Distinguishable staining with neutral red for GFP-marked and GFP-nonmarked Fusarium oxysporum strains simultaneously colonizing root surfaces

Green fluorescent protein (GFP)-marked Fusarium oxysporum f. sp. melonis and nonmarked F. oxysporum f. sp. fragariae were stained with neutral red. The neutral red stained vacuoles of the fungi without disturbing GFP fluorescence in the cytoplasm. GFP-marked fungi showed fluorescent hyphae with dark-stained vacuoles, whereas nonmarked fungi were detected as nonfluorescent hyphae with dark-dotted vacuoles. Root colonization by these two fungi was monitored using this method. Microconidia attached similarly to the root surface and elongated vegetative hyphae. Only the pathogenic fungi invaded, causing necrosis at the inoculation site. Thus, the present method enabled us to track simultaneously the various formae speciales of F. oxysporum colonizing the root surface.