Real-time RT-PCR for detection of Raspberry bushy dwarf virus, Raspberry leaf mottle virus and characterizing synergistic interactions in mixed infections

2012 ◽  
Vol 179 (1) ◽  
pp. 38-44 ◽  
Author(s):  
Diego F. Quito-Avila ◽  
Robert R. Martin
Keyword(s):  
Real Time ◽  
Dwarf Virus ◽  
Mottle Virus ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Synergistic Interactions ◽  
Raspberry Bushy Dwarf Virus

scholarly journals Effect of Raspberry bushy dwarf virus, Raspberry leaf mottle virus, and Raspberry latent virus on Plant Growth and Fruit Crumbliness in ‘Meeker’ Red Raspberry

Plant Disease ◽  
2014 ◽  
Vol 98 (2) ◽  
pp. 176-183 ◽  
Author(s):  
Diego F. Quito-Avila ◽  
Danielle Lightle ◽  
Robert R. Martin
Keyword(s):  
British Columbia ◽  
The United States ◽  
Fruit Weight ◽  
Rubus Idaeus ◽  
Latent Virus ◽  
Dwarf Virus ◽  
Mottle Virus ◽  
Mixed Infections ◽  
Red Raspberry ◽  
Raspberry Bushy Dwarf Virus

Raspberry crumbly fruit in red raspberry (Rubus idaeus), widespread in the Pacific Northwest of the United States and British Columbia, Canada, is most commonly caused by a virus infection. Raspberry bushy dwarf virus (RBDV) has long been attributed as the causal agent of the disease. Recently, the identification of two additional viruses, Raspberry leaf mottle virus (RLMV) and Raspberry latent virus (RpLV), in northern Washington and British Columbia, suggested the existence of a possible new virus complex responsible for the increased severity of the disease. Virus testing of crumbly fruited plants from five fields in northern Washington revealed the presence of RLMV and RpLV, in addition to RBDV. Plants with less severe crumbly fruit symptoms had a much lower incidence of RLMV or RpLV. Field trials using replicated plots of ‘Meeker’ plants containing single and mixed infections of RBDV, RLMV, or RpLV, along with a virus-free control, were developed to determine the role of RLMV and RpLV in crumbly fruit. Field evaluations during establishment and two fruiting years revealed that plants infected with the three viruses or the combinations RBDV+RLMV and RBDV+RpLV had the greatest reduction in cane growth, or fruit firmness and fruit weight, respectively. Quantitative reverse transcription–polymerase chain reaction (RT-PCR) showed that the titer of RBDV was increased ~400-fold when it occurred in mixed infections with RLMV compared to RBDV in single infections. In addition, a virus survey revealed that RLMV and RpLV are present at high incidence in northern Washington; whereas the incidence in southern Washington and Oregon, where crumbly fruit is not as serious a problem, was considerably lower.

scholarly journals Barley yellow dwarf virus and Cereal yellow dwarf virus Quantification by Real-Time Polymerase Chain Reaction in Resistant and Susceptible Plants

2003 ◽  
Vol 93 (11) ◽  
pp. 1386-1392 ◽  
Author(s):  
Boovaraghan Balaji ◽  
Dennis B. Bucholtz ◽  
Joseph M. Anderson
Keyword(s):  
Real Time ◽  
Barley Yellow Dwarf Virus ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Wheat Line ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Barley Yellow Dwarf ◽  
Polymerase Chain ◽  
Yellow Dwarf Virus

Reliable detection and quantification of barley and cereal yellow dwarf viruses (YDVs) is a critical component in managing yellow dwarf diseases in small grain cereal crops. The method currently used is enzyme-linked immunosorbent assay (ELISA), using antisera against the coat proteins that are specific for each of the various YDVs. Recently, quantitative real-time reverse-transcription polymerase chain reaction (Q-RT-PCR) has been used to detect bacterial and viral pathogens and to study gene expression. We applied this technique to detect and quantify YDVs using primers specific for Barley yellow dwarf virus-PAV (BYDV-PAV) and Cereal yellow dwarf virus-RPV (CYDV-RPV) coat protein genes because of the higher sensitivity of RT-PCR and the advantage of using a real-time PCR instrument. This Q-RT-PCR was used to detect BYDV and CYDV, and to examine disease development in a resistant wheatgrass, a resistant wheat line, a susceptible wheat line, and a susceptible oat line. BYDV-PAV and CYDV-RPV were detected as early as 2 and 6 h, respectively, in susceptible oat compared with detection by ELISA at 4 and 10 days postinoculation. BYDV-PAV RNA accumulated more rapidly and to a higher level than CYDV-RPV RNA in both oat and wheat, which may account for PAV being more prevalent and causing more severe viral disease than CYDV. Q-RT-PCR is reproducible, sensitive, and has the potential to be used for examining yellow dwarf disease and as a rapid diagnostic tool for YDVs.

scholarly journals Occurrence of Lily mottle virus on Lilium in Italy

Plant Disease ◽  
2012 ◽  
Vol 96 (5) ◽  
pp. 771-771 ◽  
Author(s):  
D. Rizzo ◽  
L. Stefani ◽  
M. Paoli ◽  
S. Lazzereschi ◽  
B. Nesi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Leaf Tissue ◽  
Flowering Plant ◽  
Mottle Virus ◽  
Rt Pcr ◽  
Cdna Synthesis ◽  
Vein Clearing ◽  
Cp Gene ◽  
Necrotic Spots

Lily mottle virus (LMoV), a member of the genus Potyvirus, is one of the main viruses infecting lily. Symptoms on lily differ according to the susceptibility and sensitivity of different cultivars and hybrids. They range from leaf mottle or mosaic, vein clearing, chlorotic and yellow streaking, leaf curling, and necrotic spots, to milder forms of leaf symptoms. Plants may even be symptomless at some stages of growth. A varietal collection of Lilium from the early 1990s is held in Pistoia Province (Tuscany, Italy) and is composed of Asian hybrids obtained from intraspecific breeding of commercial cultivars. During a survey conducted from May to June 2010, several plants showing vein clearing, leaf mottle, leaf mosaic, and reddish brownish necrotic spots were observed. Leaf samples from 60 symptomatic or symptomless lily plants, belonging to 20 cultivars, were collected and tested for the presence of LMoV. Samples were assayed by double-antibody sandwich (DAS)-ELISA and eight of them, belonging to four different cultivars, tested positive. Total RNA was extracted from 2 g of leaf tissue of every collected sample according to the protocol described earlier (3) and cDNA synthesis was performed with an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Samples were tested by reverse transcription (RT)-PCR and real-time PCR assays using primers LMoV1 (5′-GCAAATGAGACACTCAATGCTG-3′) and LMoV2 (5′-CGTGCGTGAAGTAACTTCATAG-3′) designed to amplify 651 bp of the coat protein (CP) gene of LMoV (1). Results obtained with RT-PCR and real-time PCR exactly matched those achieved with ELISA assay, and the eight positive samples showed amplicons of the expected size. PCR products from five infected samples were directly sequenced from both directions and submitted in GenBank (Accessions Nos. JQ655106 to JQ655110). Our isolates share more than 99% nucleotide identity among each other. Comparison with other LMoV-CP gene sequences present in GenBank showed nucleotide identities ranging from 93 to 94% with LMoV isolates from South Korea (GenBank Accession Nos. GQ150683 to GQ150686), China (GenBank Accession Nos. EU348826, AJ748256, AJ564636, and AJ564637), Australia (GenBank Accession No. JN127341), and Japan (GenBank Accession No. AB570195). To our knowledge, this is the first report of LMoV on Lilium in Italy where this virus was already reported to infect escarole (2). Considering the economic importance of Lilium production as a flowering plant in Pistoia Province, and in several other areas of Italy, the report of LMoV present on lilies suggests the use of healthy propagation material and the adoption of preventive measures to avoid its diffusion. References: (1) J.-H. Lim et al. Korean J. Microbiol. 45:251, 2009. (2) V. Lisa et al. Plant Dis. 86:329, 2002. (3) D. J. MacKenzie et al. Plant Dis. 81:222, 1997.

scholarly journals IDENTIFICATION AND EFFECTS OF MIXED INFECTION OF Potyvirus ISOLATES WITH Cucumber mosaic virus IN CUCURBITS

2016 ◽  
Vol 29 (4) ◽  
pp. 1028-1035 ◽  
Author(s):  
GRAZIELA DA SILVA BARBOSA ◽  
JOSÉ ALBERSIO DE ARAÚJO LIMA ◽  
MANOEL ABILIO DE QUEIRÓZ ◽  
RITA DE CÁSSIA SOUZA DIAS ◽  
CRISTIANO SOUZA LIMA
Keyword(s):  
Host Range ◽  
Cucumber Mosaic Virus ◽  
Mosaic Virus ◽  
Cucurbita Pepo ◽  
Protein Gene ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Synergistic Interactions ◽  
Virus Interaction ◽  
Biological Differences

ABSTRACT Mixed infections in cucurbits are frequently observed in natural conditions between viruses from the Potyvirus genus and Cucumber mosaic virus (CMV), which significantly decreases productivity. The objectives of the present study was to compare the host range of PRSV-W, WMV, and ZYMV isolates and evaluate the effects of mixed infections with CMV in zucchini plants (Cucurbita pepo L.). Host range studies comprising 23 plant species confirmed some similarities and biological differences among the isolates of PRSV-W, ZYMV, and WMV. RT-PCR confirmed the amplification of DNA fragments of the PRSV-W, WMV, and ZYMV coat protein gene (cp) and cytoplasm inclusion gene (ci). The virus interaction studies in zucchini Caserta plants indicated synergistic interactions, particularly among species from the Potyvirus genus, and some CMV interference with some virus combinations.

Development of SYBR Green I-Based One-Step Real Time RT-PCR Assay for Quantifying Southern rice black-streaked dwarf virus in Rice

2013 ◽  
Vol 162 (1) ◽  
pp. 26-32 ◽  
Author(s):  
Tong Zhou ◽  
Linlin Du ◽  
Ying Lan ◽  
Feng Sun ◽  
Yongjian Fan ◽  
...  
Keyword(s):  
Real Time ◽  
Sybr Green ◽  
Sybr Green I ◽  
Dwarf Virus ◽  
Rt Pcr ◽  
Pcr Assay ◽  
One Step

scholarly journals First Report of Raspberry bushy dwarf virus on Red Raspberry and Grapevine in Slovenia

Plant Disease ◽  
2003 ◽  
Vol 87 (9) ◽  
pp. 1148-1148 ◽  
Author(s):  
I. Mavrič ◽  
M. Viršček Marn ◽  
D. Koron ◽  
I. Žežlina
Keyword(s):  
Polyclonal Antiserum ◽  
Rubus Idaeus ◽  
Dwarf Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Natural Occurrence ◽  
Red Raspberry ◽  
Rt Pcr ◽  
First Report ◽  
Raspberry Bushy Dwarf Virus ◽  
Das Elisa

In 2002, severe vein yellowing and partial or complete yellowing of leaves was observed on some shoots of red raspberry (Rubus idaeus) cvs. Golden Bliss and Autumn Bliss. Sap of infected plants of cv. Golden Bliss was inoculated onto Chenopodium quinoa and Nicotiana benthamiana. Faint chlorotic spots were observed on inoculated leaves of C. quinoa approximately 14 days after inoculation but no systemic symptoms appeared. No symptoms were observed on N. benthamiana. Raspberry bushy dwarf virus (RBDV) was detected in the original raspberry plant using double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with polyclonal antiserum (Loewe Biochemica, Sauerlach, Germany). Systemic infections of inoculated C. quinoa and N. benthaminana were confirmed using DAS-ELISA. In 2001 and 2002, unusual virus symptoms were observed on grapevine grafts (Vitis vinifera) of cv. Laški Rizling. Symptoms appeared as curved line patterns and yellowing of the leaves. No nepoviruses were found in symptomatic plants, but RBDV was confirmed using DAS-ELISA. RBDV infection was later confirmed in grapevine cv. Štajerska Belina with similar symptoms. RBDV was transmitted mechanically from grapevine to C. quinoa where it was detected by immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR). IC-RT-PCR was used to amplify a part of the coat protein gene of the virus from raspberry and grapevine, and the amplification products were sequenced (1). The obtained sequence shared at least 93% nucleotide sequence identity with other known RBDV sequences, which confirmed the serological results. To our knowledge, this is the first report of the natural occurrence of RBDV in grapevine and also of RBDV infection of red raspberry in Slovenia. Reference: (1) H. I. Kokko et al. Biotechniques 20:842, 1996.

scholarly journals Occurrence and molecular analysis of quarantine virus in lily cultivation areas in Brazil

2016 ◽  
Vol 51 (5) ◽  
pp. 615-622 ◽  
Author(s):  
Eliana Borges Rivas ◽  
Estevão Cardoso de Almeida Bôdi ◽  
Ricardo Harakava ◽  
Fabio Gregori ◽  
Marcos Cesar Gonçalves
Keyword(s):  
Mottle Virus ◽  
Mixed Infections ◽  
Coat Proteins ◽  
Rt Pcr ◽  
Tree Reconstruction ◽  
Specific Primers ◽  
Lily Symptomless Virus ◽  
Tulip Breaking Virus ◽  
Genes Encoding ◽  
Prevalent Virus

Abstract: The objective of this work was to describe the occurrence of quarantine Tulip breaking virus (TBV, synonym Lily mottle virus - LMoV) and Lily symptomless virus (LSV), and their respective molecular analyses, to provide data for supporting TBV removal from the Brazilian A1 quarantine pest list, since this virus has spread among the main commercial lily crops in Brazil. The occurrence of these viruses was detected in 12 cultivation areas through multiplex reverse transcription (RT-PCR), using specific primers to genes encoding the respective coat proteins (CP). Eight fragments of 800 nucleotides (nt) obtained from the LMoV-infected lilies and nine fragments of 600 nt from LSV-infected lilies were sequenced. Phylogenetic tree reconstruction showed a robust branch containing the LMoV Brazilian sequences, other LMoV isolates, TBV, and Tulip band breaking virus, suggesting that all are LMoV isolates, although they are clustered into two subgroups. Phylogenetic analysis also showed a robust branch supporting all Brazilian and other LSV sequences, except for an LSV Japanese isolate. Recombination analyses also showed an LMoV recombinant isolate, whereas no recombination events were found among LSV isolates. Lily mottle virus is the prevalent virus in lily crops in Brazil, in single and mixed infections with LSV or Cucumber mosaic virus (CMV).

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

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1259-1259 ◽  
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.

scholarly journals Optimized Yellow Dwarf Virus Multiplex PCR Assay Reveals a Common Occurrence of Barley yellow dwarf virus-PAS in Kansas Winter Wheat

2018 ◽  
Vol 19 (1) ◽  
pp. 37-43 ◽  
Author(s):  
Alma G. Laney ◽  
Rodolfo Acosta-Leal ◽  
Dorith Rotenberg
Keyword(s):  
Winter Wheat ◽  
Barley Yellow Dwarf Virus ◽  
The United States ◽  
Yellow Dwarf ◽  
Dwarf Virus ◽  
Mixed Infections ◽  
Rt Pcr ◽  
Barley Yellow Dwarf ◽  
Field Samples ◽  
Yellow Dwarf Virus

Barley yellow dwarf is an aphid-transmitted virus disease caused by yellow dwarf virus (YDV) species in the family Luteoviridae. Previous partial sequencing efforts conducted in Kansas revealed that Barley yellow dwarf virus-PAS (PAS) occurs in winter wheat fields, and currently available YDV multiplex reverse-transcription PCR (RT-PCR) assays do not detect this species. To enable precise determination of YDV species for research, disease diagnostic, and plant breeding programs, this study enhanced and validated the utility of a multiplex RT-PCR protocol to discriminate six YDV species, including PAS, in archived and fresh field samples. From a representative subset of samples collected from commercial and variety trial locations across nine wheat growing regions of Kansas, PAS and Barley yellow dwarf virus-PAV (PAV) were equally prevalent in single or mixed infections, and other YDVs occurred in mixed infections with PAS and/or PAV in low numbers. The optimized multiplex assay provided robust and specific detection of YDVs and showed promise as a diagnostic tool for determining species occurrence and composition of YDVs in an intensive wheat cropping region of the United States.

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