Study On The Secondary Mechanism Of Streptomyces HN6 Metabolite YY3 Against Fusarium Oxysporum F.Sp. Cubense

Banana wilt disease is caused by Fusarium oxysporum f.sp.Cubeme(FOC)infestation,and is the main disease in the global banana planting industry.The research on the prevention and control of banana wilt disease is of great significance to ensuring the healthy development of the banana industry system.In the early stage of the research group,the high antifungal active macrolide compound YY3 was isolated from Streptomyces griseus HN6,The compound has a broad spectrum of antifungal activity,and its antimycotic activity against banana withering is especially high.However,the mechanism of action is not yet clear,which limits the further development and application of this compound.In this study,Fusarium oxysporum f.sp.Cubeme race 4(Foc4)was used as the target fungi to study the antifungal mechanism of this compound in order to lay a theoretical foundation for clarifying the mechanism of action of this compound and provide scientific reference for green prevention and control of banana wilt disease.The specific research content is as follows:1.The effect of YY3 on the mycelium growth and spore germination of Foc4 was measured.The results showed that the compound could significantly inhibit the growth of mycelia of the target fungi with an EC50 of 20.8 ?g/mL,and significantly inhibited the spore germination of the target fungi with an EC50 of 12.62 ?g/mL.2.The effect of YY3 on the structure of Foc4 cells was determined.Ultrastructural observation revealed that the cell wall of the target pathogen became thinner after YY3 treatment;the organelles in the mycelia foamed and disintegrated and the mitochondria abnormally increased;the cytoplasmic density increased.Finally,cell tissue collapsed.Physiological and biochemical tests showed that after treatment with YY3 at a concentration of 50 ?g/mL,the N-acetylglucosamine content in the mycelia increased 1.62 fold,indicating that the compound induced the activation of the target fungal chitinase and interfered with the cell wall synthesis of the target bacteria.However,the compound had no significant effect on ?-1,3-glucanase;the conductivity of the target fungi increased by 25.43%after treated with YY3 at a concentration of 50 ?g/mL,and the compound could destroy the cell membrane structure of the target fungi.3.The effect of metabolite YY3 on the biosynthesis of Foc4 was determined.The results showed that the content of total sugar,protein and fat in the target fungi gradually decreased with the increase of the treatment concentration of the drug.At the dose of 50 ?g/mL,the total sugar content decreased by 29.7%,the protein content decreased by 22.9%,and the fat content was 39.6%.It can be seen that under this concentration treatment,fat is most affected.4.The effect of metabolite YY3 on the biooxidation of Foc4 was measured.The results showed that at the concentration of 50 pg/mL,YY3 had no effect on the activity of complex enzyme ?,?,? of mitochondrial respiratory chain,but it significantly inhibited the activity of mitochondrial complex III and complex enzyme V,in which complex enzyme In activity was reduced by 60.49%and compound enzyme V activity was reduced by 51.5%;at the same time,the compound could also reduce the activity of succinate dehydrogenase(SDH),a key enzyme in the Krebs cycle,by 56.6%.The compound had no significant effect on malate dehydrogenase(MDH).In conclusion,YY3 has a significant effect on Foc4 biooxidation.5.The effect of metabolite YY3 on the apoptosis of Foc4 was determined.At the test concentration of 50 ?g/mL,the membrane potential on the mitochondrial inner membrane was significantly reduced after the mycelium was treated for 2 h.Meanwhile,Reactive oxygen species(ROS)gradually accumulated in the Foc4 hypha,and the burst occurred after 6 h incubation of the drug.which was 3.23 times that of the control.After the Foc4 mycelium was incubated with YY3 for 12 h,the Foc4 mycelial DNA strands of the treated group were found to have been broken by TUNEL assay.In this study,we investigated the antifungal mechanism of Streptomyces HN6 secondary metabolite YY3 on Foc4 from the perspective of cell structure and ultramicromorphology,biosynthesis,biooxidation,and apoptosis.Finally,we determined the antifungal mechanism of YY3 against Foc4,including the destruction of the plasma membrane and mitochondrial dysfunction,eventually induce apoptosis of cells.