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Abstract
The combination of confocal laser scanning microscopy (CLSM) and staining with vital fluorescence dyes has promising potential for studying subcellular structures in living filamentous fungi and oomycetes. CLSM offers high resolution images and the possibility of optical sectioning of the specimen. Vital dyes make it possible to do this while the cells are still alive and with minimum disturbance of cellular processes. In this study confocal microscopy and the vital dyes Nile red, SYTO 13, DIOC7(3), and carboxy SNARF-1 were used on several species. Subsequently the technique was used to detect effects of the fungal antagonist viscosinamide. These fluorescent dyes were expected to stain hydrophobic elements, nuclei, mitochondria, and cytoplasm respectively. Nile red and DIOC7(3) were found to be especially well suited for confocal imaging. Nile red stained the vacuolar membranes in Rhizoctonia solani and demonstrated the presence of transvacuolar tubules. In addition, the stain showed that viscosinamide caused deformation of the hyphae. DIOC7(3) staining indicated that mitochondria lost their structural orientation upon exposure to viscosinamide. In conclusion, viscosinamide has effects on hyphae which can be detected with vital fluorescent dyes. The effects may be related to induction of cation channels.