Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil

Abstract

Pectobacterium brasiliense (Pb) is one of the causal agents of soft rot and blackleg diseases and has become a pathogen of economic importance in many potato production regions worldwide. Accurate, sensitive and timely identification of Pb is critical for improved management of the pathogen to mitigate yield losses. This study describes the development and validation of a TaqMan probe-based quantitative real-time PCR assay for rapid and specific detection of Pb in plant material and soil. A primer-pair that amplifies a 125-bp fragment was designed from the 16-23 s intergenic spacer region ribosomal RNA and the tRNA-Glu gene region. The specificity of the assay was evaluated with 24 isolates representative of nine different Pectobacterium and Dickeya species associated with soft rot and blackleg of potatoes. The designed Pb species-specific primers and FAM-labelled TaqMan probe were specific for detection of Pb in all the assays performed and it did not detect other Pectobacterium and Dickeya species. The TaqMan PCR assay could detect Pb DNA quantities as low as 10 fg/µl and DNA from a concentration of cells as low as 103 colony forming units/ml. The assay was capable of identifying and quantifying Pb in potato tubers and in field soils without the interference of inhibitors. The developed TaqMan PCR assay can be used for routine Pb diagnostics, surveillance and epidemiological studies.