An Improved Method for Measurement of the Resting Spore Density of Plasmodiophora brassicae from Infested Soils

Abstract

It is important to measure the resting spore density of Plasmodiophora brassicae in soil in order to establish a countermeasure control for clubroot disease. The fluorescence microscopic method (Takahashi & Yamaguchi) has been the conventional method. But, when using this method, a low recovery percentage of the resting spore artificially infested in the Andosol and its subsoil was obtained. It was thought that the cause of the low recovery percentage is electrical adsorption of the resting spore with negative charges on the surface of allophane soils, which have positive charges. An almost satisfactory recovery percentage of the resting spore was obtained by using a sodium hexametaphosphate solution as a soil-dispersing agent, pH adjustment of soil suspension to above 9, and ultrasonic treatment for dispersion of soil particles. The improved method for measurement of the resting spore density is as follows. Take 20 g of air-dried soil sample into a 500 mL bottle. Add 400 mL of 2.0 g L^<-1> sodium hexametaphosphate solution, and shake for 1 min by hand. Adjust the pH of soil suspension to 10 with 1.0 mol L^<-1> sodium hydroxide solution, and disperse the soil particles by ultrasonic treatment using 180 W for 5 min. After sonication, readjust the pH to 9 with 1.0 mol L^<-1> sodium hydroxide solution and shake for 1 min. Take 40 mL of the soil suspension, and pass it through a 38 μm mesh using distilled water. Fill up to 100 mL. Mix equal volumes of the final soil suspension and 200 mg L^<-1> calcofluor white M2R and 100 mg L^<-1> ethidium bromide solution for staining. Measure the number of resting spores using fluorescence microscopy.