Tomato Chlorosis Virus Isolates Research Articles

Metagenomic analysis for detection and discovery of plant viruses in wild Solanum spp. in South Africa

AbstractWild plant species play an important role in plant virus epidemiology, mainly by harbouring viruses pathogenic to cultivated plant species. The genus Solanum includes important crop species as well as some highly abundant and widely distributed wild plant species. The aim of the present study was to profile the viromes of 11 wild Solanum spp. found in five provinces of South Africa. Metatranscriptomic analysis of pooled RNA samples from each of the Solanum spp. detected viruses belonging to 13–19 families. Reverse transcription PCR confirmed the detection of potato virus Y (PVY), potato leaf roll virus (PLRV), tomato torrado virus (ToTV) and tomato chlorosis virus (ToCV) for the first time in some of the Solanum spp. studied. Phylogenetic analyses indicated that the PVY and PLRV strains from the wild Solanum spp. were divergent from those reported in cultivated crops, while there was limited divergence among ToTV and ToCV isolates. Viruses detected for the first time in South Africa included tobacco mild green mosaic virus, tomato matilda virus, a pepper enamovirus from Rwanda and a cytorhabdovirus from potato in Kenya. Potentially novel viruses were also detected in S. lichtensteinii, S. mauritianum and S. viarum. This study demonstrates the role of Solanum spp. in harbouring viruses pathogenic to solanaceous crops, and in the emergence of new virus strains. It may therefore inform strategies for virus disease management by control of wild solanums that harbour viruses pathogenic to important Solanum crops.

Differential reaction of sweet pepper to infection with the crinivirus tomato chlorosis virus probably depends on the viral variant

AbstractThe tomato chlorosis virus (ToCV), transmitted by whitefly species of the genera Bemisia and Trialeurodes in a semipersistent manner, causes significant losses in solanaceous crops including tomato (Solanum lycopersicum) and sweet pepper (Capsicum annuum). Worldwide reports of natural and experimental infection of sweet pepper plants with ToCV are contradictory, raising the question of whether the critical factor determining infection is related to the susceptibility of sweet pepper cultivars or the genetics of virus isolates. In this work, ToCV isolates obtained from different hosts and geographical origins were biologically and molecularly analysed, transmitted by B. tabaci MEAM1 and MED, and the reaction of different sweet pepper cultivars was evaluated under different environmental conditions. Brazilian ToCV isolates from tomato, potato (S. tuberosum), S. americanum, and Physalis angulata did not infect plants of five sweet pepper cultivars when transmitted by B. tabaci MEAM1. Temperatures did not affect the sweet pepper susceptibility to tomato‐ToCV isolates from São Paulo, Brazil, and Florida, USA. However, sweet pepper‐ToCV isolates from Spain and São Paulo, Brazil, were transmitted efficiently to sweet pepper plants by B. tabaci MEAM1 and MED. Although the results indicated that ToCV isolates from naturally infected sweet pepper plants seem to be better adapted to plants of C. annuum, phylogenetic analyses based on the complete nucleotide sequences of RNA1 and RNA2 as well as the p22 gene did not reveal significant nucleotide differences among them. Additional studies are needed to identify intrinsic characteristics of ToCV isolates that favour infection of sweet pepper plants.

First Report of Tomato Chlorosis Virus Infecting Tomato in Pakistan

Tomato chlorosis virus (ToCV) is a bipartite crinivirus (Closteroviridae) consisting of two positive-sense single-stranded RNAs: RNA1 and RNA2 (Fiallo-Olive and Navas-Castillo 2019). It is a phloem-limited virus transmitted by whiteflies (Bemisia tabaci and Trialeurodes vaporariorum) in a semipersistent manner (Fiallo-Olive and Navas-Castillo 2019; Lee et al. 2018). In March 2019, a survey of the tomato crop (cv. Richman) was conducted in the Multan district of Pakistan; 35% of plants in the crop showed virus-like symptoms such as interveinal chlorosis, brittleness, and general yellowing in the older and middle leaves. These symptoms were similar to those caused by ToCV and tomato infectious chlorosis virus (TICV) (Wisler et al. 1998). Tomato plants were infested by dense (20/leaf) whitefly (B. tabaci) populations. Thirty-six samples were collected from different plants (30 symptomatic and six asymptomatic) and were tested for ToCV and TICV. Total RNA was extracted from leaf samples using TRIzol reagent and assayed by RT-PCR, targeting the ToCV movement protein gene with primers ToCV-RNA2-IF and ToCV-RNA2-1R (Lee et al. 2018) and the TICV heat shock protein homolog gene with primers TICV-32 and TICV532 (Vaira et al. 2002). RT-PCR was also performed to target the clathrin adaptor complexes medium subunit (CAC), a reference protein gene of tomato, by using the forward primer 5′-CCTCCGTTGTGATGTAACTGG-3′ and the reverse primer 5′-ATTGGTGGAAAGTAACATCATCG-3′ (amplification size: 173 bp) (Gonzalez-Aguilera et al. 2016). Twenty-seven of 30 samples yielded the 827-bp ToCV product, whereas no amplification was obtained in any of the asymptomatic samples. TICV was not detected in any of the analyzed samples. Whiteflies (B. tabaci biotype B) collected from disease-free cotton plants and reared on cucumber plants (nonhost of ToCV) under greenhouse conditions were used in the transmission assay. One hundred nonviruliferous whiteflies were given an acquisition access period (AAP) of 48 h on an infected ToCV tomato plant and then transferred onto 20 healthy tomato seedlings (hybrid Richman, 5 whiteflies/seedling in one cage) for a 48-h inoculation access period (IAP). To ascertain that the whiteflies were nonviruliferous, 100 whiteflies were transferred onto five healthy tomato plants (20 whiteflies/plant) for an IAP of 48 h. Sixteen of 20 inoculated plants showed typical ToCV symptoms 28 days postinoculation, and ToCV was detected by RT-PCR using the ToCV-specific primers. No symptoms were observed on the negative control plants, confirming the nonviruliferous nature of whiteflies. The ToCV-positive tomato plants were used as a source for back inoculation to an indicator host, Nicotiana benthamiana. About 200 nonviruliferous whiteflies were given a 48-h AAP on the ToCV-infected tomatoes and then transferred to four healthy N. benthamiana plants (50 whiteflies/plant) for a 48-h IAP, and two healthy N. benthamiana plants were controls (fed on by nonviruliferous whiteflies). All ToCV-inoculated N. benthamiana plants showed distinctive symptoms of yellowing, interveinal chlorosis, and leaf brittleness; no symptoms were observed on the control plants. RT-PCR confirmed ToCV in symptomatic indicator plants and its absence in the control plants. The amplicons (movement protein gene) of two selected ToCV isolates from the field plants were sequenced in both directions. The resulting sequences were submitted to NCBI (MN385628 and MN385629). BLASTn analysis of the sequences revealed that the newly identified isolates are 99.88% identical to isolates reported from Turkey (KY419528), China (KJ815045), and Greece (EU284744). To our knowledge, this is the first report of ToCV infecting tomato in Pakistan. The wide occurrence of ToCV and its whitefly vectors in tomato crops in the region indicates that effective integrated management measures are needed to minimize losses caused by the virus in tomato.

Host range and natural infection of tomato chlorosis virus in weeds collected in Central Brazil

Tomato chlorosis virus (ToCV), transmitted by whiteflies, is an important virus that infect tomato plants worldwide. The incidence of ToCV in tomato crops in Brazil is usually high, especially in the Distrito Federal and the state of Goias, in the central part of the country. We conducted a survey in central Brazil to detect natural infections of ToCV in weeds near tomato plants with typical criniviral symptoms to determine the possible causes of its high incidence. We also conducted an experimental host range study using a Brazilian ToCV isolate, ToCV-BR. ToCV was only detected in three out of 19 weed species collected under natural conditions: Solanum americanum, Nicandra physaloides and Amaranthus viridis. Although these species are the most important weeds, occurring at high frequencies associated with tomato crops in Brazil, the number of weed species infected by ToCV was low in the vicinity of tomato fields. In experimental trials of whitefly (Bemisia tabaci) transmission, the ToCV-BR isolate infected 10 out of 50 plant species tested. The natural abundance of both whiteflies and ToCV-susceptible plants in the field throughout the year may affect the incidence of ToCV in tomato in central Brazil.

First report of Tomato chlorosis virus infecting tomato in Kenya

This study investigated Tomato chlorosis virus (ToCV) present in plants collected in September 2017, in Kenya. We collected tomato leaves that exhibited severe interveinal yellowing symptoms and used reverse transcription polymerase chain reaction (RT-PCR) to confirm that 15 of the 36 samples were infected with ToCV. One sample collected in Kiambu County was used for molecular analyses. Partial sequences of the genome of RNA1 (including RNA-dependent RNA polymerase gene) and RNA2 (includes coat protein, minor coat protein, and heat shock protein 70 homolog genes) were amplified using specific primers. Nucleotide sequences of these segments showed the highest similarity with segments from all ToCV isolates other than the Taiwan isolate. Genetic and molecular phylogenetic analyses showed similar results. This is the first report of ToCV in Kenya.

First Report of Tomato Chlorosis Virus Infecting Tectona grandis Associated With Infestation of Bemisia tabaci Mediterranean in Central Brazil

The commercial production of teak (Tectona grandis L. f., family Lamiaceae) has been increasing in Brazil due to the adaptation of this timber species to the predominant environmental conditions. Symptoms of interveinal yellowing in older leaves similar to that induced by species of the genus Crinivirus (family Closteroviridae) were observed (≈90% incidence) in 100-day-old teak seedlings in one orchard in Brasilia–DF in 2018. Moreover, a severe whitefly infestation was observed in association with these symptomatic plants. Fifteen leaf samples collected at random from distinct symptomatic plants were tested for the presence of tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), two criniviruses reported infecting a wide range of crops in Brazil (Fonseca et al. 2016). Leaf samples from asymptomatic teak plants (n = 10) were also collected and employed as negative controls. Total RNA extraction was performed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA) from all samples of symptomatic and asymptomatic plants. RT-PCR assays were conducted using the universal primer pair HS-11/HS-12 (which amplifies a 587-bp RNA-2 segment encompassing the HSP70h protein gene), followed by a nested-PCR performed with the primer pair ToCV- (ToC-5/ToC-6) and TICV-specific primers (TIC-3/TIC-4) (Dovas et al. 2002). All 15 symptomatic teak samples were positive only for ToCV (≈463-bp amplicons). PCR product from one randomly selected sample was directly sequenced. The obtained sequence (GenBank MH688047) displayed 99.7% identity with the equivalent sequence of a tomato-infecting ToCV isolate reported in Brazil (EU868927). Five symptomatic samples were also positive in dot-blot hybridization assays using a coat protein-derived RNA probe (436-nt fragment of RNA-2; primers MA-380/MA-381) (Fortes et al. 2012) labeled with digoxigenin (DIG)-11-UTP and with the chemiluminescent substrate CDP Star kit (Roche Diagnostics). Total DNA extraction and PCR assays were also conducted using pooled (three pools of n = 10 each) whitefly samples collected on symptomatic teak plants. A segment of the mitochondrial cytochrome oxidase I gene (mtCOI) was amplified using the specific primers C1-J-2195-FW and L2-N-3014- RV (Simon et al. 1994), yielding ≈860 bp amplicons. Direct sequencing of three amplicons obtained from the whitefly samples (MH688048) indicated high identity (99%) to B. tabaci Mediterranean (MED) species (= Q biotype). ToCV-transmission assays were performed using B. tabaci MED adults. Thirty viruliferous whiteflies derived from healthy whiteflies fed on ToCV-infected teak plants were confined in separated cages with healthy teak (n = 6) and tomato cv. Santa Clara (n = 6) seedlings. Aviruliferous whiteflies were also confined to healthy teak (n = 6) and tomato (n = 6) plants (negative controls). After 72 h, the whiteflies were eliminated by insecticide spraying. Thirty days after the feeding access period, only teak and tomato plants exposed to viruliferous B. tabaci MED exhibited crinivirus-like symptoms and were positive to ToCV in RT-PCR assays. To our knowledge, this is the first report of T. grandis as a host of ToCV as well as the first report of virus infection in this woody plant in Brazil. The introduction of B. tabaci MED into southern Brazil was recently reported (de Moraes et al. 2017). Our results also confirm the presence of B. tabaci MED in central Brazil and its association with ToCV infection of T. grandis seedlings. Although expressing overall mild symptoms, the potential negative effects of ToCV in teak plants need to be investigated in more detail.

Prevalence, epidemiology and molecular studies of Tomato chlorosis virus (ToCV) in South Africa.

Criniviruses accumulate in the phloem tissue and damage crops by reducing chlorophyll which is essential for plant growth and development. Tomato chlorosis crinivirus (ToCV) is vectored by several whitefly species that damage tomato crops throughout the world. In South Africa, ToCV is a poorly studied pathogen of global economic importance. Therefore, a national survey was initiated to investigate the occurrence and distribution of criniviruses infecting tomato crops in South Africa. Whitefly infested tomato crops exhibiting interveinal leaf chlorosis and chlorotic flecking symptoms were assayed for crinivirus infections using a multiplex reverse transcription polymerase reaction (RT-PCR) approach to assess for the presence of crinivirus species that are known to infect solanaceous hosts. Next-generation sequencing (NGS) was used to generate the complete genome of ToCV from South Africa. Results from the survey indicated that ToCV is presently the only crinivirus species infecting tomatoes in South Africa. Blast analysis showed that the RNA-1 segment of ToCV from South Africa (ToCR1-186) matched 99% to Spanish isolates. On the other hand, the RNA-2 (ToCR2-186) segment matched 98% to a South Korean isolate and three Spanish isolates. Although recombination events were not detected, phylogenetic studies showed inconsistencies in the grouping of RNA-1 and RNA-2 segments for some of the ToCV isolates analyzed in this study. Therefore, we suggest the possibility of intraspecific reassortment. This is the first comprehensive study and full genome sequence of ToCV from South Africa. The information generated from this study is intended to raise awareness of ToCV infections on tomato crops in South Africa.

Genetic diversity and phylogenetic analyses of tomato chlorosis virus isolates using the coat protein gene sequences

Since the first report of tomato chlorosis virus (ToCV) in 2008, yellowing symptoms were observed in major tomato production areas of Turkey. Therefore, surveys were conducted in Antalya, Burdur, Isparta and Mugla provinces where 329 symptomatic tomato samples were collected from greenhouses and open fields. Testing all samples by RT-PCR using primers specific for ToCV coat protein (CP) gene revealed that 113 of 329 tomato samples resulted in positive amplification of this virus. While ToCV was not detected in Isparta and Burdur, more than 50% of the samples collected from Antalya tested positive. The CP genes of 12 selected ToCV isolates from different areas were cloned, sequenced and compared with each other and the full-length CP genes of isolates from other countries. Genetic diversity and phylogenetic analysis showed that the Turkish ToCV isolates have low genetic diversity and they are closely related, clustering in the same phylogenetic group. On the other hand, the CP genes of ToCV isolates from different countries showed greater diversity than the Turkish isolates and were phylogenetically divided into three groups.

Biochemical, Serological, Molecular and Natural Host Studies on Tomato Chlorosis Virus in Egypt.

Tomato Chlorosis Virus (ToCV) is a white fly-transmitted and phloem-limited crinivirus reported in this study for the first time in Egypt. ToCV caused drastic reduction in tomato yield since 2013. The aim of this study is to characterize the virus incidence using biological, serological and molecular tools. The B. tabaci MEAM1 white fly was used for virus isolation and propagation. Identity of ToCV , its natural hosts were confirmed with RT-PCR using a specific primer pair for ToCV-heat shock protein 70 homologue (HSP70h) gene, sequencing and phylogenetic studies. ToCV was purified using the innovative electro-elution technique. The induced antiserum for the Egyptian isolate of the virus (ToCV-Giza) was used for DAS-ELISA and dot blotting immuno-assays to evaluate the virus presence in tomato and other natural hosts. The ToCV-Giza isolate was donated an accession number "MH667315.1" from the GenBank. Blastx sequence analysis of the HSP70h gene indicated 97-99% of amino acid similarities with many tested ToCV isolates. Phylogenetic studies showed the clustering of all ToCV isolates including ToCV-Giza in a separate group from the other tested criniviruses. The virus had a UV spectrum of a nucleoprotein with Amax and Amin at 260 and 240 nm, respectively and A260/280 ratio of 1.33. Out of 52 different tested plant species within 22 families, 44 were positive hosts for ToCV. Thirty seven out of these 44 plant species were considered as new hosts for ToCV in the present study. These included Ammi majus and Coriandrum sativum (Apiaceae), cabbage (Brassicaceae), sweet potato (Convolvulaceae), melon, cucumber, luffa (Cucurbitaceae), soybean, cowpea, faba bean (Fabaceae), Egyptian and American Cotton (Malvaceae). Several ornamentals either herbal type or woody trees belonging to Acanthaceae, Amaranthaceae, Euophorbiaceae, Moraceae and Rubiaceae were also recognized for the first time as hosts for ToCV. The obtained results confirmed the wide distribution of ToCV in its natural hosts in Egypt. Hygienic measures including control of the virus vector and removing of natural hosts should be strictly implicated.

Phylogenetic Characterization of Tomato chlorosis virus Population in Korea: Evidence of Reassortment between Isolates from Different Origins.

Tomato chlorosis virus (ToCV) is a whitefly-transmitted and phloem-limited crinivirus. In 2013, severe interveinal chlorosis and bronzing on tomato leaves, known symptoms of ToCV infection, were observed in greenhouses in Korea. To identify ToCV infection in symptomatic tomato plants, RT-PCR with ToCV-specific primers was performed on leaf samples collected from 11 tomato cultivating areas where ToCV-like symptoms were observed in 2013 and 2014. About half of samples (45.18%) were confirmed as ToCV-infected, and the complete genome of 10 different isolates were characterized. This is the first report of ToCV occurring in Korea. The phylogenetic relationship and genetic variation among ToCV isolates from Korea and other countries were also analysed. When RNA1 and RNA2 are analysed separately, ToCV isolates were clustered into three groups in phylogenetic trees, and ToCV Korean isolates were confirmed to belong to two groups, which were geographically separated. These results suggested that Korean ToCV isolates originated from two independent origins. However, the RNA1 and RNA2 sequences of the Yeonggwang isolate were confirmed to belong to different groups, which indicated that ToCV RNA1 and RNA2 originated from two different origins and were reassorted in Yeonggwang, which is the intermediate point of two geographically separated groups.

Identification of Eight Solanum (subgenus Leptostemonum) Species as Novel Natural Hosts of Tomato chlorosis virus in Brazil

HomePlant DiseaseVol. 102, No. 8Identification of Eight Solanum (subgenus Leptostemonum) Species as Novel Natural Hosts of Tomato chlorosis virus in Brazil PreviousNext DISEASE NOTES OPENOpen Access licenseIdentification of Eight Solanum (subgenus Leptostemonum) Species as Novel Natural Hosts of Tomato chlorosis virus in BrazilL. S. Boiteux, M. F. Lima, M. E. N. Fonseca, J. L. Mendonça, A. F. Costa, J. G. Silva-Filho, M. G. Fontes, and M. González-ArcosL. S. Boiteux†Corresponding author: L. S. Boiteux; E-mail: E-mail Address: leonardo.boiteux@embrapa.brhttp://orcid.org/0000-0002-5791-8002Search for more papers by this author, M. F. LimaSearch for more papers by this author, M. E. N. FonsecaSearch for more papers by this author, J. L. MendonçaSearch for more papers by this author, A. F. CostaSearch for more papers by this author, J. G. Silva-FilhoSearch for more papers by this author, M. G. FontesSearch for more papers by this author, and M. González-ArcosSearch for more papers by this authorAffiliationsAuthors and Affiliations L. S. Boiteux † M. F. Lima M. E. N. Fonseca J. L. Mendonça A. F. Costa J. G. Silva-Filho , Embrapa Vegetable Crops (CNPH), Brasília–DF, Brazil M. G. Fontes , Dept. Fitopatologia, UnB, Brasília–DF, Brazil M. González-Arcos , INIA, Salto Grande, Uruguay. Published Online:24 May 2018https://doi.org/10.1094/PDIS-11-17-1718-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Leaves of Solanum (subgenus Leptostemonum) species with crinivirus-like symptoms (interveinal to overall chlorosis and reduced vigor) were collected during field surveys in Brasília–DF, Brazil (2012 to 2017). Samples were obtained from Solanum stramoniifolium Jacq. (CR-073 and CR-076 isolates), S. sessiliflorum Dunal (CR-075), S. subinerme Jacq. (CR-079), S. jamaicense Mill. (CR-082), S. mammosum L. (CR-081), S. scuticum M. Nee (CR-198), and S. velleum Roem & Schult. (CR-199). Prevalence of symptomatic plants was high (80 to 100%), with mild symptoms in most of the sampled species except for early-infected S. stramoniifolium and S. mammosum, in which symptoms were severe. A symptomatic S. paniculatum L. plant (found as a weed in a tomato field) was collected (CR-114) in Venturosa (Pernambuco State, Brazil). Leaves of tomato plants with crinivirus-like symptoms were also obtained (CR-115) in the same field in Venturosa. All samples were tested for the presence of two criniviruses known to infect solanaceous plants, Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV). Total nucleic acids were extracted using TRIzol from tissues of symptomatic and healthy controls (i.e., seedlings of all Solanum species maintained under controlled conditions). The universal primer pair HS-11/HS-12 (which amplifies a 587-bp RNA-2 segment encompassing the HSP70h protein gene) was used in the RT-PCR step (Dovas et al. 2002). A nested-PCR assay was performed with ToCV- (ToC-5/ToC-6) and TICV-specific (TIC-3/TIC-4) primers (Dovas et al. 2002). Only ToCV-specific amplicons (∼463-bp) were detected in all symptomatic plants. Neither TICV- or ToCV-specific amplicons were detected in the healthy controls. Purified PCR products were directly sequenced at the Genomic Analysis Laboratory (CNPH, Brazil). Alignment of the sequences (419 nt) obtained for the eight Solanum isolates (GenBank accession nos. KY400116 to KY400119, KT727959, KX398666, KY400124, KY400132, and KY400133) and the tomato isolate CR-115 (KY400125) showed 99.2 to 100% identity among them and with a tomato-infecting ToCV isolate from Brazil (EU868927). ToCV infection was confirmed via dot-blot hybridization with a coat protein-derived RNA probe (436 nt fragment of RNA-2; primers MA-380/MA-381) (Fortes et al. 2012) labeled with digoxigenin (DIG)-11-UTP and with the chemiluminescent substrate CDP Star kit (Roche Diagnostics). Nucleic acid extracts of all isolates and their respective healthy controls were included in the dot-blot assays. Positive hybridization results were observed only in symptomatic samples. ToCV isolates from S. stramoniifolium and S. mammosum were transmitted to tomato ‘TX468 RG’ in controlled assays using adults of Bemisia tabaci MEAM1 as vectors. Even though Koch’s postulates were not fulfilled for ToCV as causal agent of the disease syndrome observed in all the Solanum species sampled, this virus has already been reported infecting members of the subgenus Leptostemonum (Fonseca et al. 2016), suggesting a widespread susceptibility of the plants from this taxon. These host species are widespread in South America either as cultivated and weed species or as members of the native flora with occurrence near many tropical and semitropical tomato-producing areas across the entire continent (Arruabarrena et al. 2014; 2015). Hence, these Solanum species (with long vegetative cycle) might have a significant epidemiological role as they might function as year-round reservoirs of ToCV.

Molecular Characterization and Detection of a Genetically Distinct Tomato Chlorosis Virus Strain in Taiwan.

The whitefly-transmitted tomato chlorosis virus (ToCV) belonging to the genus Crinivirus (family Closteroviridae) affects tomato production worldwide. ToCV was first recorded in Taiwan in 1998 affecting tomato production. In this study, a local virus isolate XS was obtained, after serial whitefly transmissions from a diseased tomato plant displaying general chlorosis were collected in central Taiwan. The whole genome sequence of XS was determined from cDNA fragments amplified by reverse transcription (RT)-PCR, first using the degenerate primers for viruses of Closteroviridae and followed by degenerate and specific primers designed on available sequences of the ToCV isolates. The nucleotide (nt) sequences of RNA-1 and RNA-2 of the XS shared low identities of 77.8 to 78% and 78 to 78.1%, respectively, with genome segments of other ToCV isolates. Nevertheless, the viral RNA-dependent RNA polymerase (RdRp), heat shock protein 70 homolog (Hsp70h), and major capsid protein (CP) shared 88.3 to 96.2% amino acid (aa) identities with other ToCV isolates, indicating that XS is a new strain of this virus. Phylogenetic analyses of these three proteins indicated that all ToCV isolates from different counties outside Taiwan are closely related and clustered in the same clade, whereas the XS isolate is distinct and forms a unique branch. A one tube RT-PCR assay using primers designed from the genomic sequence of the XS was able to detect the ToCV-XS in infected tomato plants and in individual whiteflies. A field survey during 2013 to 2016 revealed a high ToCV-XS prevalence of 60.5% in 172 tested tomato samples, demonstrating that ToCV-XS is becoming an emerging threat for tomato production in Taiwan.

Genetic diversity and silencing suppression activity of the p22 protein of Tomato chlorosis virus isolates from tomato and sweet pepper.

As for other bipartite criniviruses (genus Crinivirus, family Closteroviridae), the genome of Tomato chlorosis virus encodes an RNA silencing suppressor, the protein p22, in the 3'-proximal region of RNA1. This protein has been reported as having one of the longest lasting local suppressor activities when transiently expressed in Nicotiana benthamiana. Here, we examined the genetic diversity of the p22 gene in ToCV isolates from tomato and sweet pepper. The p22 gene sequences clearly grouped into two separated clades. However, functional analysis of both types of p22 proteins indicated no evident differences in suppressor activity. Our findings provide experimental evidence that the presence of a "strong" silencing suppressor is a conserved feature of ToCV isolates.

First Report of Tomato chlorosis virus Infecting Tomato Crops in Uruguay.

A survey of viral diseases was carried out during 2012 to 2013 in two major tomato (Solanum lycopersicum L.) producing regions in Uruguay (Salto and Canelones). Lower leaves of fruit-bearing plants were observed displaying yellowing and interveinal chlorosis under greenhouse conditions. The symptoms were similar to those associated with magnesium deficiency. However, the chlorosis displayed a tendency to move up affecting medial and apical leaves and prevailed even after supplementary magnesium applications to the soil, indicating potential infection by either Tomato chlorosis virus (ToCV) or Tomato infectious chlorosis virus (TICV) (3). Four leaf samples were collected from two sites in Canelones and 28 samples were collected from distinct commercial fields in Salto. Whiteflies (Bemisia tabaci biotype Q and Trialeurodes vaporariorum) were present in all sampling sites. Total RNA was extracted from symptomatic and healthy (control) plants and used for cDNA preparation with the HS-11/HS-12 primer pair followed by PCR amplification using the same primer pair. The 587-bp amplicon, corresponding to a highly conserved region of the heat shock protein (HSP-70) homolog gene reported in both TICV and ToCV genomes, was observed only in the symptomatic samples. These PCR products were then subjected to nested PCR using the ToCV specific primer pair (ToC-5/ToC-6) and TICV specific primer pair (TIC-3/TIC-4) (1). The expected 463-bp ToCV-specific amplicon was observed in all symptomatic plants but not in the healthy controls. The 223-bp amplicon corresponding to TICV was not observed in any sample, indicating the sole presence of ToCV. The amplicon of one Uruguayan ToCV isolate from Salto (named as CRS03) was purified and directly sequenced (GenBank KC626018). BLAST analysis revealed 99% identity of CRS03 with one Spanish isolate (AF233435.1) (2). Virus-free B. tabaci biotype Q adults were exposed to symptomatic plants infected with the CRS03 isolate for a 24-h period and then cage-confined with 10 healthy tomato plants (line 'LT17') for a 48-h period. Symptoms were reproduced in all tested plants after a 65-day period and ToCV infection was confirmed via PCR assays and by sequence analysis of the gel-purified amplicons. This is the first formal report of ToCV infecting tomatoes in Uruguay. Incidence of symptomatic plants in tomato crops varied from 30 to 100%, even under low whitefly pressure. Epidemiological information needs to be generated in order to evaluate the impact of ToCV in the fresh-market tomato yield and quality.

Tobacco: A New Natural Host of Tomato chlorosis virus in Spain.

In March 2013, symptoms of mild leaf curling, mosaic, and interveinal yellowing were observed in tobacco (Nicotiana tabacum) plants grown in a row surrounding the exterior of a greenhouse containing a tomato crop in Guía de Isora, Tenerife (Canary Islands, Spain). The tobacco plants were found lightly infested by the whitefly (Hemiptera: Aleyrodidae) Bemisia tabaci. The greenhouses in this area are devoted to the commercial production of tomato. The farmers grow some tobacco plants inside and outside of them as a reservoir of parasitoids and depredators of B. tabaci. This insect is the natural vector of the main viruses severely affecting tomato in the Canary Islands, the begomovirus Tomato yellow leaf curl virus and the crinivirus Tomato chlorosis virus (ToCV). ToCV was detected in Spain in 1997 (2) and has become established in most of the coastal provinces of eastern and southern continental Spain and in the Canary Islands. Approximately 50% of the tomato plants grown inside the greenhouse close to the tobacco plants showed typical ToCV symptoms, and infection by this virus was confirmed in the seven plants tested by reverse transcription (RT)-PCR using specific coat protein gene (CP) primers (see below). Total RNA was extracted with TRIzol Reagent (Invitrogen) from leaves of five tobacco plants showing the symptoms mentioned above and analyzed by dot-blot hybridization using digoxigenin-labeled RNA probes to the CP gene of ToCV. Positive signal was obtained for all five plants. RT-PCR reactions were performed with specific primers for the detection of ToCV, MA380(+) (5'-GTGAGACCCCGATGACAGAT-3') and MA381(-) (5'-TACAGTTCCTTGCCCTCGTT-3'), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5'-TGGTCGAACAGTTTGAGAGC-3') and MA397(-) (5'-TGAACTCGAATTGGGACAGA-3'), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected size (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. The amplified product of the RdRp gene fragment from one sample was directly sequenced (Macrogen Inc., South Korea) and resulted closely related to ToCV isolates from Sudan (GenBank Accession No. JN411686, 99.6% nt identity) and Spain (DQ983480, 99.4% nt identity), thereby confirming the infection by this virus. Partial sequence of the ToCV isolate from tobacco was deposited in GenBank under accession no. KJ175084. In addition, all five plants resulted positive when analyzed by ELISA for Tomato spotted wilt virus and Potato virus Y and by PCR for Tomato yellow leaf curl virus (data not shown), all three viruses reported to infect naturally tobacco. Although tobacco has been reported as an experimental host of ToCV (4), to our knowledge, this is the first report of this species as a natural host of this virus. The finding of ToCV infecting tobacco raises the question of whether this virus could emerge as a pathogen of this crop and questions the use that farmers make of tobacco as reservoirs of natural enemies for whitefly control in tomato.

Insights into the genetic diversity and the phylogenetic analysis of Tunisian isolates of Tomato chlorosis virus

A one-step RT-PCR was used to identify Tomato chlorosis virus (ToCV) (Crinivirus members) infections on some Tunisian cultures. Results provided evidence that the ToCV is the most prevalent virus on Tunisian tomato crops. Findings were validated with specific primers. Genetic analysis of ToCV isolates was explored based on sequence data of viral segments within the coat protein (CP), the heat shock protein (HSP70h) and the polymerase protein (RdRp). Synonymous (dS) and non-synonymous (dN) substitution rates and their ratio were analyzed. The patterns of mutations were shaped depending on the considered fragment from the three viral regions. Selective neutrality test was significantly negative, suggesting a recent expansion of ToCV isolates. Pairwise mismatch distribution gave a bimodal pattern and pointed to the clustering of ToCV isolates into two distinct geographical clades. Genetic haplotype network provided evidence of the existence of two distinct clusters. The star-like shaped pattern confirmed recent expansion of ToCV isolates.

The Complete Nucleotide Sequence of the RNA 1 of a Chinese Isolate of Tomato chlorosis virus

AbstractInterveinal leaf chlorosis, brittleness, limited necrotic flecking or bronzing developed on greenhouse‐grown tobacco and tomato plants at Nanjing Agricultural University from 2010 to 2013. A positive RT‐PCR using a pair of degenerate primers for Crinivirus confirmed the diseased plants were infected with Tomato chlorosis virus (ToCV). The complete RNA 1 genomic sequence of this ToCV isolate was determined; it comprises of 8596 nucleotides with four open reading frames. Phylogenetic analysis of ToCV isolates from diverse geographical regions categorized the ToCV isolates into two main groups. Group one consisted of Chinese, American‐Florida, Greek and Brazilian isolates, while Group two contained only the Spanish isolate. The first group had two subgroups, one of Chinese and American‐Florida isolates, while the other subgroup had Greek and Brazilian isolates. This is the first study of the complete nucleotide sequence of the RNA 1 of ToCV isolated from China.

First Report of Tomato chlorosis virus in China.

In October 2012, a severe yellowing disease was found on greenhouse and plastic house tomato (Solanum lycopersicum) plants in Beijing, China. The disease incidence varied from 5 to 80% in each of six fields across Haidian and Daxing districts. The lower leaves showed symptoms of interveinal chlorosis, leaf brittleness, and limited brown necrotic flecks, similar to symptoms induced by Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) (two members of genus Crinivirus, family Closteroviridae) (4). A large number of whiteflies (Bemisia tabaci) were also observed. Leaf samples were taken from eight symptomatic and two asymptomatic tomato plants in two plastic houses in the Haidian district and total RNA was isolated from the 10 samples using TRIzol reagent (Tiangen, Beijing, China). Nested reverse transcription (RT)-PCR was performed to test the presence of ToCV and TICV with degenerate primers HS-11 and HS-12 and specific primers ToC-5/ToC-6 or TIC-3/TIC-4 for ToCV or TICV, respectively (1). With ToCV primers, a 463-bp specific fragment was amplified from eight symptomatic samples but not from two asymptomatic samples, and there was no amplification with TICV primers from any sample. Sequence analysis of the amplified fragment showed 99% nucleotide sequence identity with the heat shock protein 70 homolog (HSP70h) gene of ToCV isolates from Japan (GenBank Accession No. AB513442), Spain (DQ136146), Florida (AY903448), and Greece (EU284744). The presence of ToCV was confirmed by amplification of a 848-bp fragment covering the coat protein (CP) gene of ToCV with primers CP-F (5'-GAATCTTTTAGAAGCTTTGGTTTAAGG-3') and CP-R (5'-GATCCTCTTGATCCTCATAGATTTC-3') (3). The CP had 97 to 99% amino acid sequence identity to the above-mentioned four ToCV isolates. A sequence of the CP gene obtained from one isolate was deposited at GenBank (KC311375). Additionally, virions were isolated from 25 g of symptomatic samples followed Klaassen's method (2) and their lengths were estimated to be about 800 to 850 nm by transmission electronic microscopy To our knowledge, this is the first report of ToCV on tomato in mainland China. Tomato is one of the most widely cultivated crops in China and the spread of ToCV in China may cause significant economic losses. Further information on the prevalence and incidence of ToCV is required to assess the potential impact of this virus.

Low Genetic Diversity Suggests a Single Introduction and Recent Spread of Tomato chlorosis virus in Brazil

By comparing the partial nucleotide sequences of the heat shock protein HSP70 homologue gene, we assessed the genetic diversity of Brazilian tomato isolates of Tomato chlorosis virus (ToCV), as well as their relationship with other ToCV isolates found worldwide. The Brazilian ToCV isolates shared 99.9–100% nucleotide identity, which indicates low genetic diversity. Brazilian ToCV isolates showed a closer evolutionary relationship to those from Mediterranean countries. Based on these results, the origin of Brazilian ToCV isolates and the possible number of introductions of the virus into Brazil are discussed.

First Report of Tomato chlorosis virus Infecting Tomato in Sudan.

In March 2011, interveinal yellowing and necrosis symptoms on middle and lower leaves were observed in tomato (Solanum lycopersicum L., cv. Castle Rock) plants grown in three adjacent greenhouses of the Agricultural Research Corporation at Wad Medani (Gezira State, Sudan). These symptoms resembled those caused by Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) (4) (genus Crinivirus, family Closteroviridae). Whitefly (Bemisia tabaci) infestation was also observed in these greenhouses. Total RNA was extracted by TRIzol Reagent (Invitrogen, Carlsbad, CA) from symptomatic leaves and analyzed by dot-blot hybridization with digoxigenin-labelled RNA probes to the coat protein (CP) gene of ToCV and to the minor coat protein (CPm) gene of TICV. Positive signal was obtained only with the ToCV probe. Reverse transcription (RT)-PCR reactions were performed with two pairs of primers specific for the detection of ToCV, MA380(+) (5'-GTGAGACCCCGATGACAGAT-3') and MA381(-) (5'-TACAGTTCCTTGCCCTCGTT-3'), specific to the CP gene (ToCV RNA 2) (3), and MA396(+) (5'-TGGTCGAACAGTTTGAGAGC-3') and MA397(-) (5'-TGAACTCGAATTGGGACAGA-3'), specific to the RNA-dependent RNA polymerase (RdRp) gene (ToCV RNA 1) (1). DNA fragments of the expected sizes (436 and 763 bp, respectively) were obtained, thus supporting the presence of ToCV in the symptomatic samples. Amplified DNA fragments were cloned in pGEM-T Easy vector (Promega, Madison, WI) and one clone per amplicon was sequenced (Macrogen Inc., Seoul, South Korea). The highest nucleotide sequence identity of the CP gene fragment obtained (GenBank Accession No. JN411685) was 99.2% related with North American ToCV isolates from Florida (DQ234674), Colorado (DQ234675), and Georgia (HQ879842), while the RdRp gene fragment (JN411686) was more closely related (99.0%) to the Spanish AT80/99 isolate (DQ983480). Although yellowing symptoms similar to those reported here have been observed sporadically during the last few years in open-field tomato crops in the state of Gezira, additional studies are needed to determine the prevalence and economic impact of ToCV infections in tomato cultivation in Sudan. To our knowledge, ToCV has been found in continental Africa only in Morocco and South Africa, in the Mediterranean climate areas in the northern and southern edges of the continent, respectively (2). The finding of ToCV infecting tomato in Sudan raises the question of whether this virus is emerging also in other tropical areas of the continent and illustrates the need to monitor whitefly-infested areas within Africa for the presence of ToCV.