Microsporum gypseum

All tested fungi were cultured yeast-nitrogen base growth medium (Sigma-Aldrich, St. Louis, MO, USA). Stock solutions (5000 μg/mL) of C. winterianus (leaves and root) EOs were prepared in DMSO and diluted as above. The freshly harvested fungi with approximately 7.5 × 107 CFU/mL final concentrations were added to each well of 96-well microdilution plates and were incubated at 35 °C for 24 h. DMSO and amphotericin B (Sigma-Aldrich, St. Louis, MO, USA) were negative and positive antifungal controls, respectively, as previously described [56 (link)]. Seven fungal strains were used: Aspergillus niger (ATCC-16888), Candida albicans (ATCC-18804), Microsporum canis (ATCC-11621), Trichophyton mentagrophytes (ATCC-18748), Aspergillus fumigatus (ATCC-96918), Microsporum gypseum (ATCC-24102), and Trichophyton rubrum (ATCC-28188).

For the antifungal evaluation, standardized strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, and CEREMIC (CCC), Centro de Referencia en Micología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531-(2000)-Rosario, Argentina, were used: C. albicans ATCC 10231, S. cerevisiae ATCC 9763, C. neoformans ATCC 32264, Aspergillus flavus ATCC 9170, Aspergillus fumigatus ATTC 26934, Aspergillus niger ATCC 9029, Trichophyton rubrum CCC 113, Trichophyton mentagrophytes ATCC 9972, and Microsporum gypseum CCC 115. Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 °C, maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid, Cambridge, UK), and subcultured every 15 days to prevent pleomorphic transformations. Inocula of cell or spore suspensions were obtained according to reported procedures [26 ,27 ] and adjusted to 1–5 × 10³ cells/spores with colony forming units (CFU)/mL.