Tomato Spotted Wilt Virus Resistance Research Articles

Fruit melanotic ringspot (FMRS) – a disease of resistant Capsicum genotypes infected with Tomato spotted wilt virus (TSWV) on the fruits

Etiology of pepper fruit melanotic ringspot (FMRS) disease (Salamon, 2009) was studied on fruit samples collected in forced pepper populations. It was noticed that in spite of heavy thrips (Frankliniella occidentalis) infestations and of TSWV epidemy detected in the forcing houses, FMRS occurred only in plants having healthy foliage. Symptomatological surveys strongly suggested that FMRS appeared exclusively in specific pepper genotypes. The size of melanotic ringspots has been observed to grow at room temperature during postripening of diseased fruits. A mechanically transmitted plant virus was isolated from symptomatic parts of 9 white pepper fruits affected by FMRS. On test plants each of the virus isolates caused systemic symptoms characteristic to TSWV. Using cDNA/PCR technique and TSWV N-gene specific primers a ca. 300 bp long DNA fragment has been amplified from total nucleic acid extracted from symptomatic tissues but never from asymptomatic parts of the fruits showing FMRS. Plant progenies grown from seeds of FMRS diseased fruits segregated in respect of resistance and/or susceptibility to TSWV infection. TSWV was also detected in and isolated from three fruits showed non-melanotic yellow rings (one of them was infected with a tobamovirus, too). Seedlings derived from these fruits proved to be susceptible to TSWV. Based on the above results we could conclude that the FMRS disease developed on fruits of “cecei” type white peppers that carry a TSWV resistance gene, most likely the Tsw gene in heterozygous form. These fruits were infected with thrips transmitted TSWV and FRMS appeared as a hypersentive reaction (HR) manifested in fruits.

Modified expression of alternative oxidase in transgenic tomato and petunia affects the level of tomato spotted wilt virus resistance.

BackgroundTomato spotted wilt virus (TSWV) has a very wide host range, and is transmitted in a persistent manner by several species of thrips. These characteristics make this virus difficult to control. We show here that the over-expression of the mitochondrial alternative oxidase (AOX) in tomato and petunia is related to TSWV resistance.ResultsThe open reading frame and full-length sequence of the tomato AOX gene LeAox1au were cloned and introduced into tomato 'Healani' and petunia 'Sheer Madness' using Agrobacterium-mediated transformation. Highly expressed AOX transgenic tomato and petunia plants were selfed and transgenic R1 seedlings from 10 tomato lines and 12 petunia lines were used for bioassay. For each assayed line, 22 to 32 tomato R1 progeny in three replications and 39 to 128 petunia progeny in 13 replications were challenged with TSWV. Enzyme-Linked Immunosorbent Assays showed that the TSWV levels in transgenic tomato line FKT4-1 was significantly lower than that of wild-type controls after challenge with TSWV. In addition, transgenic petunia line FKP10 showed significantly less lesion number and smaller lesion size than non-transgenic controls after inoculation by TSWV.ConclusionIn all assayed transgenic tomato lines, a higher percentage of transgenic progeny had lower TSWV levels than non-transgenic plants after challenge with TSWV, and the significantly increased resistant levels of tomato and petunia lines identified in this study indicate that altered expression levels of AOX in tomato and petunia can affect the levels of TSWV resistance.

Evaluation of tomato (Lycopersicon esculentum Mill.) plants with natural and transgenic resistance against Tomato spotted wilt virus (TSWV) isolates occurring in the Republic of South Africa (RSA)

Tomato spotted wilt virus (TSWV) infections causes significant economic losses in the commercial production of tomato (Lycopersicon esculentum Mill.). This study was undertaken to evaluate tomato with natural and transgenic resistance when inoculated with TSWV isolates occurring in the Republic of South Africa (RSA). The Stevens cultivar which has natural resistance to TSWV conferred by the Sw-5 gene and the transgenic 13-1 line which expresses the nucleocapsid (N) protein gene of the TSWV-BL isolate were used as test plants. Six TSWV isolates collected from Gauteng, KwaZulu-Natal, North West, Limpopo and Mpumulanga provinces were mechanically inoculated onto test plants. The trial was arranged in a general treatment structure with randomized block design and repeated once. Plants were assessed for TSWV resistance based on a disease severity rating scale and measurements of virion accumulation levels (A405nm) using Enzyme linked immunosorbent assay (ELISA)[A1]. There were no significant differences among the reactions produced by the six TSWV isolates on the test plants. Although both plants were susceptible to the TSWV isolates from RSA by exhibiting similar high viral accumulation levels, the transgenic tomato line showed milder disease severity than the natural resistant cultivar. Results suggest that transgenic resistance is a more viable approach in the control of TSWV in RSA. Key words: Tomato spotted wilt virus (TSWV), Republic of South Africa, virus resistance, transgenic, tomato (Lycopersicon esculentum Mill.).

Identification of molecular markers for Sw-5 gene of tomato spotted wilt virus resistance

Tomato spotted wilt virus (TSWV) is one of the most destructive viral diseases that threaten worldwide tomato (Solanuum lycopersicum L. syn Lycopersicon esculentum Mill.) production. Use of host resistance appears to be the best way to control the disease. Marker assisted selection (MAS) has become very important and useful in the selection of TSWV resistance genes. The objective of this research was to identify Sw-5 gene-specific PCR-based molecular markers and gene-derived single nucleotide polymorphism (SNP) markers for MAS in tomato breeding. A gene-specific PCR-based marker and two gene-derived SNP markers specific for TSWV resistance Sw5-b allele were identified and validated in different tomato germplasm. It is expected that the present findings will be helpful in advancing the MAS for TSWV resistance in tomato.

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes.

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F1 hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F1 hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.

TSWV resistance in DH lines of tobacco (Nicotiana tabacum L.) obtained from a hybrid between ‘Polalta’ and ‘Wiślica’

With 2 tables Abstract Nicotiana tabacum cv. ‘Polalta’ is reported as having a hypersensitive type of Tomato spotted wilt virus (TSWV) resistance. In ‘Polalta’ hybrids with susceptible cultivars, morphological deformations (thickened and ribbon‐shaped leaves, irregular venation, tumours) appear. Acceptable DH lines derived from hybrids of ‘Polalta’ with ‘Wiślica’ were obtained by a method of in vitro culture. Those DH lines were evaluated for TSWV resistance. Tests for resistance were conducted under greenhouse conditions using one TSWV isolate from a local source. The presence of the virus was verified using DAS‐ELISA. The DH lines PW‐833, PW‐834, and PW‐900 showed a near correct morphology and stably inherited dominant monogenic resistance to TSWV.

Field evaluation for the combination of white mould and tomato spotted wilt disease resistance among peanut genotypes

White mould, stem rot, and southern blight are caused by the same soilborne fungus, Sclerotium rolfsii Sacc. and when combined with tomato spotted wilt caused by Tomato spotted wilt virus (TSWV), result in major disease problems in the cultivated peanut ( Arachis hypogaea L.). These disease problems are universal and occur not only in the southeast U.S.A. but also in most peanut production areas. Consequently, there is an increasing need for genotypic resistance as the most cost effective way to reduce the financial loss due to the combination of these two diseases. During five years (2004–08), several advanced breeding lines and recently released peanut cultivars from the Georgia peanut breeding program were evaluated for combined resistance to both pathogens. The best combination of white mould and TSWV resistance and highest yield over multiple years was consistently found in the recently released medium-maturity, runner-type peanut cultivars, Georgia-07W, Georgia-03L, and AP-3. However, all of the other Georgia cultivars and advanced Georgia breeding lines were also found to be either similar or significantly better than the check cultivars C-99R and Florida-07.

The N protein of Tomato spotted wilt virus (TSWV) is associated with the induction of programmed cell death (PCD) in Capsicum chinense plants, a hypersensitive host to TSWV infection

In sweet pepper, the Tsw gene, originally described in Capsicum chinense, has been widely used as an efficient gene for inducing a hypersensitivity response (HR) derived Tomato spotted wilt virus (TSWV) resistance. Since previously reported studies suggested that the TSWV-S RNA mutation(s) are associated with the breakdown of Tsw mediated TSWV resistance in peppers, the TSWV genes N (structural nucleocapsid protein) and NSS (non-structural silencing suppressor protein) were cloned into a Potato virus X (PVX)-based expression vector, and inoculated into the TSWV-resistant C. chinense genotype, PI 159236, to identify the Tsw-HR viral elicitor. Typical HR-like chlorotic and necrotic lesions followed by leaf abscission were observed only in C. chinense plants inoculated with the PVX-N construct. Cytopathological analyses of these plants identified fragmented genomic DNA, indicative of programmed cell death (PCD), in mesophyll cell nuclei surrounding PVX-N-induced necrotic lesions. The other constructs induced only PVX-like symptoms without HR-like lesions and there were no microscopic signs of PCD. The mechanism of TSWV N-gene HR induction is apparently species specific as the N gene of a related tospovirus, Tomato chlorotic spot virus, was not a HR elicitor and did not cause PCD in infected cells.

Screening Two Lycopersicon peruvianum Collections for Resistance to Tomato spotted wilt virus.

The United States Department of Agriculture (USDA) Research Service and the Tomato Genetics Resource Center (TGRC) Lycopersicon peruvianum germplasm collections (16,335 plants from 285 accessions) were screened with the Tomato spotted wilt virus (TSWV) isolates TSWV6 from Hawaii, and Anwa-1 from Western Australia. Using TSWV6 to screen for resistance, 10,634 L. peruvianum plants from 280 accessions were screened for resistance, resulting in 168 (60%) accessions with 1,437 (14%) plants indicating resistance, with all 1,404 89S (Sw-5+/Sw-5+) and 1,456 89R (Sw-5/Sw-5) controls infected. When using Anwa-1 for screening, 864 (15%) of 5,701 L. peruvianum plants were uninfected from 106 of the 181 accessions tested, and 472 (95%) of the 495 89S and 421 (73%) of the 574 89R controls were infected. Of the 172 accessions tested with both isolates, 54 were resistant to one isolate but not the other. Additionally, more accessions from the USDA than from the TGRC collection indicated resistance. TSWV-resistant accessions were somewhat equally distributed throughout the L. peruvianum geographic range, with an observation that northern Chile and southern Peru seemed to have an unusually high portion of accession indicating resistance. The value of Sw-5 is discussed in relationship to potential additional sources of TSWV resistance.

AFLP and SCAR Markers Linked to Tomato Spotted Wilt Virus Resistance in Tobacco

Tomato spotted wilt virus (TSWV) is a serious disease in tobacco (Nicotiana tabacum L.). The breeding line ‘Polalta’ contains a single dominant gene conferring resistance to TSWV that was introgressed from N. alata Link & Otto. The resistance is tightly associated with an abnormal plant type, however, and traditional backcrossing has been ineffective in producing normal plants with TSWV resistance. A potential strategy to overcome this problem is to use molecular markers to select against alien chromatin in backcross progeny. Amplified fragment length polymorphism (AFLP) technology and bulked segregant analysis were applied to identify markers linked to the resistance gene. The DNA bulks from susceptible and resistant doubled haploid lines derived from a cross between susceptible cultivar K326 and Polalta were analyzed to identify linked markers. An F2 population and the doubled haploids were used to construct a 2.5‐cM map of this locus containing 17 coupling phase and seven repulsion phase markers. By selecting for resistant plants with an improved plant type and marker profile in a BC3 population, four plants were identified that were missing 1 to 14 of the AFLP markers associated with the alien chromatin, demonstrating that rare recombination events can be identified. Four AFLP fragments were successfully converted to sequence characterized amplified region markers suitable for large‐scale screening. This approach may facilitate development of TSWV‐resistant tobacco cultivars.

A New Dominantly Inherited Source of TSWV Resistance in Tomato Derived from L. chilense, which Resists Isolates that Overcome Sw-5

Tomato spotted wilt virus (TSWV), a tospovirus, is a thrips-vectored disease infecting more than 1000 species of both monocots and dicots, including many species of agriculture importance. TSWV is the limiting factor for tomato (Lycopersicum esculentum Mill.) production in several areas of the world. For a number of years, the Sw-5 gene (derived from L. peruvianum Mill.) has provided acceptable control of this disease. Recently, Sw-5 derived resistance has been overcome by virulent pathogen isolate(s) in tomato production areas such as Spain and Italy. In earlier studies, we identified a potential new source of resistance to TSWV derived from L. chilense Dun. accession LA 1938. In a set of recent field studies, it was demonstrated that this putative new source of resistance was highly resistant to TSWV in Hawaii, Florida/Georgia, and South Africa. Furthermore, greenhouse screening trials have clearly demonstrated that the L. chilense source of TSWV resistance is resistant to isolates that overcome tomatoes homozygous for Sw-5. In these same greenhouse and field studies, there is uniform evidence that this resistance is dominant. Subsequent greenhouse studies suggest that this resistance is controlled by a single gene. Studies have been initiated to verify the inheritance of the gene(s) and to develop linked molecular markers. Furthermore, studies are under way in Australia to test this resistance on non-TSWV tospoviruses. If the data demonstrate that this is a single dominant gene we suggest this gene be designated Sw-7.

Genetic variability of Tomato spotted wilt virus in Australia and validation of real time RT‐PCR for its detection in single and bulked leaf samples

SummaryThe potential for large‐scale use of a sensitive real time reverse transcription polymerase chain reaction (RT‐PCR) assay was evaluated for the detection of Tomato spotted wilt virus (TSWV) in single and bulked leaf samples by comparing its sensitivity with that of DAS‐ELISA. Using total RNA extracted with RNeasy® or leaf soak methods, real time RT‐PCR detected TSWV in all infected samples collected from 16 horticultural crop species (including flowers, herbs and vegetables), two arable crop species, and four weed species by both assays. In samples in which DAS‐ELISA had previously detected TSWV, real time RT‐PCR was effective at detecting it in leaf tissues of all 22 plant species tested at a wide range of concentrations. Bulk samples required more robust and extensive extraction methods with real time RT‐PCR, but it generally detected one infected sample in 1000 uninfected ones. By contrast, ELISA was less sensitive when used to test bulked samples, once detecting up to 1 infected in 800 samples with pepper but never detecting more than 1 infected in 200 samples in tomato and lettuce. It was also less reliable than real time RT‐PCR when used to test samples from parts of the leaf where the virus concentration was low. The genetic variability among Australian isolates of TSWV was small. Direct sequencing of a 587 bp region of the nucleoprotein gene (S RNA) of 29 isolates from diverse crops and geographical locations yielded a maximum of only 4.3% nucleotide sequence difference. Phylogenetic analysis revealed no obvious groupings of isolates according to geographic origin or host species. TSWV isolates, that break TSWV resistance genes in tomato or pepper did not differ significantly in the N gene region studied, indicating that a different region of the virus genome is responsible for this trait.

Tomato spotted wilt virus resistance among high and normal O/L ratio peanut cultivars with and without irrigation

In recent years, several high oleic (O) and low linoleic (L) fatty acid ratio peanut ( Arachis hypogaea L.) cultivars with varying levels of disease resistance have been developed and released for commercial production. Improved disease resistance has become of even greater importance, particularly resistance to tomato spotted wilt virus (TSWV) which causes spotted wilt disease in peanut. The objective of this study was to compare these high and normal O/L ratio peanut cultivars with and without irrigation for yield and TSWV resistance. For three consecutive years (1997–1999) the normal O/L ratio cultivar ‘Georgia Green’ and the high O/L ratio cultivar ‘Georgia Hi-O/L’ were found to have the highest pod yield, and a comparably high level of resistance to TSWV when compared to 11 other cultivars with and without irrigation. Georgia Hi-O/L was also found to have a significantly higher O/L ratio than the other high and normal O/L ratio cultivars for improved peanut oil quality both with and without irrigation. In general, irrigation appeared to increase expression of plant symptoms for TSWV disease incidence and had little effect upon O/L ratios among the 13 peanut cultivars evaluated.

Tomato Spotted Wilt Virus (TSWV) resistance in tomato derived from Lycopersicon chilense Dun. LA 1938

Tomato spotted wilt virus (TSWV) resistance wasidentified in Y118 (Fla 925-2), an F1BC1S6 tomato line(Lycopersicon esculentum Mill.), derived from a crosswith L. chilense Dun. (LA 1938). This line waspreviously selected for tomato mottle virus (ToMoV)resistance in Florida. Progeny from crosses betweenFla 925-2 and three different TSWV susceptible L.esculentum parents were used in TSWV resistancestudies. A total of 75 F1 and 596 F2 plants from allthree crosses were screened for TSWV resistance. ForF2 plants free of TSWV symptoms, evaluations were madeusing enzyme-linked immunosorbent assay (ELISA). TenF3 populations used for further greenhouse and fieldscreenings were selected from F2 plants found to befree of the virus using visual and ELISA criteria ateach evaluation. One F1 and four F3 lines werestudied under field conditions (Stellenbosch, SouthAfrica) in which 100% of the `Flora-Dade' susceptiblecontrols were severely infected with TSWV. Theresults of the field study clearly establish that TSWVfield resistance is present in the Fla 925-2 (Y118)derived lines. Studies conducted on these linesrevealed that this resistance has the distinctcharacteristic of often `recovering' from initiallyhigh levels of virus titer in the tissue to levelsbelow detection with ELISA.

High temperature effects on hypersensitive resistance to Tomato Spotted Wilt Tospovirus (TSWV) in pepper (Capsicum chinense Jacq.)

The tomato spotted wilt virus resistance in pepper conferred by the Tsw gene is less stable at 32 °C continuous temperature. Continuous high temperatures for at least nine days lead to systemic spread and necrotic symptoms in plants that are totally resistant at a lower temperature (22 °C). We show that continuous high temperatures destabilize this resistance in young plants, but older inoculated plants rarely develop systemic symptoms. Populations segregating for the Tsw gene showed that heterozygosity at the Tsw locus increased the chance of inoculated seedlings to develop systemic necrotic symptoms. Genetic background was less significantly involved in the thermosensitivity of the resistant response. As a consequence, it would be preferable to grow cultivars homozygous at the Tsw locus in high temperature conditions.

Genetics of tomato spotted wilt virus resistance coming from Lycopersicon peruvianum

New resistance sources coming from Lycopersicon peruvianum, especially those introgressed in UPV 32 line, are studied. UPV 32 resistance is controlled by a single gene. Resistance and dominance levels of this gene are conditioned by thrips transmission and isolate aggressiveness. A partial overcoming of resistance occurs due to the incomplete penetrance of the gene. Incomplete dominance of resistance also happens, which suggests gene dosage dependence. UPV 32 gene segregates independently of both Sw-5 gene and UPV 1 resistance gene, also coming from Lycopersicon peruvianum. It is proposed to name Sw-6 this new locus from UPV 32. Sw-5 gene and UPV 1 resistance gene show higher resistance than Sw-6. Heterozygotes for UPV 1 resistance gene were more resistant than heterozygotes for Sw-5. The lower dependence of UPV 1 resistance gene on the gene dosage effect makes it very useful for the development of commercial hybrids.

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato.

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones.

Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomatoes using RAPD and RFLP analyses

The Sw-5 locus confers dominant resistance to tomato spotted wilt virus (TSWV). To map the location and facilitate the identification of markers linked to Sw-5 we developed a pair of near-isogenic lines (NILs) and an F2 Lycopersicon esculentum x L. pennellii population segregating for resistance to TSWV. DNA from the NILs was analyzed using 748 random 10-mer oligonucleotides to discern linked molecular markers using a random amplified polymorphic DNA (RAPD) approach. One random primer (GAGCACGGGA) was found to produce a RAPD band of about 2200 bp that demonstrates linkage to Sw-5. Data from co-segregation of resistance and restriction fragment length polymorphisms (RFLPs) in a F2 interspecific population position Sw-5 between the markers CT71 and CT220 near the telomere of the long arm of chromosome 9.