This research investigated the inhibitory effects of essential oils (EOs) extracted from four Citrus sinensis cultivars (Thomson navel, Jaffa sweet, Sanguine blood, and Valencia) against Alternaria spp., a pathogen affecting oranges. Among the cultivars studied, the EO from the Sanguine blood cultivar exhibited the highest antifungal activity when added into the culture medium and it was selected for further investigations. Sanguine blood EO reduced melanin in the fungal hyphae and completely inhibited vegetative growth of Alternaria species at 550–750 μg mL−1 concentrations and inhibited spore germination at 800–1000 μg mL−1 concentrations, which were similar to the fungicide Mancozeb. Separation of cytoplasm from cell wall, hyphal emptying, destruction of cell wall, plasma membrane, organelles such as nucleus and mitochondria, hyphal lysis and cell death were observed in all three fungal species tested. Activities of cell wall degrading enzymes produced by the pathogens were significantly reduced in the fungi treated with the EO. Reduction of the disease index was observed in all four cultivars, similar to the effect of fungicide. Gas chromatography-mass spectrometry analysis revealed presence of D-limonene, β-myrcene, and β-linalool as the main components of the EO obtained from the Sanguine blood cultivar. In conclusion, the EOs as natural plant protectants could control Alternaria spp. on orange under both in vitro and in vivo conditions.

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