First Report of Tomato spotted wilt virus in Sweet Pepper in Taiwan

A new disorder on pepper showing symptoms of chlorosis and chlorotic spots on leaves was observed in sweet pepper (Capsicum annuum cv. Andalus) fields in Ren-Ai Township, Nantou County in July, 2009. The disorder occurred in more than 30% of the pepper plants, with a height of approximately 40 cm (1.5 feet), which was approximately one-half the size of the asymptomatic ones. Symptomatic plants bore much smaller fruits with abnormal shapes. Three symptomatic sweet pepper plants were collected and tested for potential viruses. Reverse transcription (RT)-PCR was performed for the detection using three degenerate primer pairs, gL3637/gL4435c for tospoviruses (2), Hrp5/Pot1 for potyviruses (1,3), and Tob-Uni1/Tob-Uni2 for tobamoviruses (4), and specific primers, FJJ2001-7/FJJ2001-8 (5′-TATGTCCATGGACAAATCCGAATCA and 5′-TCTCTGGATCCACGAGTTCAAACTGGGAG) for the coat protein gene of Cucumber mosaic virus (CMV). An 819-nt DNA fragment containing the partial L RNA of tospovirus was amplified from the total RNA isolated from each of these three samples by RT-PCR with primer pair gL3637/gL4435c. One amplified fragment was cloned and sequenced. A homology search in GenBank indicated that the new pepper-infecting virus in Taiwan was Tomato spotted wilt virus (TSWV) since the partial L RNA shared more than 94.5% nucleotide and 98.2% amino acid identity with five TSWV isolates (Accession Nos. AB190813, AB198742, AY070218, D10066, and NC_002052). No DNA fragment was obtained by RT-PCR using primer pairs for CMV, potyviruses, or tobamoviruses. A virus culture (TwPep1) isolated from one of the symptomatic sweet pepper plants was then established in Nicotiana tabacum cv. White Burley and N. benthamiana through triple single-lesion isolation. TWPep1 reacted positively only to the antiserum against TSWV by indirect-ELISA but not to those of Watermelon silver mottle virus, Capsicum chlorosis virus, Tobacco mosaic virus, Tomato mosaic virus, and CMV. Partial L RNA and the full-length nucleocapsid (N) gene of TWPep1 were obtained by RT-PCR with primer pairs gL3637/gL4435c and FJJ2002-74/FJJ2002-75 (5′-GCGCGCGGATCCTAATTTAACTTACARCTGCT 5′-TGCTGCCTCGAGCATACGGTCAAAGCATATAA), respectively. The 819-nt L RNA conserved region of TwPep1 (Accession No. GU222652) shared 94.4 to 97.7% nucleotide and 98.2 to 100% amino acid identity with those available in GenBank. The 777-bp N gene of TwPep1 (Accession No. GU222651) shared 96.7 to 99.1% nucleotide and 97.3 to 99.6% amino acid identity with 37 TSWV isolates available in GenBank. Sequence comparisons indicated that TwPep1 is an isolate of TSWV. TSWV was later detected by RT-PCR in all 10 symptomatic samples of sweet pepper plants collected from five fields in August 2009. To our knowledge, this is the first report of TSWV in sweet pepper in Taiwan. This is also the first demonstration of isolation and characterization of TSWV in Taiwan although TSWV was once detected in lisianthus (Eustoma rusellianum) by RT-PCR (1) but the isolation was not successful then. The occurrence of TSWV in pepper will have a direct economic impact on the important vegetable and floral industry in Taiwan because TSWV reportedly comprises a wide host range. References: (1) C. C. Chen et al. Bot. Stud. 947:369, 2006. (2) F. H. Chu et al. Phytopathology 91:361, 2001. (3) D. Colinet and J. Kummert. J. Virol. Methods 45:149, 1993. (4) B. Letschert et al. J. Virol. Methods 106:1, 2002.

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First Report of Tomato spotted wilt virus on Brugmansia sp. in Serbia

Plant Disease ◽

10.1094/pdis-10-12-0937-pdn ◽

2013 ◽

Vol 97 (6) ◽

pp. 850-850 ◽

Cited By ~ 3

Author(s):

D. Nikolić ◽

I. Stanković ◽

A. Vučurović ◽

D. Ristić ◽

K. Milojević ◽

...

Keyword(s):

Mosaic Virus ◽

Tomato Spotted Wilt Virus ◽

N Gene ◽

Broad Host Range ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Plant Pathol ◽

Negative Controls ◽

Wilt Virus

Brugmansia (Brugmansia spp.), also known as Angel's trumpet, is a perennial shrub in the Solanaceae that is a popular landscape plant in the tropics and subtropics, and potted plant in temperate regions. In April 2012, virus-like symptoms including chlorotic leaf patterns and curling followed by necrosis and distortion of leaves were observed on five outdoor-grown brugmansia plants in a private garden in Mackovac, Rasina District, Serbia. Symptomatic leaves were tested for the presence of several common ornamental viruses including Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), Cucumber mosaic virus (CMV), and Tobacco mosaic virus (TMV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extract from healthy brugmansia leaves were included in each ELISA. TSWV was detected serologically in all five brugmansia samples and all tested samples were negative for INSV, CMV, and TMV. The virus was mechanically transmitted from an ELISA-positive sample (41-12) to five plants of each Petuina × hybrida and Nicotiana glutinosa. Inoculated P. × hybrida plants showed local necrotic lesions and N. glutinosa showed mosaic and systemic necrosis 4 and 12 days post-inoculation, respectively, which were consistent with symptoms caused by TSWV (1). For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the OneStep RT-PCR (Qiagen, Hilden, Germany) using a set of TSWV-specific primers, TSWV CP-f and TSWV CP-r (4), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs from naturally infected brugmansia and symptomatic N. glutinosa plants were extracted using the RNeasy Plant Mini Kit (Qiagen). Total RNAs obtained from the Serbian tobacco isolate of TSWV (GenBank Accession No. GQ373173) and healthy brugmansia plants were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic brugmansia and N. glutinosa but not from healthy tissues. The amplified product derived from the isolate 41-12 was sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen), deposited in GenBank (JX468080), and subjected to sequence analysis by MEGA5 software (3). Sequence comparisons revealed that the Serbian isolate 41-12 shared the highest nucleotide identity of 99.9% (99.5% amino acid identity) with an Italian TSWV isolate P105/2006RB (DQ915946) originating from pepper. To our knowledge, this is the first report of TSWV on brugmansia in Serbia. Due to the increasing popularity and economic importance of brugmansia as an ornamental crop, thorough inspections and subsequent testing for TSWV and other viruses are needed. This high-value ornamental plant may act also as reservoir for the virus that can infect other ornamentals and cultivated crops, considering that TSWV has a very broad host range (2). References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

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Development of Reverse Transcription Thermostable Helicase-Dependent DNA Amplification for the Detection of Tomato Spotted Wilt Virus

Journal of AOAC International ◽

10.5740/jaoacint.16-0132 ◽

2016 ◽

Vol 99 (6) ◽

pp. 1596-1599 ◽

Cited By ~ 4

Author(s):

Xinghai Wu ◽

Chanfa Chen ◽

Xizhi Xiao ◽

Ming Jun Deng

Keyword(s):

Reverse Transcriptase ◽

Mosaic Virus ◽

Reverse Transcription ◽

Tomato Spotted Wilt Virus ◽

Tomato Mosaic Virus ◽

N Gene ◽

Impatiens Necrotic Spot Virus ◽

Rt Pcr ◽

Tomato Spotted Wilt ◽

Wilt Virus

Abstract A protocol for the reverse transcription-helicase-dependent amplification (RT–HDA) of isothermal DNA was developed for the detection of tomato spotted wilt virus (TSWV). Specific primers, which were based on the highly conserved region of the N gene sequence in TSWV, were used for the amplification of virus's RNA. The LOD of RT–HDA, reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP), and reverse transcriptase-polymerase chain reaction (RT-PCR) assays were conducted using 10-fold serial dilution of RNA eluates. TSWV sensitivity in RT–HDA and RT-LAMP was 4 pg RNA compared with 40 pg RNA in RT-PCR. The specificity of RT–HDA for TSWV was high, showing no cross-reactivity with other tomato and Tospovirus viruses including cucumber mosaic virus (CMV), tomato black ring virus (TBRV), tomato mosaic virus (ToMV), or impatiens necrotic spot virus (INSV). The RT–HDA method is effective for the detection of TSWV in plant samples and is a potential tool for early and rapid detection of TSWV.

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First Report of Pepper veinal mottle virus in Tomato and Pepper in Taiwan

Plant Disease ◽

10.1094/pdis-93-1-0107a ◽

2009 ◽

Vol 93 (1) ◽

pp. 107-107 ◽

Cited By ~ 9

Author(s):

Y. H. Cheng ◽

R. Y. Wang ◽

C. C. Chen ◽

C. A. Chang ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Mosaic Virus ◽

Polyclonal Antibodies ◽

Amino Acid Identity ◽

Mottle Virus ◽

Rt Pcr ◽

Acid Identity ◽

First Report ◽

Cp Gene ◽

Pepper Veinal Mottle Virus

In May of 2006, samples from tomato plants (Solanum lycopersicum cv. Known-you 301) exhibiting necrotic symptoms on stems, petioles, and leaves were collected from Chiayi County, Taiwan. Double-antibody sandwich-ELISAs were performed using Cucumber mosaic virus, Tomato mosaic virus, Potato virus Y, Watermelon silver mottle virus, and Chilli veinal mottle virus (ChiVMV) polyclonal antibodies. Three of eight samples reacted with antibodies against ChiVMV but not with the others. Using the potyvirus degenerate primers (Hrp 5/Pot 1) (2), an expected 1.5-kb DNA fragment including the 3′-end of the NIb gene, the complete coat protein (CP) gene, and the 3′-nontranslatable region of the virus was amplified from total RNA isolated from these three samples by reverse transcription (RT)-PCR. A homology search in GenBank indicated that the new tomato-infecting virus in Taiwan belongs to Pepper veinal mottle virus (PVMV) since they shared >90% amino acid identity in the CP gene. A virus culture (Tom1) isolated from one of the diseased tomatoes was then established in Chenopodium quinoa and Nicotiana benthamiana and the CP gene was amplified and sequenced (GenBank Accession No. EU719647). Comparisons of the 807-nt CP gene with those of five PVMV isolates available in GenBank showed 81.5 to 93.1% nucleotide and 90.0 to 97.8% amino acid identity. Tom1 induced irregular necrotic lesions on stems, petioles, and leaves of tomato while inducing only mild mottle symptoms on pepper. Serological cross reaction between ChiVMV and PVMV has been observed previously (1,3) and also found in this study. To differentiate these two potyviruses by RT-PCR, primer pair CPVMVup/dw (5′-TATTC(T/C)TCAGTGTGG(A/T/C)T(T/C)CCACCAT and 5′-(T/C)C(A/T)C(A/T)(A/T/G)(A/T)AA(A/G)CCATAA(A/C)(A/C)ATA(A/G)T(T/C)T) was designed on the basis of the comparison of the CP gene and the 3′-nontranslatable region of the PVMV and ChiVMV. DNA fragments of 171 and 259 bp are expected to be amplified from ChiVMV and PVMV, respectively, by RT-PCR with primers CPVMVup/dw. In a field survey done in 2006, samples from diseased peppers (Capsicum annuum) that reacted with the polyclonal antibodies against ChiVMV were further identified by RT-PCR with primers CPVMVup/dw, indicating that both ChiVMV and PVMV infected pepper crops (Capsicum spp.) in Taiwan. A pepper isolate (Pep1) of PVMV was obtained from Nantou County through three times of single lesion passages on C. quinoa and then propagated on N. benthamiana. The CP gene of Pep1 was amplified and sequenced (GenBank Accession No. EU719646) and found to share 99.1% nucleotide and 100% amino acid identity with that of Tom1. Pep1 caused mild mottle symptoms on leaves of both tomato and pepper. To our knowledge, this is the first report of the presence of PVMV in Taiwan as well as in East Asia. References: (1) B. Moury et al. Phytopathology 95:227, 2005. (2) S. S. Pappu et al. Plant Dis. 82:1121, 1998. (3) W. S. Tsai et al. Plant Pathol. 58:408, 2008.

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First Report of Tomato spotted wilt virus on Pepper in Montenegro

Plant Disease ◽

10.1094/pdis-03-11-0167 ◽

2011 ◽

Vol 95 (7) ◽

pp. 882-882 ◽

Cited By ~ 5

Author(s):

J. Zindović ◽

A. Bulajić ◽

B. Krstić ◽

M. Ciuffo ◽

P. Margaria ◽

...

Keyword(s):

Amino Acid ◽

Tomato Spotted Wilt Virus ◽

Nucleotide Identity ◽

N Gene ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Italian Isolate ◽

Negative Controls ◽

Wilt Virus

In April 2009, chlorotic and necrotic ring spots, chlorotic line patterns, and stunting were observed on greenhouse-grown pepper plants in the vicinity of Podgorica, Montenegro. Disease symptom incidence was estimated at 40%. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV) with a commercial double-antibody sandwich (DAS)-ELISA diagnostic kit (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls were included in each ELISA. TSWV was detected serologically in 33 of 75 pepper samples. The virus was mechanically transmitted from ELISA-positive pepper samples to Nicotiana tabacum cv. Samsun using chilled 0.05 M phosphate buffer (pH 7) containing 0.1% sodium sulfite (1). Inoculated test plants produced chlorotic and necrotic concentric rings and necrotic spots, consistent with symptoms caused by TSWV on N. tabacum. For further confirmation of TSWV infection, reverse transcription (RT)-PCR was performed with the One-Step RT-PCR Kit (Qiagen, Hilden, Germany) using three sets of primers: S70-for/S890-rev (2) and S574-for/S1433-rev (3), both specific to the nonstructural (NSs) gene; and S1983-for/S2767-rev (2), specific to the nucleocapsid protein (N) gene. Total RNAs from naturally infected pepper and symptomatic N. tabacum cv. Samsun plants were extracted with the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Total RNAs obtained from the Italian isolate of TSWV (GenBank Accession No. DQ398945) and healthy tobacco plants were used as positive and negative controls, respectively. The expected sizes of the RT-PCR products (820, 859, and 784 bp) were amplified from symptomatic pepper samples but not from healthy tissues. The PCR product obtained from isolate Is-344 using primers specific to N gene was purified by a QIAquick PCR Purification Kit (Qiagen), cloned into the pGEM-T Easy Vector (Promega, Madison, WI) and sequenced in both directions using the same primer pair as in RT-PCR. The sequences amplified with the two primer pairs specific to the NSs gene were obtained by direct sequencing (Bio-Fab Research Srl, Pomezia, Italy) and joined using MEGA4 software. Sequence analysis of the complete N gene (777 bp; GenBank Accession No. GU369717) revealed that the TSWV isolate originating from Montenegro shared 98.2 to 99.7% nucleotide identity (98.1 to 100% amino acid identities) with corresponding TSWV sequences deposited in GenBank. The Montenegrin isolate Is-344 was most closely related to Italian isolates from tomato (GU369725) and eggplant (GU369720). The partial (1,257 bp) nucleotide sequence of NSs gene (GU369737) showed 96 to 99.8% nucleotide identity (96.9 to 100% amino acid identity) with previously reported TSWV sequences, and in this case the highest identity was with French isolates from tomato (FR692835) and lettuce (FR692831). To our knowledge, this is the first report on the occurrence of TSWV in Montenegro. Data of this study sheds light on the importance of further survey studies and inspections of TSWV-susceptible crops cultivated in Montenegro. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) W. P. Qiu et al. Virology 244:186, 1998. (3) M. Tsompana et al. Mol. Ecol. 14:53, 2005.

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First Report of Tomato spotted wilt virus on Gloxinia in Bosnia and Herzegovina

Plant Disease ◽

10.1094/pdis-08-12-0777-pdn ◽

2013 ◽

Vol 97 (3) ◽

pp. 429-429 ◽

Cited By ~ 2

Author(s):

V. Trkulja ◽

J. Mihić Salapura ◽

B. Ćurković ◽

I. Stanković ◽

A. Bulajić ◽

...

Keyword(s):

Bosnia And Herzegovina ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Identity ◽

N Gene ◽

Impatiens Necrotic Spot Virus ◽

Post Inoculation ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

In June and July 2012, symptoms resembling those caused by a tospovirus infection were observed on the greenhouse-grown gloxinia (Sinningia speciosa Benth. and Hook.) in the Lijevče polje, in the vicinity of Banja Luka (Bosnia and Herzegovina). Infected plants exhibited chlorotic ring spots and chlorotic and necrotic patterns followed by necrosis and distortion of leaves. Disease symptom incidence was estimated at 30% out of 400 inspected plants. Symptomatic leaves were collected and tested by double-antibody sandwich (DAS)-ELISA test using commercial polyclonal antisera (Bioreba AG, Reinach, Switzerland) for two of the most important tospoviruses in the greenhouse production of ornamentals: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). TSWV was detected serologically in 27 out of 30 tested gloxinia samples, and all were negative for INSV. Symptomatic leaves of five selected ELISA-positive gloxinia plants were separately ground in chilled 0.01 M phosphate buffer (pH 7) containing 0.1% w/v sodium sulphite and were mechanically inoculated on five plants of Petunia × hybrida. All inoculated plants produced typical symptoms of TSWV (1), necrotic spots on inoculated leaves in 2 to 5 days post-inoculation. For further confirmation of TSWV infection, total RNAs were extracted using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from all 27 infected gloxinia plants and tested by reverse transcription (RT)-PCR assay. A 738-bp fragment of TSWV nucleocapsid (N) gene was amplified with One-Step RT-PCR Kit (Qiagen) using primer pairs TSWV CP-f and TSWV CP-r (4). Total RNAs from Serbian tobacco TSWV isolate (GenBank Accession No. GQ373173) and RNA extract from healthy gloxinia plants were used as positive and negative controls, respectively. Amplicons of the expected size were obtained from all 27 naturally infected gloxinia plants, while no amplification products were obtained from the healthy control. After the purification with QIAquick PCR Purification Kit (Qiagen), the RT-PCR product obtained from one selected isolate 160-12 was sequenced directly in both directions and submitted to GenBank (JX468079). Sequence analysis of the partial N gene, conducted by MEGA5 software (3), from isolate 160-12 showed the highest nucleotide identity of 99.7% (100% amino acid identity) with eight pepper isolates of TSWV from Spain (FR693229, FR693231, FR693152-153, FR693078, FR693081, FR693089, and FR693092). To our knowledge, this is the first report on the occurrence of TSWV in Bosnia and Herzegovina. The presence of this harmful pathogen into a new area could have a serious threat to intensive and increasing production of ornamentals and numerous other TSWV susceptible species in Bosnia and Herzegovina. The discovery of TSWV on gloxinia should prompt more surveys, thorough inspections, and subsequent testing of other TSWV susceptible plants cultivated in Bosnia and Herzegovina. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

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First Report of Zantedeschia mosaic virus Infecting a Zantedeschia sp. in New Zealand

Plant Disease ◽

10.1094/pdis-92-8-1253a ◽

2008 ◽

Vol 92 (8) ◽

pp. 1253-1253 ◽

Cited By ~ 3

Author(s):

T. Wei ◽

M. N. Pearson ◽

D. Cohen ◽

J. Z. Tang ◽

G. R. G. Clover

Keyword(s):

New Zealand ◽

Amino Acid ◽

Mosaic Virus ◽

Amino Acid Identity ◽

Cut Flowers ◽

Rt Pcr ◽

Acid Identity ◽

Virus Particles ◽

Blast Search ◽

Cp Gene

In February 2004, leaf yellowing, mottling, and mosaics were observed on a few plants of a Zantedeschia sp. (calla lily) growing in Rangiora, Canterbury, New Zealand. Zantedeschia spp. are known to be susceptible to at least 13 virus species (1). No symptoms were observed on Chenopodium amaranticolor, C. quinoa, Cucumis sativus, Gomphrena globosa, Nicotiana benthamiana, N. clevelandii, N. occidentalis, or N. tabacum when inoculated with sap from symptomatic plants. However, electron microscopy of crude sap preparations from a symptomatic Zantedeschia sp. and inoculated N. clevelandii plants revealed the presence of flexuous, filamentous virus particles approximately 700 nm long and 12 nm wide. No virus particles were seen in the other inoculated indicator species. Nucleic acid was extracted from leaves of the infected Zantedeschia sp. and N. clevelandii plants and tested in reverse transcription (RT)-PCR using published potyvirus-specific primers (4). PCR amplicons of the expected size (327 bp) were obtained from both plant species and sequenced directly. The products were identical, and a BLAST search in GenBank showed 99% nucleotide identity with a Taiwanese isolate of the species Zantedeschia mosaic virus (ZaMV) (GenBank Accession No. AY026463). A product of 1,531 bp (GenBank Accession No. EU544542) was amplified from symptomatic Zantedeschia by RT-PCR using novel forward (5′-GCACGGCAGATAAACACGAC-3′) and reverse (5′-GTGGGCAACCTTCAACTGTG-3′) primers designed to amplify the 3′ untranslated region (3′UTR), coat protein (CP), and partial nuclear inclusion b protein (NIb) genes. The product was sequenced and had 94% nucleotide identity with a South Korean ZaMV isolate (GenBank Accession No. AB081519), with 95% nucleotide (97% amino acid) identity in the CP gene. A second crop of Zantedeschia spp. in Tauranga, New Zealand (approximately 700 km north of Rangiora) was observed to have similar disease symptoms. Symptomatic plants tested positive in ELISA using a potyvirus-specific monoclonal antibody (Agdia Inc., Elkhart, IN). Nucleic acid was extracted from leaves of symptomatic plants and tested in RT-PCR using potyvirus-specific primer pairs, PV2I/T7 and D335 and U335 and PV1/SP6, which amplify overlapping regions within the 3′UTR, CP, and NIb genes (2,3). The products were sequenced and a consensus sequence of 1,793 bp was generated (GenBank Accession No. EU532065). A BLAST search showed that the sequence had 78% nucleotide (88% amino acid) identity with Zantedeschia mild mosaic virus (ZaMMV) (GenBank Accession No. AY626825). However, the sequences had only 73% nucleotide (79% amino acid) identity in the CP gene, and therefore, this second virus may be a distinct species. To our knowledge, this is the first report of ZaMV in New Zealand. Cut flowers are an increasingly important commodity in New Zealand and Zantedeschia is one of the most important crops; in 2005, exports of rhizomes and cut flowers of the genus were worth NZ$10.9 million. These viral diseases may require management to ensure that the quality of production is maintained. References: (1) C. H. Huang et al. Plant Pathol. 56:183, 2007. (2) S. A. Langeveld et al. J. Gen. Virol. 72:1531, 1991. (3) A. M. Mackenzie et al. Arch. Virol. 143:903, 1998. (4) V. Marie-Jeanne et al. J. Phytopathol. 148:141, 2000.

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First Report of Capsicum chlorosis virus Infecting Tomato in Taiwan

Plant Disease ◽

10.1094/pdis-04-10-0275 ◽

2010 ◽

Vol 94 (10) ◽

pp. 1263-1263 ◽

Cited By ~ 7

Author(s):

C.-H. Huang ◽

Y.-X. Zheng ◽

Y.-H. Cheng ◽

W.-S. Lee ◽

F.-J. Jan

Keyword(s):

Amino Acid ◽

Amino Acid Identity ◽

N Gene ◽

Rt Pcr ◽

Degenerate Primers ◽

Tomato Plants ◽

Acid Identity ◽

First Report ◽

Conserved Region ◽

Capsicum Chlorosis Virus

In December 2009, two samples from tomato plants (Solanum lycopersicum cv. Known-you 301) showing symptoms of chlorosis and necrosis on leaves were collected from two different fields that exhibited 5% disease incidence in Wufeng Township, Taichung County. Reverse transcription (RT)-PCR was applied to detect the presence of potential viruses in collected samples using three degenerate primers (3), gL3637/gL4435c for tospoviruses, Tob-Uni1/Tob-Uni2 for tobamoviruses, and Hrp5/Pot1 for potyviruses, and one specific primer, FJJ2001-7/FJJ2001-8, for the coat protein gene of Cucumber mosaic virus (3). An 816-nt DNA fragment was amplified from each of these two field samples by RT-PCR with the tospovirus degenerate primers, gL3637/gL4435c, designed from the conserved region of L RNA. One of the amplified fragments was cloned and sequenced. A homology search indicated that the new tomato-infecting virus in Taiwan might belong to Capsicum chlorosis virus (CaCV) since the partial L RNA shared more than 87% nucleotide and 99.6% amino acid identity with two CaCV isolates from Thailand (GenBank Accession Nos. DQ256124 and NC_008302). A virus culture isolated from the symptomatic tomato was established in Chenopodium quinoa through triple single-lesion isolation and designated as TwTom1. The partial L RNA and full-length nucleocapsid (N) gene of TwTom1 were obtained by RT-PCR with primer pairs gL3637/gL4435c and FJJ 2010-2 (5′-TTAAAT(C/T)ACAC(C/T)TCTATAGA)/N3534c (1), respectively. The 816-nt L RNA conserved region of TwTom1 (Accession No. HM021140) also shared 87% nucleotide and 99.6% amino acid identity with those of the above mentioned two CaCV isolates available in GenBank. The 828-nt N gene of TwTom1 (Accession No. HM021139) shared 85 to 98.1% nucleotide and 92 to 100% amino acid identity with those of 26 CaCV isolates available in GenBank. TwTom1 shared the highest N gene nucleotide and amino acid identity, 98.1 and 100%, respectively, with a gloxinia isolate (Accession No. AY312061). Sequence analysis results indicated that TwTom1 is an isolate of CaCV. The TwTom1 isolate was back inoculated onto three tomato (cv. Known-you 301) plants for pathogenicity test. The inoculated tomato plants showed symptoms of chlorosis at 13 days postinoculation (dpi) and symptoms of chlorosis plus necrosis on leaves at 20 dpi, which were similar to that observed in the field. A protein band measuring approximately 30 kDa in the crude sap of the TwTom1-infected tomato was observed in western blotting using the antiserum against the N protein of CaCV. In addition, CaCV was later detected by RT-PCR in two symptomatic tomato samples collected from another field. CaCV was first found in Australia, then Thailand, Taiwan, China, and India (2). Although CaCV was found to infect several species of ornamental crops in Taiwan, to our knowledge, this is the first report of CaCV that could naturally infect tomato, a nonornamental plant in Taiwan. References: (1) Y. H. Lin et al. Phytopathology 95:1482, 2005. (2) H. R. Pappu et al. Virus Res. 141:219, 2009. (3) Y.-X. Zheng et al. Plant Dis. 94:920, 2010.

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First Report of Tomato spotted wilt virus in Blackberry Lily in North America

Plant Disease ◽

10.1094/pdis.2003.87.1.102c ◽

2003 ◽

Vol 87 (1) ◽

pp. 102-102 ◽

Cited By ~ 3

Author(s):

S. Adkins ◽

L. Breman ◽

C. A. Baker ◽

S. Wilson

Keyword(s):

North America ◽

Tomato Spotted Wilt Virus ◽

Amino Acid Sequences ◽

South Florida ◽

N Gene ◽

Enzyme Linked Immunosorbent Assay ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Wilt Virus

Blackberry lily (Belamcanda chinensis (L.) DC.) is an herbaceous perennial in the Iridaceae characterized by purple-spotted orange flowers followed by persistent clusters of black fruit. In July 2002, virus-like symptoms including chlorotic ringspots and ring patterns were observed on blackberry lily leaves on 2 of 10 plants in a south Florida ornamental demonstration garden. Inclusion body morphology suggested the presence of a Tospovirus. Tomato spotted wilt virus (TSWV) was specifically identified by serological testing using enzyme-linked immunosorbent assay (Agdia, Elkhart, IN). Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579 base pair region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. Since these 2-year-old plants were grown on-site from seed, they were likely inoculated by thrips from a nearby source. Together with a previous observation of TSWV in north Florida nursery stock (L. Breman, unpublished), this represents, to our knowledge, the first report of TSWV infection of blackberry lily in North America although TSWV was observed in plants of this species in Japan 25 years ago (2). References: (1) S. Adkins, and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) T. Yamamoto and K.-I. Ohata. Bull. Shikoku Agric. Exp. Stn. 30:39, 1977.

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First Report of Tomato spotted wilt virus on Chrysanthemum in Serbia

Plant Disease ◽

10.1094/pdis-08-12-0778-pdn ◽

2013 ◽

Vol 97 (1) ◽

pp. 150-150 ◽

Cited By ~ 7

Author(s):

I. Stanković ◽

A. Bulajić ◽

A. Vučurović ◽

D. Ristić ◽

K. Milojević ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Disease Incidence ◽

N Gene ◽

Impatiens Necrotic Spot Virus ◽

Maximum Parsimony Tree ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Negative Controls ◽

Wilt Virus

In July 2011, greenhouse-grown chrysanthemum hybrid plants (Chrysanthemum × morifolium) with symptoms resembling those associated with tospoviruses were observed in the Kupusina locality (West Bačka District, Serbia). Disease incidence was estimated at 40%. Symptomatic plants with chlorotic ring spots and line patterns were sampled and tested by double antibody sandwich (DAS)-ELISA using polyclonal antisera (Bioreba AG, Reinach, Switzerland) against the two of the most devastating tospoviruses in the greenhouse floriculture industry: Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (2). Commercial positive and negative controls and extracts from healthy chrysanthemum tissue were included in each ELISA. TSWV was detected serologically in 16 of 20 chrysanthemum samples and all tested samples were negative for INSV. The virus was mechanically transmitted from ELISA-positive chrysanthemum samples to five plants each of both Petunia × hybrida and Nicotiana tabacum ‘Samsun’ using chilled 0.01 M phosphate buffer (pH 7) containing 0.1% sodium sulfite. Inoculated plants produced local necrotic spots and systemic chlorotic/necrotic concentric rings, consistent with symptoms caused by TSWV (1). The presence of TSWV in ELISA-positive chrysanthemum plants and N. tabacum‘Samsun’ was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using primers TSWVCP-f/TSWVCP-r specific to the nucleocapsid protein (N) gene (4). A Serbian isolate of TSWV from tobacco (GenBank Accession No. GQ373173) and RNA extracted from a healthy chrysanthemum plant were used as positive and negative controls, respectively. An amplicon of the correct predicted size (738-bp) was obtained from each of the plants assayed, and that derived from chrysanthemum isolate 529-11 was purified (QIAqick PCR Purification Kit, Qiagen) and sequenced (JQ692106). Sequence analysis of the partial N gene, conducted with MEGA5 software, revealed the highest nucleotide identity of 99.6% (99% amino acid identity) with 12 TSWV isolates deposited in GenBank originating from different hosts from Italy (HQ830186-87, DQ431237-38, DQ398945), Montenegro (GU355939-40, GU339506, GU339508), France (FR693055-56), and the Czech Republic (AJ296599). The consensus maximum parsimony tree obtained on a 705-bp partial N gene sequence of TSWV isolates available in GenBank revealed that Serbian TSWV isolate 529-11 from chrysanthemum was clustered in the European subpopulation 2, while the Serbian isolates from tomato (GU369723) and tobacco (GQ373172-73 and GQ355467) were clustered in the European subpopulation 1 denoted previously (3). The distribution of TSWV in commercial chrysanthemum crops is wide (2). To our knowledge, this is the first report of TSWV infecting chrysanthemum in Serbia. Since chrysanthemum popularity and returns have been rising rapidly, the presence of TSWV may significantly reduce quality of crops in Serbia. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) Daughtrey et al. Plant Dis. 81:1220, 1997. (3) I. Stanković et al. Acta Virol. 55:337, 2011. (4) A. Vučurović et al. Eur. J. Plant Pathol. 133:935, 2012.

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Tomato spotted wilt virus Identified in Desert Rose in Florida

Plant Disease ◽

10.1094/pd-89-0526c ◽

2005 ◽

Vol 89 (5) ◽

pp. 526-526 ◽

Cited By ~ 5

Author(s):

S. Adkins ◽

C. A. Baker

Keyword(s):

Tomato Spotted Wilt Virus ◽

The United States ◽

N Gene ◽

Enzyme Linked Immunosorbent Assay ◽

Insect Vector ◽

Rt Pcr ◽

Tomato Spotted Wilt ◽

Wilt Virus ◽

Symptomatic Plant ◽

Das Elisa

Desert rose (Adenium obesum (Forssk.) Roem. & Schult), a member of the family Apocynaceae, is characterized by fleshy stems and leaves and colorful flowers. This exotic ornamental, originally from southeast Africa, is propagated vegetatively and is a perennial in warm climates. Virus-like foliar symptoms, including chlorotic ring and line patterns, were observed in the fall of 2004 on one of five stock plants being maintained in a greenhouse in Fort Pierce, FL. Inclusion body morphology suggested the presence of a Tospovirus in the symptomatic plant, and Tomato spotted wilt virus (TSWV) was specifically identified in this plant using a commercially available double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA; Agdia, Elkhart, IN). TSWV was not detected in symptomless desert rose plants nor was Impatiens necrotic spot virus detected in any of the plants using DAS-ELISA. Graft transmission of TSWV to other desert rose plants was successful. Sequence analysis of a nucleocapsid (N) protein gene fragment amplified by reverse transcription-polymerase chain reaction (RT-PCR) with primers TSWV723 and TSWV722 (1) from total RNA of the symptomatic plant confirmed the diagnosis. Nucleotide and deduced amino acid sequences of a 579-bp region of the RT-PCR product were 95 to 99% and 95 to 100% identical, respectively, to TSWV N-gene sequences in GenBank. No product was amplified from symptomless plants. Since these 3-year-old plants were grown on-site from seed and only expressed symptoms 2 months following damage to the greenhouse by hurricanes Frances and Jeanne, it is likely that viruliferous thrips were introduced from local vegetable or ornamental production areas during or following the storms. To our knowledge, this is the first report of TSWV infection of desert rose in Florida, although TSWV was observed in this plant in Europe approximately 10 years ago (3,4). Because of the wide distribution of TSWV in the United States, the increasing popularity of desert rose, and the recent identification of Cucumber mosaic virus in this host (2), attention to sanitation and insect vector management is merited during desert rose propagation and production. References: (1) S. Adkins and E. N. Rosskopf. Plant Dis. 86:1310, 2002. (2) C. A. Baker et al. Plant Dis. 87:1007, 2003. (3) J. Mertelik et al. Acta Hortic. 432:368, 1996. (4) J. Th. J. Verhoeven and J. W. Roenhorst. Acta Hortic. 377:175, 1994.

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