Utilization of dropped Citrus reticulata Blanco fruit as a botanical fungicide to control foot rot disease in rice caused by Fusarium fujikuroi

Citrus reticulata Blanco shows substantial fruit drop due to diverse environmental factors leading to agricultural waste. However, such waste can be utilized as a safe and cheape source of botanical fungicide. The present work aimed to evaluate the antifungal potential of C. reticulata fruits dropped in June against Fusarium fujikuroi Nirenberg causing foot rot disease in rice as a natural and eco-friendly fungicide. The methanol extract of dropped citrus fruits was prepared by Soxhlet extraction and was further fractionated by column chromatography using petroleum ether, dichloromethane, ethyl acetate, methanol, ethanol and distilled water as solvents. Among all the treatments, the ethyl acetate fraction exhibited the maximum antifungal potential against F. fujikuroi having the least median effective dose at 650 µg/ml. The fraction (1500 µg/ml) showed significantly less disease incidence than the untreated plants in the nursery and transplanted plants of basmati rice cultivar Pusa Basmati 1121 during the kharif seasons 2021 and 2022. The fraction also caused a significant increase in cell constituents release in F. fujikuroi as compared to the control. Scanning electron microscopy analysis also showed that the treated hyphae were shrunken and broken at several points. This finding suggests that the ethyl acetate fraction might act on the fungal cell membrane leading to loss of cell membrane permeability that ultimately leads to cell death. GC–MS analysis of the fraction showed the presence of 3',4',5,6,7,8–hexamethoxyflavone (29.58%), 2-methoxy-4-vinylphenol (22.41%) and 4',5,6,7,8–pentamethoxyflavone (16.78%) as major compounds. Molecular docking of these major compounds with α-β-tubulin (PDB Code: 4FFB) revealed a binding score in the range of -6.1 to -5.3 kcal/mol respectively. Hence, the dropped citrus fruits which are generally discarded as waste may be used as botanical fungicides to control foot rot disease in rice.