Study On The Inhibition Mechanism Of Chelerythrine And Its Derivatives Against Plant Pathogens

Plant diseases seriously threaten to global food security and ecosystems.Chemical pesticides have become the most effective method to control crop diseases.However,repeated use of chemical pesticide over a long period of time may result in pesticide resistance and harmful residues to human health and food safety.Therefore,it is necessary to develop new pesticides to control crop diseases.Plant secondary metabolites have been paid more and more attention in research,which are used to control pest and disease in agriculture.Among them,alkaloids possess several biological activities,including antiviral,anticancer and antifungal.Research and development of botanical fungicides is of far-reaching ecological significance.Chelerythrine(CHE)is one of the most important alkaloids in Chelidonium maj us L.,and CHE is a natural benzophenanthridine alkaloid.Dihydrochelerythrine(DHCHE)is a reductive product of CHE,which is a derivative of CHE.Rice false smut is a serious disease in rice,this study aiming at the two alkaloids in Chelidonium majus L.,Ustilaginoidea virens(U.virens)for the antimicrobial test indoor and outdoor field control test,found chelerythrine base to U.virens fungus spore germination inhibition rate is as high as 86.7%,two kinds of alkaloid induced the rice germ cell apoptosis.The main research content is as follows:In this paper,the antibiotic activities of these two alkaloids were studied,including the following aspects:(1)Extract of Chelidonium majus L.was extracted by ultrasonic extraction method and the extract was identificed by high performance liquid chromatography(HPLC),infrared radiation(IR)spectroscopy and nuclear magnetic resonance(NMR).It was proved that the extract was CHE.The antibiotic activity of CHE against pathogens was researched in vitro.It was found that the minimum inhibitory concentration of citrulline against Xanthomonas oryzae(X.oryzae)was up to 7.5×10-3 mg/mL,and it was revealed that CHE inhibited U.virens with 50%effective concentrations(EC50)of 6.53×10-3 mg/mL.CHE displayed the obvious inhibitory activity against U.virens at the concentration of 7.5×10-3mg/mL,compared with the other derivatives and commercial fungicide validamycin,with a inhibition rate of 56.1%,which was about twice that of validamycin.It was found by accident that DHCHE had better bacteriostatic effect.Moreover,CHE(4×10-3 mg/mL)exhibited the greatest efficacy in inhibiting spore of U.virens growth with an inhibition rate as high as 86.7%.(2)After treating U.virens with CHE,the morphological changes of fungal hyphae and spores were observed.Twisted and atrophied mycelia were observed by optical microscopy.Scanning electron microscopy results demonstrated narrow and locally fractured mycelium.Transmission electron microscopy observations showed that the cell wall had become thin and broken.CHE affected the cell membrane permeability.At 280 nm,the light absorption value of each group treated with CHE was significantly higher than that of the control group.Flow cytometry detection found that with the increase of CHE concentration,reactive oxygen species(ROS)continuously accumulated,mitochondrial membrane potential decreased,and the proportion of cell damage increased.A total of proteins were identified in CHE-treated U.virens by Tandem Mass Tags(TMT)quantitative proteomics and 823 proteins were identified as differentially expressed proteins(DEPs).The corresponding bioinformatics analysis was presented,including Gene Ontology(GO)and KEGG(Kyoto Encyclopedia of Genes and Genomes).It was found that SNARE in vesicle transport pathway and oxidative phosphorylation pathway had significant effect on funngal apoptosis.Although current studies have shown that the CHE causes protein differential expression,further exploration is necessary.(3)The antifungal activity of DHCHE against five fungal pathogens was researched in vitro,revealing that the EC50 of DHCHE inhibited U.virens was 6×10-3mg/mL.DHCHE exhibited the greatest efficacy in inhibiting mycelium growth with an inhibition rate as high as 68.8%to U.virens at the concentration of 7.5×10-3 mg/mL,compared with the other four isoquinoline alkaloids and commercial fungicide validamycin,which was more or less 2.4 times of that of validamycin(28.7%).The inhibiting rate of spore germination increases with increasing concentrations of DHCHE.As the DHCHE concentration was 4×1 0-3 mg/mL,the inhibition rate reached 62.70%to U.virens.At the same time,the ultrastructure of mycelia and spores after treatment was observed.Mycelia appeared atrophied and collapsed with their outer structure became fractured and rough under scanning electron microscopy.Transmission electron micrographs observation revealed that DHCHE caused incomplete organelles,malformed cell walls and nuclear membranes,and irregular lipid spheres.Then,reactive oxygen species(ROS)and mitochondrial membrane potential were detected by flow cytometry.ROS accumulation was significantly higher than that of the control group,and the dose-dependent increase of DHCHE induced damaged cells was observed.All the proteins were identified in DHCHE-treated U.virens by Tandem Mass Tags(TMT)quantitative proteomics and 311 proteins were identified as differentially expressed proteins(DEPs).The corresponding bioinformatics analysis was presented.It was found that oxidative phosphorylation,mismatch repair and ubiquitin pathway had great influence on apoptosis.Although current studies help to elucidate the mechanisms associated with antifungal and apoptosis of DHCHE on U.virens,further explorations are essential,thereby aiding in the potential development of novel pesticides.(4)The field control effect of dispersible oil fungicides prepared with CHE as the original drug on Pseudomonas syingae pv.tabaci and U.virens was evaluated.The control effect of Pseudomonas syingae pv.tabaci was significant,and the control effect of 20%treatment group B was up to 73.34%.It has a certain field control effect on U.virens,and the control effect is close to 50%after spraying with the agent 2000 times liquid with 20%content.Further research will be carried out in order to provide theoretical basis for the control of plant diseases and the processing and production of plant pesticides.