Validation of the qPCR technique for the detection of Plasmodiophora brassicae in horticultural soils of Colombia

Abstract

Clubroot of crucifers caused by the soil-borne pathogen Plasmodiophora brassicae is a very destructive disease worldwide. The pathogen survives for many years in soil as resting spores (RS), which are formed in large numbers within the clubbed roots. RS can accumulate in the soil when brassicas are repeatedly grown, leading to increased levels inoculum for subsequent crops. The rapid spread of the pathogen in all cruciferous vegetable production areas in Colombia put the economy of farmers and the availability of these foods in the local market at risk. DNA-based techniques can be useful in determining the presence and concentration of pathogen in soil, making them valuable diagnostic tools for disease management and decision-making. Although the presence and the concentration of P. brassicae spores in soils from cruciferous producing areas in Colombia were determined using qPCR technique in 2017, to our knowledge no other works have addressed the use of diagnosis tools for management of clubroot in the country. No symptoms of clubroot were observed in horticultural areas in the Nariño department according to the mentioned report, despite finding pathogen DNA in the soil samples. However, significant yield losses caused by clubroot have been reported in Nariño since 2020, and many farms are currently growing alternative crops such as potato, carrot, and non-cruciferous crops because of the high P. brassicae pressure. To assess the risk of growing cruciferous crops, this work aims to validate the qPCR technique to detect and quantify P. brassicae in soil samples from the field. We used a model bioassay of pathogenicity test with various concentrations of P. brassicae and with a known susceptible cultivar of broccoli to standardize the methodology. Soil samples from horticultural fields were analyzed to validate the protocol. The bioassay allowed us to determine that a low concentration of 10 spores g⁻¹ is enough to cause disease symptoms in the host and a concentration of 10² spores of P. brassicae is the limit of detection by the qPCR technique. In the presence of the host, the RS concentration of P. brassicae in the soil increased between five and nine times in a single cycle, demonstrating the high rate of propagation of the pathogen. The Nariño department had the highest pathogen concentrations compared to Cundinamarca and Boyacá departments, which was associated to the lack of risk assessments, control measurements and the consecutive growing of susceptible cruciferous crops in infected fields.