Assessment of pathogenicity of resting spores of Plasmodiophora brassicae in soil by fluorescence microscopy.

Abstract

A fluorescence microscopic method was applied to assess directly the pathogenicity of resting spores of Plasmodiophora brassicae in soil. Infested soils were stained with a mixture solution of two fluorochromes, calcofluor white M2R and ethidium bromide, and were observed by fluorescence microscopy (phase contrast, UV filter set, oil immersion). Spores could be readily identified in soil by the strong blue fluorescence of the wall layer and classified into two groups, i.e. spores with a non-fluorescing cytoplasm (blue spores) and spores with a red fluorescing cytoplasm (red spores). When the infested soils were heated for 10 days at 40, 45 or 50C, both the percentage of blue spores in the soils and the disease severity in plants grown in the soils decreased with the time of incubation at all the temperatures tested, and the decrease was more rapid and appreciable at higher temperatures. Reduction in both the percentage of blue spores and the disease severity by heat treatment of the infested soil with a moisture ratio of 40% was more pronounced than that in the soil with a moisture ratio of 2.5% upon heating for 10 days at 45C. A positive correlation between the percentage of blue spores and the disease severity was also observed in the infested soils prepared by using the spores which were obtained from galls stored for different periods of time. Throughout these experiments, the percentage of blue spores showed a highly significant positive correlation with the pathogenicity of the spores in soil reflected by the disease severity. The results obtained indicate that the pathogenicity of the spores in a given soil sample can be directly assessed by the determination of the percentage of blue spores in the soil sample. The variations in the percentage of blue spores were generally less than those in the disease severity. This method is not only simple and rapid but also precise, and may enable to quantify the spore activity.