Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

Seddas, A.; Meignoz, R.; Daire, Xavier; Boudon‐Padieu, E.

doi:10.1007/bf01877150

Cited by 20 publications

(8 citation statements)

References 23 publications

(36 reference statements)

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“… Early serological methods including ELISA and PCR‐ELISA (Seddas et al ., 1996; EPPO/CABI, 2003). PCR‐based methods including nested and multiplex‐PCR that amplify either ribosomal or non‐ribosomal phytoplasmic DNA (e.g.…”

Section: Introductionmentioning

confidence: 99%

Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

et al. 2007

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A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence dorée (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.

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Section: Introductionmentioning

confidence: 99%

Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

et al. 2007

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“…Several methods for detection of non-culturable FD or BN Phytoplasmas have been developed: (i) early serological methods including ELISA and PCR-ELISA (Seddas et al 1996, EPPO/CABI 2003. (ii) PCR-based methods including nested and multiplex-PCR that amplify either ribosomal or non-ribosomal phytoplasmic DNA (Daire et al 1997, Clair et al 2003, Lee et al 1998).…”

Section: Existing Detection Methodsmentioning

confidence: 99%

Real-Time PCR Detection Methods for Economically Important Grapevine Related Bacteria

Hren

Dreo

Erjavec

et al. 2010

Methodologies and Results in Grapevine Research

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Preventive measures are extremely important for control of diseases that are caused by plant pathogenic bacteria on economically important plants. For this reason, fast and reliable detection methods are required. Along with the time-consuming conventional detection methods such as isolation and culturing of bacteria on media, PCR-based methods have been introduced as supplementary tests for better diagnosis of plant pathogenic bacteria. In the case of non-culturable bacteria, such as Phytoplasmas, PCR-based tests even became indispensable. Real-time PCR has become a widely used platform in nucleic acid detection and quantification in diagnostics. Despite the general guidelines for some of the steps, there are no common guidelines or standard operating procedures for introduction of real-time PCR-based detection systems for pathogens for routine laboratory use. Four cases of detection systems for grapevine pathogenic bacteria that were developed at the National Institute of Biology are presented and discussed here, providing a practical overview of the whole process from the initial assay design to the implementation of the assay into the laboratory.

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“…In other phytoplasmas several proteins may be strongly antigenic. In the case of Fluorescence dorèe and elm yellows phytoplasmas, three and four immunodominant proteins respectively were identified which are serologically related (Seddas et al 1996). Immunoassays employing poly-or monoclonal antibodies have been shown to be more reliable and sensitive in detecting the phytoplasmas in plants and for establishing relationships among phytoplasmas (Clark et al 1989;Lin et al 1993).…”

Section: Immunoassaysmentioning

confidence: 96%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

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Generation and characterization of monoclonal antibodies to Flavescence dorée phytoplasma: Serological relationships and differences in electroblot immunoassay profiles of Flavescence dorée and elm yellows phytoplasmas

Cited by 20 publications

References 23 publications

Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

Real-Time PCR Detection Methods for Economically Important Grapevine Related Bacteria

Detection of Bacterial and Phytoplasmal Pathogens

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