Restricted Spread of Tomato spotted wilt virus in Thrips-Resistant Pepper

ABSTRACT Spread of Tomato spotted wilt virus (TSWV) and population development of its vector Frankliniella occidentalis were studied on the pepper accessions CPRO-1 and Pikante Reuzen, which are resistant and susceptible to thrips, respectively. Viruliferous thrips were released on plants of each accession (nonchoice tests) or on plants in a 1:1 mixture of both accessions (choice tests) in small cages containing 8 or 16 plants. Significantly fewer CPRO-1 plants became infected in the primary infection phase in both tests. In the nonchoice test, virus infection of the resistant plants did not increase after the initial infection, but all plants eventually became infected when mixtures of both cultivars were challenged in the secondary infection phase. Secondary spread of TSWV from an infected resistant or susceptible source plant was significantly slower to resistant plants than to susceptible plants, independent of source plant phenotype. The restricted introduction and spread of TSWV in the thrips-resistant cultivar was confirmed in a large-scale greenhouse experiment. The restricted and delayed TSWV spread to plants of the resistant accession in both the cage and the greenhouse experiment was explained by impeded thrips population development. The results obtained indicate that thrips resistance may provide a significant protection to TSWV infection, even when the crop is fully susceptible to the virus.     

Acquisition of Tomato spotted wilt virus by Adults of Two Thrips Species

ABSTRACT Only larval thrips that acquire Tomato spotted wilt virus (TSWV), or adults derived from such larvae, transmit the virus. Nonviruliferous adults can ingest virus particles while feeding on TSWV-infected plants, but such adult thrips have not been shown to transmit TSWV. Immunofluorescence microscopy was used to show that thrips 1, 5, 10, and 20 days after adult emergence (DAE) fed on TSWV-infected plants acquired TSWV with virus replication and accumulation occurring in both epithelial and muscle cells of Frankliniella fusca (tobacco thrips [TT]) and F. occidentalis (western flower thrips [WFT]), as indicated by immunodetection of the nonstructural (NSs) protein encoded by the small RNA and the nucleocapsid (N) protein, respectively. Adult WFT acquired TSWV more efficiently than TT. There was no significant effect of insect age on TSWV acquisition by TT. In contrast, acquisition by adult WFT at 1 and 5 DAE was higher than acquisition at 10 and 20 DAE. Subsequent transmission competence of adult cohorts was studied by vector transmission assays. All adult thrips tested that had an acquisition access period as an adult were unable to transmit the virus. These results indicate the susceptibility of adult TT and WFT to infection of midgut cells by TSWV and subsequent virus replication and confirm earlier studies that adult thrips that feed on virus-infected plants do not transmit the virus. The role of a tissue barrier in TSWV movement and infection from midgut muscle cells to the salivary glands is discussed.     

Synergistic Interaction Between Tomato chlorosis virus and Tomato spotted wilt virus Results in Breakdown of Resistance in Tomato

ABSTRACT Multiple viral infections frequently are found in single plants of cultivated and wild hosts in nature, with unpredictable pathological consequences. Synergistic reactions were observed in mixed infections in tomato plants doubly infected with the positive-sense and phloem-limited single-stranded RNA (ssRNA) crinivirus Tomato chlorosis virus (ToCV) and the negative-sense ssRNA tospovirus Tomato spotted wilt virus (TSWV). Synergism in a tomato cultivar susceptible to both viruses resulted in a rapid death of plants. A pronounced enhancement of ToCV accumulation mediated by TSWV co-infection was observed with no evident egress of ToCV from phloem tissues. No consistent alteration of TSWV accumulation was detected. More remarkable was the synergism observed in tomato cultivars which carry the Sw-5 resistance gene, which are resistant to TSWV. Pre-infection with ToCV resulted in susceptibility to TSWV, whereas co-inoculations did not. This suggested that a threshold level or a time lapse is needed for ToCV to interfere or downregulate the defense response in the TSWV-resistant plants.     

Assessing the susceptibility of chrysanthemum cultivars to tomato spotted wilt virus

Three methods were compared to assess the susceptibility of vegetatively propagated chrysanthemum to tomato spotted wilt tospovirus (TSWV): mechanical and thrips-mediated inoculation of whole plants, and a leaf-disc assay. As symptom expression was often poor or even absent, TSWV infections and subsequent susceptibility to TSWV were determined by ELISA. All 15 chrysanthemum cultivars tested were susceptible to TSWV, irrespective of their degree of vector resistance (based on feeding-scar damage rates). Thrips-mediated inoculation using different numbers of thrips revealed that 100% infection was obtained when plants were challenged by six thrips per plant, whereas 80 and over 50%, respectively, of the plants became infected when inoculated by a single male or female thrips. However, false negatives were scored even after intensive sampling because of erratic, cultivar-specific and time-dependent virus distribution after inoculation in the plants. Labour-intensive samplings and long incubation periods could be overcome by a readily applicable leaf-disc assay. This assay was as reliable as thrips-mediated inoculation of whole plants, and its use is therefore favoured to assess chrysanthemum cultivars for TSWV susceptibility.     

Thrips Resistance in Pepper and Its Consequences for the Acquisition and Inoculation of Tomato spotted wilt virus by the Western Flower Thrips

ABSTRACT Different levels of thrips resistance were found in seven Capsicum accessions. Based on the level of feeding damage, host preference, and host suitability for reproduction, a thrips susceptible and a resistant accession were selected to study their performance as Tomato spotted wilt virus (TSWV) sources and targets during thrips-mediated virus transmission. Vector resistance did not affect the virus acquisition efficiency in a broad range of acquisition access periods. Inoculation efficiency was also not affected in short inoculation periods, but was significantly lower on plants of the thrips resistant accession during longer inoculation access periods. Under the experimental conditions used, the results obtained show that transmission of TSWV is little affected by vector resistance. However, due to a lower reproduction rate on resistant plants and a lower preference of thrips for these plants, beneficial effects of vector resistance might be expected under field conditions.     

Characterization of a vector-non-transmissible isolate of Tomato spotted wilt virus

The characteristics of a thrips‐non‐transmissible isolate of Tomato spotted wilt virus (TSWV), designated TSWV‐M, were compared with those of a thrips‐transmissible isolate, designated TSWV‐T. TSWV‐M showed a narrower host range than TSWV‐T. Adult thrips failed to transmit TSWV‐M, although the vector acquired the virus during the larval stages. TSWV‐M was detected by RT‐PCR in adult thrips bodies, but not in thrips heads, suggesting that loss of thrips transmissibility was the result of the absence of virus in adult thrips salivary glands. Whereas N (nucleoprotein), NSs (non‐structural protein) and G_C (the C‐terminal portion of the glycoprotein precursor protein) were present in similar amounts in leaf tissue from TSWV‐M‐ or TSWV‐T‐infected plants, G_N (the N‐terminal portion of the glycoprotein precursor protein) was present at much lower amounts in TSWV‐M‐ than in TSWV‐T‐infected plants. SDS‐PAGE and immunoblotting analysis of TSWV‐M and TSWV‐T virion preparations with G_N‐ and G_C‐specific antibodies revealed similar amounts of the G_N and G_C glycoproteins in TSWV‐T virions, but lower amounts of G_N than G_C in TSWV‐M virions. This resulted in a statistically significant reduction in the G_N/G_C ratio in TSWV‐M virions. In affinoblots, the G_C and G_N glycoproteins of TSWV‐M exhibited weak binding with lectins showing affinity for N‐linked oligosaccharide structures. Sequence analysis of M RNA (medium segment of the TSMV genome) revealed no deletions or frameshift mutations in the G_N/G_C precursor of TSWV‐M. However, five amino acid changes were detected in the G_N/G_C precursor. A single, relatively conservative amino acid substitution (V→I) was observed in the NSm protein. Sequence analysis of S RNA (small portion of the TSMV genome) revealed a large intergenic region with no changes in the N protein and with three amino acid changes in the NSs protein.     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

番茄斑萎病毒N基因的VIGS载体构建

 番茄斑萎病毒(tomato spotted wilt orthotospovirus,TSWV)严重危害多种经济作物和园艺植物,其N基因与病毒的侵染力密切相关,将N基因直接构建到VIGS载体上研究其致病功能目前鲜见报道。本研究将TSWV N基因构建到pTRV-PTV00载体上,注射本氏烟,通过qRT-PCR定量分析发现先接种TSWV后注射沉默载体的植株抗TSWV效率达57.60%,先注射沉默载体后接种TSWV的植株抗TSWV效率达99.14%。结果表明先注射载体后接种TSWV的N基因沉默效率更高。TSWV N基因VIGS载体的构建可为研究N基因在病毒致病等方面的功能提供前期材料,并为TSWV抗病育种和田间绿色防控提供一定的理论依据。     

Effects of Latent Infection of Stock Plants and Abundance of Thrips on the Occurrence of <Emphasis Type="Italic">Tomato spotted wilt virus </Emphasis>in Chrysanthemum Fields

DAS-ELISA proved to be reliable enough to detect a latent infection by Tomato spotted wilt virus (TSWV) in asymptomatic stock plants of chrysanthemum. A high density of Frankliniella occidentalis, the predominant vector, in the presence of latently infected stock plants resulted in a high incidence of disease in the chrysanthemum production field. The incidence of disease was low when the vector thrips were not abundant in spite of the presence of latently infected stock plants. These results suggest that an infestation of the vector thrips causes severe secondary spread of TSWV originating from latently infected stock plants in chrysanthemum production fields. Received 27 July 2001/ Accepted in revised form 27 November 2001     

Overwintering of Frankliniella fusca (Thysanoptera: Thripidae) on Winter Annual Weeds Infected with Tomato spotted wilt virus and Patterns of Virus Movement Between Susceptible Weed Hosts

ABSTRACT Overwintering of tobacco thrips, Frankliniella fusca, was investigated on common winter annual host plants infected with Tomato spotted wilt virus (TSWV). Populations of tobacco thrips produced on TSWV-infected plants did not differ from those produced on healthy plants, whereas populations varied greatly among host plant species. The mean per plant populations of F. fusca averaged 401, 162, and 10 thrips per plant on Stellaria media, Scleranthus annuus, and Sonchus asper, respectively, during peak abundance in May. Adult F. fusca collected from plant hosts were predominately brachypterous throughout the winter and early spring, but macropterous forms predominated in late spring. Weed hosts varied in their ability to serve as overwintering sources of TSWV inoculum. Following the initial infection by TSWV in October 1997, 75% of Scleranthus annuus and Stellaria media retained infection over the winter and spring season, whereas only 17% of Sonchus asper plants remained infected throughout the same interval. Mortality of TSWV-infected Sonchus asper plants exceeded 25%, but mortality of infected Stellaria media and Scleranthus annuus did not exceed 8%. TSWV transmission by thrips produced on infected plants was greatest on Stellaria media (18%), intermediate on Scleranthus annuus (6%), and lowest on Sonchus asper (2%). Very few viruliferous F. fusca were recovered from soil samples collected below infected wild host plants. Vegetative growth stages of Stellaria media, Sonchus asper, and Ranunculus sardous were more susceptible to F. fusca transmission of TSWV than flowering growth stages, whereas both growth stages of Scleranthus annuus were equally susceptible. In a field study to monitor the spatial and temporal patterns of virus movement from a central source of TSWV-infected Stellaria media to adjacent plots of R. sardous, the incidence of infection in R. sardous plots increased from <1% in March to >42% in June 1999. Infection levels in the Stellaria media inoculum source remained high throughout the experiment, averaging nearly 80% until June 1999 when all Stellaria media plants had senesced. Dispersal of TSWV from the inoculum source extended to the limits of the experimental plot (>37 m). Significant directional patterns of TSWV spread to the R. sardous plots were detected in April and May but not in June. R. sardous infections were detected as early as March and April, suggesting that overwintering inoculum levels in an area can increase rapidly during the spring in susceptible weed hosts prior to planting of susceptible crops. This increase in the abundance of TSWV inoculum sources occurs at a time when vector populations are increasing rapidly. The spread of TSWV among weeds in the spring serves to bridge the period when overwintered inoculum sources decline and susceptible crops are planted.     

Biological and molecular analyses of the acibenzolar-S-methyl-induced systemic acquired resistance in flue-cured tobacco against Tomato spotted wilt virus

Tomato spotted wilt virus (TSWV) is an economically important virus of flue-cured tobacco. Activation of systemic acquired resistance (SAR) by acibenzolar-S-methyl (ASM) in flue-cured tobacco was studied under greenhouse conditions by challenge inoculation with a severe isolate of TSWV. ASM restricted virus replication and movement, and as a result reduced systemic infection. Activation of resistance was observed within 2 days after treatment with ASM and a high level of resistance was observed at 5 days onward. Expression of the pathogenesis-related (PR) protein gene, PR-3, and different classes of PR proteins such as PR-1, PR-3, and PR-5 were detected at 2 days post-ASM treatment which inversely correlated with the reduction in the number of local lesions caused by TSWV. Tobacco plants treated with increased quantities of ASM (0.25, 0.5, 1.0, 2.0, and 4.0 g a.i./7,000 plants) showed increased levels of SAR as indicated by the reduction of both local and systemic infections by TSWV. The highest level of resistance was at 4 g a.i., but this rate of ASM also caused phytotoxicity resulting in temporary foliar spotting and stunting of plants. An inverse correlation between the TSWV reduction and phytotoxicity was observed with the increase of ASM concentration. ASM at the rate of 1 to 2 g a.i./7,000 plants activated a high level of resistance and minimized the phytotoxicity. Use of gibberellic acid in combination with ASM reduced the stunting caused by ASM. Present findings together with previous field experiments demonstrate that ASM is a potential option for management of TSWV in flue-cured tobacco.     

Tospoviruses infecting vegetable crops in Israel

Symptoms of vein clearing, stem necrosis, curling, necrotic spots and rings on the leaves associated with infection by tomato spotted wilt tospovirus (TSWV) were documented among vegetable crops growing in commercial glasshouses and open fields in Israel. Plants exhibiting symptoms were collected, from 1994-01 to 1998-12. Among cultivated vegetable crops analysed for TSWV by ELISA, the following plants were found to be infected: tomato, capsicum, aubergine, lettuce, cabbage and cucumber. These incidences of the virus were all correlated with the occurrence in high population of Frankliniella occidentalis. Transmission of the virus from infected Datura stramonium to Petunia leaf discs, by F. occidentalis , was up to 26%. TSWV antigens were readily detected by ELISA in seeds harvested from naturally infected vegetable crops. However, we failed to show virus transmission to the progeny plants. Iris yellow spot tospovirus (IYSV) was detected in onion. High incidence of the disease was associated with large populations of Thrips tabaci.     

Development of an assay system using thrips-mediated inoculation to evaluate resistance of Capsicum spp. to Tomato spotted wilt virus

Considerable losses in pepper production by Tomato spotted wilt virus (TSWV) have been reported worldwide. In breeding programs, an assay for resistance that accurately estimates field occurrence of TSWV during pepper production is critical because the virus is vector transmitted. Here, we establish an assay system of TSWV-resistant Capsicum spp. using insect-mediated inoculation within an acrylic chamber in which environmental conditions such as temperature, light intensity, and nutrient supply are controlled. This chamber enables transmission of TSWV from viruliferous plants to plants used in the resistance assay with Frankliniella occidentalis safely, quickly, and precisely.     

Interaction of Tomato Spotted Wilt Tospovirus (TSWV) Glycoproteins with a Thrips Midgut Protein, a Potential Cellular Receptor for TSWV

ABSTRACT Interactions between viral and cellular membrane fusion proteins mediate virus penetration of cells for many arthropod-borne viruses. Electron microscope observations and circumstantial evidence indicate insect acquisition of tomato spotted wilt virus (TSWV) (genus Tospovirus, family Bunyaviridae) is receptor mediated, and TSWV membrane glycoproteins (GP1 and GP2) serve as virus attachment proteins. The tospoviruses are plant-infecting members of the family Bunyaviridae and are transmitted by several thrips species, including Frankliniella occidentalis. Gel overlay assays and immunolabeling were used to investigate the putative role of TSWV GPs as viral attachment proteins and deter mine whether a corresponding cellular receptor may be present in F. occidentalis. A single band in the 50-kDa region was detected with murine monoclonal antibodies (MAbs) to the TSWV-GPs when isolated TSWV or TSWV-GPs were used to overlay separated thrips proteins. This band was not detected when blots were probed with antibody to the non-structural protein encoded by the small RNA of TSWV or the TSWV nucleocapsid protein, nor were proteins from nonvector insects labeled. Anti-idiotype antibodies prepared to murine MAbs against GP1 or GP2 specifically labeled a single band at 50 kDa in Western blots and the plasmalemma of larval thrips midguts. These results support the putative role of the TSWV GPs as viral attachment proteins and identified potential cellular receptor(s) in thrips.     

Incidence of weed reservoirs and vectors of tomato spotted wilt tospovirus on southern Tasmanian lettuce farms

Thrips species and tomato spotted wilt virus (TSWV) alternate weed hosts were surveyed on two lettuce farms in southern Tasmania during 1994 and 1995. Only one known vector species, Thrips tabaci, was found at either site, comprising on average 36.8% of the total monthly catch. A major peak of thrips activity in the summer corresponded with an increase of disease in autumn harvested lettuce. Two thrips species new for Tasmania were recorded, Pseudanaphothrips achaetus and Tenothrips frici . Infection patterns within the crop indicated that localized weed infestations were the most likely reservoir of virus. ELISA testing showed that TSWV was present in a range of dicotyledonous weed species, although usually infecting only a low percentage of the plants. Arctotheca calendula appeared to be the single most important reservoir host species at one property, whilst this species and Sonchus oleraceus, Malva sylvestris, Brassica rapa ssp. silvestris, Erodium moschatum and Trifolium sp. were probably the most important reservoirs at the other property. Two new natural TSWV host species were recorded, Erodium moschatum and Brassica rapa ssp. silvestris . The property with the highest incidence of TSWV-infected lettuce had a relatively higher proportion of virus-infected weeds but less thrips activity during the infection period.     

北京辣椒和番茄上番茄斑萎病毒的鉴定

北京大兴区某蔬菜种植基地的辣椒和番茄发现疑似病毒感染,造成较大经济损失。经症状分析和RT-PCR检测,证实其感染番茄斑萎病毒(tomato spotted wilt virus,TSWV)。基于TSWV序列特征对该地区TSWV进化及来源进行分析,结果显示其与北京顺义区已经报道TSWV毒株进化关系较近,但较国内其他地区株系更接近欧美种群,表明其可能是近几年由国外引种传入。     

Occurrence of Tomato spotted wilt virus (TSWV) in Jordan

Tomato spotted wilt virus was recorded for the first time in Jordan on tomato plants. Severe disease symptoms were observed in different tomato farms in the Jordan Valley. Using a specific primer pair a fragment of the capsid protein gene of the virus has been amplified by RT-PCR and IC-RT-PCR. The amplified PCR product was cloned and sequenced. Sequence analysis revealed that the Jordanian isolate of TSWV shared high nucleotide similarities with other isolates from different countries. The sequence of the capsid protein gene was deposited in GenBank under the accession number AY646682 . The response of different tomato breeding lines and hybrids, previously developed for resistance against Tomato yellow leaf curl virus (TYLCV) were tested for their reaction to TSWV infection. All tested lines and hybrids were susceptible to TSWV infection. This has been confirmed at the molecular level by using the SCAR 421 marker linked to the TSWV resistance gene Sw-5 .     

青蒿中番茄斑萎病毒和烟草花叶病毒的分子鉴定及相关序列分析

番茄斑萎病毒(tomato spotted wilt virus, TSWV)和烟草花叶病毒(tobacco mosaic virus, TMV)是2种重要的植物病原病毒, 对多种经济作物的产量和品质均造成严重影响。2021年-2022年, 在云南省丽江市烟草种植区不同烟区采集叶片黄化、皱缩以及无症状的青蒿Artemisia caruifolia样品共计14份, 利用免疫金标速测卡和RT-PCR对其病原病毒进行检测。利用免疫金标速测卡检测结果显示, 在所检样品中有9份样品检测出TSWV, 检出率为64.28%, 有3份样品检测出TMV, 检出率为21.43%, 2种病毒复合侵染的检出率同样为21.43%;利用RT-PCR对复合侵染的3份样品进行分子检测, 结果显示, 在3份复合侵染青蒿样品中获得3条TSWV N基因序列、3条TMV cp基因序列和2条TMV RdRp部分序列。TSWV青蒿分离物与分离自云南的TSWV-2分离物相似性最高, 为99.6%;TMV青蒿分离物与分离自辽宁的TMV-Shenyang分离物和分离自云南的TMV-Yongren-1相似性最高, 均大于99.4%。这是首次发现TSWV和TMV 2种不同属病毒复合侵染青蒿。     

Role of weed hosts and the western flower thrips, Frankliniella occidentalis, in epidemiology of Tomato spotted wilt virus in the Çukurova region of Turkey

Surveys of thrips and Tomato spotted wilt virus (TSWV) on weeds were conducted in the eastern Mediterranean region of Turkey during the years 2004–2006. Thrips species were collected by vigorously shaking weedy plants into a white container for 15 sec. Plant material collected during field surveys and plants which were used for mechanical inoculation of TSWV, were tested by DAS–ELISA. The weed species Ranunculus muricatus, Melilotus officinalis, Sinapis arvensis and Portulaca oleracea were used for the virus transmission experiments in an enclosed high plastic tunnel and in cage experiments. Western flower thrips, Frankliniella occidentlis (Pergande) (Thysanoptera: Thripidae), was the most common thrips, inhabiting 80 of the 82 weed species sampled. Adults of F. occidentalis and thrips larvae were significantly more abundant on S. arvensis than on the other weed species sampled (P<0.05). Adult and larval thrips showed peak densities on most weeds in April or May. Summer annual weeds were not good hosts for reproduction of the thrips. A total of 90 samples from 17 plant species belonging to 11 plant families were ELISA positive for TSWV. No TSWV was detected on 65 weed species belonging to 26 plant families. High numbers of plant samples infected by TSWV were obtained in P. oleracea (21 samples) and in R. muricatus (15 samples). In field surveys symptoms of TSWV were detected on only R. muricatus. Incidence of the TSWV on weeds ranged between 5% and 25%. Transmission rates of TSWV by F. occidentalis to the weeds ranged from 33% to 83% in the pepper plastic tunnel and cage experiments.     

北京地区发现番茄斑萎病毒

通过采用特异快速试纸条以及进一步的RT-PCR和序列测定证实,在北京局部地区的辣椒和茄子上已发生番茄斑萎病毒,这两种作物上同时发生的蓟马种类主要为西花蓟马,并且携带该病毒。番茄斑萎病毒病是极其危险的一种病害,可对作物造成毁灭性灾害。建议有关部门立即采取相应的预防与管理措施,防止其扩散蔓延。