Confocal imaging of living fungal hyphae challenged with the fungal antagonist viscosinamide

The combination of confocal laser scanning microscopy (CLSM) and staining w ith vital fluorescence dyes has promising potential for studying subcellula r structures in living filamentous fungi and oomycetes. CLSM offers high re solution 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 micr oscopy and the vital dyes Nile red, SITO 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 wer e expected to stain hydrophobic elements, nuclei, mitochondria, and cytopla sm respectively . Nile red and DIOC7(3) were found to be especially well su ited for confocal imaging. Nile red stained the vacuolar membranes in Rhizo ctonia solani and demonstrated the presence of transvacuolar tubules. In ad dition, the stain involved that viscosinamide caused deformation of the hyp hae. DIOC7(3) staining indicated that mitochondria lost their structural or ientation upon exposure to viscosinamide. III conclusion, viscosinamide has effects on hyphae which call be detected with vital fluorescent dyes. The effects may be related to induction of cation channels.