Field survey of ranunculus mild mosaic virus, tomato spotted wilt virus and cucumber mosaic virus infections in Ranunculus asiaticus L. in Japan by newly developed multiplex RT-PCR

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.

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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 Broad bean wilt virus 2 and Ranunculus mild mosaic virus in Ranunculus asiaticus in Southern Italy

Plant Disease ◽

10.1094/pdis-05-15-0563-pdn ◽

2016 ◽

Vol 100 (1) ◽

pp. 235-235 ◽

Cited By ~ 2

Author(s):

M. Minutolo ◽

R. Sorrentino ◽

V. Masenga ◽

D. Alioto

Keyword(s):

Mosaic Virus ◽

Southern Italy ◽

Broad Bean ◽

Mild Mosaic ◽

First Report ◽

Ranunculus Asiaticus ◽

Mild Mosaic Virus ◽

Wilt Virus ◽

Broad Bean Wilt Virus

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First Report of Ranunculus mild mosaic virus on Ranunculus asiaticus in Yunnan Province, China

Plant Disease ◽

10.1094/pdis-92-11-1585a ◽

2008 ◽

Vol 92 (11) ◽

pp. 1585-1585 ◽

Cited By ~ 1

Author(s):

J. H. Wang ◽

S. Zhao ◽

X. M. Yang

Keyword(s):

Mosaic Virus ◽

Yunnan Province ◽

Disease Incidence ◽

Viral Disease ◽

Mild Mosaic ◽

Rt Pcr ◽

Potential Threat ◽

First Report ◽

Ranunculus Asiaticus ◽

Mild Mosaic Virus

In June 2007, a new viral disease occurred in commercial fields of Ranunculus asiaticus in the Yunnan Province of China. Infected plants exhibited mosaic symptoms and growth abnormalities. Viral disease incidence for this ornamental crop host in the Yunnan Province was estimated to range from 10 to 20%. Electron microscopic examination of negatively stained leaf-dip preparations from symptomatic plants identified long, flexuous linear particles (approximately 800 nm). The samples were tested using indirect antigen-coated plate (ACP)-ELISA. ACP-ELISA results showed that the leaf samples from symptomatic plants reacted positively to the potyvirus group antibody (Agdia Inc., Eklhart, IN). Total nucleic acid extracted from symptomatic plants was tested using reverse transcription (RT)-PCR with primers (S 5′-GGNAAAAYAGYGGNCARCC-3′; M4: 5′-GTTTTCCCAGTCACGAC-3′ [N = A, G, C, or T; Y = C or T; and R = A or G]) designed to amplify the 3′ terminal region of genomic RNA of the genus Potyvirus (1). RT-PCR produced a 1,650-bp amplification product that was cloned and sequenced (GenBank Accession No. EU684747). The sequenced portion showed 90 and 99% identity with the Ranunculus mild mosaic virus (RMMV) isolates (GenBank Accession Nos. DQ152191 and EF445546) from Italy and Israel, respectively (2). To our knowledge, this is the first report of RMMV in China. Infection from this virus may cause losses for cut-flower production of Ranunculus asiaticu and it is also a potential threat for international trade of Ranunculus germplasm. References: (1) J. Chen and J. P. Chen. Chin. J. Virol. 18:371, 2002. (2) M. Turina et al. Phytopathology 96:560, 2006.

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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 in Brugmansia suaveolens in Korea

Plant Disease ◽

10.1094/pdis-02-14-0173-pdn ◽

2014 ◽

Vol 98 (9) ◽

pp. 1283-1283

Author(s):

S.-K. Choi ◽

I.-S. Cho ◽

G.-S. Choi ◽

J.-Y. Yoon

Keyword(s):

Mosaic Virus ◽

Tomato Spotted Wilt Virus ◽

Korean Isolate ◽

Rt Pcr ◽

First Report ◽

Tomato Spotted Wilt ◽

Pcr Product ◽

Brugmansia Suaveolens ◽

Plant Pathol ◽

Wilt Virus

Brugmansia suaveolens, also known as angel's trumpet, is a semi-woody shrub or a small tree. Because flowers of B. suaveolens are remarkably beautiful and sweetly fragrant, B. suaveolens is grown as ornamentals outdoors year-round in the tropics and subtropics, and as potted plants in temperate regions (1). In February 2013, virus-like symptoms including mosaic symptoms followed by distortion of leaves were observed in a potted B. suaveolens in a nursery in Chung-Nam Province, Korea. Symptomatic leaves were analyzed for the presence of several ornamental viruses including Cucumber mosaic virus (CMV), Tobacco mosaic virus (TMV), Tomato bush stunt virus (TBSV), and Tomato spotted wilt virus (TSWV) by immune-strip diagnostic kits that were developed by our laboratory. Positive controls and extract from healthy leaves of B. suaveolens as a negative control were included in each immune-strip assay. TSWV was detected serologically from the naturally infected B. suaveolens, but CMV, TBSV, and TMV were not detected from the B. suaveolens. The presence of TSWV (named TSWV-AT1) was confirmed by commercially available double-antibody sandwich (DAS)-ELISA kits (Agdia, Elkhart, IN). TSWV-AT1 was mechanically transmitted from the ELISA-positive B. suaveolens to Capsicum annuum and Nicotiana glutinosa, respectively. Inoculated C. annuum showed chlorotic rings in the inoculated leaves and inoculated N. glutinosa produced mosaic and systemic necrosis in the inoculated leaves after 7 days inoculation, respectively, which were consistent with symptoms caused by TSWV (2). To confirm further TSWV-AT1 infection, reverse transcription (RT)-PCR was performed using the One-Step RT-PCR (Invitrogen, Carlsbad, CA) with TSWV-specific primers, TSWV-NCP-For and TSWV-NCP-Rev (3), designed to amplify a 777-bp cDNA of the nucleocapsid protein (NCP) gene. Total RNAs from naturally infected B. suaveolens, symptomatic C. annuum, and N. glutinosa were extracted using RNeasy Plant Mini Kit (Qiagen, Valencia, CA). Total RNAs obtained from a Korean isolate of TSWV (Accession No. JF730744) and healthy B. suaveolens were used as positive and negative controls, respectively. The expected size of the RT-PCR product was amplified from symptomatic B. suaveolens, C. annuum, and N. glutinosa but not from healthy leaves of B. suaveolens. The amplified RT-PCR product from TSWV-AT1 was directly sequenced using BigDye Termination kit (Applied Biosystems, Foster City, CA). Multiple alignment of the TSWV-AT1 NCP sequence (AB910533) with NCP sequences of other TSWV isolates using MEGA5 software (4) revealed 99.0% aa identity with an Korean TSWV isolate (AEB33895) originating from tomato. These results provide additional confirmation of TSWV-AT1 infection. It is known that high-value ornamentals may act also as reservoirs for TSWV that can infect other ornamentals and cultivated crops, because TSWV has a very broad host range (2). Elaborate inspections for TSWV and other viruses are necessary for production of healthy B. suaveolens, since the popularity and economic importance of this ornamental plant is increasing. To our knowledge, this is the first report of TSWV in B. suaveolens in Korea. References: (1) Anonymous. OEPP/EPPO Bull. 34:271, 2004. (2) G. Parrella et al. J. Plant Pathol. 85:227, 2003. (3) B.-N. Chung et al. Plant Pathol. J. 28:87, 2012. (4) K. Tamura et al. Mol. Biol. Evol. 28:2731, 2011.

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Caracterização do Tomato chlorotic spot virus isolado de jiló no Vale do Paraíba, Estado de São Paulo

Fitopatologia Brasileira ◽

10.1590/s0100-41582002000300008 ◽

2002 ◽

Vol 27 (3) ◽

pp. 285-291 ◽

Cited By ~ 9

Author(s):

MARCELO EIRAS ◽

ALEXANDRE L. R. CHAVES ◽

ADDOLORATA COLARICCIO ◽

RICARDO HARAKAVA ◽

JANSEN DE ARAUJO ◽

...

Keyword(s):

Tomato Spotted Wilt Virus ◽

Sao Paulo ◽

Ringspot Virus ◽

São Paulo ◽

Rt Pcr ◽

Spot Virus ◽

Chlorotic Spot ◽

Tomato Spotted Wilt ◽

Wilt Virus ◽

Das Elisa

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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