Tomato Infectious Chlorosis Virus Research Articles

Natural occurrence of tomato chlorosis virus on tomatillo (Physalis philadelphica) in the United States.

Tomatillo (Physalis philadelphica L.) is an annual plant native to Mexico and Guatemala, and cultivated in other tropical and subtropical regions. In October 2023, tomatillo plants with interveinal yellowing of leaves, marginal chlorosis, leaf thickening, and leaf rolling symptoms (Figure 1) were observed at Colquitt and Tift County, Georgia, US. The disease incidence ranged from 80-100 % which reduced fruit quality and marketability. Twenty tomatillo leaves exhibiting severe symptoms were collected, and, sub-sampled of the leaves were pooled into microcentrifuge tubes. Further, MagMAX 96 viral RNA isolation kit (Thermo Fisher Scientific, US), was used for the extraction of (n=4) total nucleic acid (TNA) (Kavalappara et al. 2021). Symptomatic leaves were tested for the presence of insect-transmitted viruses such as begomovirus (tomato yellow leaf curl virus, TYLCV), potyvirus (turnip mosaic virus, TuMV), crinivirus (tomato infectious chlorosis virus, TICV; tomato chlorosis virus, ToCV), and tospovirus (orthotospovirus tomatomaculae, TSWV). Polymerase chain reaction (PCR) was performed for detecting TYLCV, using gene-specific primers (Kumar et al., 2023). However, for ToCV, TuMV and TICV detection, cDNA was prepared using 100 ng of TNA as a template, followed by the PCR ( Liu et al., 2012). Moreover, the detection of TSWV was conducted using immuno-strips (Adgia, US) following the manufacturer's instructions. ToCV was detected from all the tested samples, while TuMV, TICV, TYLCV and TSWV were not detected in any symptomatic tissues. In addition, RT-PCR was performed using gene-specific primers targeting the RNA-dependent RNA polymerase (RdRP) gene and the heat-shock protein 70 (Hsp70) gene of ToCV. The PCR amplicon of 439 bp encoding Hsp70 and 643 bp corresponding to RdRP was gel-purified and Sanger sequenced (Azenta Life Sciences, US). BLASTn analysis shows RdRP gene from ToCV-tomatillo (OR905600) has 100 % identity with ToCV of RNA1 segment (RdRP, GenBank accession no. AY903447, Florida, US), while Hsp70 gene (OR900219) has 100 % identity with ToCV of RNA2 segment (Hsp70, GenBank accession no. LC778246, Cairo, Egypt). In addition, the symptomatic tomatillo leaves were studied for transmission assay using tomato, employing non-viruliferous whiteflies (Bemisia tabaci) with 48 h of acquisition access period. Further, two weeks post-infection, the presence of ToCV was detected from the test plants while other whitefly-transmitted viruses remins undetected. In 2023, ToCV is widespread in tomato-growing counties, infecting commercially grown tomato cultivars with intermediate resistance against TYLCV-IL (Israel strain). However, tomatillo plants infected with TuMV in California (Liu et al., 2012), TSWV in Georgia, (Díaz-Pérez and Pappu 2000) and TYLCV in Mexico (Gámez-Jiménez et al. 2009) were reported. This study suggests that tomatillo could be a permissive host for ToCV while restrictive to other prevalent viruses in the region. A recent investigation speculates a potential synergistic interaction between ToCV and TYLCV-IL, exacerbating the breakdown of host resistance in tomato (Fiallo-Olivé et al. 2019, Kumar et al. 2023). To the best of our knowledge, this is the first report for the natural incidence of ToCV on tomatillo within the US. The findings will contribute to developing more effective management strategies against emerging viral threats.

Development of a RT-LAMP assay for real-time detection of criniviruses infecting tomato

Yellowing symptoms caused by tomato chlorosis virus (ToCV) and tomato infectious chlorosis virus (TICV), both assigned to the genus Crinivirus, resemble nutrient deficiencies. Therefore, early diagnosis of infections will prevent crop damage and the spread of the viruses. In this study, we established a rapid detection method for ToCV and TICV by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). We first designed primer sets for RT-LAMP specific for ToCV and TICV. Next, by selecting the optimum primer set and determining the optimum conditions for the RT-LAMP reaction, each virus was detected within 50 min by piercing the diseased area of a tomato leaf with a toothpick, immersing the toothpick in the reaction solution, and conducting the RT-LAMP reaction. To verify the accuracy of the procedure, 61 tomato leaf samples showing disease symptoms were collected from five regions of Indonesia, and the RT-LAMP results for the samples were identical to those obtained with the commonly used reverse transcription-polymerase chain reaction.

First Report of Tomato Brown Rugose Fruit Virus on Greenhouse Tomato in Syria.

Tomato (Solanum lycopersicum L.) is an important vegetable crop worldwide. In spring and autumn 2017, virus-like symptoms were observed on greenhouse grown tomato plants in the east of Akkar plain (south of coastal region, Tartous governorate, Syria). These symptoms were: mild to severe mosaic on the apical leaves, brown necrosis on sepals, receptacle and flower's cluster carrier, and severe symptoms of brown rugose and discoloration on fruit. During next growing seasons, disease spread was observed in most of Syrian coastal region with disease incidence ranged from 40% to 70% by 2020. Tomato brown rugose fruit virus (ToBRFV) was suspected as a main causal agent of the disease, especially since its first report in Jordan, a neighboring country (Salem et al. 2016), Palestine (Alkowni et al. 2019), Turkey (Fidan et al. 2019), Germany (Menzel et al. 2019), Italy (Panno et al. 2019), America (Camacho-Beltrán et al. 2019), Egypt (Amer and Mahmoud, 2020), and recently in Spain (Alfaro-Fernandez et al. 2021). In November and December 2020, seventy-one leaf samples from symptomatic plants (59 from Tartous and 12 from Lattakia governorates) and seven from asymptomatic ones (5 from Tartous and 2 from Lattakia) were collected and tested for the presence of ToBRFV by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using ToBRFV-commercial kit (LOEWE® Biochemia, Germany) following the manufacturer's instructions. Results showed, forty-three of symptomatic samples reacted positively (38 in Tartous and 5 in Lattakia) and none of asymptomatic ones. On the other hand, sap mechanical inoculation of 10 tomato cv. Mandaloun F1 (Enza Zaden, the Netherlands) plants using a positive tomato isolate gave systemic mosaic symptoms in all plants identical to those observed in the original plants in the field, after 13 days of inoculation, and necrotic local lesions on 10 plants of Nicotiana tabacum after 5 days, indicating the presence of a tobamovirus in general. ToBRFV infection was confirmed in all mechanically-inoculated plants by DAS-ELISA. Further tests were necessary to investigate ToBRFV presence, because of its serological relationships with another tobamoviruses. Six representative symptomatic samples (ELISA-positive) and two asymptomatic (ELISA-negative) samples were subjected to total RNA extraction using the SV-Total RNA Extraction kit (Promega, U.S.A.) following the manufacturer's instructions. The samples were tested by two-step reverse transcription-polymerase chain reaction (RT-PCR) using species-specific primers and protocols for most common tomato-infecting viruses, including: tomato chlorosis virus and tomato infectious chlorosis virus (Dovas et al. 2002), pepino mosaic virus (PepMV) and tomato torrado virus (Wieczorek et al. 2013), alfalfa mosaic virus (Parrella et al. 2000), tomato spotted wilt virus (Salem et al. 2012) and a pair of primers: ToBRFV-F2 (5'-CATATCTCTCGACACCAGTAAAAGGACCCG-3') and ToBRFV-R2 (5'-TCCGAGTATAGGAAGACTCTGGTTGGTC-3') targeting a region of the RNA dependent RNA polymerase (RdRp), of the ToBRFV genome (KT383474; Salem et al. 2016). First-strand cDNA synthesis was carried out using Moloney murine leukemia virus reverse transcriptase (M-MLV RT; Promega) and random primer according to the manufacturer's protocol, then followed by PCR with the seven species-specific primers. Only ToBRFV was detected among all tested viruses in symptomatic samples (ELISA-positive), and none of the tested viruses was detected in the asymptomatic plants. To confirm the presence of ToBRFV, two selected RdRp-specific PCR amplicons (872 bp) were purified and ligated into pGEM T-Easy Vector (Promega), and three clones were sequenced (GenBank accession nos. MZ447794 to 96). BLASTn analysis showed that the nucleotide sequences are 99.77-100% identical and shared around 99% identity to RdRp of ToBRFV isolate (MT118666) from Turkey available in the GenBank. Accordingly, the presence of ToBRFV was confirmed by bioassays on indicator plants, DAS-ELISA, RT-PCR, and further sequencing. To our knowledge, this is the first report of ToBRFV infecting tomato in Syria, and this requires special emphasis for further investigations because of the virus severity, easy transmission ability and absent of commercial resistance varieties till now.

Simultaneous detection of 10 viruses in globe artichoke by a synthetic oligonucleotide-based DNA polyprobe

Most of the 26 viruses that infect globe artichoke are detrimental to the crop’s production, quality, and nutritional value, hamper the development of a nursery activity in the respect of current EU legislation and pose risks for the new plantings. Worldwide movement of virus-controlled plant propagative material require fast, accurate and feasible methods to guarantee reliable results and detection of multiple viruses in a single assay to reduce time and costs in routine tests. Here we describe the use of a digoxigenin-labelled polyprobe assembled from synthetic 50 nt single-stranded DNA sequences derived from genomes of the most commonly occurring viruses in globe artichoke. These included artichoke Italian latent virus (AILV), artichoke latent virus (ArLV), artichoke mottled crinkle virus (AMCV), turnip mosaic virus (TuMV), tomato infectious chlorosis virus (TICV), bean yellow mosaic virus (BYMV), cucumber mosaic virus (CMV), pelargonium zonate spot virus (PZSV), tomato spotted wilt virus (TSWV) and tobacco mosaic virus (TMV). The polyprobe described herein represent the first cheap and reliable example of a technology for broad-spectrum surveys in globe artichoke.

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.

Multiplex RT-PCR Assay for Crinivirus Detection Using RNA Prepared from Three Extraction Methods on Tomato Plant

Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) are members of the Crinivirus genus that induces yellowing symptoms in tomato plants. Detection of both viruses is generally carried out singly, thus it is necessary to develop a fast, accurate and efficient detection method to detect multiple viruses simultaneously in an effort to determine the suitable disease management strategies. This study was aimed to detect both viruses using the multiplex RT-PCR method and evaluate three methods of total RNA preparation used from tomato plants as RT-PCR templates. The methods evaluated were simple direct tube (SDT), simple dsRNA, and commercial kit (RNeasy Plant Mini Kit) as a comparison. The total source of RNA came from Crinivirus symptomatic tomato leaves from Kopeng, and Ketep (Central Java); Pakem (Yogyakarta); Malang (East Java); and Bogor (West Java). Single RT-PCR and multiplex RT-PCR using specific primers CPd I/CPd II and ToCV CF/ToCV CR with DNA band targets of 760 bp and 360 bp. The SDT and dsRNA methods have been successful in obtaining total RNA and viral RNA from tomato leaf samples. Total RNA RT-PCR with simple SDT and dsRNA methods followed by multiplex RT-PCR produces specific DNA band intensities that are comparable to Kit. RNA preparation with SDT and simple dsRNA methods is a simple, fast, easy and affordable method in providing templates for RT-PCR. Multiplex RT-PCR technique using two pairs of specific primers CPd I/CPd II and ToCV CF/ToCV CR is suitable for simultaneous testing of TICV and ToCV.

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.

Occurrence of Tomato yellow leaf curl virus and Tomato chlorosis virus mixed infections in protected tomato plants, Antalya, Turkey

During the autumn of 2011, upward leaf cupping, curling, chlorosis on the upper leaves, and interveinal chlorosis on the lower leaves of some protected tomato plants was observed in Antalya, Turkey. These symptoms indicated possible virus infection in tomato plants. Due to the symptoms, these plants could possibly have Tomato yellow leaf curl virus (TYLCV), Tomato infectious chlorosis virus (TICV), and/or Tomato chlorosis virus (ToCV) infections. Consequently, 300 greenhouses were surveyed. Because there are two cultivation periods, spring and autumn, in Antalya, the study was conducted over two periods. No mixed infections were detected during the spring season, however in autumn cultivation symptomatic tomato leaf samples from 17 greenhouses, tested with enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), were found to have mixed infections of TYLCV and ToCV. This indicated that TYLCV/ToCV mixed infection was present in about 9.3% of the greenhouses. The symptoms ranged from moderate (slight curling in upper leaves and slight leaf chlorosis in lower leaves) to severe (severe curling and extensive chlorosis in upper leaves, stunting, greatly reduced fruit set, and conspicuous chlorosis in lower leaves). This is the first report of TYLCV/ToCV mixed infections in Turkey.

The p27 open reading frame of tomato infectious chlorosis virus encodes a suppressor of RNA silencing

Tomato infectious chlorosis virus (TICV) belongs to the genus Crinivirus in the family Closteroviridae. We used Agrobacterium co-infiltration leaf patch assays to investigate the suppression of RNA silencing by p27, which is encoded by TICV-RNA1 ORF2 in a genomic location similar to that of viral suppressor of RNA silencing (VSR) ORF in other criniviruses. Additionally, Nicotiana benthamiana was inoculated with a potato virus X vector (pGR106) carrying the p27 sequence (pGR106–p27) to examine whether p27 is a VSR. Plants infiltrated with pGR106–p27 exhibited more severe necrosis and mosaic symptoms. This is the first report describing a TICV VSR.

A Protocol for Producing Virus-Free Artichoke Genetic Resources for Conservation, Breeding, and Production

The potential of the globe artichoke biodiversity in the Mediterranean area is enormous but at risk of genetic erosion because only a limited number of varieties are vegetatively propagated and grown. In Apulia (southern Italy), the Regional Government launched specific actions to rescue and preserve biodiversity of woody and vegetable crops in the framework of the Rural Development Program. Many globe artichoke ecotypes have remained neglected and unnoticed for a long time and have been progressively eroded by several causes, which include a poor phytosanitary status. Sanitation of such ecotypes from infections of vascular fungi and viruses may be a solution for their ex situ conservation and multiplication in nursery plants in conformity to the current EU Directives 93/61/CEE and 93/62/CEE that enforce nursery productions of virus-free and true-to-type certified stocks. Five Apulian ecotypes, Bianco di Taranto, Francesina, Locale di Mola, Verde di Putignano and Violetto di Putignano, were sanitized from artichoke Italian latent virus (AILV), artichoke latent virus (ArLV) and tomato infectious chlorosis virus (TICV) by meristem-tip culture and in vitro thermotherapy through a limited number of subcultures to reduce the risk of “pastel variants” induction of and loss of earliness. A total of 25 virus-free primary sources were obtained and conserved ex situ in a nursery.

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.

FIRST REPORT OF TOMATO INFECTIOUS CHLOROSIS VIRUS IN GLOBE ARTICHOKE ECOTYPES IN APULIA, SOUTHERN ITALY

Apulia (southern Italy) hosts a rich variety of globe artichoke ecotypes of high interest for breeding programs and industrial production. Most of the ecotypes are at risk of genetic erosion because of the high incidence of plant pathogens, which include infections from taxonomically different viruses. In the early autumn of 2014 and 2016 five samples were collected from each of the early-flowering ecotypes, Brindisino, Carciofo di Lucera, Francesina di Brindisi, Francesina di Trinitapoli, Locale di Mola, Nero di Ostuni, Tricasino, Troianella, Verde di Putignano, Violetto di Putignano and the late-flowering ecotypes Bianco di Taranto and Locale di Mola tardivo, and tested for viral infections by dot blot hybridization with Dig-labeled DNA probes and RT-PCR (Minutillo et al., 2012). In the five samples of the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella the hybridization signal revealed the presence of tomato infectious chlorosis virus (TICV) that in the ecotype Troianella was in mixed infection with artichoke Italian latent virus and turnip mosaic virus. TICV infection was confirmed by RT-PCR with TICV-specific primer pair for 5'- TCAGTGCGTACGTTAATGGG-3' and rev 5'-CACAGTATACAGCAGCGGCAG-3' (Minutillo et al. 2012). Amplicons from the ecotypes Verde di Putignano and Troianella were sequenced. Nucleotide sequence analyses revealed 99% identity with a TICV isolate from USA (accession No. FJ542306). Previous surveys detected TICV in globe artichoke in Liguria, Sardinia, Latium, Campania and Sicily but this is the first report of TICV in artichoke in Apulia and in the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella. Artichoke plants infected by TICV did not show symptoms and thus may act as perennial TICV reservoirs.

MOLECULAR CHARACTERIZATION AND NATURAL HOST RANGE OF TOMATO CHLOROSIS VIRUS IN SAUDI ARABIA

Tomato yellows disease (TYD) is a serious problem in tomato crops worldwide, in which two criniviruses are implicated: Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV). During the growing season 2014-2015, survey of tomato-growing greenhouses conducted in different geographical locations of Riyadh region in Saudi Arabia in order to identify the criniviruses implicated in TYD. In total, 228 leaf samples were collected from tomato plants showing typical TYD symptoms, as well as 98 from other symptomatic crops and 283 arable weeds belonging to 38 species in 22 different families. Nucleic acids extracts prepared from the samples were tested by nested RT-PCR for the simultaneous detection of TICV and ToCV. In addition, 34 adult whiteflies were collected from the surveyed tomato greenhouses and analyzed using real time PCR. Results revealed that ToCV was the only virus associated with TYD as detected in 328 (53%) of a total of 609 tested samples (including all hosts tested), while it showed an expanded host range, as it was reported for the first time in bean, bitter gourd, eggplant and pepper in Saudi Arabia. In addition, ToCV was detected in 14 weed species belonging to 8 families. TICV was not detected in any of the tested samples. All collected whiteflies were identified as Bemisia tabaci biotype B (MEAM1), an efficient vector of ToCV. Sequencing analysis of 40 selected sequences of ToCV from different hosts clustered Saudi Arabian isolates into two subgroups. Almost all Saudi Arabian isolates shared high nucleotide identity among themselves (98.0-100%) while 97.1-100% with those published in NCBI.

Occurrence of artichoke‐infecting viruses in Tunisia

Crops of globe artichoke (Cynara scolymus) in Manouba and Bizerte regions, Tunisia, were surveyed in spring and autumn 2012 for the presence of key artichoke viruses. A total of 336 leaf samples were collected randomly from plants that were either asymptomatic or showing virus‐like symptoms. These were tested for Artichoke Italian latent virus (AILV), Cucumber mosaic virus (CMV), Artichoke mottled crinkle virus (AMCV), Pelargonium zonate spot virus (PZSV), Artichoke latent virus (ArLV), Bean yellow mosaic virus (BYMV), Turnip mosaic virus (TuMV), Potato virus X (PVX), Tomato infectious chlorosis virus (TICV) and Tomato spotted wilt virus (TSWV). Results from dot‐blot hybridization and reverse transcription polymerase chain reaction (RT‐PCR) showed the occurrence of AILV and BYMV for the first time in Tunisia, and confirmed that of TICV on artichoke crops. ArLV and AILV were the most frequent viruses detected, whereas infections of AMCV, CMV and PVX had a lower incidence. PZSV, TuMV and TSWV were not detected in any of the samples collected. Notable differences were observed between results from spring and autumn surveys, probably as a consequence of the different vegetatives status of the plants and different leaf sample conditions (fresh versus dried). Consequences of the import of planting material with a poor sanitary status and of the overall phytosanitary status of globe artichoke in Tunisia are discussed.

False Yellowhead (Dittrichia viscosa), A Banker Plant as Source of Tomato infectious chlorosis virus in Greece

False Yellowhead (<i>Dittrichia viscosa</i>), A Banker Plant as Source of <i>Tomato infectious chlorosis virus</i> in Greece

EKSPRESI GEN PROTEIN SELUBUNG TOMATO INFECTIOUS CHLOROSIS VIRUS PADA ESCHERICHIA COLI

Expression of tomato infectious chlorosis virus coat protein gene on Escherichia coli. Tomato infectious chlorosis virus (TICV) is the causal agent of chlorotic disease of tomato. Detection of TICV can be carried out by RT-PCR and serological test. Titer of TICV in plant tissue is very low because TICV is limited to phloem. Serological detection of TICV requires antiserum which is not available in Indonesia. Producing antibody through cloning and coat protein gene (TICV CP gene) expression is a promising approach in producing antiserum. The objective of this study was to express TICV CP gene as antigen for antiserum production. TICV CP gene was amplified using RT-PCR from total RNA extracted from TICV infected leaves collected from Cipanas, Cianjur, West Java. The amplified CP gene was then sequenced and sub-cloned into pET 21b expression vector, transformed into Escherichia coli strain BL21 DE3(pLysS) and induced expression using IPTG 1 mM overnight at 37 °C. CP that contains 6xhistag was purified using NiNTAspin column and then confirmed by SDS-PAGE. The size of TICV CP gene was 750 bp and the gene was expressed on pET 21 b vector and SDS-PAGE showed a 29 kDa band.

Tomato infectious chlorosis virus. [Distribution map].

Abstract A new distribution map is provided for Tomato infectious chlorosis virus. Closteroviridae: Crinivirus. Main host: tomato ( Solanum lycopersicum ). Information is given on the geographical distribution in Europe (Bulgaria, France, Greece, Crete, Italy, Sardinia, Sicily and Spain), Asia (Indonesia, Japan, Honshu, Jordan and Taiwan), Africa (Tunisia) and North America (Mexico, USA, California and North Carolina).

IDENTIFIKASI TOMATO INFECTIOUS CHLOROSIS VIRUS PENYEBAB PENYAKIT KLOROSIS PADA TANAMAN TOMAT DI CIPANAS JAWA BARAT MELALUI PERUNUTAN NUKLEOTIDA GEN PROTEIN SELUBUNG UTAMA

Identification of tomato infectious chlorosis virus, the causal agent of chlorosis disease on tomato in Cipanas West Java by sequencing of main coat protein gene nucleotide. Tomato infectious chlorosis virus (TICV) causes chlorosis on tomato. Tomatoes infected by this virus shows interveinal yellowing, necrotic, bronzing, brittleness, and declining in productivity. This study aims to identify the causal agent of chlorotic disease on tomato by sequencing the coat protein gene. The methods involve collecting infected plants, total RNA extraction, cDNA synthesis, DNA amplification, visualization of the results of reverse transcription polymerase chain reaction (PCR), and phylogenetic analysis using BLAST, clustal w, Bioedit v 7.0.5.3, MEGA v 6:06. RT-PCR using spesific primers (CP-F TICV Bam and TICV R-Hind) amplified a DNA band of 792 bp, which has been successfully sequenced and identified as TICV. Nucleotide sequences homology analysis showed that TICV Indonesia_TWJ isolate Cipanas is the same strain as TICV from other countries (99.4 – 100%), such as Spain, Greece, USA, France, and Italy.

First Report of Tomato chlorosis virus (ToCV) in Tomato Crops in Saudi Arabia

During January 2014, open field and greenhouse tomato (Solanum lycopersicum L.) crops in the peripheral areas of Riyadh region (Al-Aflaj, Al-Kharj, Al-Waseel, and Al-Dalam), Saudi Arabia, were surveyed. In all surveyed tomato crops, yellowing symptoms were observed on the lower leaves, possibly infected by a whitefly transmitted crinivirus (family Closteroviridae) such as Tomato chlorosis virus (ToCV) and/or Tomato infectious chlorosis virus (TICV). Dense population of whiteflies (Bemisia tabaci G.) were present in all affected plants. Incidence of the yellowing disease varied between four greenhouses and three open field tomato crops, but in the majority of the tomato crops surveyed, symptoms typical of Begomovirus infection such as severe stunting, degeneration, upward cupping, distortion and interveinal yellowing of upper leaves, and flower abortion were also observed. Tomato yellow leaf curl virus (TYLCV) is endemic in Saudi Arabia causing severe crop losses (1). Twenty-six leaf samples from 24 symptomatic and two asymptomatic plants from four fields (three greenhouses and one open field crop) were collected and were processed in the lab at King Saud University. Whitefly transmission on tomato indicator plants was carried out using B. tabaci to fulfill Koch's postulates. Two hundred virus-free B. tabaci adults were confined to one of the collected symptomatic tomato sample singly infected with ToCV for a 48-h acquisition access period, followed by a 48-h inoculation access period on five healthy tomato plants Hybrid Super Strain B, using 40 whiteflies per plant. Crinivirus detection following transmission was conducted by RT-PCR. Total RNA was extracted from 26 collected leaf samples using the Total RNA Purification Kit and analyzed by SCRIPT One-Step RT-PCR Kit (Jena Bioscience). First, the degenerate primers HS-11/HS12 were used for amplification of a 587-bp fragment of the HSP70 gene of ToCV and TICV (3). Second, the RT-PCR product was subjected to a nested PCR using specific primers TIC-3/TIC-4 and TOC-5/TOC-6, for the detection of both TICV and ToCV, respectively (2). Finally, degenerate primers (AV494/AC1048) were used for detection of begomoviruses (4). No fragment was amplified by TIC-3/TIC-4 primer whereas TOC-5/TOC-6 amplified a size of 463 bp in all 24 symptomatic tested samples, including one mixed infection with TYLCV detected by AV494/AC1048. Asymptomatic samples did not produce any amplicon regarding TICV, ToCV, and Begomovirus detection. The amplicons of four positive fragments, each from one field, were further sequenced in both directions and all obtained sequences (KJ433488, KJ433489, KJ433490, and KJ433491) analyzed with BLAST and revealed 99% identity with the most closely deposited sequences in NCBI from Japan (AB513442) and Brazil (JQ952601). In the transmission tests, ToCV was detected to all tomato indicator plants which revealed yellowing symptoms 6 weeks post inoculation, whereas no transmission was obtained when non-viruliferous whitefly adults fed on two asymptomatic tomato leaves. To our knowledge, this is the first report of ToCV infecting tomato crops in Saudi Arabia. Further studies are being carried out to study epidemiology and genetic diversity of this virus associated with yellowing diseases of tomato in different regions of Saudi Arabia. This finding is important for the tomato crops and possibly other virus hosts as may cause serious epidemics and crop losses.

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.