Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tube
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Development of a TaqMan PCR assay for specific detection and quantification of Pectobacterium brasiliense in potato tubers and soil N. Muzhinji & J. P. Dube & E. G. de Haan & J. W. Woodhall & J. E. van der Waals
Received: 7 May 2020 / Revised: 6 August 2020 / Accepted: 10 August 2020 # Koninklijke Nederlandse Planteziektenkundige Vereniging 2020
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. Keywords Pectobacteriaceae . Seed tuber . Soft rot . Latent infection . qPCR
Introduction
: J. P. Dube : J. E. van der Waals
N. Muzhinji (*) Department of Plant and Soil Sciences, University of Pretoria, Private Bag X20, Hatfield, Pretoria 0001, South Africa e-mail: [email protected] N. Muzhinji Department of Applied and Natural Sciences, Namibia University of Science and Technology, Private Bag, Windhoek 13388, Namibia E. G. de Haan Dutch General Inspection Service for Agricultural Seed and Seed Potatoes (NAK), PO Box 1115, Randweg 14, Emmeloord 8300 BC, Netherlands J. W. Woodhall Parma Research and Extension Center, University of Idaho, 29603 U of I Lane, Parma, ID 83660, USA
Pectobacterium and Dickeya species are economically important pathogens of potato as well as other vegetable crops worldwide (Czajkowski et al. 2011; Sławiak et al. 2013). Pectobacterium species produce pectinolytic and cellulolytic enzymes that degrade cell walls resulting in infiltration and maceration of plant organs and tissues causing diseases like blackleg, stem rot and tuber soft rot on potatoes (Czajkowski et al. 2011; Kettani-Halabi et al. 2013; Lee et al. 2014). Of importance is blackleg and tuber soft rot that cause extreme yield losses to potato production in the field, during transit
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