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1.
Impatiens necrotic spot tospovirus (INSV) virions were purified using a procedure devised for tomato spotted wilt tospovirus (TSWV) from systemically infectedNicotiana benthamiana plants grown at 33 °C day/26 °C night and a photoperiod of 14 hours. With plants grown at 24/18 ° C purification was unsuccessful. In SDS-PAGE the protein pattern of INSV was similar to that reported for TSWV, except the appearance of a single G2 protein band. A polyclonal antiserum, prepared against virions, reacted in Western blots with INSV nucleoprotein and glycoproteins but only with TSWV glycoproteins. In DAS ELISA the antiserum reacted with both INSV and TSWV infected plant sap and, after absorption with TSWV, only with INSV. In TAS ELISA the antiserum trapped both INSV and TSWV nucleoproteins and glycoproteins as detected by specific monoclonal antibodies, and, after absorption with TSWV, only the homologous proteins. This appears to be the first report of the purification of INSV virions and the production of an antiserum reacting with both nucleoprotein and glycoprotein antigens.  相似文献   

2.
A putative virus-induced disease showing chlorotic ringspots on leaves of Phalaenopsis orchids has been observed in Taiwan for several years. A virus culture, 91-orchid-1, isolated from a Phalaenopsis orchid bearing chlorotic ringspot symptoms was established in Chenopodium quinoa and Nicotiana benthamiana, and characterized serologically and biologically. The virus reacted slightly with the antiserum of Watermelon silver mottle virus (WSMoV) but not with those of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV) and Groundnut ringspot virus (GRSV). Isometric particles measuring about 70–100 nm were observed. Inoculation with isolated virus was conducted to confirm that 91-orchid-1 is the causal agent of chlorotic ringspot disease of Phalaenopsis orchids. To determine the taxonomic relationships of the virus, the conserved region of L RNA and the complete nucleocapsid gene (N gene) were cloned and sequenced. The sequence of conserved region of L RNA shares 83.8, 82.5, 64.4 and 64.9% nucleotide identities and 96.5, 97.7, 67.3 and 67.6% amino acid identities with those of Peanut bud necrosis virus (PBNV), WSMoV, TSWV and INSV, respectively, indicating that 91-orchid-1 is a tospovirus related to WSMoV. The complete nucleotide sequence of the N gene determined from a cDNA clone was found to be 828 nucleotides long encoding 275 amino acids. Sequence analyses of the N gene showed that 91-orchid-1 is an isolate of Capsicum chlorosis virus (CaCV) which has been reported to infect tomato and capsicum plants in Australia and Thailand. 91-orchid-1 is therefore designated as CaCV-Ph. To our knowledge, this is the first formal report of a tospovirus infecting Phalaenopsis orchids.  相似文献   

3.
Chu FH  Yeh SD 《Phytopathology》1998,88(4):351-358
ABSTRACT Double-stranded genomic RNAs (dsRNAs) extracted from Chenopodium quinoa infected with watermelon silver mottle virus (WSMV) were similar to those of tomato spotted wilt virus (TSWV, serogroup I) and impatiens necrotic spot virus (INSV, serogroup III), except that the S dsRNA of WSMV is 0.75 and 0.6 kbp longer than those of TSWV and INSV, respectively. The complete nucleotide sequence of the genomic M RNA of WSMV was determined from cDNA clones generated from separated M dsRNA. The M RNA is 4,880 nucleotides in length with two open reading frames (ORFs) in an ambisense organization. The M RNA-encoded nonstructural (NSm) ORF located on the viral strand encodes a protein of 312 amino acids (35 kDa), and the G1/G2 ORF located on the viral complementary strand encodes a protein of 1,121 amino acids (127.6 kDa). The RNA probe corresponding to the NSm or G1/G2 ORF of WSMV failed to hybridize with the M dsRNAs of TSWV and INSV. Comparison of M and S RNAs of WSMV, TSWV, INSV, and peanut bud necrosis virus (PBNV, serogroup IV) revealed a consensus sequence of eight nucleotides of 5'-AGAGCAAU...-3' at their 5' ends and 5'-...AUUGCUCU-3' at their 3' ends. The low overall nucleotide identities (56.4 to 56.9%) of the M RNA and the low amino acid identities of the NSm and G1/G2 proteins (30.5 to 40.9%) with those of TSWV and INSV indicate that WSMV belongs to the Tospovirus genus but is phylogenetically distinct from viruses in serogroups I and III. The M RNA of WSMV shares a nucleotide identity of 79.6% with that of PBNV, and the two viruses share 83.4 and 88.7% amino acid identities for their NSm and G1/G2 proteins, respectively. It is concluded that they are two related but distinct species of serogroup IV. In addition to the viral or viral complementary full-length M RNA, two putative RNA messages for the NSm gene and the G1/G2 gene, 1.0 and 3.4 kb, respectively, were detected from the total RNA extracted from WSMV-infected tissue of Nicotiana benthamiana. The 1.0- and 3.4-kb RNAs were also detected in the viral RNAs extracted from purified nucleocapsids, suggesting that the putative messages of the M RNA of WSMV can also be encapsidated by the nucleocapsid protein.  相似文献   

4.
Chu FH  Chao CH  Peng YC  Lin SS  Chen CC  Yeh SD 《Phytopathology》2001,91(9):856-863
ABSTRACT To clarify the serological relationship of Peanut chlorotic fan-spot virus (PCFV) with other tospoviruses, antisera were produced against the nucleocapsid (N) proteins of this virus and tospoviruses from four serogroups including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Groundnut ringspot virus (GRSV), and Watermelon silver mottle virus (WSMoV). In immunodiffusion tests, the antisera only reacted with their homologous antigens. Similar results were noticed in indirect enzyme-linked immunosorbent assay and immunoblot tests, with the exception that strong cross-reactions were observed in heterologous combinations between TSWV and GRSV. The results indicated that the N protein of PCFV is not serologically related to those of the tospoviruses from the four serogroups. To further characterize the virus, viral S double-stranded RNA was extracted from PCFV-infected Chenopodium quinoa and used for cDNA cloning and sequencing. The full-length viral strand of the S RNA was determined to be 2,833 nucleotides, with an inverted repeat at the 5' and 3' ends and two open reading frames in an ambisense arrangement. The 3'-terminal sequence (5'-AUUGCUCU-3') of the viral S RNA is identical to those of other tospoviruses, indicating that PCFV belongs to the genus Tospovirus. The N and the NSs proteins of PCFV share low amino acid identities (22.3 to 67.5% and 19.3 to 54.2%) with those of reported tospoviruses, respectively. The phylogenetic dendrogram of the N gene of PCFV compared with those of other tospoviruses indicates that PCFV is distinct from other tospoviruses. In hybridization analyses, an N gene cDNA probe of PCFV did not react with viral RNAs of TSWV, GRSV, INSV, and WSMoV, and vice versa. Thus, based on these results, we conclude that PCFV is a new tospovirus species.  相似文献   

5.
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   

6.
Infection by two isolates of impatiens necrotic spot tospovirus (INSV) under temperature regimes of 25/18°C (day/night) or 33°C (continuous) was studied in Capsicum annuum (systemically susceptible to tomato spotted wilt tospovirus, TSWV), C. chinense PI 152225 and PI 159236 (reacting hypersensitively to TSWV) and Nicotiana benthamiana (systemically susceptible to both tospoviruses). At 25/18°C infection was systemic in all hosts tested. At 33°C infection in N. benthamiana was systemic whereas in C. annuum and C. chinense it was restricted to the inoculated leaves. The result differed from that reported for TSWV, where high temperature made plants more susceptible, or caused no difference. Exchanging temperature regimes 6h to 4 days after inoculation did not affect the final results one month later, with plants being only locally infected at 33°C continuous regime, or systemically infected at 25/18°C alternate regime. The two INSV isolates were biologically and serologically stable for 5 passages in N. benthamiana held continuously at 33°C.  相似文献   

7.
The rapid immunofilter paper assay (RIPA) was developed to detect Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV), and Tomato chlorotic spot virus (TCSV) using antisera against recombinant nucleocapsid (N) proteins of each tospovirus. The two-step RIPA was sensitive enough to detect each pecies specifically in only 30min. This technique is proposed as an excellent tool for routine Tospovirus diagnosis and field epidemiological studies.  相似文献   

8.
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.  相似文献   

9.
In 2009, chlorotic mottle and necrosis were observed on chrysanthemums (cv. Jimba) in Aomori Prefecture, Japan. A virus was isolated from the chrysanthemum plants by serial local-lesion transfer. The symptoms exhibited by the test plants, the particle morphology, the features of the protein and the potential for transmission by thrips were similar to those for Impatiens necrotic spot virus (INSV). The partial nucleotide sequences of the nucleocapsid protein gene and the 3′-untranslated sequence of the S RNA shared 99% identity with that of an INSV isolate. This report is the first of INSV infection of chrysanthemums in Japan.  相似文献   

10.
 应用DAS-ELISA和RT-PCR方法从褪绿和银色斑驳的西瓜叶片中检测到病毒分离物（WSMoV-YN）,感病样品能与WSMoV/GBNV复合抗血清（Agdia）呈阳性反应。获得WSMoV N蛋白的多克隆抗体,抗体能与WSMoV血清组成员CaCV和TZSV反应,但不能与INSV、TSWV、HCRV和GYSV反应。为明确引起该病害的病毒种类,采用Tospovirus通用引物对样品的总RNA进行RT-PCR扩增,获得长度为3 554 nt的S RNA全序列,经Blastn比对分析与WSMoV中国台湾分离物同源性最高,为95.8%,其N和NSs蛋白氨基酸序列同源性分别为99%和97.6%。构建系统进化树发现,西瓜银灰斑驳病毒云南分离物（WSMoV-YN）与其他WSMoV聚为一支。确定引起云南西瓜病害的病毒为WSMoV。  相似文献   

11.
Wheat streak mosaic virus (WSMV) is an economically important pathogen of wheat (Triticum aestivum) causing major yield losses in regions where severe infection occurs. To detect the presence of any new virus or new WSMV isolates, green foxtail (Setaria viridis) plants exhibiting virus-like symptoms were sampled in a summer-fallowed wheat field at the Agricultural Research Center-Hays, Kansas State University, Hays, Kansas. These plants were tested serologically for four wheat viruses: WSMV, Triticum mosaic virus (TriMV), High Plains wheat mosaic virus (HPWMoV) and Foxtail mosaic virus (FoMV). Among 38 plant samples exhibiting virus-like symptoms, 29 contained WSMV as indicated by ELISA. Four isolates from samples with relatively strong reactions were transferred to healthy wheat seedlings by mechanical inoculation in a growth chamber for pathogenicity testing. Three isolates were avirulent to a wheat variety RonL, which contains Wsm2, a gene providing temperature-sensitive resistance to currently prevalent isolates of WSMV. However, one isolate, KSH294, was able to infect RonL and showed more virulence on two other varieties/lines containing Wsm2. Further sequence and phylogenetic analysis of KSH294 confirmed that this isolate displays a sequence homology with WSMV, but has sequence differences making it distinct from previously identified WSMV isolates included in the phylogenetic analysis.  相似文献   

12.
The effect of verbena as a trap crop on the occurrence of western flower thrips, Frankliniella occidentalis, and the incidence of Tomato spotted wilt virus (TSWV) in chrysanthemums were investigated. Verbena cvs. Pink Parfait and/or Fancy Parfait were cultivated alongside chrysanthemum cv. Jimba in a greenhouse in the proportion of 17%–25% of the chrysanthemum plants. Verbena plants attracted vector thrips, reducing western flower thrips colonization of chrysanthemum until flower bud initiation, and markedly suppressing TSWV incidence on chrysanthemums until flowering. Significant quantities of linalool oxide pyran were produced by the flower of cv. Fancy Parfait; and the ratio of cis-linalool oxide pyran, an attractant for vector thrips, to the trans-type was approximately 1 : 5. Our results suggest that cultivation of verbena as a trap crop may be useful in integrated pest management programs as a control for thrips-transmitted TSWV in chrysanthemums.  相似文献   

13.
A study was conducted to determine the identity and prevalence of viruses in 455 greenhouses in the main Spanish green bean growing area. Directed surveys were conducted in 422 crops from 2000–2004 to collect samples from diseased plants displaying symptoms that could be attributed to viruses. The samples were analysed to detect any virus by means of dsRNA extraction, mechanical inoculation to test plants, as well as ELISA and/or RT-PCR tests to detect potyviruses, geminiviruses and viruses previously known to infect beans in Spain. Random surveys were conducted in the years 2002 and 2005 (in 21 and 12 greenhouses, respectively) to study the actual incidence of known viruses in the area. Symptoms were recorded in 23,108 plants from which 664 plants were collected and analysed by ELISA or RT-PCR. The results of the directed surveys showed that all the analyzed crops carried the cryptic virus Phaseolus vulgaris endornavirus (PVuV), whereas phytopathogenic viruses appeared in smaller percentages of the crops: Tomato yellow leaf curl virus (TYLCV) 20.4%, Southern bean mosaic virus (SBMV) 9.0%, Tomato spotted wilt virus (TSWV) 4.0%, and the new species Bean yellow disorder virus (BnYDV) that broke out in 2004 with occurrence values higher than 34.3% that year. From 2000–2004 an important decrease in TYLCV was observed, along with a slight increase in SBMV and a consistently low occurrence of TSWV. The results of the random surveys confirmed the increased occurrence of virus detected during the directed surveys, and furthermore demonstrated the percentage of incidence for each virus.  相似文献   

14.
Chen TC  Huang CW  Kuo YW  Liu FL  Yuan CH  Hsu HT  Yeh SD 《Phytopathology》2006,96(12):1296-1304
ABSTRACT The NSs protein of Watermelon silver mottle virus (WSMoV) was expressed by a Zucchini yellow mosaic virus (ZYMV) vector in squash. The expressed NSs protein with a histidine tag and an additional NIa protease cleavage sequence was isolated by Ni(2+)-NTA resins as a free-form protein and further eluted after sodium dodecyl sulfate-polyacrylamide gel electrophoresis for production of rabbit antiserum and mouse monoclonal antibodies (MAbs). The rabbit antiserum strongly reacted with the NSs crude antigen of WSMoV and weakly reacted with that of a high-temperature-recovered gloxinia isolate (HT-1) of Capsicum chlorosis virus (CaCV), but not with that of Calla lily chlorotic spot virus (CCSV). In contrast, the MAbs reacted strongly with all crude NSs antigens of WSMoV, CaCV, and CCSV. Various deletions of the NSs open reading frame were constructed and expressed by ZYMV vector. Results indicate that all three MAbs target the 89- to 125-amino-acid (aa) region of WSMoV NSs protein. Two indispensable residues of cysteine and lysine were essential for MAbs recognition. Sequence comparison of the deduced MAbs-recognized region with the reported tospoviral NSs proteins revealed the presence of a consensus sequence VRKPGVKNTGCKFTMHNQIFNPN (denoted WNSscon), at the 98- to 120-aa position of NSs proteins, sharing 86 to 100% identities among those of WSMoV, CaCV, CCSV, and Peanut bud necrosis virus. A synthetic WNSscon peptide reacted with the MAbs and verified that the epitopes are present in the 98- to 120-aa region of WSMoV NSs protein. The WSMoV sero-group-specific NSs MAbs provide a means for reliable identification of tospoviruses in this large serogroup.  相似文献   

15.
In 1997, virus-like symptoms were observed in dimorphotheca plants(Dimorphotheca sinuata) at two different locations in Greece. In a greenhouse near Heraklion (Crete), plants showed chlorotic patches mainly in the older leaves, and a mild stunting; near Katerini (Macedonia), garden plants showed chlorotic rings. Sap inoculation of healthy dimorphotheca plants with extracts from diseased plants from the two regions, reproduced each of the two diseases. Examination by electron microscopy (EM) of samples from diseased plants from the first region revealed filamentous particles, whereas samples from the second region showed quasi-spherical particles. The filamentous particles were decorated in EM with antibodies specific to lettuce mosaic potyvirus (LMV). Samples from the second location reacted with antibodies specific to tomato spotted wilt tospovirus (TSWV) in EL1SA tests. It is concluded that LMV and TSWV are the causal agents of the disease noticed in Heraklion and in Katerini, respectively. http://www.phytoparasitica.org posting May 31, 1999.  相似文献   

16.
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17.
Three isolates of Chrysanthemum stem necrosis virus (CSNV) were obtained from chrysanthemum plants in distinct regions of Japan in 2006 and 2007. All the original host plants showed severe necrotic symptoms on the leaves and stems. Amino acid sequence data of the nucleocapsid protein genes of the three isolates (CbCh07A, TcCh07A, and GnCh07S) showed high identities with those of two other CSNV isolates, HiCh06A L1 from Japan and Chry1 from Brazil. Furthermore, for the first time the complete nucleotide sequence of the S RNA was determined for CSNV (isolate HiCh06A). In phylogenetic analysis based on the non-structural protein genes from the genus Tospovirus, HiCh06A L1 was placed in the same genetic group as Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus. Host range examination for isolates HiCh06A L1 and CbCh07A showed that green pepper (cv. ‘Kyoyutaka’, ‘Saitamawase’, ‘Tosakatsura’, ‘L3 sarara’ and ‘L3 miogi’) and tomato (cv. ‘Sekaiichitomato’) were systemically susceptible hosts, whereas TSWV-resistant Solanaceae species, Capsicum chinense, Lycopersicon peruvianum and a TSWV-resistant cultivar of green pepper (cv. TSR miogi), were resistant.  相似文献   

18.
Virus interactions between Tomato spotted wilt virus (TSWV) and Potato virus X (PVX) containing the nucleocapsid protein (N) gene sequences were examined to evaluate the capacity of the N gene sequences from TSWV to promote RNA-mediated cross-protection. Plants simultaneously inoculated with TSWV and PVX containing the 3 96bp of the N gene were highly resistant to TSWV infection, whereas no such resistance was observed in plants inoculated with TSWV and PVX containing the 5 96bp. These results suggest that the 3 portion of the N gene has a higher capacity for promoting RNA-mediated cross-protection of TSWV.  相似文献   

19.
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   

20.
A putative virus-induced disease showing chlorotic spots on leaves of Phalaenopsis orchids was observed in central Taiwan. A virus culture, phalaenopsis isolate 7-2, was isolated from a diseased Phalaenopsis orchid and established in Chenopodium quinoa and Nicotiana benthamiana. The virus reacted with the monoclonal antibody (POTY) against the potyvirus group. Potyvirus-like long flexuous filament particles around 12–15 × 750–800 nm were observed in the crude sap and purified virus preparations, and pinwheel inclusion bodies were observed in the infected cells. The conserved region of the viral RNA was amplified using the degenerate primers for the potyviruses and sequence analysis of the virus isolate 7-2 showed 56.6–63.1% nucleotide and 44.8–65.1% amino acid identities with those of Bean yellow mosaic virus (BYMV), Beet mosaic virus (BtMV), Turnip mosaic virus (TuMV) and Bean common mosaic virus (BCMV). The coat protein (CP) gene of isolate 7-2 was amplified, sequenced and found to have 280 amino acids. A homology search in GenBank indicated that the virus is a potyvirus but no highly homologous sequence was found. The virus was designated as Phalaenopsis chlorotic spot virus (PhCSV) in early 2006. Subsequently, a potyvirus, named Basella rugose mosaic virus isolated from malabar spinach was reported in December 2006. It was found to share 96.8% amino acid identity with the CP of PhCSV. Back-inoculation with the isolated virus was conducted to confirm that PhCSV is the causal agent of chlorotic spot disease of Phalaenopsis orchids in Taiwan. This is the first report of a potyvirus causing a disease on Phalaenopsis orchids.  相似文献   

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