| Ailanthus altissima,belonging to the family Simaroubaceae,is a fast-growing deciduous tree that is native to China.Alkaloids are the main bioactive substances found in A.altissima,and have attracted extensive attention in agriculture for their antifungal activity.However,the antifungal components in the alkaloids of A.altissima and their mechanism are still unclear.In this study,canthin-6-one was isolated and purified by analyzing the inhibitory effect of crude alkaloids on plant pathogenic fungi,and its extraction process,antifungal activity,stability and biological control were further discussed.The antifungal mechanism of canthin-6-one was explained from the aspects of mycelial growth,cell membrane changes,physiological metabolism and proteomics,etc.Finally,in order to evaluate the application of canthin-6-one as a fungicide,the effect of canthin-6-one on plant growth physiology were studied.The main results are as follows:1.The mycelial growth rate method was used to analyze the antifungal effects of alkaloids isolated from A.altissima on 5 kinds of plant pathogenic fungi.Vacuum liquid chromatography(VLC)was used to isolate the antifungal components.The results showed that the crude alkaloid exhibited strong inhibitory effects on Fusarium oxysporum f.sp.cucumerinum,Fusarium graminearum,Fusarium oxysporum f.sp.benincasae,and Gloeosporium sp.A single alkaloid compound was separated from the crude alkaloids by vacuum liquid chromatography technology.After recrystallization and purification,the purity of this compound was over 98%detected by high performance liquid chromatograph(HPLC).This compound was finally identified as canthin-6-one,and its antifungal effect was better than that of crude alkaloids.2.A method for the determination of canthin-6-one in the stem bark of A.altissima was established by HPLC.In addition,the ultrasonic-assisted extraction process parameters of canthin-6-one were optimized by central composite design-response surface methodology.The results showed that the method established by HPLC to determine the content of canthin-6-one in the stem bark of A.altissima had good precision and simplicity.Optimum extraction conditions were as follow:ethanol concentration 68%,liquid-solid ratio 32 mL·g-1,ultrasound temperature 54?,extraction time 38 min;under these conditions,yield of canthin-6-one was 26.19?g·g-1,3.The antifungal activity of canthin-6-one against Fusarium oxysporum f.sp.cucumerinum was evaluated by micro-dilution method,mycelial growth rate method,and slide germination method.In addition,the influence of external factors such as ultraviolet radiation,temperature,acid-base conditions on the antifungal stability of canthin-6-one was analyzed.The results showed that canthin-6-one exhibited excellent competences for suppressing the growth of the pathogens with the MIC of 32 ?g·mL-1.This compound could obviously inhibit the growth of mycelium and spore germination,and the EC50 value was 4.2 ?g·mL-1 and 14.6?g·mL-1,respectively.The antifungal activity of canthin-6-one was not significantly altered at lower temperatures(4?50?).In contrast,canthin-6-one was sensitive to ultraviolet irradiation.Canthin-6-one maintained a high antifungal activity in neutral and acidic environments,and decreased slightly in the dark at 25? within 6 months.Cucumber seeds were treated with canthin-6-one to investigate the occurrence of Fusarium wilt.The results showed that canthin6-one significantly reduced the incidence rate and disease index of cucumber fusarium wilt.At the concentration of canthin-6-one 32 ?g·mL-1,the incidence rate was reduced by 53.6%,and the disease index was reduced by 70.7%.4.The antifungal mechanism of canthin-6-one was analyzed by detecting the activity of relative enzymes and the content of some substances.Furthermore,label-free quantitative proteomics and parallel reaction monitoring(PRM)techniques were applied to analyze the antifungal mechanism at protein level.The results showed that canthin-6-one induced the tested fungus to produce reactive oxygen species(ROS),which disturbed the dynamic balance and damaged the integrity of the membrane system.Canthin-6-one disturbed the synthesis of sugar and protein,and inhibited the activities of succinate dehydrogenase(SDH)and malate dehydrogenase(MDH)in respiratory metabolism.In addition,it interfered with ATP synthesis and ATPase activity in energy metabolism.Proteomic analysis showed that the expression of 203 proteins altered significantly after canthin-6-one treatment.These differentially expressed proteins were mainly involved in amino acid biosynthesis and nitrogen metabolism pathways.Therefore,it affects nitrogen nutrients and disturbs the normal physiological processes of fungi,and ultimately leads to the death of pathogens.5.Cucumber seeds were treated with canthin-6-one to detect the physiological indexes of cucumber seedling growth.The results showed that the normal growth of cucumber seedlings can be maintained within the treatment concentration range.Canthin-6-one did not significantly affect seed germination,seedling plant height and fresh weight.The content of vitamin C and total polyphenols increased after canthin-6-one treatment,catalase(CAT)and ascorbate peroxidase(APX)activities increased significantly,while superoxide dismutase(SOD)and peroxidase(POD)remained stable.The activities of polyphenol oxidase(PPO)and phenylalanine ammonia-lyase(PAL)were also significantly increased compared to the control,which maybe improve the disease resistance of seedlings.In summary,the alkaloid compound canthin-6-one was successfully isolated from the stem bark of A.altissima,which has the characteristics of strong inhibitory ability against plant pathogenic fungi,antifungal stability and easy degradation.The low-concentration treatment of this compound did not have a significant effect on plant growth,but improved the plant's disease resistance.The antifungal mechanism of canthin-6-one was explained from the physiological,biochemical and protein levels of the tested fungi,which provided a new idea and theoretical basis for the further development and utilization of alkaloids in A altissima. |
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