Whitefly-transmitted Crinivirus Research Articles

First report of paprika as a natural host plant for tomato chlorosis virus in Korea.

Paprika (Capsicum annuum var. grossum (L.) Sendtn.), also called bell pepper or sweet pepper, is a valuable greenhouse crop that is mostly consumed as fresh fruit in Korea. In 2022, it was cultivated on 726 ha in Korea and 82,042 tons were produced (KOSIS, 2023). In April 2023, interveinal chlorosis and yellowing were observed on the lower and middle leaves of greenhouse paprika in Jinju and Haman, South Korea, and many whiteflies (Bemisia tabaci) were present. The disease incidence was 30-40% on five farms (approximately 4.5 ha) in the two areas. The typical symptoms and presence of whiteflies in greenhouses indicated potential infection by the whitefly-transmitted crinivirus, tomato chlorosis virus (ToCV). Six samples collected from symptomatic plants were examined by leaf dip using transmission electron microscopy (TEM) and revealed filamentous virus particles about 800 nm long. To confirm the TEM results, six symptomatic paprika leaf samples were subject to RT-PCR using the specific primers ToCV-M-4F and ToCV-M-4R to detect ToCV (Choi, 2023). All tested samples were positive for ToCV. To confirm the presence of other viruses and obtain the complete genome sequences, one of the six ToCV-positive samples (ToCV-PAP-JJ6) was subject to high-throughput sequencing (HTS). Total RNA was extracted from symptomatic leaves using the RNeasy Plant Mini Kit (QIAGEN, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to standard protocols. HTS was performed on an Illumina NovaSeq 6000 system (Macrogen, Korea). De novo transcriptome assembly of the 85,668,854 reads with Trinity software (r20140717) yielded 817,303 contigs of 201 to 38,987 nucleotides (nt). BLASTn and BLASTx analysis of the contigs against the NCBI viral reference database showed that three large contigs were virus sequences, two of which were homologous to ToCV-RNA1 and -RNA2 and one to bell pepper alphaendornavirus (BPEV). The ToCV-RNA1 (genome 8,594 nt, 11,212 mapped reads, mean read coverage 196.2 times) and ToCV-RNA2 (genome 8,242 nt, 40,122 mapped reads, mean read coverage 734.9 times) contigs showed 100% coverage and 99% base pair matching (8,583/8,594 and 8,233/8,242, respectively) with known genome sequences of ToCV-RNA1 (MG813908) and ToCV-RNA2 (KP114534) isolated from tomato in Korea (Lee et al. 2018). The sequences were deposited in GenBank as isolates ToCV-PAP-JJ6 RNA1 (OR865222) and ToCV-PAP-JJ6 RNA2 (OR865223). The BPEV contig (genome 14,728 nt, 2,928,519 mapped reads, mean read coverage 15,012.4 times) was detected at very high read depth and RT-PCR for BPEV confirmed that all six samples were coinfected with ToCV. BPEV was recently reported in paprika and red pepper in Korea (Jo et al., 2022). The BPEV-infected pepper cultivars have been reported not to produce any symptoms (Escalante & Valverde 2019). ToCV was first reported in tomato greenhouses in Korea in 2013 and is a problematic virus in tomato crops, along with tomato yellow leaf curl virus (Jo et al. 2023; Lee et al. 2018). Due to the continuous presence of ToCV, whiteflies, and natural weed hosts (Kil et al. 2015) in tomato greenhouses, ToCV has spread to other crops, including paprika. To the best of our knowledge, this is the first report of ToCV infecting paprika in Korea. A nationwide study is needed to prevent the spread of ToCV, a new threat to paprika farms.

Enhanced Susceptibility to Tomato Chlorosis Virus (ToCV) in Hsp90- and Sgt1-Silenced Plants: Insights from Gene Expression Dynamics.

The emerging whitefly-transmitted crinivirus tomato chlorosis virus (ToCV) causes substantial economic losses by inducing yellow leaf disorder in tomato crops. This study explores potential resistance mechanisms by examining early-stage molecular responses to ToCV. A time-course transcriptome analysis compared naïve, mock, and ToCV-infected plants at 2, 7, and 14 days post-infection (dpi). Gene expression changes were most notable at 2 and 14 dpi, likely corresponding to whitefly feeding and viral infection. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses revealed key genes and pathways associated with ToCV infection, including those related to plant immunity, flavonoid and steroid biosynthesis, photosynthesis, and hormone signaling. Additionally, virus-derived small interfering RNAs (vsRNAs) originating from ToCV predominantly came from RNA2 and were 22 nucleotides in length. Furthermore, two genes involved in plant immunity, Hsp90 (heat shock protein 90) and its co-chaperone Sgt1 (suppressor of the G2 allele of Skp1) were targeted through viral-induced gene silencing (VIGS), showing a potential contribution to basal resistance against viral infections since their reduction correlated with increased ToCV accumulation. This study provides insights into tomato plant responses to ToCV, with potential implications for developing effective disease control strategies.

Dot-blot immunoassay for detection of tomato chlorosis virus and reaction of potato genotypes to virus infection

Tomato chlorosis virus (ToCV) is a whitefly-transmitted crinivirus that causes yield losses, mainly in tomato (Solanum lycopersicum) and potato (S. tuberosum) crops. In this work, a polyclonal antiserum for detecting the virus using a dot-blot immunoassay was developed and the responses of crinivirus-infected potato genotypes were evaluated. The virus was purified using infected tomato leaves and the antiserum was obtained by intramuscular injection of purified viral preparations in rabbit. Tomato and potato tissue samples were used to validate the polyclonal antiserum for detection of ToCV, previously analyzed by RT-PCR. A total of 81 tomato and potato samples were analyzed by RT-PCR and dot-blot immunoassay (DBIA). All samples were positive in RT-PCR, but three of them did not react in DBIA, showing of 96.3% efficiency compared with that in RT-PCR. All potato genotypes inoculated with ToCV by Bemisia tabaci MEAM1 were susceptible to infection. Potato plants of cv. Camila infected by ToCV showed the lowest virus titer and were asymptomatic.

Natural Occurrence of Tomato chlorosis virus in Cowpea (Vigna unguiculata) in China

Tomato chlorosis virus (ToCV), a phloem-limited and whitefly-transmitted crinivirus (family Closteroviridae) has been reported recently in China (Zhao, et al., 2015). In the summers of 2014 and 2016, leaves of cowpea (Vigna unguiculata) plants exhibiting interveinal chlorotic yellowing, dark green veins and curling were observed in greenhouses that were growing cowpeas and tomatoes in Liaocheng prefecture, Shandong Province, where natural infection of ToCV in tomato is widespread (Zhao et al., 2015). Tomato plants (cv.Oudun) with interveinal chlorotic yellowing symptoms and heavy whitefly (Bemisia tabaci) infestations were observed in the sampling sites. The disease incidence was around 87% for tomato plants and 5% for cowpea plants. To identify the pathogen, total RNA was extracted from leaves of six symptomatic cowpea and eight symptomatic tomato plants, with asymptomatic plants being used as controls. RT-PCR were carried out using primers ToCV-CP-F/ToCV-CP-R (5'-CCTCAAAGAGCTAAACTGGAC-3'/5'- CACATCACCGG...

Field evaluation of Cucurbit yellow stunting disorder virus transmission by Bemisia tabaci

Cucurbit yellow stunting disorder virus (CYSDV) is a whitefly-transmitted Crinivirus (Closteroviridae) that impacts melon production in many parts of the world including the USA. It has been responsible for melon crop loss in the southwestern U.S. since 2006 when it was first identified. Control strategies have revolved mainly around chemical control, but research to identify suitable products and approaches to implementing them have lagged. The current study investigated the performance of four systemic insecticides in the field while concurrently tracking CYSDV disease progression after controlled and natural whitefly inoculation of young melon plants. Assessments of virus incidence were made using two different visual observation methods in concert with ELISA analyses of leaf disks samples collected biweekly. Infection rates were consistently lowest in plots treated with the butenolide insecticide flupyradifurone while dinotefuran was second in efficacy measures. Flupyradifurone also held whitefly densities to their lowest numbers relative to the other treatments. Two other insecticides, imidacloprid and cyantraniliprole, exacerbated virus incidence in multiple trials. Further investigation into the anomalous finding of increased virus incidence due to insecticide application is ongoing.

Identification of natural weed hosts of Tomato chlorosis virus in Korea by RT-PCR with root tissues

Tomato chlorosis virus (ToCV) is a whitefly-transmitted crinivirus that causes interveinal chlorosis and bronzing on tomato leaves. Although outbreaks of ToCV have been reported in many countries, the virus was not reported in Korea until 2013. To identify weed hosts of ToCV that may serve as virus reservoirs, we analyzed various weeds that were growing together with ToCV-infected tomatoes and viruliferous Bemisia tabaci in a tomato greenhouse. We performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of root samples from 148 samples of 61 species from 24 families of plants grown in the greenhouse to avoid possible ToCV contamination by whiteflies on leaves. Seventeen weed species were identified as ToCV hosts based on RT-PCR results of root samples: Conyza canadensis, Erigeron annuus, Sonchus asper, Youngia japonica, Trigonotis peduncularis, Cardamine flexuosa, Cerastium glomeratum, Stellaria media, Chenopodium album, Ipomoea hederacea, Quamoclit coccinea, Vicia angustifolia var. segetilis, V. tetrasperma, Phytolacca americana, Mazus pumilus, Solanum americanum and S. nigrum. The amplicons obtained by RT-PCR were confirmed as ToCV by sequence analysis. Life cycle analysis of the weeds indicated that each weed could play an important role as a “green bridge” or virus reservoir between tomato cultivation seasons. This is the first report documenting the possible occurrence of ToCV in tomato plants based on weed life cycles.

Whitefly-transmitted criniviruses of cucurbits: current status and future prospects.

In the past decade, crinviruses have gained interest due to their rapid widespread and destructive nature for cucurbit cultivation. Several members of the genus Crinivirus are considered emerging viruses. Currently, four criniviruses: Beet pseudo-yellows virus, Cucurbit chlorotic yellows virus, Cucurbit yellow stunting disorder virus and Lettuce infectious yellows virus have been reported to infect field- or greenhouse- grown cucurbits. Apart from their cucurbit hosts, criniviruses infect other cash crops and weeds. Criniviruses are exclusively transmitted by whiteflies. The virion titer and the vector genus or species complex are predominant factors affecting virus transmission. These criniviruses maintain genetic stability with limited intra-species variability. They share similar core genome structure and replication strategies with some variations in the non-core proteins and downstream replication processes. Management of the diseases induced by criniviruses relies on integrated disease management strategies and on resistant varieties, when available. This review will cover their epidemiology, molecular biology, detection and management.

Molecular characterization reveals Brazilian Tomato chlorosis virus to be closely related to a Greek isolate

Tomato chlorosis virus (ToCV, genus Crinivirus, family Closteroviridae) is a whitefly-transmitted crinivirus with a bipartite RNA genome. This virus is emerging as a serious threat to tomato crops worldwide. To date, only three complete genomic sequences of ToCV have been described from North America, Spain, and Greece isolates. In this study, we present the fourth complete nucleotide sequence of the RNA 1 (8594 nt) and RNA 2 (8242 nt) components of a Brazilian ToCV isolate (ToCV-BR). The complete genome sequences of RNA 1 and RNA 2 have been deposited in the GenBank database under the accession numbers JQ952600 and JQ952601, respectively. The sequences of RNA 1 and RNA 2 shares the highest nucleotide identity of 99.6% and 99.5%, respectively, with the Greek isolate sequences. Phylogenetic analysis confirmed that both RNA 1 and RNA 2 of the Brazilian isolate are most closely related to the Greek isolate of that virus. These results suggest that ToCV may have been recently introduced to Brazil from Europe.

Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR

Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100–1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.

Resistance in melon to Cucurbit chlorotic yellows virus, a whitefly-transmitted crinivirus

Fifty-one melon (Cucumis melo) accessions that originated from India, Pakistan and Bangladesh were evaluated for resistance to Cucurbit chlorotic yellows virus (CCYV), a newly emerged species in the genus Crinivirus. CCYV was inoculated using sweet potato whitefly (Bemisia tabaci) biotype Q. Accessions, JP 138332, JP 216154, JP 216155, JP 216751 and JP 91204, showed no or faint symptoms, although CCYV was detected from the non-inoculated upper leaves by reverse transcription-polymerase chain reaction (RT-PCR). The five accessions were subjected to quantitative RT-PCR to analyze relative accumulation of CCYV RNA. All accessions except JP 138332 had levels of CCYV RNA accumulation comparable to the commercial variety, ‘Earl’s Seine’, which was used as a control. JP 138332 showed a much lower CCYV RNA accumulation. Numbers of B. tabaci biotype Q on JP 138332 did not differ from ‘Earl’s Seine’, in antixenosis tests, and the result suggested the resistance to CCYV was not due to antixenosis. Consequently, five accessions are of interest for development of resistant varieties. In particular, JP 138332 possesses a promising resistant trait for CCYV, which might be associated with inhibition of virus multiplication.

Sweetpotato Viruses: 15 Years of Progress on Understanding and Managing Complex Diseases.

Sweetpotato is a member of the morning glory family that is thought to have originated in Central or South America but also has a secondary center of diversity in the southwest Pacific islands. It is grown in all tropical and subtropical areas of the world and consistently ranks among the 10 most important food crops worldwide on the basis of dry weight produced, yielding about 130 million metric tons per year on about 9 million hectares. Sweetpotato is an important crop for food security. It has been relied on as a source of calories in many circumstances. Vines and/or storage roots can be used for direct human consumption or animal feed. Growing awareness of health benefits attributed to sweetpotato has stimulated renewed interest in the crop. Orange-fleshed cultivars, a source of vitamin A, were introduced to developing countries with hope that they would replace the white-flesh varieties and help alleviate vitamin A deficiencies. In East Africa, sweetpotato virus disease, which is caused by the synergistic interaction of the whitefly-transmitted crinivirus and the aphid-transmitted potyvirus, can cause losses of 80 to 90% in many high-yielding genotypes. During the past 15 years, as molecular methods have been adopted, much has been learned about the composition of the sweetpotato virus complexes, the effects of virus diseases on production systems, the biology of the virus–plant interaction, and management approaches to sweetpotato virus diseases. This article is intended to summarize what has been learned since earlier reviews, integrate knowledge gleaned from experiences in tropical and temperate production systems, and suggest courses of action to develop sustainable management programs for these diseases.

Newly Discovered Natural Hosts of Tomato chlorosis virus in Costa Rica.

Tomato chlorosis virus (ToCV) is an emerging whitefly-transmitted crinivirus (2). In Costa Rica in 2007, ToCV was detected in field-grown and greenhouse tomato (Solanum lycopersicum L.) plants causing symptoms of severe yellowing and foliar chlorosis (1). To identify alternative hosts that may serve as virus reservoirs, 78 samples were collected from multiple species of common weeds growing adjacent to tomato nurseries in the Cartago Province, where ToCV was previously identified, during the autumn of 2008 and summer of 2009. The weeds were collected on the basis of the presence of whiteflies and/or symptoms of interveinal chlorosis, but not all samples were symptomatic for infection by ToCV. Total RNA was extracted from leaf tissue with TRI Reagent (Molecular Research Inc., Cincinnati, OH). Reverse transcription (RT)-PCR reactions were performed with the Qtaq One-Step qRT-PCR SYBR Kit (Clontech Laboratories, Mountain View, CA) and primers specific for the ToCV HSP70h gene (3). A 123-bp DNA fragment was amplified in five weeds, which were identified taxonomically as Ruta chalepensis (Rutaceae), Phytolacca icosandra (Phytolacaceae), Plantago major (Plantaginaceae), a Brassica sp. (Brassicaceae) (two samples), and a single plant of Cucurbita moschata (Cucurbitaceae) growing next to those weeds. The amplified DNA fragments were sequenced and BLAST analysis showed 100% nucleotide sequence identity with the HSP70h gene of the Florida ToCV isolate (GenBank Accession No. AY903448). To confirm the presence of ToCV in these six weed samples, conventional RT-PCR reactions were performed using primers specific for the ToCV CPm and p22 genes as described previously (1). Nucleotide sequence analysis of the amplified DNA fragments verified their identity as ToCV, with 100% sequence identity to the CPm of the ToCV isolate of Florida (Accession No. AY903448) and the p22 gene of the Cartago, Costa Rican isolate (Accession No. FJ809714). Although the number of samples analyzed is not sufficient to allow a determination of the role of weed reservoirs in ToCV epidemics in Costa Rican tomato crops, this report on the wider natural host range of ToCV in Costa Rica may lead to a better understanding of the epidemiology of this virus and be useful in the development of disease management strategies. To our knowledge this is the first report of these weeds as natural hosts of ToCV.

Production of antiserum and immunodetection of Cucurbit chlorotic yellows virus, a novel whitefly-transmitted crinivirus

Cucurbit chlorotic yellows virus (CCYV), a whitefly-transmitted virus, is a new member of the Crinivirus genus, that causes major economic losses in cucumber, melon and watermelon crops. To develop immunodiagnostic methods for CCYV, we produced an antiserum by immunizing rabbits with bacterially expressed recombinant coat protein of CCYV and detected CCYV from CCYV-infected plants with western blotting and immunoelectron microscopy. CCYV in extracts from infected plants was also readily detected by double antibody sandwich enzyme-linked immunosorbent assay with high sensitivity and specificity. A tissue blot immunoassay indicated that CCYV localizes in the phloem tissues of the petiole and is distributed in tiny spots within the leaf lamina.

Interactions Among Sweet potato chlorotic stunt virus and Different Potyviruses and Potyvirus Strains Infecting Sweetpotato in the United States.

Sweet potato virus disease (SPVD) is the most serious viral disease of sweetpotato globally. This disease is caused by the synergistic interaction between the aphid-transmitted potyvirus Sweet potato feathery mottle virus (SPFMV) and the whitefly-transmitted crinivirus Sweet potato chlorotic stunt virus (SPCSV). In sweetpotato, titers of SPFMV have been shown to be significantly enhanced when coinfecting with SPCSV. In this study, the effect of SPCSV on titers of different potyviruses and potyvirus strains infecting sweetpotato in the United States was investigated using real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). No significant difference was observed in titers of the russet crack strain of SPFMV (SPFMV-RC), Ipomoea vein mosaic virus (IVMV), and Sweet potato virus G (SPVG) between single and mixed infections. Titers of all potyviruses and potyvirus strains evaluated were enhanced in the presence of SPCSV, suggesting that a conserved mechanism may underlie these interactions. Titers of the common strain of SPFMV (SPFMV-C), which did not cause SPVD-like symptoms when coinfecting with SPCSV, were also significantly enhanced in the presence of SPCSV. Furthermore, titers of SPCSV were found to be lower in treatments involving pairwise infections compared with plants infected by SPCSV alone. The degree of potyvirus titer enhancement did not correspond to the severity of symptoms observed in certain treatments involving pairwise infections.

Cucurbit yellow stunting disorder crinivirus

<i>Cucurbit yellow stunting disorder crinivirus</i>

First Report of Tomato chlorosis virus and Tomato infectious chlorosis virus in Tomato Crops in France.

Since 2002, yellowing symptoms associated with high levels of white-fly populations have been observed in plants of protected tomato crops in France. Symptomatic plants exhibited interveinal yellowing areas in older leaves, followed by generalized yellowing. Symptoms were not observed in young plants or fruits. Trialeurodes vaporariorum populations were generally abundant in spring, and Bemisia tabaci (established in France for approximately 10 years) became predominant in summer and fall. To check for the presence of Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV), two whitefly-transmitted criniviruses known to induce yellowing symptoms, 696 samples were collected in the major tomato-growing areas; 573 samples from southern France and 123 samples from northern France. Total RNA was extracted from each sample and analyzed using reverse transcription-polymerase chain reaction (RT-PCR). Primers specific to ToCV (2) and TICV (1,3) were used to amplify either part of the heat-shock-like protein gene HSP70h (both viruses) or part of the diverged coat protein gene (CPd), (TICV only). A 439-bp DNA fragment was obtained with ToCV primers in 178 samples from southern France collected mainly from mid-spring to early fall from 2002 to 2004. Three RT-PCR products amplified from samples collected from diverse growing areas were sequenced and showed 99 to 100% sequence identity with published ToCV sequences from Spain (GenBank Accession Nos. AF215818, AF233435, and AF215817), Portugal (GenBank Accession No. AF234029), Sicily (GenBank Accession No. AY048854), and the United States (GenBank Accession No. AF024630). Considering the high frequency of ToCV-infected samples (41 positive samples of 112 samples collected in 2002, 71 of 295 collected in 2003, and 66 of 166 collected in 2004), this virus appears to be well established in southern France but remains absent in the northern regions. The presence of TICV was tested in 485 samples using the CPd-specific primers or the HSP70h-specific primers. The virus was detected in only two samples from Nice (southeastern France) in 2003 with both primer pairs. The CPd DNA fragment (700 bp) from one of these samples was sequenced, showing 98.9% sequence identity with a TICV Japanese isolate (AB085603). Results of these assays suggest that in contrast to ToCV, TICV is not yet broadly established in France. This difference could be associated with the specificity of the vectors, since ToCV is transmitted by B. tabaci and T. vaporariorum, while TICV is transmitted only by T. vaporariorum (4).

Current Status and Newly Discovered Natural Hosts of Tomato infectious chlorosis virus and Tomato chlorosis virus in Spain.

Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) are emergent whitefly-transmitted criniviruses. ToCV and TICV were detected in Spain in 2000 (2) and 2001 (1), respectively. Both viruses infect tomato (Lycopersicon esculentum Mill) crops and cause symptoms of foliar chlorosis. ToCV is prevalent along the southern and eastern regions of Spain (provinces of Sevilla, Málaga, Almería, Murcia, Alicante, and Castellón), Balearic (Mallorca), and the Canary Islands (Tenerife and Gran Canaria). However, TICV only has been detected in the provinces of Murcia, Alicante, and Castellón in Spain. During the summer and autumn of 2002, abnormal interveinal reddening, yellowing symptoms, or both, were observed in plants of Chenopodium album L., C. murale L., and Solanum nigrum L. growing in or around tomato fields in Murcia and Almería provinces. To study the alternative hosts that may serve as virus reservoirs in areas where these viruses are prevalent, 62 samples of 42 common weed species were analyzed by reverse transcription-polymerase chain reaction using specific primers for ToCV and TICV (1). The 439-bp ToCV-specific DNA fragment was amplified in two S. nigrum samples from Alicante and Murcia provinces, and the 501-bp TICV-specific DNA fragment was amplified in one C. murale sample from Murcia, as well as in three C. album samples from Murcia and Alicante provinces. The DNA fragment amplified from the ToCV isolate was sequenced and showed 99 to 98% identity with the ToCV isolates (GenBank Accession Nos. AY048854 and AF234029) from Italy and Portugal, respectively. The DNA fragment amplified from TICV isolates were sequenced and showed 98% identity with the TICV isolate from Spain (GenBank Accession No. AF479662), confirming the diagnosis. Although the number of samples is not sufficient to conclude that we know, in a precise way, the role of weed reservoirs in TICV and ToCV epidemics in Spain, this study might contribute to a better understanding of the epidemiology of these viruses. To our knowledge this is the first report of these weeds as natural hosts of ToCV and TICV in Spain.

First Report of Tomato infectious chlorosis virus in Spain.

During the summer and autumn of 2001, symptoms of interveinal yellowing, bronzing, brittleness, and rolling of lower leaves were observed in greenhouse- and field-grown tomato (Lycopersicon esculentum) plants in Castellon Province in eastern Spain. Symptoms resembled those caused by the whitefly-transmitted criniviruses (1,2). Total RNA was extracted from 28 samples of symptomatic leaves collected in three greenhouses and one field and analyzed by reverse transcription-polymerase chain reaction using primers specific for Tomato chlorosis virus (ToCV) (1) and Tomato infectious chlorosis virus (TICV) (2). The 501-bp TICV-specific DNA fragment was amplified in four samples collected during the summer in three greenhouses and one field, and the 439-bp ToCV-specific DNA fragment was amplified in 15 samples collected during the autumn in the same three greenhouses; no mixed infections were found. The DNA fragments amplified from TICV were sequenced and showed 99 to 100% identity with the TICV isolates (GenBank Accession Nos. U67449 and AY048855) from the United States and Italy, respectively, confirming the diagnosis. One sequence was deposited as GenBank Accession No. AF479662. To our knowledge, this is the first report of TICV in Spain and the second in Europe.

First Report of Tomato chlorosis virus in Puerto Rico.

Symptoms of interveinal chlorosis, necrotic flecking, thickening, and rolling of leaves were observed on leaves of field-grown tomato (Lycopersicon esculentum) plants in Jauna Diaz, Puerto Rico. These symptoms are indicative of those produced by the whitefly-transmitted criniviruses, Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) (1). Samples collected from two symptomatic plants were examined by leaf dip and were found to contain long flexuous rods approximately 800 nm in length, characteristic of criniviruses. Symptomatic leaves were used for extraction of total nucleic acid and for whitefly transmission studies. The greenhouse whitefly, Trialeurodes vaporariorum (Westwood), is a highly efficient vector of TICV, but an inefficient vector of ToCV, whereas the banded wing whitefly, T. abutilonea (Haldeman), is an efficient vector of ToCV but does not transmit TICV (2). Whiteflies of both species were allowed to feed separately on symptomatic tomato leaves for 24 h and then transferred to healthy Physalis wrightii and Nicotiana benthamiana indicator plants. Symptoms characteristic of ToCV infection developed on 3 of 3 P. wrightii plants and 2 of 3 N. benthamiana plants following transmission by T. abutilonea. Only 1 of 3 P. wrightii plants developed such symptoms following transmission by T. vaporariorum, while no N. benthamiana plants developed symptoms, suggesting that the virus responsible for the tomato disease was ToCV. Dot blot hybridizations were performed on total nucleic acids extracted from 0.1 g of symptomatic leaves of field samples using probes specific for TICV or ToCV (2), as well as probes specific for four additional criniviruses. Symptomatic and asymptomatic leaves of plants in transmission tests, as well as comparable leaves from control plants, were also tested by dot blot. Although no criniviruses could be detected by dot blot in the original tomato tissue, these hybridizations identified ToCV in all symptomatic plants from the transmission experiments, confirming the presence of ToCV in Puerto Rico. No additional criniviruses were detected in any samples, and negative controls were virus-free. This is the first time a tomato crinivirus has been detected in the Caribbean, outside of the continental United States. The ability of ToCV to be transmitted by four different whitefly species increases the potential for this virus to spread throughout the Caribbean Basin.

Particle Lengths of Whitefly-Transmitted Criniviruses.

An improved method for particle length measurement was used for six members of the genus Crinivirus. Particle measurements were conducted with a CCD-72S solid state camera, which was interfaced with a Zeiss EM 109 electron microscope, and analyzed using the analysis 2.1 Image Analysis Software. In comparisons of specimen preparation methods, the leaf dip method is more representative and reproducible than the antibody capture method or preparation from purified virions. Particle length (nm) ranges of whitefly-transmitted criniviruses are: Abutilon yellows virus (AYV), 800 to 850; Cucurbit yellow stunting disorder virus (CYSDV), 750 to 800; Lettuce chlorosis virus (LCV), 800 to 850; Lettuce infectious yellows virus (LIYV), 700 to 750; Tomato chlorosis virus (ToCV), 800 to 850; and Tomato infectious chlorosis virus (TICV), 850 to 900.