Antifungal and Iron Supplement Assay

Four sets of YEPD broth were prepared with 18, 36, 44, and 54 μg/ml of catecholate siderophore, inoculated with 1 ml of spore suspension containing 10⁶ fungal spores and incubated under standard conditions. YEPD agar plates were prepared with similar catecholate concentrations (except 54 μg/ml) in three sets; without supplemented iron, with supplement of 2 and 4 mM FeCl₃. Plates were inoculated with fungal spores and observed for 48 h after incubation. Total biomass of fungal culture with and without siderophore treatment was measured with modifications in protocol of Granade et al. (1985). For this purpose, fungal cultures from YEPD broths were filtered using Whatman filter paper no. 1. Mycelia thus obtained were then washed thoroughly in PBS (pH 7.4). The washed mycelia were then dried in oven overnight at 60°C and weighed. For measurement of filament length, 0.25 g of fungal filaments grown on each YEPD agar plate with varying concentrations of siderophore were stained with lactophenol cotton blue (LPCB; Leck, 1999) and observed at 40× magnification under a Nikon Eclipse TiU inverted microscope. Filament length was measured using Nikon NIS element software.