Development of a Detection System for Viruses of Woody Plants Based on PCR Analysis of Immobilized Virions

Rowhani, Adib; Maningas, M. A.; Lile, L. S.; Daubert, Steve; Golino, Deborah A.

doi:10.1094/phyto-85-347

Cited by 114 publications

(61 citation statements)

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“…First-strand cDNA synthesis by Superscript II reverse transcriptase (Invitrogen), followed by PCR amplification with Pfu DNA polymerase (Stratagene) and the direct sequencing of PCR-amplified products, was performed by using primers LIYV 10, LIYV 11, LIYV 12, and LIYV 13 (12). Immunocapture RT-PCR was done according to a method described previously (18), with modifications. Following the precoating of PCR tubes with LIYV virion-specific IgG at ϳ2.3 g/ml for 30 min at 37°C and rinsing with phosphatebuffered saline plus 0.05% Tween 20 (PBST), tubes were incubated with a blocking buffer (PBST with 2% polyvinyl pyrrolidone and 2% bovine serum albumin) for 30 min at 37°C.…”

Section: Methodsmentioning

confidence: 99%

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

Stewart

Medina

Tian

et al. 2010

J Virol

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The Lettuce infectious yellows virus (LIYV) RNA 2 mutant p1-5b was previously isolated from Bemisia tabaci-transmitted virus maintained in Chenopodium murale plants. p1-5b RNA 2 contains a single-nucleotide deletion in the minor coat protein (CPm) open reading frame (ORF) that is predicted to result in a frameshift and premature termination of the protein. Using the recently developed agroinoculation system for LIYV, we tested RNA 2 containing the p1-5b CPm mutant genotype (agro-pR6-5b) in Nicotiana benthamiana plants. We showed that plant infection triggered by agro-pR6-5b spread systemically and resulted in the formation of virions similar to those produced in p1-5b-inoculated protoplasts. However, virions derived from these mutant CPm genotypes were not transmitted by whiteflies, even though virion concentrations were above the typical transmission thresholds. In contrast, and as demonstrated for the first time, an engineered restoration mutant (agro-pR6-5bM1) was capable of both systemic movement in plants and whitefly transmission. These results provide strong molecular evidence that the full-length LIYV-encoded CPm is dispensable for systemic plant movement but is required for whitefly transmission.

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Section: Methodsmentioning

confidence: 99%

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

Stewart

Medina

Tian

et al. 2010

J Virol

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“…Freeze-dried plant tissue was macerated in extraction buffer (500 mM Tris-HCl, pH 8.3, containing 2% PVP-40, 1% polyethylene glycol 6000, 140 mM NaCl and 0.05% Tween 20) at 1/200 dilution (Rowhani et al 1995). Extracts were clarified by centrifugation (10,000 g for 10 min at 4C).…”

Section: Virus Detectionmentioning

confidence: 99%

“…For this reason we used the extraction buffer, suggested by Rowhani et al (1995), for woody species that contain these compounds. This buffer proved to be effective in this research with nectarine samples.…”

Section: Virus Detectionmentioning

confidence: 99%

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine

et al. 2003

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The plum pox virus (PPV) and prunus necrotic ringspot virus (PNRSV) cause serious disease problems in stone-fruit trees. In this work, the possibility of obtaining plant material free from these viruses through thermotherapy and meristem-tip culture from infected nectarine shoots (Prunus persica var. nectarina Max, cv. 'Arm King') was studied. In addition, the detection of these viruses in in vitro cultures and young acclimatized plantlets with double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) and multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) was studied. Meristem-tip explants (0.8-1.3 mm) derived from sprouted buds of winter wood and spring shoots from field grown plants had a 2-5% regeneration response. However, application of thermotherapy to potted nectarine trees (3 weeks at a maximum temperature of 35 degrees C) facilitated excision of longer meristem tips (1.3-2.0 mm) that resulted in a significantly higher regeneration response (38%) in woody plant medium (WPM) without plant growth regulators. Such explants formed multiple shoots with the addition of 8 microM benzylaminopurine and 0.8 microM indoleacetic acid. When they were tested for the presence of PPV and PNRSV, 86% and 81% were found to be virus-free as detected by DAS-ELISA and multiplex RT-PCR, respectively. Individual shoots excised from virus-free cultures readily rooted in vitro (half-strength WPM plus 2 microM indolebutyric acid) and grew to plantlets. The combination of an efficient protocol for virus elimination and the establishment of highly sensitive diagnostics resulted in the production of nectarine plants free from PPV and PNRSV.

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“…The standard sample extraction procedure for RT-PCR detection of ASGV is based on nucleic acid isolation (total RNA) (7,8,11), which is time-consuming and not amenable for processing large numbers of samples. Potentially improved sample processing procedures for plant virus detection by PCR have been reported and include immunocapture (24), direct binding (19), print capture (17), or direct application of clarified plant extracts in reaction tubes (25). This paper reports the design of specific primers based on the comparison of partial nucleotide sequences established for four European ASGV isolates and that of the Japanese isolate published by Yoshikawa et al (26).…”

mentioning

confidence: 99%

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

et al. 1998

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Partial nucleotide sequences of amplification products obtained from four European apple stem grooving virus (ASGV) isolates using degenerate primers showed 80 to 85% similarity with the published ASGV sequence of a Japanese strain but 98 to 100% identities among themselves. Based on these sequences, two ASGV-specific primers (ASGV4F-ASGV4R) were designed to amplify a 574-bp fragment located in the putative viral RNA polymerase. With these primers, six European and five American ASGV isolates, maintained in herbaceous hosts (Chenopodium quinoa, Nicotiana glutinosa, and N. occidentalis) or in apple trees, were readily detected by reverse transcription-polymerase chain reaction (RT-PCR). Using these specific ASGV primers, dsRNA preparations have been shown to constitute good templates for reliable amplification of ASGV sequences from leaves and bark tissues of apple trees, both in a two-step RT-PCR protocol and in the one-step Titan One-Tube RT-PCR. System. Furthermore, the one-step RT-PCR system allowed a specific amplification of ASGV sequences directly from clarified crude extracts of leaves and bark tissues of apple trees during both active growth and the dormant season.

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Development of a Detection System for Viruses of Woody Plants Based on PCR Analysis of Immobilized Virions

Cited by 114 publications

References 0 publications

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

A Mutation in the Lettuce Infectious Yellows Virus Minor Coat Protein Disrupts Whitefly Transmission but Not In Planta Systemic Movement

Elimination of PPV and PNRSV through thermotherapy and meristem-tip culture in nectarine

Detection of Apple Stem Grooving Virus in Dormant Apple Trees with Crude Extracts as Templates for One-Step RT-PCR

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