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1.
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.  相似文献   

2.
ABSTRACT If acquisition access feeding (AAF) is first given after adult eclosion, none of the nine thrips species able to serve as tospovirus vectors can become infective. The previous cellular investigations of this phenomenon, carried out only in Frankliniella occidentalis, suggested that infectivity was prevented because the type member of the tospoviruses, Tomato spotted wilt virus (TSWV), was unable to enter the midgut of adult thrips. The present study extends a cellular view of tospovirus-thrips interactions to a species other than the western flower thrips, F. occidentalis. Our findings show that TSWV enters and replicates within the midgut of adult Thrips setosus, but does not infect cells beyond the midgut epithelia. After AAF as adult, TSWV replicated in T. setosus midgut cells as indicated by significant increases in nucleocapsid (N) protein detected by double-antibody sandwich enzyme-linked immunosorbent assay, and the presence of inclusions containing the S RNA-encoded nonstructural and N proteins revealed by microscopic observations. Electron microscopic observations of adult insects showed that no infection occurred in cells beyond the midgut epithelia, and insects subsampled from the same cohorts could not transmit TSWV. In contrast, electron microscopy observations of larval T. setosus revealed that TSWV infected the midgut and muscle cells, and adult insects developing from these cohorts had infected salivary glands and were able to transmit TSWV. Mature virions were observed only in the salivary glands of adults developing from infected larvae. Our findings suggest that the barrier to infectivity in T. setosus adults differs from that shown for F. occidentalis adults.  相似文献   

3.
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.  相似文献   

4.
ABSTRACT Arrhenotokous and thelytokous populations of Thrips tabaci from tobacco or leek plants were evaluated for their ability to transmit Tomato spotted wilt virus (TSWV) and for their host preference. Transmission efficiencies were comparatively studied using leaf disks of Petunia hybrida, Datura stramonium, and Nicotiana tabacum cv. Basmas. Adults of arrhenotokous populations collected on infected tobacco plants in the field were efficient transmitters (up to 48.5% transmission) and remained so when maintained on tobacco for several generations. Arrhenotokous T. tabacipopulations from leek plants were poor transmitters (up to 3.1% transmission), whereas no transmission was obtained with thelytokous populations from leek. All populations could infest leek, however none of the arrhenotokous and thelytokous populations from leek plants was able to infest tobacco. TSWV could be acquired by both first and second larval instars of a T. tabacipopulation from tobacco. However, the transmission by adults decreased with the age at which the virus was acquired by larvae. The highest efficiencies (61% of males and 51% of females transmitted) were obtained when newborn (0- to 24-h old) larvae acquired the virus. The majority of thrips started to transmit after becoming adult and rates were positively correlated with the temperature at which the thrips were kept. The median latent period values found for adults decreased with increasing temperature. The median acquisition access period (AAP50) of the population was 41 min, whereas the AAP(50) was 65 min for males and 35 min for females. The median inoculation access period of males was 246 and 365 min on tobacco and petunia, respectively, and 96 and 345 min for females. The results show that T. tabaci forms a complex in terms of host preference, reproductive strategy, and ability to transmit TSWV. The transmission parameters show that the thrips of arrhenotokous populations infesting tobacco are highly efficient vectors.  相似文献   

5.
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 GC (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, GN (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 GN‐ and GC‐specific antibodies revealed similar amounts of the GN and GC glycoproteins in TSWV‐T virions, but lower amounts of GN than GC in TSWV‐M virions. This resulted in a statistically significant reduction in the GN/GC ratio in TSWV‐M virions. In affinoblots, the GC and GN 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 GN/GC precursor of TSWV‐M. However, five amino acid changes were detected in the GN/GC 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.  相似文献   

6.
为明确云南省昆明市露地栽培条件下番茄斑萎病毒病的发生流行特征,于2014—2015年采用病害系统调查法结合病毒ELISA及RT-PCR检测方法研究露地栽培条件下由番茄斑萎病毒(tomato spotted wilt virus,TSWV)引起的病毒病发生规律及其重要寄主种类,并研究利用防虫网隔离蓟马对番茄斑萎病毒病的防控效果。结果表明:番茄斑萎病毒病在露地番茄主要种植期3—10月普遍发生,番茄苗期和移栽初期是该病毒病防控的关键期,带毒种苗调运是该病毒病的主要传播途径;田间多种茄科和菊科植物是TSWV的重要中间寄主。在田间,菊科寄主植物油麦菜、莴苣、鬼针草、牛膝菊上TSWV的检出率均较高,在42.53%~81.63%之间;茄科寄主植物中辣椒上TSWV的检出率最高,为41.99%,其次为马铃薯,TSWV检出率为27.78%,在番茄上TSWV的检出率为19.02%,因此生产中应对这些TSWV重要中间寄主给予更多关注和防控。应用防虫网能有效隔离蓟马,使番茄斑萎病毒病发病率和病情指数较对照分别降低了6.44个百分点和5.31,可有效降低番茄苗期及定植期斑萎病毒病的发生。  相似文献   

7.
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.  相似文献   

8.
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 .  相似文献   

9.
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.  相似文献   

10.
ABSTRACT Using protein blot assays, a 94-kDa thrips protein was identified that exhibited specific binding to tomato spotted wilt virus (TSWV) particles. Renaturation of the 94-kDa protein, which is conserved among the two major vector species of TSWV, Frankliniella occidentalis and Thrips tabaci, was crucial for its virus-binding properties, whereas under the same conditions no specific binding was observed with aphid (Myzus persicae) proteins. The 94-kDa protein species was present in all developmental stages of both vectoring thrips, whereas it was present mainly in the adult stage of a nonvectoring thrips species, Parthenothrips dracenae. Using antibodies against the different TSWV structural proteins, the G2 envelope glycoprotein was identified as the viral determinant involved. Because the virus-binding protein is present throughout the thrips body, but not in the gut, it may represent a receptor protein involved during circulation of the virus through its vector but probably not during viral uptake in the midgut.  相似文献   

11.
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.  相似文献   

12.
Studies were carried out on a population ofThrips tabaci Lindeman (Thysanoptera: Thripi-dae) from Liguria to assess its sex-ratio and its ability to transmit four tospoviruses: tomato spotted wilt (TSWV), impatiens necrotic spot, tomato chlorotic spot and groundnut ringspot. The population was composed of females only (therefore thelytokous). The first instar larvae were allowed to acquire the virus for 48 h on infected leaves of datura, basil or pepper, and then reared on cucumber until emergence, which medially occurred 9.5 days after hatching. Transmission capacity was checked using two inoculation access periods (lAPs) of 48 h each on pepper leaf disks.T. tabaci was able to transmit TSWV isolate P105 with an efficiency of 16.7% and 4.4% in the first and second IAP, respectively, and TSWV isolate BR-01 with an efficiency of 2.0%. The onion thrips did not transmit the three other tospoviruses. During the IAPs, almost all adults fed on the leaf disks, producing evident silvery scars. The presence of tospovirus nucleocapsids in thrips was assayed by Triple Antibody Sandwich (TAS) and cocktail ELISA. Not all adults that had transmitted TSWV were positive in the tests, whereas some non-transmitter individuals proved positive. For each of the other tospoviruses, some thrips were positive in at least one test, although none was able to transmit the virus.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
Qiu W  Moyer JW 《Phytopathology》1999,89(7):575-582
ABSTRACT Pathogen- and host-derived resistance have been shown to suppress infection by many plant viruses. Tomato spotted wilt tospovirus (TSWV) is among these systems; however, it has easily overcome nearly all host resistance genes and has recently been shown to overcome resistance mediated by the TSWV N gene. To better understand the resistance-breaking mechanisms, we have chosen TSWV N gene-derived resistance (TNDR) as a model to study how plant viruses defeat resistance genes. A defined viral population of isolates TSWV-D and TSWV-10, both suppressed by TNDR, was subjected to TNDR selection by serial passage in an N-gene transgenic plant. The genotype analysis demonstrated that the mixed viral population was driven to form a specific reassortant, L(10)M(10)S(D), in the presence of TNDR selection, but remained as a heterogeneous mixture in the absence of the selection. A genotype assay of 120 local lesion isolates from the first, fourth, and seventh transfers confirmed the shift of genomic composition. Further analysis demonstrated that the individual L(10), M(10), and S(D) RNA segments were each selected independently in response to TNDR selection rather than to a mutation or recombination event. Following the seventh transfer on the N-gene transgenic plants, TSWV S RNA remained essentially identical to the S RNA from TSWV-D, indicating that no intermolecular recombination occurred between the two S RNAs from TSWV-10 and TSWV-D nor with the transferred N gene. These results support the hypothesis that TSWV utilizes genome reassortment to adapt to new host genotypes rapidly and that elements from two or more segments of the genome are involved in suppression of the resistance reaction.  相似文献   

16.
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.  相似文献   

17.
Squash blotting on nitrocellulose membranes was used to detect tomato spotted wilt virus (TSWV) in individual Frankliniella occidentalis adult thrips using a specific polyclonal antiserum. This method offers a simple and reliable procedure to test a large number of thrips and was less time-consuming than alternative techniques available. A 95% agreement was found between the results of squash-blot and plant transmission assay. Based on this agreement, this technique was used to study the relative number of viruliferous thrips in F. occidentalis field populations collected from a farm in Catalonia where TSWV causes important economic losses. The percentages of viruliferous individuals detected from a total of 1509 collected thrips increased from 0% to 2% in the different surveys conducted in the 1993 growing season. Squash-blot proved to be a rapid and inexpensive technique to screen TSWV viruliferous thrips from field populations. The results obtained by this method could be used together with other epidemiological data to forecast TSWV-induced diseases.  相似文献   

18.
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.  相似文献   

19.
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.  相似文献   

20.
ABSTRACT The effect of Tomato spotted wilt virus (TSWV) infection on plant attractiveness for the western flower thrips (Frankliniella occidentalis) was studied. Significantly more thrips were recovered on infected than were recovered on noninfected pepper (Capsicum annuum) plants in different preference tests. In addition, more offspring were produced on the virus-infected pepper plants, and this effect also was found for TSWV-infected Datura stramonium. Thrips behavior was minimally influenced by TSWV-infection of host plants with only a slight preference for feeding on infected plants. Offspring development was positively affected since larvae hatched earlier from eggs and subsequently pupated faster on TSWV-infected plants. These results show a mutualistic relationship between F. occidentalis and TSWV.  相似文献   

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