Supplementary file 1_Plumbagin elicits evidence of apoptosis-like cell death and inhibits conidial germination and mycelial growth in ginger rot isolate Fusarium strain GI-FS1.zip

Ginger (Zingiber officinale) is an economically important spice crop widely cultivated for its culinary and medicinal values. However, its productivity is severely constrained by soft rot disease, causing substantial yield losses, quality, and persistent challenges in ginger cultivation. In this study, the major pathogenic fungus GI-FS1 was isolated from Z. officinale with typical symptoms and was identified as Fusarium species through morphological and molecular identification. The antifungal potential of plumbagin, a plant-derived naphthoquinone from Plumbago rosea, noted for antibacterial, antioxidant, and anticancer properties, was evaluated against GI-FS1. Plumbagin treatment significantly inhibited conidial germination and mycelial growth with Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values of 10 µg/mL and 15 µg/mL, respectively. Conidial germination assays, microscopy, onion peel epidermis penetration confirmed inhibition, as plumbagin-treated spores failed to germinate and penetrate epidermis. 4’,6-Diamidino-2-phenylindole (DAPI) staining showed chromatin condensation and Acridine orange/ethidium bromide (AO/EB) revealed membrane disruption, coupled with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) detected Reactive oxygen species (ROS) accumulation, signifying oxidative stress-induced cell death by plumbagin. Elevated electrolyte leakage and DNA fragmentation supported apoptosis-like mechanisms underlying fungal growth inhibition; however, further studies on apoptotic factor gene expression are required to confirm this mechanism. Scanning Electron Microscopy (SEM) analysis revealed spore shrinkage and thinner, collapsed, rough mycelia, indicating loss of cell integrity on treatment with plumbagin. Overall, these findings provide the first report on the targeted mechanisms of plumbagin in inhibiting Fusarium conidial germination and inducing apoptotic-like cell death. By disrupting fungal viability through oxidative stress and apoptosis-related pathways, the study highlights plumbagin’s potential as a natural antifungal agent for the sustainable management of soft rot disease in ginger.