bun:06], that showed a defined geographic segregation. Naturally occurring defective DNA molecules containing partial, rearranged segments of the native DNA-A, were present in one isolate. To our knowledge, this is the first report of an incursion of a begomovirus into Australia, and the first report of the characterisation of naturally occurring defective DNAs of TYLCV. Additional keywords: diagnostics, Geminiviridae, rolling-circle amplification, virus host range.

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

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

Suppressor of RNA silencing encoded by Tomato yellow leaf curl virus-Israel

The Israeli isolate of Tomato yellow leaf curl geminivirus (TYLCV-Is) is a major tomato pathogen, causing extensive crop losses both in the New and Old World. Surprisingly, however, little is known about the molecular mechanisms of TYLCV-Is interactions with tomato cells. Here, we have identified a TYLCV-Is protein, V2, which acts as a suppressor of RNA silencing and which is unrelated to presently known viral suppressors. Specifically, V2, but not other proteins of TYLCV-Is, inhibited RNA silencing of a reporter transgene, GFP. This inhibition elevated the cellular levels of the GFP transcript and the GFP protein, but it had no apparent effect on the accumulation of GFP-specific short interfering RNAs (siRNAs), suggesting that TYLCV-Is V2 targets a step in the RNA silencing pathway which is subsequent to the Dicer-mediated cleavage of dsRNA. Visualization of the sub-cellular localization of TYLCV-Is V2 in plant protoplasts and tissues showed that this protein is associated with cytoplasmic strands and inclusion bodies in the cortical regions of the cell.

Truncated Rep gene originated from Tomato yellow leaf curl virus‐Israel [Mild] confers strain‐specific resistance in transgenic tomato

SummaryTransgenic tomato plants carrying a truncated replication associated protein (T‐Rep) gene of the mild strain of Tomato yellow leaf curl virus‐Israel (TYLCV‐Is [Mild]) were prepared. The transgene encoding the first 129 amino acids of Rep conferred resistance only against the virus strain from which it was derived, while these plants were susceptible to the severe strain of TYLCV‐Is. This strain‐specific effect may be the result of high sequence divergence within the N‐terminal domains of the Rep genes of the two virus isolates which share a mere 78% sequence identity at the nucleotide level and 77% at the amino acid level. Although the transgenic tomato plants were totally resistant to whitefly inoculation with the mild strain of TYLCV‐Is, agroinoculation with the same virus strain resulted in variable resistance responses in the tested plants: while 21% of plants were totally immune to the virus, 33% were susceptible and 46% expressed a wide range of intermediate resistance characteristics. The applicability of TYLCV‐Is derived resistance in tomato is discussed.

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

Summary 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.

Types of variation in DNA-A among isolates of East African cassava mosaic virus from Kenya, Malawi and Tanzania.

Complete nucleotide sequences of the DNA-A-like molecules of three East African cassava mosaic virus (EACMV) isolates from Kenya (-K, 2801 nt) and Malawi (-MH and -MK, both 2804 nt) were determined. These sequences were compared with that published for a Tanzanian isolate (-T, 2801 nt) and the partial sequence of a third Malawian isolate. Intergenic region sequences of all isolates, and deduced amino acid sequences of their AC1 (Rep) proteins, each formed a tightly related cluster that was distinct from the comparable components of other begomoviruses. Other complementary-sense genes (AC2, AC3, AC4) differed between EACMV isolates in a way consistent with the accumulation of point mutations. In contrast, virus-sense genes (CP, AV2) of isolates -MH and -MK differed (substantially for AV2) from those of other EACMV isolates but somewhat resembled those of tomato yellow leaf curl virus-Israel, suggesting they had been acquired by recombination with an unidentified begomovirus.

Taino Tomato Mottle Virus, a New Bipartite Geminivirus from Cuba.

Geminiviruses have become the most important virus group affecting tomatoes (Lycopersicon lycopersicum (L.) Karsten) in Cuba since they have been detected in all tomato-producing areas, causing serious losses. Recently, a whitefly-transmitted, monopartite geminivirus was detected in Cuba and identified as tomato yellow leaf curl virus-Israel (TYLCV-Is) (1). Samples collected from the main tomato-producing areas during the period 1995 to1996 were further analyzed by polymerase chain reaction (PCR) with degenerate primers (PAL1v1978 and PAR1c496) (2). Whereas in samples from most areas only TYLCV was detected, in some samples from the Havana area, two DNA fragments (approximately 1.4 and 1.1 kb) were amplified by PCR. The larger fragment was identified as part of the TYLCV-Is genome, confirming the previous report (1). The 1.1-kb fragment was cloned and its nucleotide sequence suggested that a new bipartite geminivirus was also present in those tomato samples. To clone the entire genome, tomato plants were inoculated by biolistics with DNA extract from field samples. After symptom expression, a viral DNA-enriched preparation from the inoculated tomatoes was independently digested with several restriction enzymes and the products were ligated into pZero plasmid (Invitrogen, San Diego, CA). Several clones in the 2.6-kb size range were characterized by restriction mapping and hybridization against component A and B heterologous probes. Two clones were selected as containing putative A and B components and their infectivity was tested by biolistic inoculation of tomato and pepper plants. The inoculated tomatoes developed a mild mottle in the younger leaves, whereas no symptoms were visible on the inoculated pepper plants. However, the presence of viral DNA was confirmed in both tomatoes and peppers by Southern blot hybridization analysis with A- and B-specific probes. Partial sequences of both components were obtained and their analysis showed that both components shared a 170-bases common region with a 95% identity. In addition, the nucleotide sequences of two open reading frames, one in each component (AC1 and BC1), were determined and compared with geminivirus sequences deposited in Gen-Bank. A dendogram generated with the CLUSTAL program and obtained with the AC1 and BC1 amino acid sequences, placed the new geminivirus in a cluster with tomato mottle virus (ToMoV; accession nos. L14460, L14461), Abutilon mosaic virus (AbMV; X15983, X15984), potato yellow mosaic virus (PYMV; D00940, D00941), and bean dwarf mosaic virus (BDMV; M88179, M88180). The percentages of identity obtained with the amino acid sequences were as follows. For AC1: ToMoV, 87%; PYMV, 79.5%; BDMV, 78.7%; and AbMV, 78%. For BC1 protein: BDMV, 92.8%; ToMoV, 89.1%; PYMV, 88.1%; and AbMV, 67.5%. In addition, the sequences were compared with partial nucleotide sequences (AC1, coat protein [CP], and common region) of a bipartite geminivirus affecting tomatoes in Jamaica (accession nos. U83855, U83854, and U83850). Interestingly, the common regions showed a higher percentage of identity (88%) than the CP and AC1 partial nucleotide sequences (86 and 74%, respectively). These data suggest that the virus reported here is a new geminivirus and the first bipartite geminivirus reported in Cuba. Thus, the name of Taino tomato mottle virus is proposed. (Taino refers to the name of the inhabitants of Cuba at the time of Columbus's arrival in the Caribbean).