Relationships between Tospovirus Incidence and Thrips Populations on Tomato in Mendoza, Argentina

The objectives of this work were to estimate the capability of local populations of thrips as vectors of groundnut ringspot virus (GRSV) and tomato spotted wilt virus (TSWV), and to determine the species composition of vectors in tomato crops. Transmission assays were performed. Incidence of tospoviruses was estimated in commercial crops. Random samples of flowers were taken from tomato for identification of thrips. Of the five species of thrips tested, Frankliniella gemina (first record), F. occidentalis and F. schultzei transmitted GRSV and TSWV. F. schultzei was a significantly more efficient vector of GRSV than F. occidentalis under controlled assay conditions. The thrips were identified on flowers from six surveyed tomato crops. F. occidentalis was the most frequently identified species (43.0%), followed by F. schultzei (35.6%) and Thrips tabaci (10.1%). The incidence of tospoviruses was low (1.1–2.8%) in crops planted during August–September and greater (9.5–61.1%) in crops planted in December. GRSV was prevalent (85%) over TSWV (11%).     

Serological relationships and purification of bud necrosis virus,a tospovirus occurring in peanut (Arachis hypogaea L.) in India*

A procedure for the purification of a tospovirus which causes bud necrosis disease (BND) of peanut in India is described. The virus contained three polypeptides of 78 kDa, 54 kDa and 31 kDa. In two ELISA procedures the virus failed to react with antisera to tomato spotted wilt virus (TSWV) obtained from different sources and with an antiserum to impatiens necrotic spot virus (INSV). Additionally, in reciprocal tests TSWV and INSV antigens failed to react with antiserum to the virus infecting peanut in India. In electro-blot immunoassay 54 kDa and 31 kDa polypeptides of the virus reacted with the homologous antiserum. None of the heterologous antisera reacted with any of the three viral polypeptides. On the basis of serological differences the virus that causes BND in India is distinct and therefore has been named bud necrosis virus (BNV). This serotype appears to be restricted to Asia.     

Peanut yellow spot virus: A distinct tospovirus species based on serology and nucleic acid hybridisation

Nucleocapsids of peanut yellow spot virus (PYSV), purified from peanut (= groundnut) plant tissue, contained a protein with a molecular mass of 29 kDa. In ELISA and immuno-blot analysis the virus did not react with tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and peanut bud necrosis virus (PBNV) antisera. PYSV contained three RNA species, a large (L) RNA (c.8900 nucleotides), a medium (M) RNA (c.4800 nucleotides) and a small (S) RNA (c.3000 nucleotides), similar to other tospoviruses. In addition, a fourth RNA species of approximately 1800 nucleotides was also present in purified preparations. Hybridisation analysis under high stringency conditions revealed no hybridisation between PYSV RNAs and cDNA probes representing the nucleocapsid (N) gene, the glycoprotein (GP) gene and the 3' half of the RNA polymerase gene of PBNV. PYSV genomic RNAs also failed to hybridise with cDNA probes from the GP genes of TSWV and INSV. In reciprocal tests, the cDNA clones of PYSV S and M RNAs did not hybridise with any of the PBNV RNAs. Based on the absence of serological relationships between PYSV and PBNV, TSWV and INSV and lack of nucleotide homology based on hybridisation studies between the PYSV RNAs and cDNA clones from PBNV, TSWV and INSV, PYSV should be considered as a distinct species of the genus Tospovirus under a new serogroup, putatively designated ‘V’.     

The nucleotide sequence of the S RNA of Impatiens necrotic spot virus, a novel tospovirus.

Impatiens necrotic spot virus (INSV) shares a number of properties with tomato spotted wilt virus (TSWV), the type species of the genus tospovirus within the family Bunyaviridae. INSV, however, differs from TSWV in plant host range and serology. In order to define the genomic structure and the taxonomic status of this TSWV-like virus, the nucleotide sequence of its genomic S RNA segment has been determined. The molecular data obtained demonstrate that, like TSWV, INSV has an ambisense S RNA molecule, encoding a non-structural protein in viral sense and the nucleocapsid protein in viral complementary sense. The level of nucleotide sequence homology between their S RNAs, as well as the divergence in amino acid sequence homology of their gene products, confirm previous conclusions from serological studies that INSV and TSWV represent distinct virus species within the newly created genus, tospovirus.     

Analysis of Tospovirus NSs Proteins in Suppression of Systemic Silencing

RNA silencing is a sequence-specific gene regulation mechanism that in plants also acts antiviral. In order to counteract antiviral RNA silencing, viruses have evolved RNA silencing suppressors (RSS). In the case of tospoviruses, the non-structural NSs protein has been identified as the RSS. Although the tomato spotted wilt virus (TSWV) tospovirus NSs protein has been shown to exhibit affinity to long and small dsRNA molecules, its ability to suppress the non-cell autonomous part of RNA silencing has only been studied to a limited extent. Here, the NSs proteins of TSWV, groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), representatives for three distinct tospovirus species, have been studied on their ability and strength to suppress local and systemic silencing. A system has been developed to quantify suppression of GFP silencing in Nicotiana benthamiana 16C lines, to allow a comparison of relative RNA silencing suppressor strength. It is shown that NSs of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSs^TYRV was just as strong as those by NSs^TSWV and NSs^GRSV, even though NSs^TYRV was expressed in lower amounts. Using the system established, a set of selected NSs^TSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream the biogenesis of siRNAs and their sequestration. The findings are discussed in light of the affinity of NSs for small and long dsRNA, and recent mutant screen of NSs^TSWV to map domains required for RSS activity and triggering of Tsw-governed resistance.     

A Survey of Tospoviruses in Bulgaria

A survey of tospoviruses present in Bulgaria was conducted during three vegetation periods. A total of 258 different virus isolates were obtained from tobacco, tomato, weeds and ornamentals in the open fields and from glasshouses. Virus isolates were identified by inoculation to test plants and serology as Tomato spotted wilt virus (TSWV). No indications were obtained that other Tospovirus species were present in Bulgaria. In addition the transmission ability and efficiency of several Bulgarian populations of Thrips tabaci (Lind.) and Frankliniella occidentalis (Perg.) was determined. The experimental results indicated that Bulgarian TSWV isolates are transmitted with high efficiency by F. occidentalis and that only arrhenotokous and not thelytokouse populations of T. tabaci could transmit TSWV.     

Transgenic tobacco plants expressing the putative movement protein of tomato spotted wilt tospovirus exhibit aberrations in growth and appearance

Within the Bunyaviridae virus family, members of the genus Tospovirus are unique in their ability to infect plants. A characteristic genetic difference between tospoviruses and the animal-infecting members of this virus family is the occurrence of an additional gene, denoted NSM, located on the genomic M RNA segment. This gene has previously been implicated in the cell-to-cell movement of this virus during systemic infection. Transgenic tobacco plants have been obtained expressing the NSM protein of tomato spotted wilt virus (TSWV), the type member of the tospoviruses, from a constitutive promoter. Detectable amounts of the NSM protein could be observed in plants from nine different lines. The protein was only detectable in fractions enriched for cell wall material. More detailed immunogold labelling studies revealed specific association of NSM protein with plasmodesmata. Plants accumulating the NSM protein to detectable levels developed aberrations in growth, resulting in a significant reduction of size and accelerated senescence. In addition, these plants are restricted in their capacity to produce flowers. The results presented provide additional evidence that the NSM protein, by modifying plasmodesmata, represents the cell-to-cell movement function of tospoviruses. Furthermore, the phenotype of the NSM transgenic plants suggests involvement of the NSM gene product in TSWV symptom expression     

Tomato spotted wilt virus (TSWV) in Greece: its incidence following the expansion of Frankliniella occidentalis, and characterisation of isolates collected from various hosts

Leaf samples were collected from plants with tospovirus‐like symptoms from various hosts in different regions of Greece where Thrips tabaci, Frankliniella occidentalis or both vectors occur. The viruses infecting these plants were identified with polyclonal antibodies raised against the N proteins of Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) by ELISA. All samples tested positive for TSWV, but not for INSV. ELISA of thirty three isolates, using monoclonal antibodies against the N protein of TSWV, revealed the existence of five epitopes on the N protein. RT‐PCR tests on a few randomly‐selected isolates, using a pair of universal primers, a pair of primers specific for the L gene and a pair of primers specific for the N gene, as well as sequence analysis of a part of the S gene of one isolate, confirmed the authenticity of the virus isolated as TSWV. Host range studies showed differences in susceptibility, especially among species belonging to the Leguminosae and Cucurbitaceae. The species Beloporone guttata and Coleus sp. are reported for the first time as hosts of the virus, whereas Solanum melongena, Celosia cristata, Dianthus chinensis, Stephanotis floribunda and Catharanthus roseus were identified as new hosts of TSWV in Greece.     

Complementation between Two Tospoviruses Facilitates the Systemic Movement of a Plant Virus Silencing Suppressor in an Otherwise Restrictive Host

Background

New viruses pathogenic to plants continue to emerge due to mutation, recombination, or reassortment among genomic segments among individual viruses. Tospoviruses cause significant economic damage to a wide range of crops in many parts of the world. The genetic or molecular basis of the continued emergence of new tospoviruses and new hosts is not well understood though it is generally accepted that reassortment and/or genetic complementation among the three genomic segments of individual viruses could be contributing to this variability since plants infected with more than one tospovirus are not uncommon in nature.

Methodology/Principal Findings

Two distinct and economically important tospoviruses, Iris yellow spot virus (IYSV) and Tomato spotted wilt virus (TSWV), were investigated for inter-virus interactions at the molecular level in dually-infected plants. Datura (Datura stramonium) is a permissive host for TSWV, while it restricts the movement of IYSV to inoculated leaves. In plants infected with both viruses, however, TSWV facilitated the selective movement of the viral gene silencing suppressor (NSs) gene of IYSV to the younger, uninoculated leaves. The small RNA expression profiles of IYSV and TSWV in single- and dually-infected datura plants showed that systemic leaves of dually-infected plants had reduced levels of TSWV N gene-specific small interfering RNAs (siRNAs). No TSWV NSs-specific siRNAs were detected either in the inoculated or systemic leaves of dually-infected datura plants indicating a more efficient suppression of host silencing machinery in the presence of NSs from both viruses as compared to the presence of only TSWV NSs.

Conclusion/Significance

Our study identifies a new role for the viral gene silencing suppressor in potentially modulating the biology and host range of viruses and underscores the importance of virally-coded suppressors of gene silencing in virus infection of plants. This is the first experimental evidence of functional complementation between two distinct tospoviruses in the Bunyaviridae family.     

Selection of resistance breaking strains of tomato spotted wilt tospovirus

In glasshouse tests, sap from plants infected with 15 different isolates of tomato spotted wilt tospovirus (TSWV) from three Australian states was inoculated to nine genotypes of tomato carrying TSWV resistance gene Sw-5 or one of its alleles. A further two resistant tomato genotypes were inoculated with four isolates each. The normal response in resistant genotypes was development of necrotic local lesions in inoculated leaves without systemic invasion, but 22/752 plants also developed systemic reactions in addition to local hypersensitive ones. Using cultures from two of these systemically infected plants and following four cycles of subculture in TSWV resistant tomato plants, two isolates were obtained that gave susceptible type systemic reactions but no necrotic spots in inoculated leaves of resistant tomatoes. When these two isolates, Da_WA-1d and To_TAS-1d, were maintained by repeated subculture for 10 successive cycles in Nicotiana glutinosa or a susceptible tomato genotype, they still induced susceptible type systemic reactions when inoculated to resistant tomato plants. They were therefore stable resistance breaking isolates as regards overcoming gene Sw-5. When resistance-breaking isolate Da_WA^-1ld multiplied together with original isolate Da_WA-l in susceptible tomato, it was fully competitive with the original isolate. However, when Da_WA-ld and To_TAS-ld were inoculated to TSWV resistant Lycopersicon peruvianum lines PI 128660R and PI 128660S and to TSWV resistant Capsicum chinense lines PI 152225, PI 159236 and AVRDC CO0943, they failed to overcome the resistance, producing only necrotic local lesions without systemic infection. Thus, although the ease of selection, stability and competitive ability of resistance breaking isolates of TSWV is cause for concern, L. peruvianum and C. chinense lines are available which are effective against them. The effectiveness of the resistance to TSWV in nine tomato genotypes was examined in a field experiment. Spread was substantial in the susceptible control genotype infecting 42% of plants. Resistance was ineffective in cv. Bronze Rebel, 26% of plants developing infection. In contrast, it held up well in the other eight resistant genotypes with only 1–3 or no plants of each becoming infected. Accumulated numbers of Thrips tabaci, Frankliniella occidentalis and F. schultzei were closely correlated with TSWV spread.     

Root thiophenes in Tagetes minuta L. accessions from Argentina: genetic and environmental contribution to changes in concentration and composition

Three wild Tagetes minuta accessions were collected from Buenos Aires, Mendoza, and Salta provinces in Argentina and were grown in the same environment to study the concentration and composition of root thiophenes, and the production of aerial biomass. The accessions showed quantitative, but not qualitative differences in thiophenes. Five thiophenes were identified. In the Buenos Aires and Salta accessions, concentrations of total thiophenes were independent of changes in aerial biomass production, but in the Mendoza accession we found a positive correlation between total thiophenes and aerial biomass. In the Mendoza accession we observed dramatic changes in the concentration of thiophenes between years. The composition of thiophenes in the Salta and Mendoza accessions was characterized by low concentrations of BBT and α-T and high concentrations of BBTOH, whereas thiophenes in the Mendoza accession had a higher concentration of BBTOH than did the Buenos Aires accession.     

Neochrysocharis formosa (Westwood) (Hymenoptera: Eulophidae), a newly recorded parasitoid of the tomato moth, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae), in Argentina

We report the first record of Neochrysocharis formosa (Westwood) parasitizing larvae of the tomato moth, Tuta absoluta (Meyrick), in tomato crops in Northern Buenos Aires Province, Argentina. Tomato moth larvae were sampled during four consecutive growing cycles, between 2003 and 2005, in 10 sites. Neochrysocharis formosa was present only in organic outdoor and protected crops, and predominantly during the late season. Parasitism rates varied from 1.5% to 5%. The finding of this species is a new record for Argentina and South America, and T. absoluta is a new host record.     

A UK Isolate of Impatiens Necrotic Spot Virus From Glasshouse-grown Cineraria

A tospovirus was detected by ELISA in a Cineraria sample from a commercial protected crop in the UK. Its seroiogical properties indicated it to be Impatiens necrotic spot virus (INSV), results confirmed by RT-PCR tests and nucleotide sequence data on the nucleoprotein-encoding region of the genome. This is the first report of the presence of INSV in a protected crop grown in the UK.     

Distinct feeding behavior between sexes of shape Frankliniella occidentalis results in higher scar production and lower tospovirus transmission by females

Feeding behavior and scar production of male and female F. occidentalis (Pergande) (Thysanoptera: Thripidae) were studied in relation to transmission of tomato spotted wilt tospovirus (TSWV). Electrical penetration graph (EPG) analysis showed that females feed more frequently and intensively than males. The feeding intensity, reflected by silvery scar production and studied by an image analysis system, demonstrated that females induced more numerous scars than males. At the same time, males transmitted TSWV with a higher efficiency than females, indicating that TSWV transmission and scar production are not positively correlated. Furthermore, males produced significantly more local lesions of TSWV than females. These quantitative differences in scar production and transmission of TSWV can be explained by the lower mobility and higher consumption rate of females. The influence of the sex-ratio on crop damage and virus transmission, and thus to the spread of TSWV, is emphasized.     

Allelic relationships between genes for resistance to tomato spotted wilt tospovirus in Capsicum chinense

Pepper (Capsicum chinense Jacq.) has been reported to be an important reservoir of resistance genes to tomato spotted wilt virus (TSWV). The genes for TSWV resistance present in three C. chinense lines (PI 152225, PI 159236 and Panca) were investigated for allelism. All resistant lines were crossed with each other. Parents, F₁, backcrosses and F₂ populations (including reciprocals) developed from those crosses were mechanically inoculated with a highly virulent TSWV isolate. Susceptible C. annuum cv Magda was used to check inoculum virulence. Fifty plants of the F₁ hybrids; Magda x PI 152225, Magda x PI 159236, and Magda x 'Panca, were also inoculated with the TSWV isolate. The resistance response in all C. chinense sources was associated with a localized, hypersensitive-like reaction that was phenotypically expressed as a prompt formation of large local lesions accompanied by premature leaf abscission. All F₁ generations presented a final score of resistant; indicating that the expression of resistance to TSWV is conditioned by a dominant gene regardless of the source. The absence of segregation for resistance to TSWV that was observed in all generations of the crosses between C. chinense lines indicated that either a tightly linked group of genes exists or that the resistance is governed by the same single major gene (probably the already described Tsw gene). Previous reports have indicated that the Tsw gene is not effective against tospovirus members of serogroup II, i.e. tomato chlorotic spot virus (TCSV) and groundnut ring spot virus (GRSV). In the assay described here, all of the C. chinense lines showed, after mechanical inoculation, an identical susceptibility response to the TCSV and GRSV isolates.     

Disease intensity of some tomato viroses in Serbia

In this paper we present the data on the disease intensity of the tomato plants grown in glass and plastic-houses, and in the open field. The infection was caused by the following viruses: Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), Tomato spotted wilt virus (TSWV), Alfalfa mosaic virus (AMV), Potato virus X (PVX), Potato virus Y (PVY), Tomato black ring virus (TBRV), Tomato ringspot virus (ToRSV), Tomato aspermy virus (TAV), and Cucumber mosaic virus (CMV). These viruses represented most frequent tomato pathogens in Serbia. According to the obtained results, it could be concluded that 92.94% of the tested tomato plants grown in glass and plastic-houses, and 89.82% grown in the open field were infected by one of the above viruses. Most of the plant samples were infected by two or more viruses. The most frequent viruses — tomato pathogens in Serbia were ToMV, PVY and TMV.     

Selection, biological properties and fitness of resistance-breaking strains of Tomato spotted wilt virus in pepper

In glasshouse tests, infective sap from plants infected with 17 different isolates of Tomato spotted wilt virus (TSWV) from four Australian states was inoculated to three Capsicum chinense accessions (PI 152225, PI 159236 and C00943) carrying single genes that confer hypersensitive resistance to TSWV. The normal response to inoculation was development of necrotic (hypersensitive) local lesions in inoculated leaves without systemic invasion, but 3/1386 infected plants also developed systemic susceptible reactions in addition to hypersensitive ones. Similarly when two isolates were inoculated to C. chinense backcross progeny plants, 1/72 developed systemic susceptible reactions in addition to localised hypersensitive ones. Using cultures from the four plants with susceptible reactions and following three to five further cycles of serial subculture in TSWV‐resistant C. chinense plants, four isolates were obtained that gave systemic susceptible type reactions in the three TSWV‐resistant accessions, and in TSWV‐resistant cultivated pepper (C. annuum). When three of these isolates were inoculated to tomato (Lycopersicon esculentum) breeding lines with single gene resistance to TSWV, resistance was not overcome. Similarly, none of the four isolates overcame partial resistance to TSWV in Lactuca virosa. When TSWV isolates were inoculated to tomato breeding lines carrying partial resistance from L. chilense, systemic infection developed which was sometimes followed by ‘recovery’. After four successive cycles of serial passage in susceptible cultivated pepper of a mixed culture of a resistance‐breaking isolate with the non resistance‐breaking isolate from which it came, the resistance‐breaking isolate remained competitive as both were still found. However, when the same resistance‐ breaking isolate was cultured alone, evidence of partial reversion to wild‐type behaviour was eventually obtained after five but not four cycles of long term serial subculture in susceptible pepper, as by then the culture had become a mixture of both types of strain. This work suggests that resistance‐breaking strains of TSWV that overcome single gene hypersensitive resistance in pepper are relatively stable. The findings have important implications for situations where resistant pepper cultivars are deployed widely in the field without taking other control measures as part of an integrated TSWV management strategy.     

Differences in tomato spotted wilt virus vector competency between males and females of Frankliniella occidentalis

Possible differences in tomato spotted wilt virus (TSWV) transmission vector competency between Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) males and females were investigated. The males of the Dutch reference population NL3 transmitted TSWV at a notably higher rate (57%) than the females (32%). The viruliferous males also transmitted more frequently within the first six days after adult emergence than the females. For both sexes, the transmission efficiency dropped with age, simultaneously with the consumption rate. The higher vector efficiency for males appeared to be a general phenomenon as this feature was also found for thirteen other F. occidentalis populations, which originated from distinct geographic regions.