A method for assessing the pathogenic activity of resting spores of Plasmodiophora brassicae by fluorescence microscopy.

A fluorescence microscopic method was developed to directly assess the pathogenic activity of resting spores of Plasmodiophora brassicae. Resting spores were stained with a mixture solution of two fluorochromes, calcofluor white M2R (CFW) and ethidium bromide (EB), and were observed by fluorescence microscopy (phase contrast, UV filter set, oil immersion). All the spores exhibited intense blue fluorescence on the wall layer, and some spores displayed red fluorescence in the cytoplasm. Spores could be distinguished into two groups, spores with a non-fluorescing cytoplasm (blue spores) and spores with a red fluorescing cytoplasm (red spores). The fluorescent staining reaction of the spores was not affected by a staining period of less than 4hr. However, when the concentration of EB solution equally mixed with CFW solution was increased, an increase in the percentage of red spores was observed. Suitable conditions for the differentiation between blue and red fluorescence corresponded to a concentration of 10 to 50μg/ml of the EB solution using 100μg/ml of CFW solution. When spores were heated for 72hr at 40C, 50C or 60C, the percentage of blue spores was constant at 40C, but decreased both at 50C and at 60C with the time of incubation, suggesting that the blue spores are active spores. Disease severity in the plants infected with heat-treated spores was closely related to the percentage of blue spores among the treated spores. This correlation was also observed for the spores stored for different periods of time. The results obtained indicate that the pathogenic activity of the resting spores can be assessed by examining the fluorescent staining reaction of the spores. In this study, the variations in the percentage of blue spores were generally less pronounced than those in the disease severity. This method is rapid, convenient and precise.