Precise Demonstration of Salt-Stress Induced Antifungal Activity of Origanum onites Essential Oil and Taraxacum officinale Extract Against Drug Resistant Isolates of Candida albicans and Aspergillus fumigatus Using Micro-Colony Method

The rise of drug-resistant fungal pathogens has intensified the need for novel antifungal agents. Plants are a significant source, although effective concentrations in extracts are often low. Accurate in vitro assays are essential for validating these compounds. This study uses the micro-colony method, measuring hyphal growth and cell diameter under a microscope with digital imaging, to assess antifungal activity quickly and precisely. We evaluated Origanum onites essential oil and Taraxacum officinale methanol extract against drug-resistant Candida albicans and Aspergillus fumigatus strains. Yeast cell pigmentation was also assessed using image processing tools. To enhance compound penetration, mono and divalent salts (100mM KCl, NaCl, CaCl2) were added to the media. In salt-free media, Origanum onites essential oil inhibited Candida albicans (MIC: 0.3 μl/ml; MFC: 0.03 μl/ml) and Aspergillus fumigatus (MIC: 0.15 μl/ml; MFC: 0.03 μl/ml), while Taraxacum officinale extract was ineffective. Salt stress increased Origanum onites activity against Aspergillus fumigatus (MIC: 0.075 μl/ml) but had minimal impact on Candida albicans. Salt stress enabled Taraxacum officinale extract to inhibit Candida albicans (EC50: 12.71 μg/ml) and reduced its pigmentation dose-dependently without affecting toxicity against Aspergillus fumigatus. These results demonstrate that the micro-colony assay effectively evaluates plant-derived antifungal compounds and detects subtle dose-response variations in pathogenic fungi