Real-time RT-PCR fluorescent detection of tomato spotted wilt virus

Os tospovírus são responsáveis por perdas significativas em diversas culturas, principalmente solanáceas. No município de São José dos Campos (SP), plantas de jiló (Solanum gilo) apresentando sintomas de mosaico, bolhosidades, nanismo e queda acentuada da produção foram coletadas para análise. Visando a caracterização do agente causador dos sintomas, testes biológicos, elétrono microscópicos, sorológicos e moleculares foram realizados. Através de inoculação mecânica em plantas indicadoras das famílias Amaranthaceae, Chenopodiaceae e Solanaceae obtiveram-se resultados típicos aos esperados para tospovírus. Ao microscópio eletrônico de transmissão, observaram-se, em contrastação negativa, partículas pleomórficas com diâmetro entre 80 e 110 nm e em cortes ultra-finos partículas presentes em vesículas do retículo endoplasmático. Através de DAS-ELISA, identificou-se o Tomato chlorotic spot virus (TCSV). A partir de RNA total extraído de folhas infetadas, amplificaram-se, via RT-PCR, fragmentos correspondentes ao gene da proteína do capsídeo (cp) os quais foram seqüenciados e comparados com outros depositados no "GenBank". A homologia de nucleotídeos e aminoácidos deduzidos foi respectivamente de 99 e 95% quando comparada com seqüências de isolados de TCSV. A comparação com as outras espécies do gênero Tospovirus apresentou valores de homologia entre 72 e 84%. Estes resultados confirmam a identidade deste vírus como pertencente à espécie TCSV, que é predominante no Estado de São Paulo e importante patógeno de outras plantas cultivadas. Além disso, variedades de jiló quando inoculadas foram susceptíveis tanto ao TCSV como às espécies Tomato spotted wilt virus (TSWV) e Groundnut ringspot virus (GRSV).

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Comparison of ELISA and RT-PCR assays for the detection of Tomato spotted wilt virus in peanut

Peanut Science ◽

10.3146/ps08-024.1 ◽

2009 ◽

Vol 36 (2) ◽

pp. 133-137 ◽

Cited By ~ 1

Author(s):

P. M. Dang ◽

D. L. Rowland ◽

W. H. Faircloth

Keyword(s):

Tomato Spotted Wilt Virus ◽

Enzyme Linked Immunosorbent Assay ◽

Infection Rates ◽

Rt Pcr ◽

Chi Square ◽

Tomato Spotted Wilt ◽

Significant Difference ◽

Pcr Assays ◽

Chi Square Analysis ◽

Wilt Virus

Abstract Diagnosis of Tomato spotted wilt virus (TSWV) in peanut can be accomplished by enzyme-linked immunosorbent assay (ELISA) or reverse transcription polymerase chain reaction (RT-PCR) but there has been no report of a direct comparison of the success of the two assays in evaluating infection rates of field-grown peanut. We collected peanut root samples from field-grown plants, 76 in 2006 and 48 in 2007, and tested these samples by both ELISA and RT-PCR assays for the presence of TSWV. Out of 124 samples, 50 (40.3%) and 57 (46.0%) were positive for TSWV by ELISA and RT-PCR respectively. In 13.7% of these samples, ELISA and RT-PCR differed in their results. However, Chi square analysis showed no significant difference between the results for these two assays. This result supports the conclusion that ELISA and RT-PCR are comparable for detecting TSWV infection rates in field-grown peanuts.

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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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Application of Multiplex RT-PCR for Simultaneous Identification of Tomato Spotted Wilt Virus and Thrips Species in an Individual Thrips on Chrysanthemum

Research in Plant Disease ◽

10.5423/rpd.2020.26.4.264 ◽

2020 ◽

Vol 26 (4) ◽

pp. 264-271

Author(s):

Ju-Yeon Yoon ◽

Jung-Beom Yoon ◽

Mi-Hye Seo ◽

Seung-Kook Choi ◽

In-Sook Cho ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Rt Pcr ◽

Tomato Spotted Wilt ◽

Thrips Species ◽

Wilt Virus ◽

Simultaneous Identification

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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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The First Report of Tomato spotted wilt virus on Gerbera and Chrysanthemun in Venezuela

Plant Disease ◽

10.1094/pdis-01-14-0007-pdn ◽

2014 ◽

Vol 98 (8) ◽

pp. 1161-1161 ◽

Cited By ~ 8

Author(s):

E. Marys ◽

A. Mejías ◽

E. Rodríguez-Román ◽

D. Avilán ◽

T. Hurtado ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Disease Incidence ◽

The United States ◽

Cut Flowers ◽

Rt Pcr ◽

Tomato Spotted Wilt ◽

Arachis Hypogaea L ◽

Pcr Products ◽

Ornamental Crops ◽

Wilt Virus

Gerbera (Gerbera jamesonii) and Chrysanthemum (family Asteraceae) are two of the top 10 cut flowers of the world, with great commercial value. Since 1998, Venezuela began a floral industry to produce and export fresh cut gerbera and chrysanthemum, with 40% of nurseries concentrated in Altos Mirandinos (Miranda State, north central region of the country). For the past 2 years, greenhouse-grown gerbera and chrysanthemum have been observed displaying symptoms resembling those associated with tospoviruses. Symptomatic plants showed concentric rings, irregular chlorotic blotches, and deformation on leaves. Disease incidence was estimated at 30%. Mechanical inoculation with extracts of symptomatic leaves reproduced the typical concentric ring symptoms on indicator plants Arachis hypogaea L. cv. San Martín, Capsicum chinense, and G. jamesonii 6 to 15 days after inoculation. In initial tests, leaves from each 30 symptomatic gerbera and chrysanthemum species from several greenhouse facilities in Altos Mirandinos reacted positively when tested by DAS-ELISA with polyclonal antisera (ATCC, Rockville, MD) raised against Tomato spotted wilt virus (TSWV). Total RNA was extracted with the RNeasy Plant Mini kit (QIAGEN, Hilden, Germany) from two gerbera and two chrysanthemum ELISA-positive samples. The TSWV coat protein gene was amplified by conventional reverse transcription (RT)-PCR using primers CP1 TSWV (TTAACTTACAGCTGCTTT) and CP2 TSWV (CAAAGCATATAAGAACTT) (1). A single DNA product of ~823 bp was amplified from all samples. RT-PCR products were directly sequenced in both orientations and sequences were deposited in GenBank (Accession Nos. KF146700 and KF146701 derived from chrysanthemum, KF146702 and KF146703 derived from gerbera). The resulting sequences showed over 99% identity with each other. and were found to be closely related (over 99%) with TSWV isolates deposited in GenBank originating from different hosts from France (FR693058, FR693055), Montenegro (GU339506, GU339508, GU355940), Italy (HQ830187), New Zealand (KC494501), South Korea (KC261967), and the United States (AY744476). To our knowledge, this is the first confirmed report of TSWV infecting gerbera and chrysanthemum in Venezuela. The relatively widespread occurrence of TSWV in Miranda State underscores the need for systematic surveys to assess its incidence and impact on ornamental crops so that appropriate management tactics can be developed. Reference: (1) R. A. Mumford et al. J. Virol. Methods 57:109, 1996.

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