Dataset Information

LAMP assay and rapid sample preparation method for on-site detection of flavescence doree phytoplasma in grapevine.

ABSTRACT: In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely sensitive, because it detected FDp in all leaf samples that were determined to be FDp infected using quantitative real-time PCR. The whole procedure of sample preparation and testing was designed and optimized for on-site detection and can be completed in one hour. The homogenization procedure of the grapevine samples (leaf vein, flower or berry) was optimized to allow direct testing of crude homogenates with the LAMP assay, without the need for DNA extraction, and was shown to be extremely sensitive.

SUBMITTER: Kogovsek P

PROVIDER: S-EPMC4480326 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine.

Kogovšek P P Hodgetts J J Hall J J Prezelj N N Nikolić P P Mehle N N Lenarčič R R Rotter A A Dickinson M M Boonham N N Dermastia M M Ravnikar M M

Plant Pathology. 20140723 2

In Europe the most devastating phytoplasma associated with grapevine yellows (GY) diseases is a quarantine pest, flavescence dorée (FDp), from the 16SrV taxonomic group. The on-site detection of FDp with an affordable device would contribute to faster and more efficient decisions on the control measures for FDp. Therefore, a real-time isothermal LAMP assay for detection of FDp was validated according to the EPPO standards and MIQE guidelines. The LAMP assay was shown to be specific and extremely ...[more]

PMID: 26146413

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Project description:The phytoplasma-borne disease flavescence dorée is still a threat to European viticulture, despite mandatory control measures and prophylaxis against the leafhopper vector. Given the economic importance of grapevine, it is essential to find alternative strategies to contain the spread, in order to possibly reduce the current use of harmful insecticides. Further studies of the pathogen, the vector and the mechanisms of phytoplasma-host interactions could improve our understanding of the disease. In this work, RNA-Seq technology followed by three de novo assembly strategies was used to provide the first comprehensive transcriptomics landscape of flavescence dorée phytoplasma (FD) infecting field-grown Vitis vinifera leaves.With an average of 8300 FD-mapped reads per library, we assembled 347 sequences, corresponding to 215 annotated genes, and identified 10 previously unannotated genes, 15 polycistronic transcripts and three genes supposedly localized in the gaps of the FD92 draft genome. Furthermore, we improved the annotation of 44 genes with the addition of 5'/3' untranslated regions. Functional classification revealed that the most expressed genes were either related to translation and protein biosynthesis or hypothetical proteins with unknown function. Some of these hypothetical proteins were predicted to be secreted, so they could be bacterial effectors with a potential role in modulating the interaction with the host plant. Interestingly, qRT-PCR validation of the RNA-Seq expression values confirmed that a group II intron represented the FD genomic region with the highest expression during grapevine infection. This mobile element may contribute to the genomic plasticity that is necessary for the phytoplasma to increase its fitness and endorse host-adaptive strategies.The RNA-Seq technology was successfully applied for the first time to analyse the FD global transcriptome profile during grapevine infection. Our results provided new insights into the transcriptional organization and gene structure of FD. This may represent the starting point for the application of high-throughput sequencing technologies to study differential expression in FD and in other phytoplasmas with an unprecedented resolution.

Project description:BackgroundFlavescence dorée (FD) is a grapevine disease caused by phytoplasma and it is one of the most destructive pathologies in Europe. Nowadays, the only strategies used to control the epidemics are insecticides against vector, but more sustainable techniques are required. Completely resistant Vitis vinifera varieties have not been uncovered yet, but differences in susceptibility among cultivars and spontaneous recovery from FD symptoms have been observed. The grapevine cultivar 'Tocai friulano' shows very low susceptibility to FD but its defence strategy to counteract the phytoplasma spread has not been deciphered yet. In this work, the mechanisms occurring within 'Tocai friulano' FD-infected plants were examined in depth to identify the phytoplasma distribution and the defence pathways involved.ResultsIn 'Tocai friulano' symptoms of FD-infection remained confined near the area where they appeared during all the vegetative season. Analyses of secondary phloem showed a total absence of FD phytoplasma (FDp) in the trunk and its disappearance in 2-year-old arms from July to November, which was different from 'Pinot gris', a highly susceptible variety. Diverse modulations of defence genes and accumulation of metabolites were revealed in 1-year-old canes of 'Tocai friulano' FD-infected plants, depending on the sanitary status. Symptomatic portions showed high activation of both jasmonate- and salicylate-mediated responses, together with a great accumulation of resveratrol. Whereas activation of jasmonate-mediated response and high content of ε-viniferin were identified in asymptomatic 1-year-old cane portions close to the symptomatic ones.ConclusionSuccessful defence mechanisms activated near the symptomatic areas allowed the compartmentation of FD symptoms and phytoplasmas within the infected 'Tocai friulano' plants. These results could suggest specific agronomical practices to be adopted during FD management of this variety, and drive research of resistance genes against FD.

Dataset Information

LAMP assay and rapid sample preparation method for on-site detection of flavescence doree phytoplasma in grapevine.

Publications

LAMP assay and rapid sample preparation method for on-site detection of flavescence dorée phytoplasma in grapevine.

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