Abstract:
Citrus is susceptible to infection by a variety of fungi during storage and transportation, leading to severe economic loss. Penicillium italicum is one of two major postharvest diseases of citrus fruits, causing blue mold. It is crucial to seek a safe and effective way to replace or reduce the use of synthetic fungicides for health and environmental concerns. Since the ammonium carbonate can be expected to inhibit P. italicum activity in recent years, it still remains unknown on the specific mechanism and the effect on the fruit quality of different citrus species. Taking the “Navel” orange, “Tribute” citrus, and “Fertile” orange as the research objects, this study aims to investigate the antifungal activity of ammonium carbonate against Penicillium italicum causing blue mold in fruits via interfering reactive oxygen metabolism. A possible mechanism of ammonium carbonate was also clarified to evaluate the storage qualities under the safe and limited growth of P. italicum after treatment. Scanning and Transmission Electron Microscope (SEM/TEM) were utilized to characterize the mycelial morphology and mitochondrial structure. The mitochondrial ATPase activities and H2O2 content were also measured to determine the inhibition of substances against the pathogens. Furthermore, the activity of antioxidant enzymes and the content of reduced glutathione were measured to further clarify the effect of ammonium carbonate on the accumulation of Reactive Oxygen Species (ROS). In addition, an in-vivo experiment was carried out to explore the effects of ammonium carbonate on the storage quality, such as soluble solids, vitamin C, titratable acid, reducing sugar, and color of citrus. The results showed that ammonium carbonate greatly inhibited the spore germination and mycelial growth of P. italicum in a dose-dependent manner. Specifically, ammonium carbonate at 0.4 and 0.8 g/L completely inhibited the spore germination and mycelial growth, respectively. The morphology observation showed that ammonium carbonate caused the growth of mycelia with sparse nodes and fewer branches. Ultrastructural observation showed that the hypha was seriously shrunk to the abnormal structure of mitochondria. Physiological and biochemical analysis indicated that ammonium carbonate treatment caused the decrease of Na+/K+-ATPase, Ca2+-ATPase, and Mg2+-ATPase activities in the mitochondria of hypha, further resulted in the loss of reduced glutathione content and glutathione reductase activity, concurrently interrupted the balance of Superoxide Dismutase (SOD), Catalase (CAT) and Peroxidase (POD) activities in the scavenging system of ROS, and finally to promote the H2O2 accumulation in hypha of P. italicum. Nevertheless, the addition of Cysteine (Cys), a scavenger of ROS, partially restored the spore germination that inhibited by ammonium carbonate. In vivo test, 16 g/L ammonium carbonate treatment significantly reduced the lesion diameter of citrus fruits inoculated with P. italicum (P<0.05), and then alleviated the disease severity in “Novel” orange, “Tribute” citrus, and “Fertile” orange. Correspondingly, the ammonium carbonate treatment can be expected to reduce the natural disease incidence without adverse effects on fruit weight loss, color, and quality parameters, including soluble solids, titratable acid, vitamin C, and reducing sugar contents. These results demonstrated that ammonium carbonate can be used to damage the mitochondrial structure and function of P. italicum, thereby promoting the accumulation of ROS for the antifungal activity. The powerful antifungal activity of ammonium carbonate against P. italicum can offer great potential application in control of postharvest decay of citrus fruits.