Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil

Abstract

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.