scholarly journals Standardization of duplex RT-PCR for simultaneous detection of Peanut bud necrosis virus and Tobacco streak virus naturally infecting peanut (Arachis hypogeae L.)

2017 ◽  
Keyword(s):  
Simultaneous Detection ◽  
Tobacco Streak Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
Necrosis Virus

Prevalence and Molecular Characterization Coat Protein Gene of Tobacco streak virus Causing Peanut Stem Necrosis Disease in Coastal and Rayalaseema Districts of Andhra Pradesh, South-India

2021 ◽  
Author(s):  
K. Saratbabu ◽  
K. Vemana ◽  
A.K. Patibanda ◽  
B. Sreekanth ◽  
V. Srinivasa Rao
Keyword(s):  
Coat Protein ◽  
Molecular Characterization ◽  
Andhra Pradesh ◽  
Disease Incidence ◽  
Tobacco Streak Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Streak Virus ◽  
Rt Pcr ◽  
Cp Gene ◽  
Stem Necrosis

Background: Peanut stem necrosis disease (PSND) caused by Tobacco streak virus (TSV) is a major constraint for groundnut production in Andhra Pradesh (A.P.). However, studies on prevalence and spread of the disease confined to only few districts of A.P. with this background current study focused on incidence and spread of the disease in entire state of A.P. Further an isolate of TSV occurring in A.P. characterized on the basis of genetic features by comparing with other TSV isolates originated from different hosts and locations from world.Methods: Roving survey was conducted during kharif 2017-18 in groundnut growing districts of Andhra Pradesh (A.P.) for peanut stem necrosis disease incidence. Groundnut plants showing PSND symptoms were collected and tested with direct antigen coating enzyme linked immunosorbent assay (DAC-ELISA). Groundnut samples found positive by ELISA once again tested by reverse transcription polymerase chain reaction (RT-PCR). The representative TSV-GN-INDVP groundnut isolate from Prakasham district was maintained on cowpea seedlings by standard sap inoculation method in glasshouse for further molecular characterization. The Phylogenetic tree for coat protein (CP) gene was constructed using aligned sequences with 1000 bootstrap replicates following neighbor-joining phylogeny.Result: Thirty-eight (52.7%) of seventy-two groundnut samples collected from different locations in A.P were given positive reaction to TSV by DAC-ELISA. For the first time, PSND incidence observed in coastal districts (Krishna, Guntur, Sri Pottisriramulu Nellore, Prakasham) of A.P. Maximum PSND incidence recorded from Bathalapalli (22.2%) and the minimum incidence in Mulakalacheruvu (4.1%). The coat protein (CP) gene of TSV-GN-INDVP groundnut isolate was amplified by RT-PCR and it shared maximum per cent nucleotide identity (97.51-98.62%) with TSV isolates from groundnut and other different crops reported in India. All Indian isolates cluster together irrespective of crop and location based on the phylogenetic analysis.

scholarly journals First Report of Blackberry chlorotic ringspot virus in Rubus sp. in the United States

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 463-463 ◽  
Author(s):  
I. E. Tzanetakis ◽  
J. D. Postman ◽  
R. R. Martin
Keyword(s):  
United States ◽  
The United States ◽  
Ringspot Virus ◽  
Tobacco Streak Virus ◽  
Rapid Decline ◽  
Streak Virus ◽  
Black Raspberry ◽  
Rt Pcr ◽  
Indicator Plants ◽  
First Report

Blackberry chlorotic ringspot virus (BCRV), genus Ilarvirus, has been found in Rubus sp. in Scotland (2) and rose in the United States (4). The possibility that BCRV infects other hosts in the United States was explored. We tested 18 accessions of Fragaria sp. and 30 of Rubus sp. maintained at the National Clonal Germplasm Repository in Corvallis, OR. Ilarviruses had been detected in these plants by reverse transcription (RT)-PCR, ELISA, or had caused symptoms typical of ilarviruses on indicator plants. The accessions were tested by RT-PCR with primers F (5′-GTTTCCTGTGCTCCTCA-3′) and R (5′-GTCACACCGAGGTACT-3′) (4) that amplify a 519 to 522 nt (depending on the isolate) region of the RNA 3 of BCRV. The virus was detected in two accessions of black raspberry (Rubus occidentalis L.): RUB433, cv. Lowden and RUB 9012, cv. New Logan. The sequences of the fragments amplified from these accessions (GenBank Accession Nos. EF041817 and EF041818, respectively) had 97% nt sequence identity to each other and 95 and 88% nt identity to the rose and Scottish isolates (GenBank Accession Nos. DQ329378 and DQ091195, respectively). Chenopodium quinoa indicator plants inoculated with isolate RUB 433 developed mild chlorotic spots on the inoculated leaves 4 days after inoculation. RT-PCR and sequencing of the amplicons verified BCRV infection of C. quinoa. RUB 9012 was used for the characterization of Black raspberry latent virus (BRLV), later thought to be an isolate of Tobacco streak virus (TSV). This accession was recently found to be infected with Strawberry necrotic shock virus (SNSV) but not TSV (3). It is possible that BRLV may be a mixture of SNSV and BCRV. SNSV is one of the most abundant viruses of Rubus sp. in the Pacific Northwest (1), and the finding of another ilarvirus, BCRV, may account in part for the rapid decline of Rubus sp. observed in several fields in Oregon and Washington. To our knowledge, this is the first report of BCRV infecting Rubus sp. outside the United Kingdom. References: (1) A. B. Halgren. Ph.D. Diss. Oregon State University, Corvallis, OR, 2006. (2) A. T. Jones et al. Ann. Appl. Biol. 149:125, 2006. (3) I. E. Tzanetakis et al. Arch. Virol. 149:2001, 2004. (4) I. E. Tzanetakis et al. Plant Pathol. 55:568, 2006.

scholarly journals First Report of Strawberry necrotic shock virus in China

Plant Disease ◽  
2011 ◽  
Vol 95 (9) ◽  
pp. 1198-1198 ◽  
Author(s):  
L. Li ◽  
H. Yang
Keyword(s):  
Nucleic Acid ◽  
Home Garden ◽  
Tobacco Streak Virus ◽  
Chinese Isolate ◽  
Streak Virus ◽  
Total Nucleic Acid ◽  
Rt Pcr ◽  
Viral Pathogen ◽  
First Report ◽  
Strawberry Vein Banding Virus

Strawberry necrotic shock virus (SNSV) is an economically important viral pathogen that infects Fragaria and Rubus spp. SNSV was first identified in the 1950s and early studies indicated that SNSV was a strain of Tobacco streak virus (TSV). Recently, it was shown that SNSV was a distinct virus based on molecular characterization (2). Currently, SNSV is a tentative member of the Ilarvirus genus in the Bromoviridae family. In 2008, a small sampling survey for SNSV was done in Heilongjiang Province of China, and 15 strawberry samples were collected from symptomless strawberries in a home garden that had more than 5 years of strawberry cultivation history. Total nucleic acid was extracted from strawberry leaflets by modified cetyltrimethylammoniumbromide methods (3). Reverse transcription (RT)-PCR was operated with the published primer pair CPbeg F/CPend R (2). Amplified DNA fragments with the predicted size were obtained only in one strawberry sample, which was further cloned and sequenced. The sequence (GenBank Accession No. HQ830017) was closely related and highly homologous (89.7 to 98.5% identity) to that of viral isolates (GenBank Accession Nos. AY363228-AY363242) from Fragaria and Rubus spp. Phylogenetic analysis based on nucleotide sequence of the coat protein gene was done with the neighbor-joining method of MEGA 4.0 software. The result showed that all the isolates of SNSV fell into two distinct clades. The Chinese isolate formed one small clade with Japanese isolate 1291. The isolate was also transmitted to Chenopodium quinoa by mechanical inoculation in the greenhouse, and the symptom of chlorotic mottling could be found in C. quinoa and detected by RT-PCR. To determine whether the sample was infected by other strawberry viruses, RT-PCR assays with the published primer pairs SVBVdeta/SVBVdetb, SMoVdeta/SMoVdetb, and SMYEVdeta/SMYEVdetb were also performed for detection of Strawberry vein banding virus, Strawberry mottle virus, and Strawberry mild yellow edge virus using total nucleic acid extracted from the SNSV-positive sample as a template (1). The result indicated that it had been also infected by Strawberry mild yellow edge virus, although no visible symptoms were observed. To our knowledge, this is the first report of SNSV in strawberry in China. Additional work is needed to elucidate the biological characterization and significance of the finding. References: (1) J. R. Thompson et al. J. Virol. Methods 111:85, 2003. (2) I. E. Tzanetakis et al. Arch. Virol. 149:2001, 2004. (3) H. Y. Yang et al. Acta Hortic. 764:127, 2007.

scholarly journals Sensitive and specific qPCR and nested RT-PCR assays for the detection of Tobacco streak virus in soybean

2021 ◽  
Author(s):  
Cristina Zambrana-Echevarria ◽  
Mitchell Roth ◽  
Ranjit Dasgupta ◽  
Thomas German ◽  
Carol Groves ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Management Strategies ◽  
Absolute Quantification ◽  
Qpcr Assay ◽  
Diagnostic Tools ◽  
Tobacco Streak Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Research Fields

Tobacco streak virus (TSV) is a re-emerging and understudied pathogen of soybean (Glycine max). Management of TSV is challenging due to the multiple modes of transmission, widespread susceptibility of commercial soybean, and lack of reliable diagnostic tests for the virus. Soybean plants with TSV-like, virus-like, or no symptoms were collected from commercial and research fields in seven counties in Wisconsin. Two sensitive assays were developed for the detection of TSV: a fluorescent dye-based quantitative RT-PCR (qPCR) assay and a nested RT-PCR (nRT-PCR). Tobacco streak virus was detected in 47% and 91% of symptomatic samples using the qPCR assay and the nRT-PCR assay, respectively, suggesting that the nRT-PCR assay has higher sensitivity for detecting TSV. The qPCR assay’s limit of detection was determined at 10 fg and the assay was used to estimate the viral load in TSV-symptomatic samples. The titer of TSV in these samples was determined by absolute quantification and ranged from 15 fg to 0.796 ng. The two assays reported here provide diagnostic tools for the rapid and accurate detection of TSV that can aid in monitoring outbreaks, assessing management strategies, or screening soybean cultivars/accessions for resistance to the virus.

scholarly journals First Report of Tobacco streak virus on Dahlia in the Czech Republic

Plant Disease ◽  
2008 ◽  
Vol 92 (3) ◽  
pp. 484-484 ◽  
Author(s):  
V. Mokra ◽  
B. Gotzova ◽  
V. Bezdekova ◽  
P. Dedic ◽  
J. Ptacek
Keyword(s):  
Czech Republic ◽  
Mosaic Virus ◽  
Rna Extraction ◽  
Chenopodium Quinoa ◽  
Spherical Particles ◽  
Tobacco Streak Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
The Czech Republic ◽  
Dahlia Mosaic Virus

Dahlia is an important ornamental crop in the Czech Republic where they have been grown for more than 150 years. New dahlia cultivars have been selected by Czech plant breeders. Virus diseases, including mosaic and stunt caused mostly by Dahlia mosaic virus, have been a problem. From 2003 to 2005, color breaking was observed in several dahlia cultivars of foreign and Czech origin. White stripes in blossoms were most frequently expressed in the second half of the flowering season. No symptoms are visible in flowers of white and yellow cultivars. It was difficult to characterize symptoms on leaves because most cultivars were infected simultaneously by Dahlia mosaic virus. Sap inoculations of Chenopodium quinoa produced local lesions after 5 to 7 days, followed by systemic chlorosis, necrosis of younger leaves, and death of the shoot apex, indicating possible Tobacco streak virus (TSV) infection (2). Spherical particles (25 to 30 nm) were observed in leaf-dip preparations of samples from experimentally infected C. quinoa plants and analyzed by using transmission electron microscopy. These particles became decorated when using immunoelectron microscopy with TSV IgG (Bioreba, Reinach, Switzerland and Neogen, Ayrshire, Scotland). Samples of 80 dahlia cultivars were tested for TSV infection by ELISA using commercially available kits (Bioreba and Neogen). Most of the samples were grown in a collection of dahlia cultivars of Czech and foreign origin and some were obtained from growers in the Czech Republic. Fifty six dahlia cultivars were shown to be TSV infected. ELISA also indicated a higher concentration of the virus in flowers. The identity of the virus isolated from symptomatic plants was confirmed by reverse transcription (RT)-PCR using total RNA extraction from symptomatic plants. RT-PCR (4), using a primer pair (1) derived from the coat protein gene sequence of TSV (3), was followed by electrophoresis on 1.0% agarose gels. Products of the predicted size (approximately 700 bp) were found in naturally infected dahlia plants (n = 10), systemically infected host plants C. quinoa (n = 10), and symptomatic Nicotina megalosiphon (n = 10) that scored as TSV positive by ELISA. No bands of this size were seen in negative controls. To our knowledge, this is the first detection of TSV in the Czech Republic. References: (1) A. I. Bhat et al. Arch. Virol. 147:651, 2002. (2) A. A. Brunt Plant Pathol. 17:119, 1968. (3) B. J. C. Cornelissen et al. Nucleic Acids Res.12:2427, 1984. (4) S. S. Pappu et al. J. Virol. Methods 4:9, 1993.

scholarly journals First Report of Columnea latent viroid (CLVd) in Tomato in Mali

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 692-692 ◽  
Author(s):  
O. Batuman ◽  
R. L. Gilbertson
Keyword(s):  
Mosaic Virus ◽  
Tobacco Streak Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
First Report ◽  
Stunted Growth ◽  
Tomato Seedlings ◽  
Leaf Veins ◽  
Dna Fragment ◽  
Columnea Latent Viroid

During surveys of tomato (Solanum lycopersicum) fields in Niono, Mali, conducted in March 2011, unusual disease symptoms, including stunted growth, epinasty, and chlorosis of leaves and necrosis of leaf veins and stems were observed in multiple fields. The incidence of these symptoms was low (~1 to 5%), but they were distinct from those associated with known diseases in the region. A representative leaf sample with these symptoms was applied to filter paper (FTA cards, Whatman), and DNA and RNA extracts were prepared according to manufacturer instructions. RT-PCR tests for Tomato spotted wilt virus, Tobacco streak virus, Tomato necrotic spot virus, Tobacco/tomato mosaic viruses, Cucumber mosaic virus, Alfalfa mosaic virus, torradoviruses, and potyviruses, and PCR tests for begomoviruses, phytoplasmas, and ‘Candidatus Liberibacter’ infection were also negative. However, virus-like symptoms developed in all 16 tomato seedlings (cv. Early Pak 7) 7 to 10 days after mechanical (sap) inoculation with inoculum prepared from the FTA sample. No symptoms developed in mock-inoculated control plants (n = 3). Symptoms induced included stunted growth and severe epinasty of leaves, followed by necrosis of leaf veins, petioles, and stems. These symptoms were similar to those observed in plants in Mali. When RNA extracts prepared from leaves of these symptomatic plants were mechanically inoculated onto 24 tomato seedlings, similar symptoms developed in all plants, suggesting the causal agent might be a viroid. RT-PCR tests with RNA from symptomatic tomato leaves and universal (3) and various specific Pospiviroid primer pairs were negative. However, equivalent RT-PCR tests conducted with the pCLV4/pCLVR4 primer pair specific for Columnea latent viroid (CLVd) (2) generated a DNA fragment of the expected size (~370 bp). The sequence of this DNA fragment (GenBank Accession No. JQ362419) was 99% identical with those of CLVd isolates from the Netherlands (AY373446 and AY372396). In host range studies, the CLVd isolate from Mali induced symptoms in all 48 mechanically-inoculated tomato plants, whereas no symptoms developed (up to 90 days after inoculation) in inoculated Chenopodium quinoa, C. amaranticolor, Nicotiana benthamiana, N. tabacum (cvs. Havana, Glurk and Turkish), N. glutinosa, Datura stramonium, common bean (cvs. Topcrop and Pinto bean), pumpkin (cv. Small Sugar), pepper (Capsicum annuum, cv. Yolo Wonder) and cucumber (cvs. Emparator and Poinsett 76) plants (results of three independent experiments with six plants per experiment). Symptomless infections were detected in pepper (24 of 30), N. benthamiana (25 of 25), and N. tabacum cv. Turkish (11 of 24) plants by RT-PCR with the pCLV4/pCLVR4 primer pair. To our knowledge, this is the first report of CLVd infecting tomato in Mali. RT-PCR tests of seeds collected from CLVd-infected tomato, pepper, and N. benthamiana plants also detected CLVd (1). Thus, it is possible that CLVd was introduced into Mali in association with seed. References: (1) O. Batuman and R. L. Gilbertson. Phytopathology 102:S4.9, 2012. (2) R. L. Spieker. Arch. Virol. 141:1823, 1996. (3) J. T. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004.

scholarly journals Dahlia mosaic virus and Tobacco streak virus in Dahlia (Dahlia variabilis) in New Zealand

Plant Disease ◽  
2008 ◽  
Vol 92 (7) ◽  
pp. 1138-1138 ◽  
Author(s):  
H. R. Pappu ◽  
K. R. W. Hammett ◽  
K. L. Druffel
Keyword(s):  
New Zealand ◽  
Mosaic Virus ◽  
Tobacco Streak Virus ◽  
Impatiens Necrotic Spot Virus ◽  
Streak Virus ◽  
Rt Pcr ◽  
Electron Microscopic ◽  
Dahlia Mosaic Virus ◽  
Dahlia Variabilis ◽  
The Impact

Dahlia (Dahlia variabilis Hort.) is a significant ornamental plant in New Zealand. Symptoms such as mosaic, ring spots, mottling, and veinal chlorosis, suggestive of a viral infection, are often seen in various dahlia collections. To better understand the incidence of viruses in dahlia in New Zealand, several popularly grown cultivars were evaluated for viruses that are known to infect dahlia. Viruses that were tested included Cucumber mosaic virus (CMV), Dahlia mosaic virus (DMV), Impatiens necrotic spot virus (INSV), Tobacco streak virus (TSV), and Tomato spotted wilt virus (TSWV). At least one symptomatic plant was tested from each of the following cultivars: Akito Dawn, Cincinnati Dancer, Hamari Accord, Hamari Rose, LeBatts Prime, LeVonne Splinter, Riverlea Tropicana, Spartacus, Tartan, Tui Connie, and Wandas Antartica. Except for DMV, initial testing was done by ELISA with commercially available kits for the above viruses. In the case of dahlia mosaic, samples were tested for DMV that was described previously (4) and two additional and distinct caulimoviruses (DMV-D10 and DMV-Holland) that were found to be associated with dahlia (1,2). Primer pairs, ORF6st: ATG GAA GAA ATT AAG GCG T and ORF6end: TTG TCT TCA TCC ATA AAG CAG; DenF1: CAG CAA GAA ACA GGA ATT GA and DenR: TTA CAG TCG AAG CTG CTA AA; and Kapht-F: ATG AGT AAT GCT TCA GCA A and Kapht-R: TGA CCA TGG CTT CTA ACT GT were used for the specific detection of DMV-D10, DMV-Holland, and DMV, respectively (1). None of the samples tested were ELISA positive for CMV, INSV, or TSWV. To verify the TSV infection, TSV-specific primers (5′-GTC CAG ACC ATC CAT CCA AC-3′ and 5′-TTG ATT CAC CAG GAA ATC TT-3′), designed based on sequences available in GenBank, were used in reverse transcription (RT)-PCR. For DMV, the diagnostic tests used were electron microscopy and PCR followed by amplicon cloning and sequencing. Electron microscopic observation of leaf-dip preparations showed near isometric virions, approximately 50 to 60 nm in all samples tested. PCR showed that all samples tested were positive for DMV-Holland and DMV-D10. While DMV-Holland is a typical caulimovirus, DMV-D10 was found to exist as an endogenous plant pararetroviral sequence in dahlia (3). One sample each from two cultivars, Spartacus and Tui Connie, were positive for TSV by ELISA, RT-PCR, followed by the sequence analysis of the cloned amplicon. The impact of TSV-infected dahlias as a potential source of inoculum remains to be seen. Our results suggested the prevalence of dahlia mosaic-associated caulimoviruses in several dahlia cultivars and the presence of TSV in New Zealand dahlias. Dahlia mosaic continues to be prevalent in several parts of the world (1), and with the current findings in New Zealand, testing for these viruses should be conducted to ensure virus-free status of the propagating material. References: (1) V. Pahalawatta et al. Plant Dis. 91:1194, 2007. (2) V. Pahalawatta et al. Arch. Virol.153:733, 2008. (3) V. Pahalawatta et al. Virology 376:253, 2008. (4) R. D. Richins and R. J. Shepherd. Virology 124:208, 1983.

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