Study On The Antibacterial Mechanism Of Griseofulvin Targeting β-tubulin Of Didymella Segeticola, The Pathogen Of Tea Leaf Spo

Due to the lack of effective control measures,tea leaf spot caused by Didymella segeticola has reduced the quality of tea in Guizhou Province.Screening efficient biogenic pesticides can effectively control the damage of disease and ensure the quality and safety of tea in Guizhou Province.This study focused on D.segeticola,a pathogen of tea leaf spot,to study the bacteriostatic mechanism of biogenic pesticide griseofulvin against D.segeticola,and the research results were as follows:（1）The bacteriostatic activity of griseofulvin against D.segeticola was determined by mycelium growth rate method.The results showed that griseofulvin could inhibit the growth of D.segeticola mycelia,and its EC₅₀ value was 0.37μg/m L.By optical microscopy,scanning electron microscopy and transmission electron microscopy,griseofulvin was found to cause mycelial deformation,which was characterized by slight curling,enlargement of the terminal,agglutination of contents in mycelia,and increased number of mycelial protuberances.Griseofulvin changes the morphology and subcellular structure of mycelia.（2）The transcriptome and proteome data of D.segeticola treated with griseofulvin showed that intracellular material transport,cell movement and mitosis were all regulated by griseofulvin.Based on the metabolic pathway,nine differentially expressed genes were randomly selected and verified by RT-q PCR,and the expression trend was consistent with that of transcriptome sequencing.（3）The interaction between griseofulvin and D.segeticolaβ-tubulin was determined by Microscale thermophoresis,the results showed that Kd_{（β-tubulin1）}=0.094±0.029,Kd_{（β-tubulin1）}=5.177±1.983 and Kd_{（β-tubulin2）}=7.301±2.005.The results indicated that Kd value of griseofulvin binding toβ-tubulin1 was significantly lower than that ofβ-tubulin2.Therefore,it is speculated that griseofulvin may interfere with chromosome separation and fungal mitosis byβ-tubulin1,thus inhibiting mycelial growth.（4）The binding site of interaction between griseofulvin and D.segeticolaβ-tubulin was simulated by molecular docking,andβ-tubulin Gly140,Val169 and Asn204were mutated to obtain mutant plasmids,and three mutant proteins were expressed and purified.Then,the results of microthermal motion-determination are as follows:Kd_{（β-tubulin）}=0.094±0.029,Kd_（G140D）=1.771±0.617,Kd_（V169F）=0.183±0.070 and Kd_（N204I）=2.208±0.691,It showed that the Kd values of griseofulvin and three mutant proteins were lower than those of wild type and griseofulvin after mutation of the 140th Gly,the 169th Val,and the 204th Asn ofβ-tubulin.It indicated that the main binding sites of griseofulvin andβ-tubulin were the 140th Gly,the 169th Val and the 204th Asn.