Induced cell death in Ceratocystis fimbriata by pro‐apoptotic activity of a natural organic compound, perillaldehyde, through Ca2+ overload and accumulation of reactive oxygen species

Abstract

Black rot caused by the fungus Ceratocystis fimbriata causes notable losses in sweet potato production. Perillaldehyde (PAE), a secondary metabolite in perilla, was studied to determine its antifungal effects on mycelial growth and spore germination of C. fimbriata. The effects of PAE on cell wall integrity and cell membrane permeability were also investigated. To elucidate the possible mechanisms of cell death triggered by PAE, sensitivity of C. fimbriata to PAE toxicity was determined by cytoplasmic and mitochondrial calcium ion concentrations ([Ca2+]c and [Ca2+]m), reactive oxygen species (ROS), mitochondrial membrane potential (MMP), cytochrome c (cyt c) release, metacaspase activation, phosphatidylserine (PS) externalization and DNA fragmentation. The results suggest that mycelial growth and spore germination were inhibited by PAE in a dose‐dependent manner. Ceratocystis fimbriata spores treated with PAE experienced dramatic Ca2+ overload and elevated ROS production. Compared to untreated controls, the proportion of fluorescent cells stained with the ROS indicator DCFH‐DA and treated with a range of PAE concentrations from 0.0625 to 0.50 μL mL−1 increased by 2.9 ± 0.79% to 27.1 ± 0.38%. Ca2+ overload and ROS accumulation induced depolarization of the MMP, contributing to mitochondrial dysfunction. Cyt c was released from the mitochondria to the cytosol, triggering metacaspase activation. The significant antifungal activity of PAE on C. fimbriata was demonstrated by these studies, suggesting that PAE has the potential for wide application to postharvest management of tuber crops, in addition to the application to above‐ground fruit and vegetables that have been previously investigated.