First Report of the Resistance Breaking (RB) Strains of Tomato Spotted Wilt Virus (TSWV) Infecting Tomatoes with the Sw-5b Resistance Gene in Alabama

Novel resistance breaking (RB) strains of tomato spotted wilt orthotospovirus (TSWV) capable of disrupting single gene resistance in tomato (Sw-5b) and pepper (Tsw) have been reported worldwide. Thrips, a supervector of TSWV, transmit these strains in a suite of specialty and staple food crops across the globe. However, transmission biology of RB strains remains virtually unexplored. We investigated various transmission parameters viz. inoculation efficiency, putative sex-specific differences in inoculation, virus accumulation, and source sink relationships to dissect these interactions. Six novel strains of TSWV, namely Tom-BL1, Tom-BL2, Tom-CA, Tom-MX, Pep-BL and Non-RB, transmitted by western flower thrips (WFT) were used and thrips were allowed four 24h consecutive inoculation accession periods (IAPs). Our results show that most strains were inoculated at all four IAPs, however, their rates differed across IAPs. Overall, WFT had highest inoculation efficiency at the first and lowest at the second IAP. Female thrips carried higher virus titers; however, males were better at inoculating TSWV. Furthermore, we did not find significant positive correlations in virus titers between the tissues used for TSWV acquisition, thrips and thrips-inoculated leaf discs. Males inoculated RB strains at 87% efficiency whereas Non-RB strain at 80% efficiency. Female thrips were 77% and 75% efficient at inoculating RB and Non-RB strains, respectively. This study furnishes new insights into the transmission biology of TSWV RB strains, especially from inoculation and thrips sex perspectives, and provides a baseline for future molecular studies surrounding ever evolving novel TSWV strains.

Tomato spotted wilt orthotospovirus (TSWV) was first reported in 2004 from paprika in South Korea (Kim et al., 2004), where it is currently widespread. TSWV infections were reported in chili pepper, tomato, weeds, and ornamental plant species in South Korea (Choi et al., 2014; Choi and Choi, 2015; Yoon et al., 2016; Yoon et al., 2018; Yoon et al., 2019). One of the best strategies for TSWV management is planting resistant cultivars containing the Tsw gene. In 2019 virus-like symptoms were observed in chili pepper (Capsicum annuum) plants bearing the Tsw gene in Anseong-si, South Korea. The infected chili peppers showed mosaic and wilting followed by necrosis on leaves and fruits in the field. To identify the causal virus, symptomatic leaf samples were analyzed using ImmunoStrip kits (Agdia, USA); we detected three pepper-infecting viruses: Pepper mild mottle virus, Cucumber mosaic virus, and TSWV. TSWV was only detected from 40 naturally infected chili pepper plants exhibiting virus-like symptoms. To further confirm the presence of TSWV (named TSWV-P1), we amplified reverse-transcription polymerase chain reaction (RT-PCR) products for L, M, and S RNA segments using tospovirus-specific and TSWV-specific primers (Batuman et al., 2014). Expected fragments of 445, 868, and 777 bp in length were amplified and sequenced. The complete genome sequences of TSWV-P1 from a symptomatic chili pepper plant were also determined using TSWV-specific primers (Choi et al., 2014; Lian et al., 2013). The complete genome sequences of TSWV-P1 were deposited to GenBank (LC549179, LC549180, and LC549181). The sequences of each fragment were identical to a consensus sequence, showing 99.1%, 98.5%, and 98.6% identity with TSWV-L, M, and S RNA (KP008132, AY744492, and KP008134), respectively. These results clearly showed only a single TSWV infection among the naturally infected chili pepper plants, without reassortment between TSWV and another tospovirus. To confirm whether TSWV-P1 is a resistance-breaking (RB) strain, Nicotiana rustica was mechanically inoculated with sap from leaves of the infected pepper samples to propagate TSWV-P1. A non-RB TSWV isolate (TSWV-Kor-lisianthus) from lisianthus was used as a control (Yoon et al., 2017). Two resistant (with Tsw) and two susceptible chili pepper cultivars (20 plants per cultivar) were mechanically inoculated with sap from leaves of the TSWV-infected N. rustica. The incidence rates of disease caused by TSWV-P1 were 90-100% for resistant and 95-100% for susceptible cultivars. In contrast, TSWV-Kor-lisianthus caused symptoms only in the susceptible pepper cultivars (90-100% incidence). TSWV infection in representative plants was confirmed using the TSWV- ImmunoStrip kit and RT-PCR. The NSs gene of TSWV-P1 consists of 1,404 nucleotides (468 amino acids); sequence analysis of the TSWV-P1 NSs gene showed high nucleotide (99.7%) and amino acid identities (99.8%) with the NSs sequences of two TSWV isolates (FR693035, CBX24121). Protein sequence analysis of TSWV-P1 NSs revealed that no amino acid mutation was associated with those of a representative TSWV RB strain, as previously described (Almási et al., 2017), suggesting that TSWV-P1 is a RB strain. Because this TSWV-P1 can overcome resistance conferred by the Tsw gene in commercially grown chili pepper cultivars, it represents a potential threat to pepper production in South Korea.

In Hungary resurgence of Tomato spotted wilt virus (TSWV) frequently causesheavy crop losses in pepper production since the mid nineties. Management ofTSWV control was first directed against the thrips (using different insecticides orplastic traps), and against weeds as host plants of the virus and the thrips. Later onTsw resistance gene was introduced from Capsicum chinense PI 152225 and PI159236 into different types of pepper. In 2010 and 2011 sporadically, but in 2012more frequently a resistance breaking (RB) strain of TSWV on resistant peppercultivars was observed in the Szentes region (South-East Hungary). The presenceof a new resistance breaking strain was demonstrated by virological (test-plant,serological and RT-PCR) methods. Previously, the non-structural protein (NSs)encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor forTsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wildtype (WT) isolates and compared to previously analyzed TSWV strains with RBproperties from different geographical origins. Phylogenetic analysis demonstratedthat the different RB strains had the closest relationship with the local WT isolatesand there was no conserved mutation present in all the NSs genes of RB isolatesfrom different geographical origins. According to these results, it is concluded thatthe RB isolates evolved separately in geographic point of view and according to theRB mechanism. In order to find new genetic sources of resistance in Capsicumspecies 89 lines from Capsicum annuum, C. chinense, C. frutescens, C. chacoense,C. baccatum var. baccatum, C. baccatum var. pendulum and C. praetermissumwere tested with the Hungarian TSWV-RB isolate.

Resurgence of Tomato spotted wilt virus (TSWV) worldwide as well as in Hungary causing heavy economic losses directed the attention to the factors contributing to the outbreak of this serious epidemics. The introgression of Tsw resistance gene into various pepper cultivars seemed to solve TSWV control, but widely used resistant pepper cultivars bearing the same, unique resistance locus evoked the rapid emergence of resistance-breaking (RB) TSWV strains. In Hungary, the sporadic appearance of RB strains in pepper-producing region was first observed in 2010-2011, but in 2012 it was detected frequently. Previously, the non-structural protein (NSs) encoded by small RNA (S RNA) of TSWV was verified as the avirulence factor for Tsw resistance, therefore we analyzed the S RNA of the Hungarian RB and wild type (WT) isolates and compared to previously analyzed TSWV strains with RB properties from different geographical origins. Phylogenetic analysis demonstrated that the different RB strains had the closest relationship with the local WT isolates and there is no conserved mutation present in all the NSs genes of RB isolates from different geographical origins. According to these results, we concluded that the RB isolates evolved separately in geographic point of view, and also according to the RB mechanism.

Use of tomato cultivars with the Sw-5 resistance gene cluster has led to the occurrence of resistance-breaking (RB) tomato spotted wilt virus (TSWV) strains globally, including California and, recently, North Carolina and Texas. We documented disease on tomato infected with either an RB strain from California (CA-RB) or a wild type (CA-WT) strain of TSWV on tomato with (cultivar Mountain Merit) or without (cultivar Mountain Fresh Plus) the Sw-5b resistance gene and detected virus incidence over time using microneedle RNA extractions and LAMP. We developed a LAMP/Cas12a assay for detection of the CA-C118Y mutation in a CA-RB strain and tested the assay with field samples. Disease in the susceptible cultivar was less severe with CA-RB than with the CA-WT strain. In contrast, the resistant cultivar had little disease when inoculated with the CA-WT strain but exhibited stunting of greater than 50% when inoculated with the CA-RB strain. In the susceptible tomatoes, the detection rates over time by LAMP reaction were higher in CA-WT than in CA-RB-inoculated plants. In resistant tomato, CA-RB remained detectable by TSWV LAMP over 14 days, whereas the WT strain was undetectable. A two-step LAMP/Cas12a assay differentiated the two strains in 1 h. Our methods were validated with samples from TSWV-infected North Carolina fields. A phylogeny of NSm gene sequences that included North Carolina field samples revealed two independent origins of the North Carolina RB isolates. The LAMP/Cas12 assay showed excellent detection of the CA-C118Y mutation. The TSWV LAMP/Cas12a assay is adaptable for in-field applications on either a smart phone platform or heat block. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Characterization of Italian resistance-breaking isolates of tomato spotted wilt virus reveals double resistance-breaking strains in tomato and pepper and new insights into the occurrence of the recently reported D122G substitution in the NSm protein.

In June of 2009, stem vascular necrosis, interveinal necrosis of upper leaves, wilting of flowers, and necrotic spots on the pods were observed on garden pea (Pisum sativum L. 'Rajnai törpe') in northeast Hungary. A mechanical transmissible plant virus designated Ps091 was isolated from leaves of severely affected plants. Pathological investigations demonstrated that Ps091 had a host range very similar to that of Tomato spotted wilt virus (TSWV). It caused necrotic local lesions on Chenopodium spp. and induced systemic yellowing and necrosis on the upper leaves of Nicotiana benthamiana, N. clevelandii, and N. glutinosa by mechanical inoculation. Typical symptoms of TSWV infection appeared on the top leaves of pepper (Capsicum annuum L. 'Albaregia') and tomato (Solanum lycopersicum 'Kecskeméti 3') inoculated with Ps091. For molecular identification, total nucleic acids were extracted from Ps091-infected tobacco with a standard phenol-chloroform extraction method (2), and reverse transcription-PCR was conducted with TSWV N-gene specific, own designed primers (TSWV-S for: 5'-CCCAGCATTATGGCAAGCC-3', TSWV-S rev: 5'-TGATCTGGTCGAGGTTTTCCGCTAGCCC-3'). A tobacco plant infected with a reference pepper isolate, TSWV-Ca1 (1), and a healthy tobacco plant served as positive and negative controls, respectively. An approximately 300-bp DNA fragment was amplified from tobacco infected with Ps091 and TSWV-Ca1. The Ps091 amplicon was cloned, sequenced in both directions, and the sequence was deposited in GenBank (Accession No. HQ615692). Blast search analysis showed that TSWV-Ps091 had the highest identity (99%) with TSWV-P170RB strain (GenBank Accession No. DQ431238) in the cognate region. Since the latter isolate is a resistance breaking (RB) strain on pepper, pathogenicity of Ps091 on TSWV resistant pepper and tomato lines was studied. Mechanically inoculated pepper (C. annuum × C. chinense TSR F4 line) and tomato (S. lycopersicum 'Stevens') genotypes carrying the Tsw and Sw5 resistance genes, respectively, reacted with necrotic local lesions, but no systemic infections were detected by applying bioassays to N. clevelandii. These results demonstrate that Ps091 does not belong to the RB strains of TSWV. Back inoculations to pea ('Rajnai törpe') resulted in necrotic local spots as well as systemic stem and top necrosis, proving the causal relationship between TSWV-Ps091 and the pea disease observed in the field. Although TSWV has been known to cause epidemy in solanaceous crops and tobacco, to our knowledge, this is the first report of its natural occurrence on a legume plant, particularly on pea in Hungary. Because of the extreme severity of the disease caused on pea and high infection pressure, TSWV is a new threat to pea production in this country, where pea is a very important crop.

Tomato spotted wilt virus (TSWV) is a highly destructive plant pathogen transmitted by thrips, including Frankliniella occidentalis, in a circulative and propagative manner. To counter viral infections, thrips activate antiviral defences through C20 oxygenated polyunsaturated fatty acids (PUFAs), known as eicosanoids. However, at later stages of infection, C18 PUFAs, including epoxyoctadecamonoenoic acids (EpOMEs), modulate immune responses by preventing excessive and unnecessary activation. Our previous study demonstrated that TSWV elevates EpOME levels in thrips to suppress antiviral responses and enhance viral replication, with its nonstructural protein S (NSs) playing a key role in this process. In this study, we investigated the impact of NSs protein variation on vector immunity and virus–vector interactions. We assessed relative TSWV titres in thrips larvae and examined the role of eicosanoids, specifically 12,13-EpOME and PGE2, in regulating viral load and apoptosis. Our results revealed that 12,13-EpOME significantly increased viral titres, whereas PGE2 reduced the viral accumulation by promoting apoptosis in the vector insect. Phylogenetic analysis identified distinct NSs variations among TSWV isolates, with resistance-breaking (RB) and WT strains, which modulated differential infection patterns in thrips gut tissues, as visualized through fluorescence in situ hybridization. RB strains exhibited significantly higher viral titres, along with increased expression of EpOME biosynthetic gene (Fo-CYP24) and decreasing expression of EpOME degradation gene (Fo-sEH2). Apoptosis assays using the terminal deoxynucleotidyl transferase dUTP nick-end labelling assay further indicated that RB strains suppressed the gut epithelial cell death in thrips by antagonizing a process regulated by PGE2. Additionally, in vivo transient expression of the NSs gene in a nontarget insect, Spodoptera exigua, demonstrated the immunosuppressive effects by inducing EpOME level through upregulation of Se-CYP expression and downregulation of Se-sEH expression. Indeed, RB strains suppressed cellular immune responses more effectively than WT strains in S. exigua. These findings provide novel insight into the role of NSs genetic variation in TSWV transmission in the insect vector as well as in the host plants.

Tomato spotted wilt orthotospovirus (TSWV) causes substantial economic loss to tomato production, and the Sw-5b resistance gene is widely deployed for management. Here, we show (i) the emergence of resistance-breaking (RB) TSWV strains in processing and fresh market tomato production in California over the past ten years, and (ii) evolutionary relationships with RB strains from other areas. A specific RT-PCR test was used to show the C118Y RB strain that emerged in Fresno County in 2016 quickly became predominant in the central production area and remained so through this study. In 2021, the C118Y strain was detected in the Northern production area, and was predominant in 2022. However, in 2023, the C118Y strain was unexpectedly detected in fewer spotted wilt samples from resistant varieties. This was due to emergence of the T120N RB strain, previously known to occur in Spain. A specific RT-PCR test was developed and used to show that the T120N RB strain was predominant in Colusa and Sutter counties (detected in 75–80% of samples), and detected in ~50% of samples from Yolo County. Pathogenicity tests confirmed California isolates of the T120N strain infected Sw-5b tomato varieties and induced severe symptoms. Another RB strain, C118F, was associated with spotted wilt samples of Sw-5 varieties from fresh market tomato production in southern California. Phylogenetic analyses with complete NSm sequences revealed that the C118Y and T120N RB strains infecting resistant processing tomato in California emerged locally, whereas those from fresh market production were more closely related to isolates from Mexico. Thus, widespread deployment of this single dominant resistance gene in California has driven the local emergence of multiple RB strains in different tomato production areas and types. These results further emphasize the need for ongoing monitoring for RB strains, and identification of sources of resistance to these strains.

Resistance-breaking (RB) isolates of Tomato spotted wilt virus (TSWV) that overcome the resistance conferred by the Sw-5 gene in tomato have had only a limited spread since they were first detected in north-eastern Spain in 2002. Symptom expression, homogeneity, stability and the transmission capacity of RB and non-resistance breaking (NRB) isolates were biologically compared. The fitness of both types of isolates infecting tomato plants was determined in competition assays. All TSWV isolates induced similar systemic symptoms in a wide range of plant species, except RB isolates in tomato carrying the Sw-5 resistance gene and pepper carrying the Tsw resistance gene . The mechanical transmission of RB isolates to tomato plants with the Sw-5 gene failed in some trials, although NRB isolates did not differ noticeably in transmission efficiency when tested with the thrips Frankliniella occidentalis . Biological clones from individual local lesions obtained by mechanically inoculating Nicotiana glutinosa in some TSWV field samples showed that they were biologically homogeneous. Mixed infections of RB and wilt-type isolates were not found. The RB isolates were relatively stable because no reversion to NRB isolates was seen after serial passages in susceptible tomato plants. In competition assays between RB and NRB isolates, after serial passages in susceptible tomato plants, the prevalence of a particular isolate was not related to its capacity to overcome Sw-5 gene resistance. The low spread of the RB isolates in Spain does not seem to be related to a loss of fitness in tomato plants or to differences in transmission capacity by thrips, but it could be related to the reduction of the selection pressure of RB isolates as consequence of the gradual replacement of susceptible tomato plants by resistant tomato plants by growers.

Tomato spotted wilt orthotospovirus (TSWV) is one of the most devastating plant viruses causing crop disease epidemics of global economic significance. A single dominant resistant gene 'Sw-5' offering a broad-spectrum resistance to multiple orthotospoviruses was introduced in tomato cultivars. However, multiple resistance-breaking strains of TSWV were reported worldwide (Ciuffo 2005; Zaccardelli et al. 2008; Batuman et al. 2017; di Rienzo et al. 2018). Symptoms suggestive of orthotospoviral infection including stunting, bronzing, and inward rolling of leaves, and concentric necrotic spots on leaves, petioles, and fruits were observed in two TSWV-resistant tomato cultivars ('BL163' and 'HT 2') planted in a tomato variety trial in Bushland, TX in 2022. Leaf tissues from 45 resistant tomato plants (symptomatic or asymptomatic) from both resistant cultivars were tested using a TaqMan probe-based qPCR assay targeting a 200bp region in nucleoprotein (N) of the TSWV (Gautam et al. 2022). While 25 of those samples tested positive for TSWV, only ten expressed characteristic disease symptoms described above. The possibility of mixed infection in those samples with other endemic viruses in the region viz., alfalfa mosaic virus, groundnut ringspot orthotospovirus, tobacco mosaic virus, tomato chlorotic spot orthotospovirus, tomato mosaic virus, tomato necrotic streak virus, tomato ringspot virus, and tomato torrado virus was discounted through RT-PCR analysis (Kumar et al. 2011; Verbeek et al. 2012; Bratsch et al. 2018). To test the RB phenotype of the observed putative TSWV-RB strains, three-week-old tomato plants from eight commercially available TSWV resistant cultivars and one non-resistant cultivar (n=10 each) were mechanically inoculated with leaf tissues collected from a single symptomatic plant from one of the field-grown resistant cultivars. The experiment was replicated twice. Hypersensitive response was observed on all inoculated leaves of resistant plants one week post inoculation. Furthermore, all eight resistant cultivars started expressing local and systemic TSW symptoms 12 to 16 days post inoculation (dpi), while non-resistant cultivar started expressing symptoms at 9 dpi. TSW incidence across all resistant cultivars was 30-70%, while in susceptible cultivar it was 90%. Symptoms exhibited by all resistant cultivars resembled those of symptoms observed in field collected plants. The expression of Sw-5 gene in all eight resistant cultivars and the lack thereof in a susceptible cultivar was confirmed using Sw-5b specific primers and using Actin as a housekeeping gene in qRT-PCR (Islam et al. 2022). The RB strains in Sw-5 resistant tomato in California (Batuman et al. 2017) had the C118Y mutation in the TSWV NSm protein, consistent with the original reporting of C118Y or T120N RB mutations in 11 TSWV isolates from Spain (NCBI accession # HM015517 & HM015518) (Lopez et al. 2011). The nucleotide and amino acid sequence analysis of NSm gene from Bushland RB isolates from four resistant cultivars (NCBI accessions # OP810513-14 [field], OQ247901-05 [mechanically inoculated]) shared 98.9 and 99.4% homology with the Californian NSm sequences of TSWV RB tomato isolate (KX898453 and ASO67371), respectively. While the Nsm C118Y or T120N RB mutations were absent in all Bushland TSWV RB isolates, they had six additional unique point mutations across the NSm (I163V, P227Q, V290I, N293S, V294I, K296Q), which could potentially be responsible for resistance breaking. Despite the lack of C118Y or T120N RB mutations, Bushland isolates were capable of disrupting Sw-5-mediated TSWV resistance in all eight commercial resistant tomato cultivars. This study suggests a new or a different class of fundamental mechanisms are likely to be responsible for resistance breaking in Sw-5b resistant tomatoes. The new RB strain/s of TSWV therefore pose a substantial threat to tomato production in TX and other tomato-growing regions of the US.

Tomato spotted wilt virus (TSWV) causes significant economic losses in the commercial culture of tomato (Lycopersicon esculentum Mill.). Culture practices and introgression of natural sources of resistance to TSWV have only been marginally effective in controlling the TSWV disease. Recently however, high levels of protection against TSWV have been obtained by transforming tobacco with a chimaeric gene cassette comprising the TSWV nucleoprotein gene. This report demonstrates the successful application of this newly-created TSWV resistance gene in cultivated tomato. Transformation of an inbred tomato line with the TSWV nucleoprotein gene cassette resulted in high levels of resistance to TSWV that were maintained in hybrids derived from the parental tomato line. Therefore, transformant lines carrying the synthetic TSWV resistance gene make suitable progenitors for TSWV resistance to be incorporated into the breeding programmes of tomato.

Widespread use of tomato cultivars with the Sw-5 resistance gene has led to the emergence of resistance-breaking (RB) strains of tomato spotted wilt virus across the globe. In June of 2022, tomato spotted wilt (TSW) symptoms were observed at two farms (A and B, within 15 miles of each other) in Rowan County, NC on several commercial TSW resistant tomato cultivars (all heterozygous for the Sw-5 gene). At farm A, ~10% of plants had symptomatic foliage with ~30% of fruit with symptoms, while at farm B, up to 50% of plants had symptomatic foliage with ~80% of fruit with symptoms. Visual symptoms included stunting, severe leaf curling and bronzing, necrotic lesions on leaves, petioles and stems, and concentric ring spots on fruit (Supplementary Fig. 1). TSWV ImmunoStrips (AgDia, Elkhart, IN) and reverse-transcription (RT)-PCR with NSm primers (di Rienzo et al 2018) confirmed the presence of TSWV in 12 symptomatic plants sampled across the two farms. Primers designed to detect Impatiens necrotic spot virus, groundnut ringspot virus, tomato chlorotic spot virus, tomato chlorosis virus, alfalfa mosaic virus, and tomato necrotic streak virus (ilarvirus, Badillo et al., 2016) failed to generate amplicons of the expected size from cDNA generated from these field samples. The amplicons from full-length NSm cDNA were sequenced from independent, single-leaflet isolates from the TSWV-positive plants (three from farm A, nine from farm B) with the expectation of finding an amino acid (aa) substitution associated with the Sw-5 RB phenotype identified previously in CA (C118Y, Batuman et al. 2017) or Spain (C118Y and T120N, Lopez et al. 2011). All three nucleotide sequences from farm A contained the NSm C118Y substitution reported in CA. All three sequences were 99% identical (including the C118Y mutation) to NCBI GenBank accession KU179600.1, a TSWV isolate collected from GA in 2014 with no cultivar information reported. The nine nucleotide sequences from farm B contained neither of the two previously reported aa substitutions associated with the RB phenotype. Instead, all contained a D122G substitution within a conserved region of the TSWV NSm protein reported to be involved in direct interaction with the Sw-5 protein (Zhu et al 2017). Likewise, Huang et al (2021) generated a D122A mutation in TSWV-NSm, resulting in failure to elicit a Sw-5 mediated hypersensitive response. Three NSm sequences retrieved from GenBank contained the D122G substitution (AY848921.1, HM015516.1, KU179582.1), however, this mutation was not implicated directly with RB phenotypes (Ciuffo et al., 2005; Lopez et al., 2011; Marshall, 2016). The RB phenotype was confirmed with the NC variants on 'Mountain Merit' (Sw-5) by two means of virus inoculation: mechanical, rub-inoculation with extracted sap from infected plants, and thrips transmission assays with lab colony-maintained, Frankliniella occidentalis, the western flower thrips. Symptomatic leaf tissue obtained from these inoculation assays tested positive for TSWV by DAS-ELISA (AgDia, Elkhart, IN) and RT-PCR with NSm primers, providing definitive evidence of the occurrence of RB-TSWV at both farms, and subsequent sequencing confirmed the C118Y and D122G substitutions. This report warrants further investigation of the putative origins, prevalence and epidemiological implications of RB-TSWV variants in NC tomato production, and the development of new sources of resistance to TSWV.

SummaryPlants use intracellular nucleotide‐binding leucine‐rich repeat immune receptors (NLRs) to recognize pathogen‐encoded effectors and initiate immune responses. Tomato spotted wilt virus (TSWV), which has been found to infect >1000 plant species, is among the most destructive plant viruses worldwide. The Sw‐5b is the most effective and widely used resistance gene in tomato breeding to control TSWV. However, broad application of tomato cultivars carrying Sw‐5b has resulted in an emergence of resistance‐breaking (RB) TSWV. Therefore, new effective genes are urgently needed to prevent further RB TSWV outbreaks. In this study, we conducted artificial evolution to select Sw‐5b mutants that could extend the resistance spectrum against TSWV RB isolates. Unlike regular NLRs, Sw‐5b detects viral elicitor NSm using both the N‐terminal Solanaceae‐specific domain (SD) and the C‐terminal LRR domain in a two‐step recognition process. Our attempts to select gain‐of‐function mutants by random mutagenesis involving either the SD or the LRR of Sw‐5b failed; therefore, we adopted a stepwise strategy, first introducing a NSmRB‐responsive mutation at the R927 residue in the LRR, followed by random mutagenesis involving the Sw‐5b SD domain. Using this strategy, we obtained Sw‐5bL33P/K319E/R927A and Sw‐5bL33P/K319E/R927Q mutants, which are effective against TSWV RB carrying the NSmC118Y or NSmT120N mutation, and against other American‐type tospoviruses. Thus, we were able to extend the resistance spectrum of Sw‐5b; the selected Sw‐5b mutants will provide new gene resources to control RB TSWV.

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.