Studies About Antifungal Mechainsm Of ?-pinene Against Penicillium Of Citrus

Citrus fruits are highly susceptible to Penicillium italicum during transportation and storage.The existing research showed that ?-pinene,the active ingredient in pine needles,has a good inhibitory effect on P.italicum.However,there were few studies on the mechanism of ?-pinene against P.italicum.Therefore it is necessary to explore the action mode of ?-pinene for developing natural product fungicides for postharvest disease control of citrus fruits.In this paper,the effects of ?-pinene on mycelia respiration,mitochondrial activity and electrpn transfer chain complexes activities and the effects of ?-pinene treatment on cell membrane lipid components and functions were evaluated,in order to reveal the mechanism of inhibitory effects of the volatile component.The main results are as follows:(1)?-pinene has a strong inhibitory effects on P.italicum mycelium respiration,in a concentration dependent and time dependent manner.By investigating the synergistic effects of ?-pinene and six inhalation inhibitors,it was found that the possible action sites of ?-pinene on P.italicum were the tricarboxylic acid cycle,glycolysis and the pathway from NADH to coenzyme Q on the electron transfer chain.To confirm this conclusion,we determined the activity changes of succinate dehydrogenase,and malate dehydrogenase,which reprensent TCA cycle activity,ATPase representing electron transport chain activity and 6-phosphate fructokinase,which represent glycolysis activity.The results showed that under the treatment of ?-pinene,the activity of malate dehydrogenase decreased and the succinate dehydrogenase increased first and then decreased,the activity of ATPase decreased and 6-phosphate fructose kinase increased significantly,which indicated that ?-pinene inhibited TCA cycle and glycolysis pathway.The contents of ATP,ADP and AMP in mycelia of P.italicum were measured,the content of ATP under the treatment of ?-pinene was significantly decreased,AMP increased significantly and the energy level decreased with the incubation time.These results demonstrated that ?-pinene incubation disturbed of P.italicum,which results in cell dysfunction.(2)Mitochondria are the main organelle of energy metabolism and play a very important role in cell function.The effects of ?-pinene on mitochondria functions of the pathogen were further investigated.The results showed that under the treatment of ?-pinene,the mitochondrial respiratory rate of P.italicum hyphae continued to decline,in a concentration dependent paterm.Further investigation on the activity of mitochondrial respiratory chain enzyme complexes of the pathogen hypae showed that ?-pinene selectively inhibited the activity of the enzyme complex I,complex II and complex IV,which indicated that the mitochondrial electron transportion was restrained,and the possible action site were in complex I,complex II and complex IV.?-pinene also induced overloading of mitochondrial matrix Ca2+,leading to mitochondrial swelling,promoted mitochondrial permeability transition pore(MPTP)opening,and triggered apoptosis of mycelia cells.The results showed that the damage of mitochondrial function is important inhibitory mechanism of ?-pinene on P.italicum.(3)Cell membrane composition and functions of mycelia of P.italicum were changed under ?-pinene treatment.The content of soluble protein and cellular nucleic acid in mycelia reduced with the incubation of ?-pinene.Nucleic acid substance in mycelium was leaked and the membrane permeability was increased which indicated the damage of function of cell membrane.The contents of ergosterol and total lilpids in mycelia were dicreased with beta pinene treatment.GC–MS analysis of the lipid composition showed that ergesterol content of the treated mycelia was much lower than the control groupn,while,the contents of squalene and lanosterol which are ergosterol precursors did not change significantly.The contents of some ergosterol relevants in the biosynthesis pathway accumulated under the effect of ?-pinene.The content of cholesterol increased slightly and the content of some cholesterol relevants reduced.These results show that cell membrane component changed significantly under the effect of ?-pinene.In conclusion,?-pinene inhibited the respiration of P.italicum and had inhibitory effects on TCA and glycolytic pathway,and the key enzymes,which caused energy metabolism disorder of P.italicum.?-pinene selectively inhibited mitochondira the activity of the enzyme complex I,complex II and complex IV,caused mitochondrial calcium overload and made mitochondrial permeability transition pore(MPTP)open.And finanly may cause apoptosis of P.italicum.