Investigation of Tomato ringspot virus (ToRSV) by Real-Time TaqMan RT-PCR in Hakkari province, Turkey

Despite the significance of grape production to the fruit industry in Tennessee (TN), no published information has been available on viruses affecting grapevines in the state. Hence, a survey was conducted during the 2016 and 2017 growing seasons to determine the status of nine major viruses of grapevines in TN vineyards by taking advantage of classical serological assays and confirmatory nucleic acid-based diagnostic approaches. A total of 349 samples from 23 grapevine cultivars mostly displaying viral-like symptoms were collected from 23 commercial vineyards. All samples were assayed by DAS-ELISA for arabis mosaic virus (ArMV), grapevine leafroll-associated virus (GLRaV)-1, GLRaV-2, GLRaV-3, GLRaV-4, grapevine fanleaf virus (GFLV), tobacco ringspot virus (TRSV), and tomato ringspot virus (ToRSV). Selected serologically positive samples were also tested by RT-PCR, followed by Sanger sequencing of the generated amplicons. Additionally, 19 grapevines displaying symptoms characteristic of grapevine red blotch virus (GRBV) were also assayed by PCR followed by confirmatory sequencing-based methods. Collectively, these assays verifiably detected GLRaV-1, GLRaV-2, GLRaV-3, ToRSV, and GRBV in TN vineyards. This is the first record of the presence of these viruses in TN vineyards. ArMV, GLRaV-4, GFLV, and TRSV were not detected. The majority of samples tested positive for a single virus, whereas mixed infections with more than one virus were detected in 37% of samples.

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Development of Real-Time RT-PCR Assay for Detection of Prunus necrotic ringspot virus in Fruit Trees

Plant Disease ◽

10.1094/pdis.2003.87.11.1344 ◽

2003 ◽

Vol 87 (11) ◽

pp. 1344-1348 ◽

Cited By ~ 19

Author(s):

S. Marbot ◽

M. Salmon ◽

M. Vendrame ◽

A. Huwaert ◽

J. Kummert ◽

...

Keyword(s):

Real Time ◽

Fruit Trees ◽

Minor Groove ◽

Diagnostic Techniques ◽

Ringspot Virus ◽

Covalent Attachment ◽

Minor Groove Binder ◽

Rt Pcr ◽

Pcr Assay ◽

Prunus Necrotic Ringspot Virus

A real-time fluorescent reverse-transcriptase polymerase chain reaction (RT-PCR) assay using a short fluorogenic 3′ minor groove binder (MGB) DNA hydrolysis probe was developed for the detection of Prunus necrotic ringspot virus (PNRSV) in stone fruit trees. The covalent attachment of the minor groove binder moiety at the 3′ end of the probe increased the probe target duplex stability and raised the melting temperature to a range suitable for real-time analysis. The real-time RT-PCR assay correlated well with conventional RT-PCR results for the detection of PNRSV. This assay reliably detects PNRSV in bark tissues of dormant cherry and plum trees. Furthermore, it is well adapted for the routine detection of PNRSV because it eliminates one risk of contamination by performing the whole test in a single closed tube. This system may replace the commonly used diagnostic techniques (e.g., woody indicators and immunological tests) to detect this virus.

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Incidence of viruses in highbush blueberry (Vaccinium corymbosum L.) in Serbia

Pesticidi i fitomedicina ◽

10.2298/pif1602045j ◽

2016 ◽

Vol 31 (1-2) ◽

pp. 45-50

Author(s):

Darko Jevremovic ◽

Svetlana Paunovic ◽

Aleksandar Leposavic

Keyword(s):

Large Scale ◽

Vaccinium Corymbosum ◽

Ringspot Virus ◽

Mottle Virus ◽

Elisa Assay ◽

Highbush Blueberry ◽

Rt Pcr ◽

Tomato Ringspot Virus ◽

Large Scale Survey ◽

Pcr Test

A large-scale survey for highbush blueberry (Vaccinium corymbosum L.) viruses in Serbia was performed from 2011 to 2015. A total of 81 leaf samples from 15 locations were collected and analyzed for the presence of 8 viruses. Serological ELISA assay was performed to determine the presence of: Blueberry scorch virus (BlScV), Blueberry shock virus (BlShV), Blueberry shoestring virus (BSSV), Blueberry leaf mottle virus (BLMoV), Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV). All samples were tested for the presence of Blueberry red ringspot virus (BRRV) by PCR and for Blueberry mosaic-associated virus (BlMaV) by RT-PCR test. The analyses confirmed the presence of BlMaV in 8 (9.9%) samples and BRRV in 1 (1.2%) sample. No BlScV, BlShV, BLMoV, BSSV, TRSV or ToRSV viruses were detected in any of the analyzed samples.

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Development of TaqMan real-time RT-PCR for sensitive detection of diverse Raspberry ringspot virus isolates

Journal of Virological Methods ◽

10.1016/j.jviromet.2020.113821 ◽

2020 ◽

Vol 278 ◽

pp. 113821

Author(s):

Joe Tang ◽

Filomena Ng ◽

Deepika Kanchiraopally ◽

Lisa Ward

Keyword(s):

Real Time ◽

Ringspot Virus ◽

Sensitive Detection ◽

Rt Pcr ◽

Virus Isolates

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First Report of Prune dwarf virus and Prunus necrotic ringspot virus on Peach in Montenegro

Plant Disease ◽

10.1094/pdis-12-12-1147-pdn ◽

2013 ◽

Vol 97 (9) ◽

pp. 1259-1259 ◽

Cited By ~ 2

Author(s):

J. Zindović ◽

C. Lanzoni ◽

C. Rubies Autonell ◽

C. Ratti

Keyword(s):

Real Time ◽

Ringspot Virus ◽

Dwarf Virus ◽

Rt Pcr ◽

First Report ◽

Cp Gene ◽

Healthy Control ◽

Prunus Necrotic Ringspot Virus ◽

Leaf Distortion ◽

Das Elisa

In September and October 2011, samples were collected from mature peach trees (~17 years old) exhibiting symptoms of chlorotic rings and spots, vein clearing, mosaic, necrosis, leaf distortion, stunting, and rosette formation in a major commercial orchard (~80 ha) near Podgorica, Montenegro. Samples were collected from nine different peach varieties (cvs. Adriana, Caldesi, Gloria, Maria Marta, May Crest, Morsiani, Rita Star, Spring Belle, and Spring Crest). Samples (n = 58) were tested using DAS-ELISA for the presence of Prune dwarf virus (PDV) and Prunus necrotic ringspot virus (PNRSV). Commercial positive and negative controls were included in each ELISA (antisera and controls supplied by BIOREBA AG, Reinach, Switzerland). Only one symptomatic sample from cv. Gloria tested positive for PDV (sample reference: 399/11), a further 11 samples (cvs. Rita Star [six], May Crest [four] and Spring Crest [one]) were positive for PNRSV. Samples were also tested for Plum pox virus (PPV) by real-time RT-PCR (1). The PDV positive sample (399/11) showing mosaic was in mixed infection with PPV, as were 6 of the 11 PNRSV samples, including sample 373/11 with yellow mottling and leaf distortion symptoms. On single-infected PNRSV, sample 368/11 chlorotic line patterns and leaf deformations were observed. To confirm the presence of PDV and PNRSV, positive samples were also tested by RT-PCR. Total RNA was extracted using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). RT-PCR was performed with primer pairs PDV2F/PDV1R (3) and MG1/MG2 (2) specific for PDV and PNRSV, respectively. Amplicons of the expected size, 173 bp for PDV and 675 bp for PNRSV, were obtained from corresponding ELISA-positive samples. Amplified products from three samples (PDV 399/11 and PNRSV 368/11 and 373/11) were cloned into pGEM-T Easy Vector (Promega, Madison, WI) then sent for sequence analysis (MWG-Biotech AG, Edersberg, Germany). Sequence data was compared to sequences published in GenBank. Analysis of sequence obtained from isolate 399/11 (cv. Gloria) corresponded to partial CP gene of PDV, with a high degree of similarity to isolates reported from other parts of the world ranging from 94.2 to 95.9%, showing highest similarity with isolate Ch 137 (L28145). Sequence analyses of CP gene from PNRSV isolates 368/11 (JX569825) and 373/11 (JX569826) proved to be 89.3 to 99.7% identical with corresponding sequences of isolates previously described. In particular, the Montenegrin PNRSV isolates were most closely related to Chilean NctCl.augl isolate from nectarine (EF565253). To demonstrate that the virus was infectious, seedlings of peach cv. GF305 were side grafted with bud-woods from PDV (sample 399/11) and PNRSV-positive samples (samples 368/11 and 373/11) and a healthy control sample. Grafted seedlings were kept in a greenhouse with a under 16-h light regime at 22 to 24°C and observed for symptom development. No symptoms were observed in grafted plants with the healthy control. All plants inoculated with virus-positive samples exhibited stunted vegetation and mild mottle with no difference in symptoms between the two viruses. Indicator plants of peach cv. GF305 inoculated with PPV dual-infected samples (399/11 and 373/11) were subsequently shown to be positive for PPV by real-time RT-PCR. Subsequent DAS-ELISA test on samples from experimentally inoculated trees using specific antisera as described above confirmed PDV and PNRSV infections as expected. These viruses have recently been reported from sour cherry (Prunus cerasus L.) in Serbia (4), ~600 km to the northeast. However, to our knowledge, this is the first report on the occurrence of PDV and PNRSV in Montenegro. References: (1) N. Capote et al. Int. Microbiol. 12:1, 2009. (2) M. Glasa et al. Ann. Appl. Biol. 140:279, 2002. (3) D. R. Parakh et al. Acta Hortic. 386:421, 1996. (4) S. Radičević et al. Genetika 44:285, 2012.

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First Report of Tomato ringspot virus in Butterfly Bush (Buddleia davidii)

Plant Disease ◽

10.1094/pdis.2003.87.1.102b ◽

2003 ◽

Vol 87 (1) ◽

pp. 102-102 ◽

Cited By ~ 5

Author(s):

P. L. Hughes ◽

S. W. Scott

Keyword(s):

United States ◽

Mosaic Virus ◽

Southeastern United States ◽

Positive Control ◽

Ringspot Virus ◽

Home Garden ◽

Line Pattern ◽

Rt Pcr ◽

First Report ◽

Tomato Ringspot Virus

Leaves displaying bright yellow or light green line pattern symptoms were collected from individual, large, mature buddleias in a home garden in Clemson, SC, a botanical garden in Knoxville, TN, and a container-grown plant on sale in a retail home and garden store in Seneca, SC. Buddleias grown in the southeastern United States frequently display virus-like symptoms, but the line pattern symptom displayed by these plants was atypical of the mosaic, mottling, and leaf deformation seen when buddleias are infected with Alfalfa mosaic virus (AMV) or Cucumber mosaic virus (CMV) (2,4). Line pattern symptoms are frequently seen in woody species infected by ilarviruses or nepoviruses (2). No ilarviruses are reported to infect buddleia and only the nepovirus, Strawberry latent ringspot virus, which is restricted mainly to Europe, is reported to infect this species (1,2). The nepoviruses Tomato ringspot virus (ToRSV) and Tobacco ringspot virus (TRSV) are frequently found infecting plants of many species in the southeastern United States (3). Total RNA was extracted from the three symptomatic plants and used in reverse transcription-polymerase chain reactions (RT-PCR) to detect ToRSV and TRSV using primer pairs developed in this laboratory, which amplify regions around the amino terminus of the coat protein of the respective viruses. The expected amplification product for ToRSV of 327 base pairs was obtained from samples tested from each plant, and the nucleotide sequence of the product showed 96% identity with the corresponding fragment of GenBank Accession No. NC_003839 that the primers were designed to amplify. Repeated attempts to isolate a virus from symptomatic leaves using sap inoculation to Chenopodium amaranticolor Coste & Reyne, C. quinoa Willd, Nicotiana clevelandii Gray, and N. tabacum L. have failed. Repeated testing by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) of leaves from the plant growing in Clemson consistently produced absorbance values at 405 nm in the range of 0.47 to 0.55 (mean of 8 separate samples per test) for symptomatic and asymptomatic leaves. The range of values for the positive control (ToRSV-G growing in N. clevelandii) was 1.3 to 1.5. The ranges of values for the noninfected controls (noninfected N. clevelandii and leaf tissue from a buddleia known to be infected with AMV and CMV but in which ToRSV or TRSV had never been detected by RT-PCR) were 0.102 to 0.104 and 0.102 to 0.106, respectively. The extraction buffer produced absorbance readings in the range of 0.098 to 0.102. RT-PCR of RNA extracted from other portions of the leaves used in the ELISA consistently amplified the 327-bp product from symptomatic leaves and from the positive control but not from noninfected control tissues. RNA from asymptomatic leaves on the infected plant also produced the 327-bp product in RT-PCR. Isolation of viruses from woody hosts is frequently difficult, and although, we have yet to succeed to confirm the association between the observed symptom and ToRSV, the evidence from PCR and ELISA would indicate ToRSV is present in these plants. To our knowledge, this is the first report of ToRSV, a member of the genus Nepovirus, in buddleia. References: (1) J. Albouy and J. C. Devergne. Maladies á Virus des Plants Ornementales. INRA Editions, Paris, 1998. (2) J. I. Cooper. Virus Diseases of Trees and Shrubs. 2nd ed. Chapman and Hill, London, 1993. (3) J. R. Edwards and R. G. Christie. Pages 352–353 in: Handbook of Viruses Infecting Legumes. CRC Press, Boca Raton, FL, 1991. (4) C. J. Perkins and R. G. T. Hicks. Plant Pathol. 38:443, 1989.

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First Report of Tomato ringspot virus Infecting Pepper in Iran

Plant Disease ◽

10.1094/pdis-07-12-0664-pdn ◽

2012 ◽

Vol 96 (12) ◽

pp. 1828-1828 ◽

Cited By ~ 2

Author(s):

Y. Sokhansanj ◽

F. Rakhshandehroo ◽

R. Pourrahim

Keyword(s):

Mosaic Virus ◽

Ringspot Virus ◽

Local Lesion ◽

Capsicum Frutescens ◽

Polymerase Gene ◽

Rt Pcr ◽

Viral Polymerase ◽

First Report ◽

Tomato Ringspot Virus ◽

Das Elisa

Chili pepper (Capsicum frutescens) represents an important crop in Iran and is under cultivation in different regions in Northern Iran. In spring 2012, commercially grown tabasco (Capsicum frutescens) peppers in Varamin, Shahriar, and Karaj districts of Tehran province developed an undescribed disease. Symptoms observed were mosaic, leaf malformations, and stunting. Fruit symptoms included chlorosis and distortion. To verify the identity of the disease, six fields were surveyed and 72 symptomatic leaves were collected and screened by double antibody sandwich (DAS)-ELISA using specific antibodies to Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), Pepper mild mottle virus (PMMV), Tomato mosaic virus (ToMV), Tobacco mosaic virus (TMV), and Arabis mosaic virus (ArMV). ToRSV was found in 23% of the samples collected. None of the samples had a positive reaction to other tested viruses. The ToRSV-positive peppers were used for mechanical transmission to Chenopodium quinoa, local lesion host, and after two cycles of single local lesion isolation, they were transferred to Cucumis sativus, Solanum esculentum, and Capsicum fructescens. Inoculations resulted in systemic mosaic and chlorotic local lesion on C. sativus; leaf distortion and mosaic on S. esculentum; and mosaic, mottle, and stunting on C. fructescens. All inoculated plants were positive for ToRSV with DAS-ELISA. To further verify ToRSV infection, reverse transcription (RT)-PCR was conducted. Two primers were designed on the basis of the highly conserved sequences of the putative viral polymerase gene available in the GenBank. RT-PCR of total RNA extract from infected peppers and inoculated plants with the designed primers RdR-R (5′-CGCCTGGTAATTGAGTAGCCC-3′) and RdR-F (5′-GAAGAGCTAGAGCCTCAACCAGG-3′), consistently amplified the 411-bp product, while no amplification products were obtained from noninfected control (healthy plants). The fragment from tabasco pepper was cloned into pTZ57R/T (Ins T/A clone PCR Cloning kit, Fermentas, St. Leon-Rot, Germany) and sequenced in both directions of three clones. The resulting nucleotide sequence (GenBank Accession No. JQ972695) had the highest identity (94%) with the polymerase gene of a ToRSV isolate from blueberry cv. Patriot (Accession No. GQ141528) and had lower identity (91%) with that of a ToRSV isolate from blueberry cv. Bluecrop (Accession No. GQ141525). Tomato ringspot virus (ToRSV) is reported to infect Capsicum spp. in the United States (1,2). Our results confirm the natural infection of pepper plants in Tehran by ToRSV. To our knowledge, this is the first report of ToRSV infection of pepper in Iran. The finding of this disease in Tehran confirms further spread of the virus within northern regions of Iran and prompts the need for research to develop more effective management options to reduce the impact of ToRSV on pepper crops. Beside, primers designed on the basis of putative viral polymerase gene sequences may improve the detection of ToRSV isolates by RT-PCR in Iran. References: (1) S. K. Green and J. S. Kim. Technical Bulletin. No.18, 1991. (2) G. P. Martelli and A. Quacquarelli. Acta Hortic. 127:39, 1983.

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