WIV, a protein domain found in a wide number of arthropod viruses, which probably facilitates infection.

The most powerful approach to detect distant homologues of a protein is based on structure prediction and comparison. Yet this approach is still inapplicable to many viral proteins. Therefore, we applied a powerful sequence-based procedure to identify distant homologues of viral proteins. It relies on three principles: (1) traces of sequence similarity can persist beyond the significance cutoff of homology detection programmes; (2) candidate homologues can be identified among proteins with weak sequence similarity to the query by using 'contextual' information, e.g. taxonomy or type of host infected; (3) these candidate homologues can be validated using highly sensitive profile-profile comparison. As a test case, this approach was applied to a protein without known homologues, encoded by ORF4 of Lake Sinai viruses (which infect bees). We discovered that the ORF4 protein contains a domain that has homologues in proteins from >20 taxa of viruses infecting arthropods. We called this domain 'widespread, intriguing, versatile' (WIV), because it is found in proteins with a wide variety of functions and within varied domain contexts. For example, WIV is found in the NSs protein of tospoviruses, a global threat to food security, which infect plants as well as their arthropod vectors; in the RNA2 ORF1-encoded protein of chronic bee paralysis virus, a widespread virus of bees; and in various proteins of cypoviruses, which infect the silkworm Bombyx mori. Structural modelling with AlphaFold indicated that the WIV domain has a previously unknown fold, and bibliographical evidence suggests that it facilitates infection of arthropods.

Spatiotemporal Patterns of Iris Yellow Spot Virus and Its Onion Thrips Vector, Thrips tabaci, in Transplanted and Seeded Onion Fields in New York.

Onion thrips, Thrips tabaci (Lindeman), transmits iris yellow spot virus (IYSV) and is one of the most important pests of Allium crops. IYSV is a member of the species Tospovirus iridimaculaflavi in the genus Orthotospovirus of the family Tospoviridae. This virus typically reduces overall onion bulb quality and weight but can also prematurely kill onion plants. IYSV is neither seed nor mechanically transmitted. Onion fields are typically established via seeds and transplants. A decade ago, onion thrips tended to colonize transplanted fields before seeded fields because plants in transplanted fields were larger and more attractive to thrips than smaller onions in seeded fields. Therefore, we hypothesized that the incidence of IYSV in transplanted fields would be detected early in the season and be spatially aggregated, whereas IYSV would be absent from seeded fields early in the season and initial epidemic patterns would be spatially random. In 2021 and 2022, IYSV incidence and onion thrips populations were quantified in 12 onion fields (four transplanted fields and eight seeded fields) in New York. Fields were scouted four times throughout the growing season (n = 96 samples), and a geospatial and temporal analysis of aggregation and incidence was conducted to determine spatiotemporal patterns in each field type. Results indicated that spatial patterns of IYSV incidence and onion thrips populations were similar early in the season, indicating that transplanted onion fields are no longer the dominant early-season source of IYSV in New York. These findings suggest the need to identify other important early-season sources of IYSV that impact New York onion fields.

Identifying Onion Fields at Risk of Iris Yellow Spot Virus in New York.

Iris yellow spot virus (IYSV) poses a significant threat to dry bulb onion, Allium cepa L., production and can lead to substantial yield reductions. IYSV is transmitted by onion thrips, Thrips tabaci (Lindeman), but not via seed. Transplanted onion fields have been major early season sources of IYSV epidemics. As onion thrips tend to disperse short distances, seeded onion fields bordering transplanted onion fields may be at greater risk of IYSV infection than seeded fields isolated from transplanted ones. Additionally, seeded onion fields planted early may be at greater risk of IYSV infection than those seeded later. In a 2-year study in New York, we compared IYSV incidence and onion thrips populations in seeded onion fields relative to their proximity to transplanted onion fields. In a second study, we compared IYSV incidence in onion fields with either small or large plants during midseason. Results showed similar IYSV incidence and onion thrips populations in seeded onion fields regardless of their proximity to transplanted onion fields, while IYSV incidence was over four times greater in large onion plants than in small ones during midseason. These findings suggest a greater risk of onion thrips-mediated IYSV infection in onion fields with large plants compared with small ones during midseason and that proximity of seeded fields to transplanted ones is a poor indicator of IYSV risk. Our findings on IYSV spread dynamics provided valuable insights for developing integrated pest and disease management strategies for New York onion growers.

Development of a reverse transcription recombinase polymerase amplification combined with lateral flow assay for equipment-free on-site field detection of tomato chlorotic spot virus.

BACKGROUND: Tomato chlorotic spot virus (TCSV) is an economically important, thrips-transmitted, emerging member of the Orthotospovirus genus that causes significant yield loss mainly in tomatoes, but also in other vegetable and ornamental crops. Disease management of this pathogen is often challenging due to the limited availability of natural host resistance genes, the broad host range of TCSV, and the wide distribution of its thrips vector. Point-of-care detection of TCSV with a rapid, equipment-free, portable, sensitive, and species-specific diagnostic technique can provide prompt response outside the laboratory, which is critical for preventing disease progression and further spread of the pathogen. Current diagnostic techniques require either laboratory-dependent or portable electronic equipment and are relatively time-consuming and costly. RESULTS: In this study, we developed a novel technique for reverse-transcription recombinase polymerase amplification combined with lateral flow assay (RT-RPA-LFA) to achieve a faster and equipment-free point-of-care detection of TCSV. The RPA reaction tubes containing crude RNA are incubated in the hand palm to obtain sufficient heat (∼36 °C) for the amplification without the need for equipment. Body-heat mediated RT-RPA-LFA is highly TCSV-specific with a detection limit as low as ∼6 pg/µl of total RNA from TCSV-infected tomato plants. The assay can be performed in 15 min in the field. CONCLUSION: To the best of our knowledge, this is the first equipment-free, body-heat-mediated RT-RPA-LFA technique developed to detect TCSV. Our new system offers a time-saving advantage for the sensitive and specific diagnostic of TCSV that local growers and small nurseries in low-resource settings can use without skilled personnel.

Genome-enabled insights into the biology of thrips as crop pests.

BACKGROUND: The western flower thrips, Frankliniella occidentalis (Pergande), is a globally invasive pest and plant virus vector on a wide array of food, fiber, and ornamental crops. The underlying genetic mechanisms of the processes governing thrips pest and vector biology, feeding behaviors, ecology, and insecticide resistance are largely unknown. To address this gap, we present the F. occidentalis draft genome assembly and official gene set. RESULTS: We report on the first genome sequence for any member of the insect order Thysanoptera. Benchmarking Universal Single-Copy Ortholog (BUSCO) assessments of the genome assembly (size = 415.8 Mb, scaffold N50 = 948.9 kb) revealed a relatively complete and well-annotated assembly in comparison to other insect genomes. The genome is unusually GC-rich (50%) compared to other insect genomes to date. The official gene set (OGS v1.0) contains 16,859 genes, of which ~ 10% were manually verified and corrected by our consortium. We focused on manual annotation, phylogenetic, and expression evidence analyses for gene sets centered on primary themes in the life histories and activities of plant-colonizing insects. Highlights include the following: (1) divergent clades and large expansions in genes associated with environmental sensing (chemosensory receptors) and detoxification (CYP4, CYP6, and CCE enzymes) of substances encountered in agricultural environments; (2) a comprehensive set of salivary gland genes supported by enriched expression; (3) apparent absence of members of the IMD innate immune defense pathway; and (4) developmental- and sex-specific expression analyses of genes associated with progression from larvae to adulthood through neometaboly, a distinct form of maturation differing from either incomplete or complete metamorphosis in the Insecta. CONCLUSIONS: Analysis of the F. occidentalis genome offers insights into the polyphagous behavior of this insect pest that finds, colonizes, and survives on a widely diverse array of plants. The genomic resources presented here enable a more complete analysis of insect evolution and biology, providing a missing taxon for contemporary insect genomics-based analyses. Our study also offers a genomic benchmark for molecular and evolutionary investigations of other Thysanoptera species.

A multiplex PCR assay for rapid identification of major tospovirus vectors reported in India.

BACKGROUND: To date, four thrips vectors have been reported to transmit five different tospoviruses in India. Their identification at an early stage is crucial in formulating appropriate pest management strategies. Since morphometric key-based thrips identification based on the adult stage is time-consuming, there is a need to develop diagnostic tools which are rapid, accurate, and independent of developmental stages. Here, we report a multiplex PCR assay to identify four major thrips vectors viz. Thrips palmi, T. tabaci, Scirtothrips dorsalis, and Frankliniella schultzei present in India. RESULTS: Cytochrome oxidase subunit III and internal transcribed spacer region 2 were utilized to design species-specific primers. Of 38 pairs of primers tested, primer pairs AG35F-AG36R, AG47F-AG48R, AG87F-AG88R, and AG79F-AG80R amplified 568 bp, 713 bp, 388 bp, and 200 bp products from the DNA templates of T. palmi, S. dorsalis, T. tabaci, and F. schultzei, respectively at same PCR conditions. The specificity of the primer pairs was validated with a large number of known specimens and no cross-reactivity was observed with other thrips species. The multiplex PCR assay with a cocktail of all the four primer pairs detected four thrips vectors efficiently and could discriminate all of them concurrently in a single reaction. CONCLUSION: The multiplex PCR reported in this study could identify the major thrips vectors reported in India. The assay will be useful in ascertaining distribution profile of major thrips vectors, disease epidemiology, screening large samples, and quarantine.

Identification and localization of Tospovirus genus-wide conserved residues in 3D models of the nucleocapsid and the silencing suppressor proteins.

BACKGROUND: Tospoviruses (genus Tospovirus, family Peribunyaviridae, order Bunyavirales) cause significant losses to a wide range of agronomic and horticultural crops worldwide. Identification and characterization of specific sequences and motifs that are critical for virus infection and pathogenicity could provide useful insights and targets for engineering virus resistance that is potentially both broad spectrum and durable. Tomato spotted wilt virus (TSWV), the most prolific member of the group, was used to better understand the structure-function relationships of the nucleocapsid gene (N), and the silencing suppressor gene (NSs), coded by the TSWV small RNA. METHODS: Using a global collection of orthotospoviral sequences, several amino acids that were conserved across the genus and the potential location of these conserved amino acid motifs in these proteins was determined. We used state of the art 3D modeling algorithms, MULTICOM-CLUSTER, MULTICOM-CONSTRUCT, MULTICOM-NOVEL, I-TASSER, ROSETTA and CONFOLD to predict the secondary and tertiary structures of the N and the NSs proteins. RESULTS: We identified nine amino acid residues in the N protein among 31 known tospoviral species, and ten amino acid residues in NSs protein among 27 tospoviral species that were conserved across the genus. For the N protein, all three algorithms gave nearly identical tertiary models. While the conserved residues were distributed throughout the protein on a linear scale, at the tertiary level, three residues were consistently located in the coil in all the models. For NSs protein models, there was no agreement among the three algorithms. However, with respect to the localization of the conserved motifs, G18 was consistently located in coil, while H115 was localized in the coil in three models. CONCLUSIONS: This is the first report of predicting the 3D structure of any tospoviral NSs protein and revealed a consistent location for two of the ten conserved residues. The modelers used gave accurate prediction for N protein allowing the localization of the conserved residues. Results form the basis for further work on the structure-function relationships of tospoviral proteins and could be useful in developing novel virus control strategies targeting the conserved residues.

Iris Yellow Spot Virus Prolongs the Adult Lifespan of Its Primary Vector, Onion Thrips (Thrips tabaci) (Thysanoptera: Thripidae).

Iris yellow spot virus (IYSV) from the genus Tospovirus, family Peribunyaviridae, reduces yield in several crops, especially Allium spp. IYSV is primarily transmitted by onion thrips (Thrips tabaci), but little is known about how IYSV impacts the biology of its principal vector. In a controlled experiment, the effect of IYSV on the lifespan and fecundity of onion thrips was examined. Larvae were reared on IYSV-infected onions until pupation. Individual pupae were confined until adults eclosed, and the lifespan and total progeny produced per adult were monitored daily. Thrips were tested for the virus in reverse-transcriptase polymerase chain reaction using specific primers to confirm the presence of IYSV. Results indicated that 114 and 35 out of 149 eclosing adults tested positive (viruliferous) and negative (nonviruliferous) for IYSV, respectively. The viruliferous adults lived 1.1-6.1 d longer (average of 3.6 d) than nonviruliferous adults. Fecundity of viruliferous and nonviruliferous onion thrips was similar with 2.0 ± 0.1 and 2.3 ± 0.3 offspring produced per female per day, respectively. Fecundity for both viruliferous and nonviruliferous thrips also was significantly positively correlated with lifespan. These findings suggest that the longer lifespan of viruliferous onion thrips adults may allow this primary vector of IYSV to infect more plants, thereby exacerbating IYSV epidemics.

Asymmetric Trimeric Ring Structure of the Nucleocapsid Protein of Tospovirus.

Tomato spotted wilt virus (TSWV), belonging to the genus Tospovirus of the family Bunyaviridae, causes significant economic damage to several vegetables and ornamental plants worldwide. Similar to those of all other negative-strand RNA viruses, the nucleocapsid (N) protein plays very important roles in its viral life cycle. N proteins protect genomic RNAs by encapsidation and form a viral ribonucleoprotein complex (vRNP) with some RNA-dependent RNA polymerases. Here we show the crystal structure of the N protein from TSWV. Protomers of TSWV N proteins consist of three parts: the N arm, C arm, and core domain. Unlike N proteins of other negative-strand RNA viruses, the TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, the N and C arms of the N protein interact with the core domains of two adjacent N proteins. By solving the crystal structures of the TSWV N protein with nucleic acids, we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. These characteristics are similar to those of N proteins of other viruses of the family Bunyaviridae Based on these observations, we discuss possibilities of a TSWV encapsidation model.IMPORTANCE Tospoviruses cause significant crop losses throughout the world. Particularly, TSWV has an extremely wide host range (>1,000 plant species, including dicots and monocots), and worldwide losses are estimated to be in excess of $1 billion annually. Despite such importance, no proteins of tospoviruses have been elucidated so far. Among TSWV-encoded proteins, the N protein is required for assembling the viral genomic RNA into the viral ribonucleoprotein (vRNP), which is involved in various steps of the life cycle of these viruses, such as RNA replication, virus particle formation, and cell-to-cell movement. This study revealed the structure of the N protein, with or without nucleic acids, of TSWV as the first virus of the genus Tospovirus, so it completed our view of the N proteins of the family Bunyaviridae.

Identification and genome analysis of tomato chlorotic spot virus and dsRNA viruses from coinfected vegetables in the Dominican Republic by high-throughput sequencing.

The Tomato chlorotic spot virus (TCSV) was first reported in the 1980s, having its occurrence limited to Brazil and Argentina. Due to an apparent mild severity in the past, molecular studies concerning TCSV were neglected. However, TCSV has disseminated over the USA and Caribbean countries. In Dominican Republic TCSV has been recently reported on important cultivated crops such as pepper and beans. In this work, we provide the first complete genome of a TCSV isolate from symptomatic plants in Dominican Republic, which was obtained by high-throughput sequencing. In addition, three dsRNA viruses from different virus families were identified coinfecting these plants Bell pepper endornavirus (BPEV), Southern tomato virus (STV) and Pepper cryptic virus 2 (PCV-2). Phylogenetic analysis showed that the Dominican Republic TCSV isolate has a close relationship with other TCSV isolates and a reassortant isolate between TCSV and Groundnut ringspot virus (GRSV), all found in USA. BPEV, STV and PCV-2 isolates from Dominican Republic were close related to corresponding American isolates. The possible biological implications of these virus-mixed infections are discussed.

Development of a microarray for simultaneous detection and differentiation of different tospoviruses that are serologically related to Tomato spotted wilt virus.

BACKGROUND: Tospoviruses, the plant-infecting genus in the family Bunyaviridae, are thrips borne and cause severe agricultural losses worldwide. Based on the serological relationships of the structural nucleocapsid protein (NP), the current tospoviruses are divided into six serogroups. The use of NP-antisera is convenient for virus detection, but it is insufficient to identify virus species grouped in a serogroup due to the serological cross-reaction. Alternatively, virus species can be identified by the N gene amplification using specific primers. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and one of the most destructive plant viruses. Eight known tospoviruses, Alstroemeria necrotic streak virus (ANSV), Chrysanthemum stem necrosis virus (CSNV), Groundnut ringspot virus (GRSV), Impatiens necrotic spot virus (INSV), Melon severe mosaic virus (MeSMV), Pepper necrotic spot virus (PNSV), Tomato chlorotic spot virus (TCSV) and Zucchini lethal chlorosis virus (ZLCV), sharing serological relatedness with TSWV in NP, are grouped in the TSWV serogroup. Most of the TSWV-serogroup viruses prevail in Europe and America. An efficient diagnostic method is necessary for inspecting these tospoviruses in Asia, including Taiwan. METHODS: A microarray platform was developed for simultaneous detection and identification of TSWV-serogroup tospoviruses. Total RNAs extracted from Chenopodium quinoa leaves separately inoculated with ANSV, CSNV, GRSV, INSV, TCSV and TSWV were used for testing purposes. The 5'-biotinylated degenerate forward and reverse primers were designed from the consensus sequences of N genes of TSWV-serogroup tospoviruses for reverse transcription-polymerase chain reaction (RT-PCR) amplification. Virus-specific oligonucleotide probes were spotted on the surface of polyvinyl chloride (PVC) chips to hybridize with PCR products. The hybridization signals were visualized by hydrolysis of NBT/BCIP with streptavidine-conjugated alkaline phosphatase. The microarray was further applied to diagnose virus infection in field crop samples. RESULTS: Amplicons of approximately 0.46 kb were amplified from all tested TSWV-serogroup tospoviruses by RT-PCR using the degenerate primer pair Pr-dTS-f/Pr-dTS-r. Virus species were identified on chips by hybridization of PCR products with respective virus-specific probes. The microarray was successfully used to diagnose TSWV infection in field pepper samples. CONCLUSIONS: In this study, a rapid, sensitive and precise microarray method has been developed to simultaneously detect and identify six TSWV-serogroup tospoviruses. The microarray platform provides a great potential to explore tospoviruses that can help researchers and quarantine staff to prevent invasions of tospoviruses.

Influence of Groundnut bud necrosis virus on the Life History Traits and Feeding Preference of Its Vector, Thrips palmi.

The effect of Groundnut bud necrosis virus (GBNV) infection on the life history traits of its vector, Thrips palmi, and its feeding preference on GBNV-infected plants were studied. A significant difference was observed in the developmental period (first instar to adult) between the GBNV-infected and healthy thrips, wherein the developmental period of GBNV-infected thrips was decreased. However, there was no effect on the other parameters such as preadult mortality, adult longevity, and fecundity. Further investigation on a settling and feeding choice assay of T. palmi to GBNV-infected and healthy plants showed that T. palmi preferred GBNV-infected cowpea plants more than the healthy cowpea plants. This preference was also noticed for leaf disks from GBNV-infected cowpea, groundnut, and tomato plants.

Homology modeling and molecular dynamics provide structural insights into tospovirus nucleoprotein.

BACKGROUND: Tospovirus is a plant-infecting genus within the family Bunyaviridae, which also includes four animal-infecting genera: Hantavirus, Nairovirus, Phlebovirus and Orthobunyavirus. Compared to these members, the structures of Tospovirus proteins still are poorly understood. Despite multiple studies have attempted to identify candidate N protein regions involved in RNA binding and protein multimerization for tospovirus using yeast two-hybrid systems (Y2HS) and site-directed mutagenesis, the tospovirus ribonucleocapsids (RNPs) remains largely uncharacterized at the molecular level and the lack of structural information prevents detailed insight into these interactions. RESULTS: Here we used the nucleoprotein structure of LACV (La Crosse virus-Orthobunyavirus) and molecular dynamics simulations to access the structure and dynamics of the nucleoprotein from tospovirus GRSV (Groundnut ringspot virus). The resulting model is a monomer composed by a flexible N-terminal and C-terminal arms and a globular domain with a positively charged groove in which RNA is deeply encompassed. This model allowed identifying the candidate amino acids residues involved in RNA interaction and N-N multimerization. Moreover, most residues predicted to be involved in these interactions are highly conserved among tospoviruses. CONCLUSIONS: Crucially, the interaction model proposed here for GRSV N is further corroborated by the all available mutational studies on TSWV (Tomato spotted wilt virus) N, so far. Our data will help designing further and more accurate mutational and functional studies of tospovirus N proteins. In addition, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.

A multilayered regulatory mechanism for the autoinhibition and activation of a plant CC-NB-LRR resistance protein with an extra N-terminal domain.

The tomato resistance protein Sw-5b differs from the classical coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR) resistance proteins by having an extra N-terminal domain (NTD). To understand how NTD, CC and NB-LRR regulate autoinhibition and activation of Sw-5b, we dissected the function(s) of each domain. When viral elicitor was absent, Sw-5b LRR suppressed the central NB-ARC to maintain autoinhibition of the NB-LRR segment. The CC and NTD domains independently and additively enhanced the autoinhibition of NB-LRR. When viral elicitor was present, the NB-LRR segment of Sw-5b was specifically activated to trigger a hypersensitive response. Surprisingly, Sw-5b CC suppressed the activation of NB-LRR, whereas the extra NTD of Sw-5b became a positive regulator and fully activated the resistance protein, probably by relieving the inhibitory effects of the CC. In infection assays of transgenic plants, the NB-LRR segment alone was insufficient to confer resistance against Tomato spotted wilt tospovirus; the layers of NTD and CC regulation on NB-LRR were required for Sw-5b to confer resistance. Based on these findings, we propose that, to counter the negative regulation of the CC on NB-LRR, Sw-5b evolved an extra NTD to coordinate with the CC, thus developing a multilayered regulatory mechanism to control autoinhibition and activation.

The complete genome of the tospovirus Zucchini lethal chlorosis virus.

BACKGROUND: Zucchini lethal chlorosis virus (ZLCV) causes significant losses in the production of cucurbits in Brazil. This virus belongs to the genus Tospovirus (family Bunyaviridae) and seems to be exclusively transmitted by Frankliniella zucchini (Thysanoptera). Tospoviruses have a tripartite and single-stranded RNA genome classified as S (Small), M (Medium) and L (Large) RNAS. Although ZLCV was identified as a member of the genus Tospovirus in 1999, its complete genome had not been sequenced until now. FINDINGS: We sequenced the full-length genome of two ZLCV isolates named ZLCV-SP and ZLCV-DF. The phylogenetic analysis showed that ZLCV-SP and ZLCV-DF clustered with the previously reported isolate ZLCV-BR09. Their proteins were closely related, except the non-structural protein (NSm), which was highly divergent (approximately 90 % identity). All viral proteins clustered similarly in our phylogenetic analysis, excluding that these ZLCV isolates have originated from reassortment events of different tospovirus species. CONCLUSION: Here we report for the first time the complete genome of two ZLCV isolates that were found in the field infecting zucchini and cucumber.

Monoclonal antibodies for differentiating infections of three serological-related tospoviruses prevalent in Southwestern China.

BACKGROUND: The thrips-borne tospoviruses Calla lily chlorotic spot virus (CCSV), Tomato zonate spot virus (TZSV) and a new species provisionally named Tomato necrotic spot associated virus (TNSaV) infect similar crops in southwestern China. The symptoms exhibiting on virus-infected crops are similar, which is difficult for distinguishing virus species by symptomatology. The sequences of nucleocapsid proteins (NPs) of CCSV, TNSaV and TZSV share high degrees of amino acid identity with each other, and their serological relationship was currently demonstrated from the responses of the previously reported monoclonal antibodies (MAbs) against the NP of CCSV (MAb-CCSV-NP) and the nonstructural NSs protein of Watermelon silver mottle virus (WSMoV) (MAb-WNSs). Therefore, the production of virus-specific antibodies for identification of CCSV, TNSaV and TZSV is demanded to improve field surveys. METHODS: The NP of TZSV-13YV639 isolated from Crinum asiaticum in Yunnan Province, China was bacterially expressed and purified for producing MAbs. Indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting were conducted to test the serological response of MAbs to 18 tospovirus species. Additionally, the virus-specific primers were designed to verify the identity of CCSV, TNSaV and TZSV in one-step reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Two MAbs, denoted MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18), were screened for test. MAb-TZSV-NP(S15) reacted with CCSV and TZSV while MAb-TZSV-NP(S18) reacted specifically to TZSV in both indirect ELISA and immunoblotting. Both MAbs can be used to detect TZSV in field-collected plant samples. The epitope of MAb-TZSV-NP(S18) was further identified consisting of amino acids 78-86 (HKIVASGAD) of the TZSV-13YV639 NP that is a highly conserved region among known TZSV isolates but is distinct from TNSaV and TZSV. CONCLUSIONS: In this study, two MAbs targeting to different portions of the TZSV NP were obtained. Unlike MAb-CCSV-NP reacted with TNSaV as well as CCSV and TZSV, both TZSV MAbs can be used to differentiate CCSV, TNSaV and TZSV. The identity of CCSV, TNSaV and TZSV was proven by individual virus-specific primer pairs to indicate the correctness of serological responses. We also proposed an serological detection platform using MAb-CCSV-NP, MAb-TZSV-NP(S15) and MAb-TZSV-NP(S18) to allow researchers and quarantine staff to efficiently diagnose the infections of CCSV, TNSaV and TZSV in China and other countries.

Diversity of Thrips Species and Vectors of Tomato Spotted Wilt Virus in Tomato Production Systems in Kenya.

Thrips have been recognized as primary vectors of tomato spotted wilt virus (TSWV) with Frankliniella occidentalis (Pergande) reported as the most important and efficient vector, while other species such as Thrips tabaci Lindeman also include populations that can vector the virus. A study was undertaken to establish the diversity of thrips and presence of vectors for TSWV in four major tomato production areas in Kenya. The cytochrome oxidase 1 (CO1) gene was used to generate sequences from thrips samples collected from tomatoes and weeds, and phylogenetic analysis done to establish the variation within potential vector populations. Ceratothripoides brunneus Bagnall was the predominant species of thrips in all areas. F. occidentalis and T. tabaci were abundant in Nakuru, Kirinyaga, and Loitokitok but not detected at Bungoma. Other vectors of tospoviruses identified in low numbers were Frankliniella schultzei (Trybom) and Scirtothrips dorsalis Hood. Variation was observed in T. tabaci, F. occidentalis, and F. schultzei. Kenyan specimens of T. tabaci from tomato belonged to the arrhenotokous group, while those of F. occidentalis clustered with the Western flower thrips G group. The detection of RNA of TSWV in both of these species of thrips supported the role they play as vectors. The study has demonstrated the high diversity of thrips species in tomato production and the occurrence of important vectors of TSWV and other tospoviruses.

Thrips Settling, Oviposition and IYSV Distribution on Onion Foliage.

Thrips tabaci Lindeman (Thysanoptera: Thripidae) adult and larval settling and oviposition on onion (Allium cepa L.) foliage were investigated in relation to leaf position and leaf length at prebulb plant growth stages under controlled conditions. In the laboratory, four and six adult females of T. tabaci were released on onion plants at three-leaf stage and six- to eight-leaf stage, respectively, and thrips egg, nymph, and adult count data were collected on each of the three inner most leaves at every 2-cm leaf segment. Thrips settling and oviposition parameters were quantified during the light period on the above ground portion of onion plants from the distal end of the bulb or leaf sheath "neck" through the tips of the foliage. Results from studies confirmed that distribution of thrips adults, nymphs, and eggs were skewed toward the base of the plant. The settling distributions of thrips adults and nymphs differed slightly from the egg distribution in that oviposition occurred all the way to the tip of the leaf while adults and nymphs were typically not observed near the tip. In a field study, the foliage was divided into three equal partitions, i.e., top, middle, basal thirds, and thrips adults by species, primarily Frankliniella fusca (Hinds) and T. tabaci, were collected from each partition to determine if there was a similar bias of all adult thrips toward the base of the plant. The results suggested that adults of different species appear to segregate along leaf length. Finally, thrips oviposition on 2-cm segments and Iris yellow spot virus positive leaf segments were quantified in the field, irrespective of thrips species. Both variables demonstrated a very similar pattern of bias toward the base of the plant and were significantly correlated.

From immunity to susceptibility: virus resistance induced in tomato by a silenced transgene is lost as TGS overcomes PTGS.

Tomato line 30.4 was obtained engineering the nucleocapsid (N) gene of tomato spotted wilt virus into plant genome, and immunity to tomato spotted wilt virus infection of its self-pollinated homozygous progeny was observed. Despite the presence of a high amount of transgenic transcripts, transgenic proteins have not been detected, suggesting a mechanism of resistance mediated by RNA. In the present study, we identify post-transcriptional gene silencing as the main mechanism of resistance, which is able to spread systemically through grafting, and show that the line 30.4 resistant plants produce both 24 and 21-22 nt N-gene specific siRNA classes. The transgenic locus in chromosome 4 shows complex multiple insertions of four T-DNA copies in various orientations, all with 3' end deletions in the terminator and part of the N gene. However, for three of them, polyadenylated transcripts are produced, due to flanking tomato genome sequences acting as alternative terminators. Interestingly, starting at the fifth generation after the transformation event, some individual plants show a tomato spotted wilt virus-susceptible phenotype. The change is associated with the disappearance of transgene-specific transcripts and siRNAs, and with hyper-methylation of the transgene, which proceeds gradually through the generations. Once it reaches a critical threshold, the shift from post-transcriptional gene silencing to transcriptional silencing of the transgene eliminates the previously well established virus resistance.