title: Development of real-time PCR (TaqMan) assays for the detection and quantification of Botrytis cinerea in planta.
abstract: Real-time PCR assays based on TaqMan chemistry have been developed for the detection and quantification of Botrytis cinerea, suitable for a wide range of different host plant species. Assays were designed to the beta-tubulin gene, the intergenic spacer (IGS) region of the nuclear ribosomal DNA and also to a previously published, species-specific sequence characterised amplified region (SCAR) marker; the assays were compared to a published method based on SYBR Green I technology. The assays designed to the IGS region and SCAR marker proved to be highly specific for B. cinerea but assays designed to the beta-tubulin gene and the previously published assay designed to the cutinase-A gene both cross-react with B. fabae. The assay designed to the IGS region was the most sensitive and was able to reliably detect and quantify as little as 20 fg of B. cinerea DNA. The method incorporates the detection of a gene from the plant host to compensate for variations in extraction efficiency and size of sample tested. The assays designed were used to follow the progression of infection of B. cinerea in plant material inoculated with spores to the point of symptom induction. They should be ideally suited to investigating infection processes in-planta and could be used to investigate aspects of infection/plant pathogenesis, by B. cinerea and are particularly suited to the detection and quantification of the pathogen prior to the development of symptoms.
authors.affiliation: Central Science Laboratory, Sand Hutton, York, YO41 1LZ, United Kingdom.
authors.forename: M Belen
authors.initials: MB
authors.lastname: Suarez
authors.forename: Kathryn
authors.initials: K
authors.lastname: Walsh
authors.forename: Neil
authors.initials: N
authors.lastname: Boonham
authors.forename: Tim
authors.initials: T
authors.lastname: O'Neill
authors.forename: Simon
authors.initials: S
authors.lastname: Pearson
authors.forename: Ian
authors.initials: I
authors.lastname: Barker
mesh_terms: D001483:Base Sequence; D020171:Botrytis / Q000235:genetics / Q000302:isolation & purification*; D004271:DNA, Fungal; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014404:Tubulin / Q000235:genetics*
chemical_list: D004271:DNA, Fungal; D014404:Tubulin