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Specific Polymerase Chain Reaction Primers for the Detection of Plasmodiophora brassicae in Soil and Water

First and third authors: Department of Applied Biology and Biotechnology, RMIT University, 124 La Trobe Street, Melbourne, Australia 3000; second author: New Zealand Institute of Crop and Food Research, Private Bag 4704, Christchurch, New Zealand; and fourth author: Agriculture Victoria, Institute for Horticultural Development, 621 Burwood Hwy., Knoxfield, Victoria, Australia 3180

The development of specific oligonucleotide primers for Plasmodiophora brassicae has led to a nested polymerase chain reaction (PCR) detection method for P. brassicae in soil and water. Initially, the PCR was used to amplify a section of the rDNA repeat. The PCR products were sequenced and the data used to design primers that were directed at the ribosomal RNA genes and internal transcribed spacer regions. Specificity was tested against more than 40 common soil organisms, host plants, and spore suspension contaminants, as well as P. brassicae isolates from around Australia and the world. Sensitivity was determined to be 0.1 fentograms (fg; 10^-15 g) for pure template and as low as 1,000 spores per g of potting mix. In soil, P. brassicae was detected in all soils where the inoculum was sufficient to result in clubroot symptoms. Also outlined is a simple method of DNA extraction from soil.