Optimization, characterization and biosafety of oregano, rosemary and mint oil mixture against Penicillium digitatum in citrus using L-optimal mixture design

The increasing demand for natural alternatives to synthetic fungicides has prompted research into natural products like essential oils for postharvest disease management. This study investigated the antifungal, antioxidant, cytotoxic, and genotoxic potential of essential oil mixtures derived from oregano, rosemary, and mint against Penicillium digitatum, the predominant fungal pathogen causing green mold in orange fruits. P. digitatum NPAGRASU 2024 was isolated and identified as the most abundant species (60.2%) from infected oranges. It was deposited in GenBank with gene accession number PP930644 and deposited in MIRCEN culture collection as EMCC 358874. The antimicrobial activity of individual essential oils was evaluated, with oregano exhibiting the highest antifungal activity (inhibition zone diameter of 4.2 cm) against P. digitatum. L-optimal mixture design of response surface methodology (RSM) optimization revealed a highly effective mixture (Run 8) comprising 46.26% oregano and 53.74% rosemary, with a 99.65% actual growth reduction. The oregano oil demonstrated potent antioxidant activity, reaching approximately 75% DPPH radical scavenging at 3.125 mg/mL. Cytotoxicity assessment using the MTT assay showed morphological changes and reduced cell viability in liver cells treated with the PEOs mixture at 300 µg/mL. However, the optimized mixture did not induce significant chromosomal aberrations compared to the control, suggesting minimal genotoxic effects. In vivo, evaluation on oranges revealed 60% inhibition of green mold by the 1% (v/v) optimized mixture for 7 days. Histological analysis indicated low toxicity to the liver at the highest tested concentration (1% mixture). GC-MS analysis identified major compounds like cavarcol, caryophyllene, eucalyptol, phenols and levomenthol in oregano oil, contributing to its bioactivities. This study demonstrates the potential of optimized essential oil mixtures as effective and eco-friendly alternatives for postharvest disease control.