First Report of Tomato spotted wilt virus on Chrysanthemum in Serbia

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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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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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 Tomato spotted wilt virus Infecting Onion and Garlic in Serbia

Plant Disease ◽

10.1094/pdis-02-12-0157-pdn ◽

2012 ◽

Vol 96 (6) ◽

pp. 918-918 ◽

Cited By ~ 5

Author(s):

I. Stanković ◽

A. Bulajić ◽

A. Vučurović ◽

D. Ristić ◽

K. Milojević ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Disease Incidence ◽

Sandwich Elisa ◽

Thrips Tabaci ◽

N Gene ◽

Pcr Analysis ◽

Rt Pcr ◽

Tomato Spotted Wilt ◽

Negative Controls ◽

Wilt Virus

In June 2011, extensive bleaching and numerous small whitish spots on leaves were observed in an onion (Allium cepa) seed crop as well as chlorotic spots and streaks in the neighboring garlic (A. sativum) bulb crop in the Aleksandrovo locality (Central Banat District, Serbia). Affected plants occurred throughout the field and disease incidence was estimated at 60% in the onion and 40% in the garlic crop. A high population of Thrips tabaci that was found in both crops, and local necrotic spots on Petunia × hybrida mechanically inoculated with infected onion or garlic sap by a chilled 0.01 M phosphate buffer, pH 7.0, containing 0.1% sodium sulfite (1), suggested the presence of a Tospovirus. For these reasons, sampled symptomatic onion and garlic plants were tested for the presence of Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) using commercial double-antibody sandwich-ELISA diagnostic kits (Bioreba AG, Reinach, Switzerland). Commercial positive and negative controls and extracts from healthy onion and garlic tissue were included in each ELISA. Of the 18 onion and 10 garlic plants tested, 16 and 7 samples, respectively, were positive for TSWV, and all were negative for IYSV. The identity of TSWV was further confirmed by conventional reverse transcription (RT)-PCR analysis. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was performed with the One-Step RT-PCR Kit (Qiagen) using TSWV-specific forward (5′-GGTTAAGCTCACTAAGAAARCA-3′) and reverse primers (5′-TTTAACYCCRAACATTTCATAGA-3′), designed to amplify a 738-bp fragment of the nucleocapsid protein (N) gene. Total RNAs obtained from plants infected with a Serbian isolate of TSWV (GenBank Accession No. GQ373173) and healthy onion garlic plants were used as positive and negative controls, respectively. An amplicon of the expected size was produced from the 16 onion and 7 garlic ELISA-positive plants, but not from healthy controls. The amplified products derived from the two selected isolates, 114-11 from onion and 115-11 from garlic, were sequenced directly after purification with the QIAquick PCR Purification kit (Qiagen); the sequences obtained were allocated GenBank Accession Nos. JQ619234 and JQ619235, respectively. Sequence analysis of the partial N gene, conducted with MEGA5 software (4), revealed 99.9% nucleotide identity (100% amino acid identity) between the two Serbian Allium isolates. Serbian onion and garlic isolates showed the highest nucleotide identities of 100% and 99.9% with Serbian summer squash isolate (JF303081) and tobacco isolate from Montenegro (GU369729), respectively. Well-established in many European countries, TSWV has been reported as an important constraint to the production of tomato, pepper, tobacco, and ornamentals (2), but the information on TSWV naturally infecting Allium spp. is limited. The presence of TSWV on onion and garlic in Serbia revealed that its known host range has expanded in Europe. To our knowledge, other than Marchoux's unpublished data (3), there are no other reports of garlic as a natural host of TSWV. The TSWV presence on Allium spp. represents a serious threat for these crops in Serbia, considering that it is prevalent in other crops in the area and its vectors are widespread. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) H. R. Pappu et al. Virus Res. 141:219, 2009. (3) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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First Report of Tomato spotted wilt virus on Gerbera hybrida in Serbia

Plant Disease ◽

10.1094/pdis-10-10-0704 ◽

2011 ◽

Vol 95 (2) ◽

pp. 226-226 ◽

Cited By ~ 9

Author(s):

I. Stanković ◽

A. Bulajić ◽

A. Vučurović ◽

D. Ristić ◽

J. Jović ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Rdrp Gene ◽

Impatiens Necrotic Spot Virus ◽

Globe Artichoke ◽

Rt Pcr ◽

Gerbera Hybrida ◽

First Report ◽

Tomato Spotted Wilt ◽

Negative Controls ◽

Wilt Virus

In May 2009, approximately 30% of plants within a greenhouse-grown Gerbera hybrida crop in Vranjska Banja (Pčinj District) in Serbia displayed chlorotic oak-leaf patterns followed by necrosis and distortion of leaves. Symptoms on naturally infected gerbera plants and local necrotic spots on Petunia × hybrida mechanically inoculated with infected gerbera sap using chilled 0.05 M phosphate buffer (pH 7) containing 1 mM Na-EDTA, 5 mM Na-DIECA, and 5 mM Na-thioglycolate (4) suggested the presence of a Tospovirus. Symptomatic leaves were tested for the presence of Tomato spotted wilt virus (TSWV), Impatiens necrotic spot virus (INSV), and Chrysanthemum stem necrosis virus (CSNV) by commercial double-antibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Sauerlach, Germany). Commercial positive and negative controls and extract from healthy gerbera tissue were included in each ELISA. All 20 tested plants were negative for INSV and CSNV. TSWV was detected serologically in 18 of 20 gerbera samples. The presence of TSWV in ELISA-positive symptomatic gerbera plants was further confirmed by conventional reverse transcription (RT)-PCR. Total RNAs were extracted with an RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) and RT-PCR was conducted with the OneStep RT-PCR Kit (Qiagen) using Serbian tobacco TSWV isolate (GQ279731) and RNA extract from healthy gerbera as positive and negative controls, respectively. Two different sets of TSWV-specific primers, L1 TSWVR/L2 TSWVF (2) and M962/M66 (3), for a 276-bp fragment of the RNA-dependent RNA polymerase (RdRp) gene and a 897-bp fragment of the NSm gene, respectively, were used for both amplification and sequencing. RT-PCR analyses of each tested plant detected the presence of amplification fragments of expected size. The amplified products corresponding to part of the RdRp and NSm genes derived from the isolate 158-Gerb were purified (QIAquick PCR Purification Kit, Qiagen) and sequenced in both directions (GenBank Accession Nos. HQ246452 and HQ246453, respectively). Sequence analysis of the partial RdRp gene, conducted using MEGA4 software, revealed 91.1 to 98% nt identity (95.1 to 98.8% amino acid [aa] identities) with corresponding sequences of TSWV L RNA deposited in GenBank. The highest identity was found with an isolate from globe artichoke (AM940436) in Greece, and isolates from tomato (GQ279732), impatiens (GQ132190), and tobacco isolates (GQ279731, FJ189392, and FJ189393) found within Serbia. Analysis of the NSm sequence of isolate 158-Gerb demonstrated nucleotide identities varying between 90.6 and 99.6% (80.9 and 99.6% aa identities) with those of previously reported TSWV isolates. The highest identity was with tobacco isolate GQ373174 from Serbia. Therefore, while gerbera is one of the principal ornamental hosts of TSWV in the EPPO region (1), to our knowledge, this is the first report infecting gerbera in Serbia, which may have a devastating influence on its production. References: (1) Anonymous. OEPP/EPPO Bull. 29:465, 1999. (2) R. A. Mumford et al. J. Virol. Methods 46:303, 1994. (3) W. P. Qiu et al. Virology 244:186, 1998. (4) P. Roggero et al. Plant Dis. 86:950, 2002.

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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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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 Tomato spotted wilt virus on Coprosma repens (Mirror Bush) in Italy

Plant Disease ◽

10.1094/pdis-91-10-1362c ◽

2007 ◽

Vol 91 (10) ◽

pp. 1362-1362 ◽

Cited By ~ 1

Author(s):

G. Polizzi ◽

M. G. Bellardi

Keyword(s):

Tomato Spotted Wilt Virus ◽

Protein A ◽

Culture Collection ◽

N Gene ◽

Impatiens Necrotic Spot Virus ◽

Rt Pcr ◽

Gardenia Jasminoides ◽

Tomato Spotted Wilt ◽

Thermal Cycler ◽

Wilt Virus

Coprosma repens A. Rich. (mirror bush, Rubiaceae) is a hardy salt tolerant shrub that is native to New Zealand where it is primarily a coastal weed. In temperate climates, many variegated varieties and hybrids of mirror bush grow extensively in gardens. In February 2007, irregular or semicircular necrotic spots, sometimes in concentric rings, were noticed on leaves of approximately 2,000 potted, 1-year-old plants of C. repens ‘Tapuata Gold’ obtained as cuttings from a nursery located in Catania Province. The symptoms were detected on approximately 60% of the plants and were localized exclusively on older leaves especially in the yellow or white border. Protein A sandwich (PAS)-ELISA showed mirror bush was positive for the Batavian lettuce strain of Tomato spotted wilt virus (antiserum to TSWV: PVAS-450 from American Type Culture Collection, Manassas, VA). Double antibody sandwich (DAS)-ELISA with polyclonal antisera to Cucumber mosaic virus, TSWV, and Impatiens necrotic spot virus confirmed the presence of only TSWV. Reverse transcription (RT)-PCR was employed to characterize the TSWV isolate. RT-PCR was carried out with primers (forward 5′-TTA ACT TAC AGC TGC TTT-3′ and reverse 5′-CAA AGC ATA TAA GAA CTT-3′) specific for the CP gene of TSWV (3). Amplification was performed in a thermal cycler (Gene Amp PCR System 24000; Perkin Elmer, Hayward, CA) by preheating at 94°C for 5 min followed by 30 cycles of 1.5 min of denaturation at 94°C, 2 min of annealing at 48°C, and 1 min for extension at 72°C. Finally, the amplified DNA was incubated at 72°C for 7 min for a final extension. All samples yielded DNA fragments of the expected size of 823 bp, which included the entire N gene. Purified PCR products were cloned and sequencing (GenBank Accession No. EU020104) was done by Sequiserve (Vatterstetten, Germany). Comparison with sequences available from the GenBank database showed 96 to 99% homology with the same region of the genome for all TSWV isolates, thus confirming the identity of the virus as an isolate of TSWV. In the Rubiaceae family, TSWV was previously detected on Galium spp., Ixora spp., Gardenia jasminoides Ellis, and Bouvardia sp. (1,2,4). To our knowledge, this is the first occurrence of this virus on a member of the genus Coprosma. The high incidence of the disease in the nursery could be due to propagation of cuttings from an infected source. References: (1) M. K. Hausbeck et al. Plant Dis. 76:795, 1992. (2) C. Jordá et al. Plant Dis. 79:358, 1995. (3) R. A. Mumford et al. J. Virol. Methods 46:303, 1994. (4) A. M. Vaira et al. Plant Pathol. 42:530,1993.

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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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First Report of Tomato Spotted Wilt Virus Identified in Tulbaghia violacea in Florida

Plant Health Progress ◽

10.1094/php-03-19-0020-br ◽

2019 ◽

Vol 20 (2) ◽

pp. 131-131

Author(s):

K. K. Dey ◽

C. Li ◽

M. C. Sellers ◽

John McVay ◽

L. Whilby ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Frankliniella Occidentalis ◽

Leaf Tissue ◽

Nucleotide Identity ◽

N Gene ◽

First Report ◽

Tomato Spotted Wilt ◽

Florida Department ◽

Consumer Services ◽

Wilt Virus

Tulbaghia violacea Harv., also known as society garlic, belongs to the genus Tulbaghia (Amaryllidaceae). It is diversely used for medicinal, culinary, and ornamental purposes. In June 2018, T. violacea plants from Lake County, FL, with typical tospovirus-like symptoms of chlorotic ring patterns, were submitted to the Florida Department of Agriculture and Consumer Services, Division of Plant Industry (Gainesville, FL) for identification. Representative leaf tissue from four plants tested positive in a serological assay and reverse transcription-polymerase chain reaction. Amplicons of the expected size were produced using the tomato spotted wilt virus (TSWV) nucleocapsid (N) gene primers. They were directly sequenced and deposited in GenBank (MK567883). The 777-bp N gene sequence had 98 to 99% nucleotide identity with the corresponding region of previously described Florida TSWV isolates and 96 to 99% nucleotide identity with all TSWV isolates in GenBank. Epidemics of TSWV occur frequently in numerous crops in Florida, chiefly tomato, pepper, tobacco, and ornamentals. The western flower thrip, Frankliniella occidentalis (Pergande), is the most efficient vector of TSWV and has become established in Florida. This is the first report of TSWV infection in T. violacea.

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