Detection of five viruses infecting dormant bulbs by TaqMan-based real-time RT-PCR

Wei, Ting; Lebas, B. S. M.; Shiller, Jason; Quinn, B. D.; Clover, G. R. G.

doi:10.1007/s13313-011-0095-1

Cited by 16 publications

(4 citation statements)

References 20 publications

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“…The extracted RNA was then retro-transcribed into cDNA using the iScript cDNA synthesis kit (Biorad, USA). Primers and probes for GLRaV-1, GLRaV-2, GLRaV-3 [ 20 ], GRSPaV, GVA [ 21 ], GFLV, GFkV [ 22 ] and ArMV [ 23 ] detection were used. For each sample, 2 μl of cDNA were amplified in a total volume of 20 μl containing 1x SsoFasto probe Master Mix (Biorad) and 0.4 μM of each primer and probes.…”

Section: Methodsmentioning

confidence: 99%

Phylogenetic analysis of viruses in Tuscan Vitis vinifera sylvestris (Gmeli) Hegi

et al. 2018

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The health status of the native grapevine Vitis vinifera subsp. sylvestris (Gmeli) Hegi in natural areas in Europe has received little attention. A survey was carried out on wild grapevines in Tuscany (Italy), where isolates of the Grapevine rupestris stem pitting virus (GRSPaV), Grapevine leafroll-associated virus 1 and 3 (GLRaV-1 and GLRaV-3) and Grapevine virus A (GVA) were detected. The complete coat protein (CP) region of these isolates was sequenced to investigate the relationship of the viral variants from Tuscan wild grapevines with isolates from different geographical origins. According to the phylogenetic analyses, GLRaV-1 and GLRaV-3 isolates from Tuscan wild grapevines clustered with isolates from cultivated grapevines with nucleotide sequence identities ranging from 66% to 87% and from 72.5% to 99% respectively, without any correlation between the distribution and geographical origin. Conversely, GRSPaV and GVA isolates clustered together with other Italian isolates from V. vinifera with nucleotide sequence identities ranging from 71.14% to 96.12% and from 73.5% to 92%, respectively. Our analysis of the whole amino acid sequences revealed a high conservation level for the studied proteins explained by a selective pressure on this genomic region, probably due to functional constraints imposed on CP, such as specific interactions with cellular receptors in the insect vectors necessary for successful transmission. In addition, analyses of genetic recombination suggest no significant point mutations that might play a significant role in genetic diversification. The dN/dS ratio also estimated a low number of non-silent mutations, highlighting the purifying selective pressure. The widespread distribution of the Rugose wood complex (GRSPaV and GVA associated disease) in comparison with the Grapevine Leafroll associated viruses (GLRaV-1 and -3) could explain the major geographical correlation found for the viral variants detected in Tuscany.

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Section: Methodsmentioning

confidence: 99%

Phylogenetic analysis of viruses in Tuscan Vitis vinifera sylvestris (Gmeli) Hegi

et al. 2018

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“…This limited sensitivity has the potential to limit the effectiveness of the Rtul assay in quantifying inoculum levels in planting stock. It is likely the decreased sensitivity of the Rtul assay associated with increased bulb tissue may be due to the presence of qPCR inhibitors in the dormant tulip bulb tissues [36]. A variety of protocols have been developed to remove inhibitors from nucleic acids before PCR [37].…”

Section: Discussionmentioning

confidence: 99%

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris

Coats

DeBauw

Lakshman

et al. 2022

JoF

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Gray bulb rot of tulips and bulbous iris is caused by the soil-borne fungal pathogen, Rhizoctonia tuliparum (Rtul). Sclerotia present in infected bulbs, as well as overwintering sclerotia in soil and field debris, are the primary sources of infection. A method for accurate and sensitive detection of Rtul from soil and infected bulbs, and estimation of inoculum threshold levels, is needed for the management of disease caused by this pathogen. We designed a unique set of primers targeting the ITS2 region of the Rtul genome and developed a highly sensitive quantitative PCR (qPCR)-based method for Rtul identification using these primers, where the threshold of detection was approximately 1 fg Rtul DNA. The assay was more sensitive with sclerotia collected from the field (natural) than with those grown in the lab, and more sensitive with natural-light than natural-dark sclerotia. Also, the detection method was more sensitive when sclerotia were extracted from soil than from bulb tissue. The qPCR method was highly specific, as no PCR amplification was detected when genomic DNA from 62 non-Rtul Rhizoctonia isolates from a wide range of anastomosis groups were tested. To understand the evolutionary relationships and genomic diversity of Rtul, we performed phylogenetics of the ITS1-5.8S-ITS2 region and ITS2-molecular morphometric characterization (MMC) of Rtul isolates. The three Rtul isolates whose ITS sequences were available in GenBank formed a distinct phylogenetic clade with Ceratobasidium anceps as the nearest relative. Furthermore, MMC analysis revealed genetic divergence among these three Rtul isolates.

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“…(European Commission directive 2005/43/EC). Diagnostic tests (real-time PCR) were carried out for grapevine fanleaf virus , arabis mosaic virus , grapevine leafroll - associated virus 1 , grapevine leafroll-associated virus 3 , and Grapevine fleck virus [ 77 , 78 , 79 ]. Both healthy and infected samples were collected from plants that had negative results in all diagnostic tests.…”

Section: Methodsmentioning

confidence: 99%

Signaling Cross-Talk between Salicylic and Gentisic Acid in the ‘Candidatus Phytoplasma Solani’ Interaction with Sangiovese Vines

Nutricati

Pascali

Negro

et al. 2023

Plants

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“Bois noir” disease associated with ‘Candidatus Phytoplasma solani’ seriously compromises the production and survival of grapevines (Vitis vinifera L.) in Europe. Understanding the plant response to phytoplasmas should help to improve disease control strategies. Using a combined metabolomic and transcriptomic analysis, this work, therefore, investigated the phytoplasma–grapevine interaction in red cultivar Sangiovese in a vineyard over four seasonal growth stages (from late spring to late summer), comparing leaves from healthy and infected grapevines (symptomatic and symptomless). We found an accumulation of both conjugate and free salicylic acids (SAs) in the leaves of ‘Ca. P. solani’-positive plants from early stages of infection, when plants are still asymptomatic. A strong accumulation of gentisic acid (GA) associated with symptoms progression was found for the first time. A detailed analysis of phenylpropanoids revealed a significant accumulation of hydroxycinnamic acids, flavonols, flavan 3-ols, and anthocyanin cyanidin 3-O-glucoside, which are extensively studied due to their involvement in the plant response to various pathogens. Metabolomic data corroborated by gene expression analysis indicated that phenylpropanoid biosynthetic and salicylic acid-responsive genes were upregulated in ‘Ca. P. solani-positive plants compared to -negative ones during the observed period.

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Detection of five viruses infecting dormant bulbs by TaqMan-based real-time RT-PCR

Cited by 16 publications

References 20 publications

Phylogenetic analysis of viruses in Tuscan Vitis vinifera sylvestris (Gmeli) Hegi

Phylogenetic analysis of viruses in Tuscan Vitis vinifera sylvestris (Gmeli) Hegi

Detection and Molecular Phylogenetic-Morphometric Characterization of Rhizoctonia tuliparum, Causal Agent of Gray Bulb Rot of Tulips and Bulbous Iris

Signaling Cross-Talk between Salicylic and Gentisic Acid in the ‘Candidatus Phytoplasma Solani’ Interaction with Sangiovese Vines

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