Studies On Antifungal Activity And Its Mechanism Of Paeonol Against Sclerotium Rolfsii In Aconitum Carmichaelii Debx.

Southern blight on Aconitum carmichaeli caused by Sclerotium rolfsii is one of the most serious soil-born fungal diseases to Aconitum carmichaeli.There are few targeted studies on the prevention and treatment of southern blight on Aconitum carmichaeli presently.The methods of agricultural control and chemical products control are widely used,but the effect is not significant.Pesticide residues can also lead to problems such as farm Land pollution,increased resistance of pathogenic fungi,and toxicity to non-target organisms.At present,the screening of plant-derived antifugal active substances is still the focus of the development of green chemical fungistat.Because of the green,natural and harm Less advantages,Chinese herbal medicines with antifugal activity have become a hot spot in the research and development of new fungistat particularly.The main active ingredient paeonol extracted from the Chinese herbal moutan cortex has a wide range of highly effective bacteriostatic activities.To this end,this paper takes paeonol as the research object.The antifugal activity of 100μg·m L^-1of paeonol on nine plant pathogenic fungi was determined,and it was found that it had the strongest inhibitory effect on S.rolfsii.The concentration for 50%of maximal effect(EC₅₀),minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）on S.rolfsii were further determined.To explore its antifungal mechanism,the changes of the ultrastructure of pathogenic fungi after treatment with paeonol were observed,and the effect of paeonol on the content of internal substances and key enzyme activities of pathogenic fungi was determined.On this basis,the differentially expressed genes of the pathogenic fungi after treatment with different concentrations of paeonol were analyzed by transcriptome technology.Finally,the effect of paeonol on the growth of target plants and the determination of disease control efficacy were carried out through indoor potting experiments.The study aimed to reveal the antifungal mechanism of paeonol on S.rolfsii,and to provide a new method and theoretical basis for the prevention and treatment of Southern blight on Aconitum carmichaeli.The results are as follows:（1）100μg·m L^-1paeonol had the best inhibitory effect on S.rolfsii,with an inhibitory rate of 86.90%.The EC₅₀ of paeonol against S.rolfsii was 42.54±2.24μg·m L^-1,the MIC was 400μg·m L^-1,and the MBC was 500μg·m L^-1.After the treatment of paeonol,the hyphae growth rate of S.rolfsii was significantly reduced,the number of sclerotia formation days was prolonged,and the number of sclerotia produced decreased.Treatment with vector band toxicity can significantly inhibit the germination of sclerotia.And the germination inhibition rate reached 100%with 2MIC(800μg·m L^-1)concentration paeonol treatment within 96 h.The 24-h immersion method had no inhibitory effect on sclerotia germination.（2）The results of scanning electron microscopy showed that the treatment of paeonol could lead to changes in the morphology of S.rolfsii,the hyphae were shriveled and thinned,and the surface was wrinkled and rough.The results of transmission electron microscopy showed that paeonol treatment could lead to malformation of somatic cells of S.rolfsii,some organelles were lysed,cytoplasm was uneven,and cell shrinkage was accompanied by plasmatic wall separation.After treatment with paeonol,the amount of nucleic acid and protein extravasation in somatic cells of S.rolfsii increased,the content of soluble proteins and reducing sugars in tissues decreased,the content of malondialdehyde and hydrogen peroxide increased,the activity of protective enzymes（catalase,peroxidase and superoxide dismutase）decreased,the content of ATP decreased,and the activity of enzymes associated with sugar metabolism（succinate dehydrogenase and malate dehydrogenase）decreased.And the degree of change was linearly related to the concentration of the paeonol.（3）Transcriptome sequencing shows that S.rolfsii produced 6 998 new genes after treatment with paeonol.There are 1 326 new genes were annotated in functional databases.By comparing with the blank control group,differentially expressed genes were screened.The results showed that there were 4 719 differentially expressed genes in CK＿vs＿1/16 MIC samples,including 2 181 up-regulated genes and 2 538down-regulated genes.CK＿vs＿1/8 MIC samples contained 6 373 differentially expressed genes,including 3 139 up-regulated genes and 3 234 down-regulated genes.CK＿vs＿1/4 MIC samples contained 7 317 differentially expressed genes,including 3 644 up-regulated genes and 3 673 down-regulated genes.CK＿vs＿1/2 MIC samples contained 7 423 differentially expressed genes,including 3 694 up-regulated genes and 3 729 down-regulated genes.GO function annotation results showed that a total of 2 988 differentially expressed genes were enriched in 1 494 GO terms,significantly enriched in metabolic process,cellular process,membrane,membrane part,cell,cell part,catalytic activity,and binding.The enrichment of the KEGG pathway metabolic pathway showed that 2050 differentially expressed genes were mapped to 125 KEGG major metabolic pathways.Up-regulated genes were significantly enriched in ribosome and fructose and mannose metabolism pathways.Down-regulated genes are significantly enriched in oxidative phosphorylation,protein processing in the endoplasmic reticulum,carbon metabolism,peroxisome,and citrate cycle pathways.Therefore,paeonol may have an inhibitory effect on S.rolfsii by destroying the integrity of cell membranes,affecting biosynthesis,inhibiting enzyme activity,and interfering with substance metabolism and energy metabolism.（4）After 7 days of root irrigation with paeonol,it was observed that the Aconitum carmichaeli plants grew healthily and were safe.The efficacy of paeonol on Southern blight on Aconitum carmichaeli can reach 64.55%.Compared with the blank control group,the disease index was reduced by about 55.56%.