Occurrence and characterization of tomato spotted wilt orthotospovirus isolated from cucumber

European Journal of Plant Pathology - Diseases associated with tomato spotted wilt orthotospovirus (TSWV) are a serious threat to vegetable production worldwide. In 2019, leaf necrosis disease was...     

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。     

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...     

Biological and molecular characterization of tomato spotted wilt virus (TSWV) resistance-breaking isolates from Argentina

Tomato spotted wilt virus (TSWV) has been present in Argentina since 1938 and had limited sweet pepper and tomato production until the introduction of resistant cultivars bearing Tsw and Sw-5b genes. However, the wide use of TSWV-resistant pepper plants in La Plata Horticultural Belt (LPHB) triggered the emergence of resistance-breaking isolates (RB), increasing the economic impact of TSWV in pepper. This work characterized 11 natural RB pepper isolates from LPHB that have overcome the Tsw resistance gene in Capsicum sp. but are unable to break the Sw-5b-mediated resistance in tomato. Phylogenetic analysis of the N gene showed that the LPHB isolates are most closely related to isolates from Asia, indicating that Argentine TSWV isolates might have emerged from the Asian continent. The NSs sequence analysis reinforces the hypothesis that the appearance of an RB phenotype is a consequence of a number of different single amino acid substitutions spread along the NSs gene that lead to multiple independent evolutionary events. These results provide information on the current situation of the tospovirus–pepper/tomato pathosystems in LPHB, which represents a fundamental prerequisite to include these RB isolates in future screening programmes in order to select new and durable sources of resistance to TSWV in pepper.     

Purification and serological analysis of tomato spotted wilt virus

A modified procedure for the purification of TSWV is described which consists of differential centrifugation, treatment with antiserum against sap from healthyN. rustica and density-gradient centrifugation in sucrose gradients. Centrifugation in the zonal-rotor proved efficient for processing large amounts of infected leaves.Antisera to purified TSWV were produced in rabbits. These sera reacted with healthyN. rustica sap. This reaction could be abolished by absorption with either healthyN. rustica sap or polysaccharides purified fromN. rustica. Unabsorbed sera reacted with the major TSWV structural proteins following their electrophoretic separation in polyacrylamide-gels. The positions of the precipitin lines corresponded with those of the structural proteins visualized by staining. The intensity of these lines was greatly diminished after absorption of the antisera with either healthyN. rustica sap or polysaccharides. It was concluded that the reaction of TSWV antiserum with the structural proteins is partly due to host-derived polysaccharides covalently bound to the virus proteins.Samenvatting Het bronsvlekkenvirus van de tomaat (TSWV) werd gezuiverd met een procedure die bestond uit een cyclus van differentiële centrifugering, behandeling van de virus-suspensie met antiserum tegen bestanddelen van gezonde planten (N. rustica) en dichtheidsgradiëntcentrifugering (Fig. 1 en Fig. 3). De zone rotor kon worden gebruikt wanneer grote hoeveelheden bladmateriaal werden gebruikt (Fig. 2).Twee antisera, die elk een verschillende activiteit tegen TSWV (Tabel 1, Fig. 4) hadden, werden bereid. Deze sera reageerden ook met sap en polysacchariden uitN. rustica (Fig. 6). Door verzadiging met polysacchariden werd de reactie tegen TSWV aanmerkelijk verzwakt. Analyse van de eiwitbandjes die na polyacrylamidegel-elektroforese waren gevormd, toonde aan dat de activiteit van de sera tegen de eiwitcomponenten van TSWV was gericht (Fig. 5) en dat deze activiteit na verzadiging met polysacchariden zwakker was. Uit deze reacties kan geconcludeerd worden dat polysacchariden van de waardplant aan de structurele eiwitten van TSWV zijn gebonden. Niet geabsorbeerd antiserum geeft met TSWV drie precipitatielijnen (Fig. 4), maar na absorptie met compleet virus trad nog slechts één precipitatielijntje op (Fig. 7). Mogelijk wordt dit lijntje gevormd door het nucleocapside-eiwit dat door de dissociatie van het virus vrij komt. Sera die tegen normale bestanddelen vanN. rustica zijn bereid, vormen geen zichtbare reactie met compleet virus; wel wordt er een lijntje gevormd indien het virus met guanidine-HCl wordt gedissociëerd (Fig. 8). Deze waarneming kan verklaren dat deze antisera kunnen worden gebruikt bij de zuivering van TSWV ondanks het feit dat dit virus polysacchariden uit de waardplant draagt.     

Comparative host reactions and Frankliniella occidentalis transmission of different isolates of tomato spotted wilt tospovirus from Spain

The abilities of different isolates of tomato spotted wilt tospovirus (TSWV) collected from northeastern and eastern Spain to infect 10 host species, and to be acquired and transmitted by the western flower thrips (WFT), Frankliniella occidentalis were compared. Two isolates of TSWV from a single source plant could be separated according to the different type of local lesions they induced in Nicotiana glutinosa. Host ranges of the studied TSWV isolates were very similar, but differences were found in the symptoms induced and in their capacity to infect specific hosts systemically. Lycopersicon esculentumDatura stramonium were evaluated for their potential as virus acquisition host species. The proportion of transmitter adult thrips obtained from WFT larvae fed on L. esculentum was greater than from D. stramonium. No differences were detected between TSWV isolates in their ability to be acquired and transmitted by WFT. No evidence was obtained of alterations in TSWV particles which could affect WFT transmissibility due to the repeated mechanical transfers used to clone the isolates. Our findings do not support the existence of pathological effects of TSWV on WFT.     

A predictive model for spotted wilt epidemics in peanut based on local weather conditions and the tomato spotted wilt virus risk index

Tomato spotted wilt virus (TSWV), a member of the genus Tospovirus (family Bunyaviridae), is an important plant virus that causes severe damage to peanut (Arachis hypogaea) in the southeastern United States. Disease severity has been extremely variable in individual fields in Georgia, due to several factors including variability in weather patterns. A TSWV risk index has been developed by the University of Georgia to aid peanut growers with the assessment and avoidance of high risk situations. This study was conducted to examine the relationship between weather parameters and spotted wilt severity in peanut, and to develop a predictive model that integrates localized weather information into the risk index. On-farm survey data collected during 1999, 2002, 2004, and 2005 growing seasons, and derived weather variables during the same years were analyzed using nonlinear and multiple regression analyses. Meteorological data were obtained from the Georgia Automated Environmental Monitoring Network. The best model explained 61% of the variation in spotted wilt severity (square root transformed) as a function of the interactions between the TSWV risk index, the average daily temperature in April (TavA), the average daily minimum temperature between March and April (TminMA), the accumulated rainfall in March (RainfallM), the accumulated rainfall in April (RainfallA), the number of rain days in April (RainDayA), evapotranspiration in April (EVTA), and the number of days from 1 January to the planting date (JulianDay). Integrating this weather-based model with the TSWV risk index may help peanut growers more effectively manage tomato spotted wilt disease.     

北京地区侵染茄子的番茄斑萎病毒分子鉴定与分析

调查发现北京地区一温室栽培茄子Solanum melongena L.出现严重病毒病。利用基于小RNA的高通量测序技术和RT-PCR方法,明确了引起茄子病害的病毒种类为番茄斑萎病毒,将其命名为TSWV-eggplant分离物。进一步克隆了该病毒的基因组全长(S RNA、M RNA、L RNA),并构建其系统发育树。结果表明,该分离物的S RNA与美国分离物亲缘关系较近,M RNA与中国分离物亲缘关系较近,而L RNA与韩国分离物亲缘关系较近。因此,本研究发现的TSWV分离物与国内已发生报道的分离物不同,该分离物是否存在不同分离物之间基因组的重组需要进一步研究。     

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.     

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.     

由美国进境生菜种子上番茄斑萎病毒的检测鉴定

采用DAS-ELISA检测方法,从美国进境的生菜种子中筛选出携带有番茄斑萎病毒(Tomato spotted wilt virus,TSWV)的疑似阳性样品,通过建立的RT-PCR方法对酶联免疫吸附测定阳性样品进行复核鉴定,并进一步测序。测序结果与GenBank中已有的番茄斑萎病毒分离物KT160282、AY744474、GU369726、KP330470的序列同源性达到96%。结果表明,TSWV可通过种子携带入境。     

Detection of tomato spotted wilt virus and transmission by Frankliniella occidentalis in France

Tomato spotted wilt virus (TSWV) was isolated from pepper, tomato, eggplant, broad bean, lettuce, basil, chrysanthemum, aster, New Guinea impatiens, anemone and gloxinia plants. Virus identification was based on host range, vector transmission, serology and electron microscopy. TSWV was readily detected by ELISA in naturally or artificially infected cultivated or weed plants. The virus was also detected in individual F. occidentalis thrips. The spread of TSWV in vegetable and ornamental plants in greenhouses and/or in the open is related to the close relationship of the virus with the vector.     

Ornamental plants and thrips populations associated with tomato spotted wilt virus in Greece

A survey was conducted in order to record the ornamental plants that are hosts of tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV) in Greece. Polyclonal antibodies prepared against the N protein of a Greek isolate of TSWV fromGerbera jamesonii (GR-34) were used. Leaf samples were taken from plants showing typical symptoms of tospovirus infection such as chlorotic and necrotic rings on the leaves and malformation and necrosis of the flowers. The samples were tested by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using polyclonal antibodies to the N proteins of TSWV and INSV (NL-07). ELIS A-positive samples were mechanically transmitted to plants ofPetunia hybrida, Nicotiana rustica andN. benthamiana to confirm infection. Although none of the samples was found infected with INSV, TSWV presence was recorded in 42 botanical species that belong to 40 genera in 27 families. Among them the speciesBeloperone guttata, Coleus barbatus, Impatiens petersiana andLilium auratum are reported for the first time as hosts of TSWV, whereasBegonia sp.,Catharanthus roseus Celosia cristata, Dianthus chinensis, Fuchsia hybrida andStephanotis floribunda are found as new hosts of the virus in Greece. Thrips collected from TSWV-infected plants were in most cases identified asFrankliniella occidentalis, except from plants ofDendranthema sp. andDianthus caryophyllus whereThrips tabaci individuals were also identified. Different percentages of transmitters were noticed when the thrips populations collected from TSWV-infected ornamental hosts were tested for transmission of TSWV.     

An electron microscope study of tomato spotted wilt virus in the plant cell

Characteristic particles of about 70 m diameter were found in tomato spotted wilt virus (TSWV) infected cells of roots, stems, leaves and petals of several plant species. These particles were similar in size and form to those found in the pellets of partially purified infective TSWV.     

Transmission of tomato spotted wilt tospovirus by Thripstabaci populations originating from leek

The transmission of tomato spotted wilt tospovirus (TSWV) by Thrips tabaci collected from leek was studied using the petunia local-lesion leaf-disc assay. After an acquisition-access period of 72 h given to newborn larvae up to 8 h old, the efficiency of transmission by adults was determined in three inoculation-access periods of 48 h. This efficiency varied for six T. tabaci populations from 0.7 to 11.6% in experiments using the Greek TSWV isolate GR-04. Males were more efficient transmitters than females (19 out of 176 versus five out of 494). Frankliniella occidentalis transmitted the same virus with a higher efficiency (34.8%). The transmission rate differed also among TSWV isolates, as shown in tests with four T. tabaci using two isolates. The virus was more efficiently acquired from infected leaf material of Datura stramonium than from that of Emilia sonchifolia . Plants of the latter species were more susceptible than Nicotiana tabacum in thrips transmission tests.     

Characterization of two Tospoviruses in Italy: tomato spotted wilt and impatiens necrotic spot

Tospovirus serogroups I and III have recently been designated as species, tomato spotted wilt virus (TSWV) and impatiens necrotic spot virus (INSV), while the species status of serogroup II isolates remains undefined. Fifteen Tospovirus isolates from ornamental and vegetable crops in Liguria, Italy, were found to belong either to TSWV (seven isolates) or to INSV (eight isolates) on the basis of test-plant reactions, serological techniques using DAS ELISA kits raised against the nucleoproteins of the type members of the two species, and cytopathology. None of them could be assigned to serogroup II using DAS ELISA kits raised against nucleoproteins of this serogroup. Italian isolates representative of the two species reacted in indirect ELISA using a polyclonal antiserum against the entire particle of a TSWV isolate, but with higher intensity for our TSWV isolates than for the INSV isolates. Western blots and dot immunobinding assays confirmed that the nucleoproteins of the two species are unrelated whereas the glycoproteins are related. The cytopathology was similar for two isolates representative of TSWV and INSV, except that the type of filaments encountered was different, and appeared to be characteristic of the species.     

Effect of plant age at time of infection by tomato spotted wilt tospovirus on the yield of field-grown tomato

Naturally infected tomato plants that expressed tomato spotted wilt virus symptoms at 24, 38, 45, 60, 67, and 74 days after transplanting were monitored for production in an experimental crop grown in the open from May to September in northeastern Spain. Plants were tagged, tested for tomato spotted wilt virus infection by enzyme-linked immunosorbent assay, and data on symptom expression and yield were individually recorded. Plants that developed symptoms at 24, 38, or 45 days after transplanting yielded significantly less and produced fewer and smaller tomatoes than those that developed symptoms at 60, 67, and 74 days after transplanting. These later infected plants showed similar patterns of production with maximum yields between 27 July and 17 August, when most fruit was harvested. Production components such as fruit number per plant, yield of mature fruit per plant, or fruit weight increased the older the plants were when first symptoms were exhibited. However, marketable fruit production was drastically decreased by tomato spotted wilt virus infection, due to abnormal ripening of mature fruit in infected plants. Little and no significantly different amounts of marketable fruit were produced, irrespectively of plant age at time of symptom expression. Implications for spotted wilt management in tomato are discussed.     

Occurrence of tomato spotted wilt and cucumber mosaic viruses in field-grown tomato crops and associated weeds in northeastern Spain

The occurrence of cucumber mosaic (CMV) and tomato spotted wilt (TSWV) viruses in the main vegetable producing regions of northeastern Spain was studied during the growing seasons of 1992 and 1993. by sampling in July and September. Both viruses were found widely distributed in field tomato crops and surrounding weeds. Coastal areas were the most affected by both CMV and TSWV, whereas inland TSWV was nearly absent. Higher levels of infection were found in autumn for TSWV in tomato crops and weeds, and for CMV in tomatoes. From the 51 weed species of 19 families collected. 25 species were found infected with CMV and 15 with TSWV. Among them, Convolvulus arvensis. Malva sylvestris , and Sonchus tenerrimus could be of special importance as local reservoirs; commonly associated with tomato crops. they were infected quite frequently with CMV and/or TSWV and being perennial, could maintain the viruses between cropping seasons.