Tomato Yellow Leaf Curl Virus Isolates Research Articles

Establish an efficient inoculation system of tomato yellow leaf curl virus to assist tomato resistance breeding

Tomato yellow leaf curl virus (TYLCV) endangers the production of tomato in the world, which is largely overcome by cultivating resistant tomato cultivars. Since virus evolves fast and may disrupt the resistance in resistant cultivars, tomato resistance breeding against emerging TYLCV isolates is a long-term task, which requires an efficient inoculation system of TYLCV to screen and identify resistant tomato plants. Previous Agrobacterium-mediated TYLCV infectious clone is to construct the viral genome in the binary vector, and does not have high infection efficiency. In this study, we integrate TYLCV genome in pTRV2GW vector containing partial genome of Tobacco Rattle virus, and generate an Agrobacterium-mediated infectious clone pTRV2GW-TYLCV. Tomato plants are infected by vacuum infiltration with the Agrobacterium culture of infectious clone. PCR analysis with all TYLCV ORFs in infected plants reveals distinct detection of each ORF at different infection stages and a final 100 % infection rate of this inoculation method. Infected tomato plants have typical TYLCV disease symptoms including yellowing, curling, and smaller leaves. Plant growth and leaf photosynthesis are repressed in infected plants compared to non-infected plants. Moreover, the inoculation system is successfully applied for differentiating TYLCV resistant and susceptible cultivars, which can assist tomato resistance breeding.

Phylogenomic and population genetics analyses of extant tomato yellow leaf curl virus strains on a global scale

Tomato yellow leaf curl virus (TYLCV) is a monopartite DNA virus with a genome size of ~ 2,800 base pairs. The virus belongs to the genus Begomovirus within the family Geminiviridae. Extant TYLCV strains are differentiated based on an established threshold of 94% genome-wide pairwise nucleotide identity. The phylogenetic relationships, diversification mechanisms, including recombination, and extent of spread within and from the center of origin for TYLCV have been reported in previous studies. However, the evolutionary relationships among strains, strains’ distribution and genomic diversification, and genetic mechanisms shaping TYLCV strains’ evolution have not been re-evaluated to consider globally representative genome sequences in publicly available sequence database, including herein newly sequenced genomes from the U.S. and Middle East, respectively. In this study, full-length genome sequences for the extant strains and isolates of TYLCV (n=818) were downloaded from the GenBank database. All previously published genome sequences, and newly sequenced TYLCV genomes of TYLCV isolates from Kuwait and USA, determined herein (n=834), were subjected to recombination analysis. To remove the ‘phylogenetic noise’ imparted by interspecific recombination, the recombinant genomes were removed from the data set, and the remaining non-recombinant genome sequences (n=423) were subjected to population genetics and Bayesian analyses. Results of the phylogeographical analysis indicated that the type strain, TYLCV-Israel, and TYLCV-Mild strain, were globally distributed, spanning Africa, America, Asia, Australia/Oceania, Europe, and New Caledonia, while the other TYLCV strains were prevalent only throughout the Middle East. The results of Bayesian evolutionary (ancestral) analysis predicted that TYLCV-Israel represents the oldest, most recent common ancestor (MRCA) (41,795 years), followed by TYLCV-Mild at 39,808 years. These were closely followed by two Iranian strains viz., TYLCV-Kerman and TYLCV-Iran at 37,529 and 36,420 years, respectively. In contrast, the most recently evolving strains were TYLCV-Kuwait and TYLCV-Kahnooj at 12,445 and 298 years, respectively. Results of the neutrality test indicated that TYLCV-Israel and TYLCV-Mild populations are undergoing purifying selection and/or population expansion, although statistically significant selection was documented for only TYLCV-Israel, based on positive selection acting on five codons.

Comparative Genome Analysis of Old World and New World TYLCV Reveals a Biasness toward Highly Variable Amino Acids in Coat Protein.

Begomoviruses, belonging to the family Geminiviridae and the genus Begomovirus, are DNA viruses that are transmitted by whitefly Bemisia tabaci (Gennadius) in a circulative persistent manner. They can easily adapt to new hosts and environments due to their wide host range and global distribution. However, the factors responsible for their adaptability and coevolutionary forces are yet to be explored. Among BGVs, TYLCV exhibits the broadest range of hosts. In this study, we have identified variable and coevolving amino acid sites in the proteins of Tomato yellow leaf curl virus (TYLCV) isolates from Old World (African, Indian, Japanese, and Oceania) and New World (Central and Southern America). We focused on mutations in the coat protein (CP), as it is highly variable and interacts with both vectors and host plants. Our observations indicate that some mutations were accumulating in Old World TYLCV isolates due to positive selection, with the S149N mutation being of particular interest. This mutation is associated with TYLCV isolates that have spread in Europe and Asia and is dominant in 78% of TYLCV isolates. On the other hand, the S149T mutation is restricted to isolates from Saudi Arabia. We further explored the implications of these amino acid changes through structural modeling. The results presented in this study suggest that certain hypervariable regions in the genome of TYLCV are conserved and may be important for adapting to different host environments. These regions could contribute to the mutational robustness of the virus, allowing it to persist in different host populations.

Genetic recombination among tomato yellow leaf curl virus isolates in commercial tomato crops in Kuwait drives emergence of virus diversity: a comparative genomic analysis

ObjectiveWhitefly-transmitted tomato yellow leaf curl virus (TYLCV) continues to be a major constraint to tomato production in Kuwait. However, very limited information is available about the population structure and genetic diversity of TYLCV infecting tomato in Kuwait.ResultsWhole genome sequences of 31 isolates of TYLCV, collected from commercial tomato crops grown in northern (Abdally) and southern (Al Wafra) parts of Kuwait, were deciphered. Eighteen isolates of TYLCV are identified as potential genetic recombinants. The isolates Abdally 6A and Abdally 3B reported in this study were identified to be potential recombinants. Compared to the 15 isolates from the Abdally area, and the three previously reported KISR isolates of Kuwait, six out of sixteen Al Wafra isolates showed an insertion of 19 extra nucleotides near the 5′-end. There are also four nucleotide variations before the 19-extra-nucleotides. The additional 19 nucleotides observed in nine isolates indicate that these isolates might have resulted from a single gene recombination/insertion event. Molecular phylogeny based on complete genome sequences of TYLCV isolates suggests transboundary movement of virus isolates due to geographic proximity. The information presented herein is quite useful for the comprehension of TYLCV biology, epidemiology and would aid in the management of disease in the long run.

First Report of Tomato Yellow Leaf Curl Virus Infecting Pepper and Tomato in Oklahoma.

Tomato yellow leaf curl virus (TYLCV), (genus Begomovirus and family Geminiviridae) is one of the devastating viruses infecting tomatoes (Solanum Lycopersicon) and could have a significant impact on tomato production worldwide (Moriones and Navas-Castillo, 2000; Rybicki, 2015; Perez-Padilla et al. 2019). TYLCV was first reported in the United States (US) in 1997 in Florida (Polston et al. 1999) and since then it has been reported in several other states but not in Oklahoma (de Sa et al. 2008). Both pepper (Capsicum spp.) and tomato are grown in >50 counties of Oklahoma for fresh produce in the local market, and for processing industries. During a survey in 2021, pepper (bell pepper cv SV3964) and tomato (cv grand marshall) plants grown in a commercial field in Bixby, Oklahoma, showed typical virus-like symptoms including yellowing, leaf curling, cupping, twisting, and mottling (Fig. 1). Estimated disease incidence was 10% in both crops. In addition, whiteflies (Bemisia tabaci) were also observed on these plants. Twenty symptomatic samples from pepper (n=16) and tomato (n=4) plants, and four asymptomatic from pepper (n=2) and tomato (n=2) plants were collected and brought to the Plant Virology Lab at the University of Tulsa for further analysis Total RNA was extracted from all samples using the Spectrum Plant Total RNA Kit (Sigma-Aldrich, LOT: SLCG7913). RNA from two symptomatic samples, one each from pepper and tomato (named BX6 and BX11, respectively) were subjected to high-throughput sequencing (HTS) on the NextSeq 500/550 high-output kit v2.5 (Illumina, USA) at the genomic facility, Oklahoma State University (Stillwater, OK, USA). A total of 22,385,404 (average length 73.6 bp) and 19,012,605 (average length 73.7 bp) trim pair-end for both samples (BX6 and BX11) were assembled using CLC Genomics Workbench (v12.0.3) (Qiagen, Inc) and subjected to BLASTn analysis. The three contigs: 393 bp (coverage 363.24X), 670 bp (coverage 489.92X), and 990 bp (coverage 112.96X) for BX6 isolate, and four contigs: 393 bp (coverage 341.64X), 668 bp (coverage 457.23X), 814 bp (coverage 107.86X), and 990 bp (coverage 93.71X), for BX11 isolate, showed from 96 to 99% nucleotide (nt) identities with movement protein, capsid protein, C3, V2, C4, AC1, AC2, AC3 protein genes of the TYLCV isolates from Australia, China, Iran, Mexico, South Korea, and the US. The HTS data did not reveal any other viral sequence in these two samples. To further confirm the presence of TYLCV in these samples, three overlapping TYLCV-specific primer pairs (Supplementary Table 1) were designed based on the alignment of complete genome sequences of TYLCV isolates present in the Genbank. Total DNA was extracted from fresh leaf tissues of BX6 and BX11 samples using the Plant DNA Kit (Omega BIO-TEK,) and used in the PCR assay. The expected PCR products (814 bp, 1,085 bp and 1,068 bp) were generated from both samples providing further evidence of TYLCV infection. To obtain the complete genome sequence, these PCR amplified DNA fragments of both isolates were gel extracted and cloned using the pGEM-T® Easy Vector system. Three independent clones of each fragment were sequenced and analyzed by Sanger sequencing. The resulting consensus sequences were used in a BLASTn search against the GenBank and had the highest identities (99-99.2%) with TYLCV sequences. The overlapping consensus nt sequence of the BX6 isolate was 2,782 nt (Accession no ON321843), whereas that of BX11 was 2,777 nt (Accession no ON785706) showed 99.2% and 99.0% nt identities with the corresponding sequence of TYLCV isolates (KX347141 and KX347142) respectively from Australia. The nt identity between BX6 and BX11 was 99.7%. Further screening by PCR of the remaining 18 symptomatic field samples (pepper, n=15 and tomatoes, n=3) revealed TYLCV infection in two pepper and all three tomato samples consistent with the greater susceptibility of tomato. None of the asymptomatic pepper and tomato samples were positive for TYLCV. DNA from both BX6 and BX11 samples was also used in the rolling circle amplification (RCA) assay (TempliPhi Amplification Kit, Cytiva) and the presence of circular TYLCV DNA was detected. Our results demonstrated the presence of TYLCV infection in these symptomatic pepper and tomato plants. This is the first report of TYLCV infecting pepper or tomato in open fields in Oklahoma. Further surveys are needed to determine the incidence of TYLCV and the presence of its vector in other counties throughout Oklahoma.

Le virus des feuilles jaunes en cuillère de la tomate favorise la tolérance au stress de la sécheresse de Solanum lycopersicum L.

Plants develop defence mechanisms in response to abiotic and biotic stresses that can have both negative and positive effects. Tomatoes grown in the field are normally exposed to abiotic stresses such as high temperatures and water shortages, as well as biotic stresses such as tomato yellow leaf curl virus (TYLCV), which greatly reduces productivity in this crop. In this study, two TYLCV Korean isolates were used as molecular and physiological tools to identify interactions between TYLCV infection and drought tolerance in tomato. The tomatoes were inoculated by TYLCV-infectious clones and exposed to drought stress, which led to wilted leaves on plants in the mock group, while those on TYLCV-inoculated plants showed no significant drought symptoms. Moreover, the average relative water content (RWC) was higher in TYLCV-infected plants than in the mock group, and genes associated to drought tolerance were pre-activated in well-watered tomato plants. These results confirm that TYLCV infection enhance drought tolerance in tomato plants and pre-inoculation with symptomless TYLCV isolates can be applied to tomato plants before being cultivated in water-deficit regions.

High-Throughput Sequencing Identified Distinct Bipartite and Monopartite Begomovirus Variants Associated with DNA-Satellites from Tomato and Muskmelon Plants in Saudi Arabia.

The studies on the prevalence and genetic diversity of begomoviruses in Saudi Arabia are minimal. In this study, field-grown symptomatic tomato and muskmelon plants were collected, and initially, begomovirus infection was confirmed by the core coat protein sequences. Four tomato and two muskmelon plants with viral infections were further evaluated for Illumina MiSeq sequencing, and twelve sequences (2.7-2.8 kb) equivalent to the full-length DNA-A or DNA-B components of begomoviruses were obtained along with eight sequences (~1.3-1.4 kb) equivalent to the begomovirus-associated DNA-satellite components. Four begomovirus sequences obtained from tomato plants were variants of tomato yellow leaf curl virus (TYLCV) with nt sequence identities of 95.3-100%. Additionally, two tomato plants showed a mixed infection of TYLCV and cotton leaf curl Gezira virus (CLCuGeV), okra yellow crinkle Cameroon alphasatellite (OYCrCMA), and okra leaf curl Oman betasatellite (OLCuOMB). Meanwhile, from muskmelon plants, two sequences were closely related (99-99.6%) to the tomato leaf curl Palampur virus (ToLCPalV) DNA-A, whereas two other sequences showed 97.9-100% sequence identities to DNA-B of ToLCPalV, respectively. Complete genome sequences of CLCuGeV and associated DNA-satellites were also obtained from these muskmelon plants. The nt sequence identities of the CLCuGeV, OYCrCMA, and OLCuOMB isolates obtained were 98.3-100%, 99.5-100%, and 95.6-99.7% with their respective available variants. The recombination was only detected in TYLCV and OLCuOMB isolates. To our knowledge, this is the first identification of a mixed infection of bipartite and monopartite begomoviruses associated with DNA-satellites from tomato and muskmelon in Saudi Arabia. The begomovirus variants reported in this study were clustered with Iranian isolates of respective begomovirus components in the phylogenetic dendrogram. Thus, the Iranian agroecological route can be a possible introduction of these begomoviruses and/or their associated DNA-satellites into Saudi Arabia.

First Report of Tomato Yellow Leaf Curl Virus Infecting Upland Cotton (Gossypium hirsutum) in Alabama, USA.

Cotton (Gossypium hirsutum L.) is used as a non-host of tomato yellow leaf curl virus (TYLCV) (family Geminiviridae, genus Begomovirus) in many studies (Ghanim and Czosnek 2000; Legarrea et al. 2015; Zeidan and Czosnek 1991), but only one reports methods used to determine host-status (Sinisterra et al. 2005), and there is one contradictory report from China stating cotton is a host of TYLCV (Li et al. 2014). In October 2018, cotton was screened for the presence of begomoviruses in Elmore, Escambia and Macon Counties, AL, where infestations of its whitefly vector (Bemisia tabaci Genn.) occurred in August. DNA was extracted from fully expanded leaves from the upper 1/3 of the canopy using a DNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany) and amplified with primers V324/C889 targeting a 575 bp coat protein fragment of begomoviruses (Brown et al. 2001). Five out of 200 cotton samples tested positive, and sequences recovered from three samples revealed 98-99% identity to TYLCV isolates in NCBI (Accession Nos. MT947801-03); sequences from the other two samples were of low quality and inconclusive. These samples were not available for additional tests, therefore, we proceeded to confirm host status using a monopartite clone of TYLCV-Israel (Reyes et al. 2013) reported in the US (Polston et al. 1999). All experiments were conducted in growth chambers with 16:8 light:dark cycle at 25.0℃ and 50% RH. Cotton seedlings (DeltaPine 1646 B2XF) at the 2-3 true leaf stage and tomatoes (Solanum lycopersicum L., var. 'Florida Lanai') at the 4 true leaf stage were agroinoculated at the stem tissue between the apical meristem and the first node (Reyes et al. 2013). Tomato served as a positive control; tomato and cotton mock inoculated with an empty vector were negative controls. A hole-punch was used to collect 4 leaf discs along midveins of the three, uppermost fully expanded leaves. DNA was extracted 28 days after inoculation as described above. A 390 bp segment of the intergenic region of TYLCV-A was amplified using primers PTYIRc287/PTYIRv21 (Nakhla et al., 1993). PCR results from agroinoculated plants confirmed (2/18) cotton plants, (5/5) tomatoes and (0/10) mock inoculated controls were infected with TYLCV. Whitefly transmission to cotton was confirmed using a leaf-disc bioassay for rapid testing (Czosnek et al. 1993). Bemisia tabaci MEAM-1 reared on eggplant (non-host of TYLCV) were placed on agroinoculated TYLCV-infected tomato/span> plants for a 96-h acquisition access period. Cohorts of 10 viruliferous B. tabaci were aspirated into 30mL cups each containing a 2.5cm healthy cotton leaf disc set in plant agar. After a 48-h inoculation access period, adults and their eggs were removed from the leaf discs. Leaf discs were held another 96-h before they were tested for TYLCV using the methods described above. TYLCV-infection was confirmed in (9/20) cotton leaf discs, demonstrating the viral load delivered by whiteflies was high enough to initiate local infection in cotton. No obvious begomovirus symptoms were observed on cotton plants in the field or laboratory. Field collection of samples was prompted by symptoms attributed to cotton leafroll dwarf virus (Avelar et al. 2017). TYLCV infection of cotton does not appear to be of economic importance. Additional information is needed to determine the frequency of infection in the field, specificity of TYLCV isolate x cotton genotype interactions leading to successful infection, and underlying causes of conflicting host-status reports in previously published studies.

Genetic Variability of Some Tomato Yellow Leaf Curl Virus Isolates and the Effect of the Virus on the Tomato (Solanum lycopersicom L.) Plant Content of Some Mineral Elements

This study was conducted to isolate and identify three isolates of Tomato yellow leaf curl virus (TYLCV), infecting tomato, using polymerase chain reaction technology (PCR) and determining the nucleotide sequences produced by PCR- amplified products to determine the genetic similarity and differences amongst the virus isolates. It also aimed to analyze the plant content of mineral elements: magnesium, calcium, sodium, and potassium to determine the effect of the virus on the plant content of these elements. The laboratory experiments mentioned in this study were carried out in the Plant Virology Laboratory of the Plant Protection Department at the College of Agriculture/ Karbala University. Analysis of the mineral elements was carried out in the Soil Laboratory, College of Agriculture/ University of Kufa. A greenhouse experiment was also carried out to investigate the response of some tomato genotypes against TYLCV during the agricultural season 2018-2019. Results of PCR amplification by the CP-F and CP-R primer pair revealed the possibility of amplifying a 789bp product from each TYLCV isolate isolated from some farms located in some desert areas in Najaf and Karbala governorates. Analysis of the sequences resulting from the PCR-amplified products obtained from the viral isolates (5, 8, and 10) by BLAST Basic Local Alignment Search Tool (BLAST) indicated that all these viral isolates diagnosed in this study belong to TYLCV. TYLCV isolates 5 and 8 obtained from Najaf province had a 100% similarity in the sequences of PCR-amplified products amplified from the TYLCV coat protein. These isolates gave a difference (96%) in the coat protein nucleotide sequence of the virus isolate 10. Furthermore, analysis of some mineral elements in plants infected with TYLCV showed a decrease in the concentrations of magnesium and calcium and an increase in the concentrations of elements sodium and potassium with a significant difference from their normal concentrations in the non-infected plants.

Association of tomato yellow leaf curl virus — Oman strain with the leaf curl and yellow mosaic symptoms on papaya and wild poinsettia in Oman

Begomoviruses (Geminiviridae) cause severe diseases in several crops, often causing significant losses due to reduced yield and quality. These viruses are easily transmitted by the whitefly (Bemisia tabaci) vector. Begomovirus-like symptoms were observed in wild poinsettia (Euphorbia cyathophora) and cultivated papaya (Carica papaya) plants in the Nizwa, Al-Dakhliya and Al-Batinah regions of Oman, where papaya is grown extensively. The aim of this study was to identify the suspected viral causal agent associated with yellow mosaic and leaf curl symptoms in the poinsettia and papaya plants. Symptomatic leaf samples were collected and subjected to DNA isolation followed by PCR amplification with begomovirus-coat protein specic primers. An amplicon of betasatellites was also amplified using betasatellite-specific primers. The amplicons were cloned and sequenced bi-directionally. The full-length begomoviral genome was determined to be 2766 and 2752 bp in length for the wild poinsettia and papaya isolates, respectively. The respective betasatellites were found to be 1354 and 1370 bp. Sequence analysis indicated that the papaya isolate, herein tomato yellow leaf curl virus (TYLCV)-Pap-Oman, shared 95.0% nucleotide identity with the wild poinsettia isolate, herein TYLCV-Poin-Oman, followed by 93.5% identity with TYLCV-Iran (GU076448). Phylogenetic analysis grouped TYLCV-Pap-Oman and TYLCV-Poin-Oman with TYLCV isolates previously reported from Iran, Saudi Arabia and Sudan, whereas the two betasatellites grouped them with TYLCB isolates previously reported from Oman. Based on the nucleotide sequence identity and phylogenetic tree analysis, the Begomovirus identified from papaya and wild poinsettia is considered an isolate of TYLCV from plant host species previously reported in the Arabian Peninsula.

Genetic diversity of tomato yellow leaf curl virus isolates and the effect of virus on the hormones content of tomato (Solanum lycopersicum) plants

Tomato yellow leaf curl virus (TYLCV) is one of the most economically important plant viruses globally and due to its continuing quick spread, now it afflicts many tomato growing countries in the Mediterranean basin, Africa, southern Asia, as well as South, Central and North America. Infection of tomato plants with TYLCV can reach 100% causing a severe reduction in tomato production, especially when plants are infected in the early stage of growth. Therefore, this study was conducted, during the growing season 2018-19 at the college of Agriculture, Kerbala University, Iraq, to isolate and diagnose three different isolates of TYLCV, infecting tomato, using the polymerase chain reaction technology (PCR) and determining the nucleotide sequences of PCR- amplified products and the effect of TYLCV infection on the plant contents of some hormones (gibberellin, cytokinin, and Indole acetic acid). Results of PCR amplification showed the possibility of amplifying a 789bp PCR product from each virus isolate (1, 2, 3, and 4). Analysis of the nucleotide sequences of PCR products using BLAST showed that all the diagnosed isolates belong to TYLCV. By comparing the sequences of nucleotide sequences, there was a genetic similarity of 100% among these isolates. However, all tomato genotypes tested in this study were found to be susceptible to TYLCV with different degrees of infection. TYLCV infection also had a significant effect in reducing the level of the hormone gibberellin in the infected genotypes. Increased levels of the cytokinin and indole acetic acid hormones were also observed in the TYLCV-infected genotypes.

First report of tomato yellow Leaf curl virus and cucumber mosaic virus infecting Huoxiang (Agastache rugosa) in CHINA

Huoxiang (Agastache rugosa, family Labiatae) is used as a traditional Chinese medicine. In 2017, Huoxiang with stunted, chlorotic, and mosaic symptoms were found in Haidian District of Beijing, China. To identify the causal agent(s), total RNA of symptomatic leaves was extracted and further analyzed by high-throughput sequencing (HTS). Sequence analyses revealed nine contigs with high nucleotide sequence identity (93%–100%) with tomato yellow leaf curl virus (TYLCV) from the genus Begomovirus, family Geminiviridae and seventeen contigs with high nucleotide sequence identity (85%–100%) with cucumber mosaic virus (CMV) from the genus Cucumovirus, family Bromoviridae. To confirm the HTS results, RT-PCR was performed with TYLCV-specific primers and CMV-specific primers, respectively. Two fragments of 651 bp (GenBank accession No. MG775386) and 657 bp (MG754206) were obtained, respectively. Sequencing and BLASTn analysis revealed that the 651 bp fragment shared 100% nucleotide identity with TYLCV isolate ZJHZ (MF590742), and the 657 bp fragment shared 98.9% nucleotide identity with CMV strain K (AF127977). To our knowledge, this is the first report of TYLCV and CMV infecting A. rugosa.

Molecular Investigation of Tomato Yellow Leaf Curl Virus (TYLCV) and Identification its Isolates Disseminated in some Tomato Protected Culture in Al-Qadisiyah Province.

This study aimed at conducting a molecular investigation of some virus isolates associated with tomato yellow leaf curl disease (TYLCD). Ten tomato samples exhibiting TYLCD are collected from fields and tunnel at certain tomato growing area in Al-Qadisiyah Province (El-HamzaEl-Sharqi district, Al-Sadeer and Al-Sunnia).. Begomoviruses are investigated by Polymerase chain reaction (PCR) using Deng a genus specific primer set in symptomatic samples. The amplified DNA fragments of expected size are sequenced, analyzed and compared to equivalent Gene Bank sequences using MEGA6 software. PCR technique using Deng primers could detect begomoviruses when amplified the 540 bp DNA fragments from 6 out of 10 symptomatic tomato samples. Sequence analysis shows that sequences obtained shared 96 - 99.5% nucleotide (nt) sequence identities with Tomato yellow leaf curl virus (TYLCV) equivalent Gene bank coat protein CP sequences from Cuba (AJ223505), USA (AY530931) and Israel (AB110217). Neighbor-Joining phylogenetic tree constructed from partial coat protein region confirmed the relatedness when grouped all sequences obtained to equivalent sequences belong to TYLCV from Gen Bank but not to other begomovirus species. Sequence comparison shows that TYLCV isolates are variable when share nt sequence 91-100% (approximated)to each other’s, which indicates that sequences isolated may belong to different TYLCV strains. The high nucleotide sequence identities suggests that TYLCV may have been introduced to Al-Qadisiyah Provence through infected plant materials, however full length sequence comparisons and biological assays are required to confirm this relatedness and resolve the possible origin of TYLCV isolates in Al-Qadisiyah Province..

Molecular evidence for the occurrence of TYLCV on Mentha longifolia in Jeddah, Saudi Arabia.

Begomoviruses are whiteflies transmitted virus causing serious disease in many important plants exhibiting variable symptoms with significant economic loss globally. Mentha is an important crop being grown here in Saudi Arabia for various purposes. The begomovirus associated disease was observed on Mentha crops during field survey which were growing near to tomato field. There is no published report available about the association of begomovirus on Mentha from this region. So, this work was conducted to identify the causative agent associated with yellow vein mosaic disease. Naturally infected samples were collected from various locations and causative agent was identified by PCR using begomovirus specific primers and further cloned and sequenced bidirectionally. The full genome had total 2785 nucleotides while betasatellite molecule had 1365 nucleotides. Based on full-genome sequence analysis, the identity matrix and phylogenetic analysis showed the highest identity (99.6%) with Tomato yellow leaf curl virus (TYLCV) reported from tomato in the Kingdom of Saudi Arabia. The identified begomovirus was observed as isolate of TYLCV.

Evaluation under diverse conditions of a differential host reaction scale to Tomato yellow leaf curl virus in tomato

Tomato yellow leaf curl virus (TYLCV) is the most widespread virus species causing Tomato yellow leaf curl disease. Accurate methods for assessment of resistance are requisite in breeding programs. Researchers at the Volcani Center (Israel) developed a scale of differential TYLCV reactions in seven non-segregating tomato host lines, with virus responses ranging from susceptible to highly resistant. In the present work, this scale has been evaluated with inoculation and assessment methods routinely used by two different research groups, in Spain and Cuba. Different TYLCV isolates, inoculation methods, plant ages, and environmental conditions were compared. Symptom scores of the tomato lines were generally lower in the conditions assayed in both locations than those originally described. Reaction ranking order of the standard tomato lines was more similar to the original description in the assays carried out in Spain. However, response of the lines to TYLCV obtained in both locations did not correspond to scale grades for most of the conditions. A large difference between symptom scores in the susceptible and the resistant lines was observed, with the range in the resistant lines being narrow. These results indicate that the number of standard lines used could be reduced, selecting the most susceptible and the most resistant lines, and one with intermediate resistance. All the factors evaluated affected symptom development. This highlights the importance of establishing a standard inoculation method, experimental conditions, evaluation period and appropriate resistance evaluation criteria, to ensure precise evaluation of genotype responses.

First complete genomic characterization and phylogeny of a new recombinant of tomato yellow leaf curl virus (genus Begomovirus, family Geminiviridae) from Kuwait.

While whitefly-transmitted begomoviruses are economically important constraints to tomato production in Kuwait, little is known about genomic features of these viruses from Kuwait. A begomovirus isolated from severely diseased tomatoes, collected over a two-year period in the main tomato-growing areas of Kuwait, was characterized at the molecular level. The complete genomic sequence of the begomovirus was determined, and phlylogeographic studies were conducted to better understand genetic diversity of the virus in the region. Based on genome properties and phylogenetic analysis, the begomovirus was found to be a strain of tomato yellow leaf curl virus (TYLCV). The virus genome was monopartite, as neither DNA B nor satellite DNA molecules were detected. Two isolates characterized in this study shared 97% and 95% nucleotide sequence identity with a previously characterized Kuwaiti isolate, TYLCV-KISR. Among TYLCV isolates with known genome sequences, the Kuwaiti isolates shared highest sequence identity (95%) with TYLCV-Almeria (Spain). Genetic diversity and phylogenetic analysis showed that the three Kuwaiti isolates formed a distinct clade that was separate from those of known TYLCV sequences. One Kuwaiti isolate (KW 1-3) could be a novel variant of TYLCV. Two recombination events were detected in the genome sequence of KW 1-3, which appeared to be a recombinant derived from TYLCV parents from Oman and Kuwait.

Molecular Characterization of Tomato Yellow Leaf Curl Virus in Korea and the Construction of an Infectious Clone

Several tomato production regions in Korea were surveyed for tomato yellow leaf curl disease (TYLCD). Tomato leaf samples showing TYLCD-like symptoms were collected from Tongyeong (To), Geoje (Gi), and Gimhae (Gh) cities of the southern part of Korea. Tomato yellow leaf curl virus (TYLCV) was detected and the full-length genomes of the isolates were sequenced. The TYLCV isolates found in Korea shared high sequence identity (> 99%) with TYLCV-IL [JR:Omu:Ng] (AB110217). Phylogenetic relationship analysis revealed that they formed two groups (with little genetic variability), and the To, Gj, and Gh isolates belonged to the TYLCV-IL group. An infectious clone of TYLCV-To (JQ013089) was constructed and agroinoculated into Nicotiana benthamiana, Nicotiana tabacum var. Xanthi, Petunia hybrida, Capsicum annuum, and Lycopersicon esculentum cv. Hausumomotaro. Agroinfection with a dimeric infectious clone of TYLCV-To induced severe leaf curling and stunting symptoms in these plants, excluding C. annuum. Tomato plants then developed typical yellow leaf curl symptoms.

FIRST REPORT OF SOLANUM ELAEAGNIFOLIUM AS NATURAL HOST OF TOMATO YELLOW LEAF CURL VIRUS SPECIES (TYLCV AND TYLCSV) IN TUNISIA

Tomato yellow leaf curl virus (TYLCV) and Tomato yellow leaf curl Sardinia virus (TYLCSV) (genus Begomovirus, family Geminiviridae), as well as recombinant variants, were recently found in tomato in Tunisia (Mnari-Hattab et al., 2014). To identify sources of infection, tomato plants and Solanum elaeagnifolium, an invasive weed, were sampled and tested for the presence of TYLCV and TYLCSV. Genomic DNA was purified from leaf samples showing curling and yellowing and subjected to PCR using primers designed in the coat protein gene of TYLCV (Mnari-Hattab et al., 2014; Wyatt and Brown, 1996). A product of ca. 580 bp was obtained from four of six S. elaeagnifolium and six of six tomato samples tested. The nucleotide sequence of one amplicon from tomato and two amplicons from S. elaeagnifolium was obtained and deposited in GenBank as accession numbers KF990598, KF990599 and KF990600. The nucleotide sequence of Tunisian TYLCV isolates was 91.2 to 98.9% identical and shared 91 to 98.3% identity with a TYLCV isolate from Turkey (AJ867487). Multiplex PCR (Davino et al., 2008) was further used to identify virus species with the amplification of a 570 bp fragment for TYLCV and a 800 bp fragment for TYLCSV. Results showed two S. elaeagnifolium and four tomato plants infected with TYLCV or TYLCSV, and two S. elaeagnifolium and one tomato plant infected with both viruses. To the best of our knowledge, this is the first report of S. elaeagnifolium as a natural host of TYLCV and/or TYLCSV in Tunisia. These findings suggest S. elaeagnifolium as a reservoir for tomato yellow leaf curl virus disease.

First report of hard watermelon syndrome in Tunisia associated with Tomato yellow leaf curl virus infection

In Tunisia, watermelon (Citrullus lanatus) is one of the most common and popular summer fruit crops, and is widely cultivated in the central and southern parts of the country. Since 1994, abnormal symptoms have been observed on fruits by the end of the cropping season grown in the regions of Kairouan, Sidi Bouzid and Gafsa. In some fields, watermelons harden precociously and, when ripe, present a white heart. Moreover, young leaves were mildly curled and stunted (Fig. 1). These symptoms were distributed randomly in the fields and were initially misdiagnosed and attributed to physiological and nutritional disorders. Since the first reports of this syndrome in 1994, leaf and fruit samples with characteristic symptoms had been tested for the occurrence of any of the viruses known to affect cucurbits in Tunisia, but none showed any positive results (Mnari-Hattab et al., 2). The frequency and distribution of the ‘hard watermelon syndrome’ suggested that virus infection might be responsible, particularly as Tomato yellow leaf curl virus (TYLCV), Tomato yellow leaf curl Sardinia virus (TYLCSV) and/or parental recombinant genus Begomovirus (family Geminiviridae) were widespread on tomato and on silverleaf nightshade (Solanum elaeagnifoli) growing in the same area (Mnari-Hattab et al., 2014; Zammouri & Mnari-Hattab, 5). In 2014, a total of 22 samples, eight samples of preserved vegetable material (i.e. leaves, peduncle and fruit bark) and 14 samples freshly collected from plants exhibiting the characteristic syndrome were tested for begomovirus infection. Genomic DNA was purified, and PCR amplification was performed using degenerate oligonucleotide primers based on the sequence in the Begomovirus coat protein genes (Wyatt & Brown, 4; Mnari-Hattab et al., 5). Amplicons circa 560 bp in size were obtained from all the tested symptom-bearing samples (plants with hardened fruit) but no amplicons were generated from symptomless plant samples. Three of the amplicons were cloned and sequenced, and the nucleotide sequences were deposited in GenBank (Accession Nos. KM042091 - KM042093). BLAST searches of the GenBank database revealed that the sequences from watermelon, which were 97.2 to 98.7% identical to one another other, shared 96% homology with a Turkish TYLCV isolate (Accession No. AJ867487). Similar TYLCV amplicons generated from tomato and silverleaf nightshade plants collected from open field tomato crops in Kairouan had similar sequences (results not shown). Further investigations using multiplex PCR amplification (Davino et al., 1) revealed that all 22 samples referred to above were infected with TYLCV, and that two of the samples were doubly infected with TYLCV and TYLCSV following generation of respectively 570 bp and 800 bp amplicons for the two viruses. To the best of our knowledge, this is the first report that associates the watermelon hardness syndrome with Tomato yellow leaf curl virus infection. Based on our findings, further targeted surveys must therefore be undertaken to advance our knowledge on tomato yellow leaf curl disease epidemics on cucurbit crops in Tunisia.

New natural hosts of Tomato yellow leaf curl virus identified in and near tomato-growing greenhouses in eastern China

Tomato yellow leaf curl virus (TYLCV) causes huge losses to tomato production worldwide. In July 2011 and July–August 2012, we screened for potential TYLCV hosts in a tomato-growing area in Shandong Province, the core vegetable-producing region in China. PCR detection showed that 5 species of plants, Zinnia elegans, Acalypha australis, Gossypium hirsutum, Abutilon theophrasti, and Nicotiana tabacum, were infected. Full genomic sequences of the new TYLCV isolates were obtained and submitted for sequence analysis. Sequence alignment and similarity analysis showed that they all belonged to the TYLCV-IL strain.