Breeding And Mechanism Of Bacillus Megaterium Antagonistic Sclerotium Rolfsii

Sclerotium rolfsii is a serious soil-borne disease that can infect more than 500plants.In this paper,Bacillus megaterium L2 was mutagenized by atmospheric pressure room temperature plasma?ARTP?.The positive mutant strains with strong antagonistic effect on Sclerotium rolfsii R-67 were screened by plate screening,shaking flask screening and genetic stability test.Then the broad-spectrum bacteriostasis,phosphorus metabolism capacity,phenylacetic acid production capacity,and environmental tolerance were found out the difference in bacteriostatic properties before and after mutagenesis of L2,and a preliminary investigation of the antibacterial mechanism of phenylacetic acid on R-67,a monomer compound isolated from L2.The results were shown as follows:1.Three positive mutant strains with strong antagonistic effect on R-67 and good genetic stability strains 30s-56,40s-22 and 60s-60 was screened by plate primary screening,liquid shaking flask screening and passage culture for 10generations.Compared with the original strain,their inhibition ability were respectively increased by 45.49%,56.70%,45.99%.2.The results of the broad-spectrum bacteriostatic experiment showed that,using the original strain as a control,the positive mutant 30s-56 had an increase of9.20%in its bacteriostatic capacity against Phytophthora nictotianae PN1201.the positive mutant 40s-22 increased the bacteriostatic ability of Sclerotium rolfsii R-67by 31.80%,and increased Raltionia solanancearum Q11-2 by 7.90%.the positive mutant 60s-60 increased the bacteriostatic ability of Alternaria alternata A-3 by85.06%and Erwinia carotoovora EC-1 by 46.36%.However,each strain had no inhibitory effect on three pathogenic bacteria:Agrobacterium tumefaciens T-37,Escherichia coli,and Staphylococcus aureus.3.Phosphorus metabolism experiment results show that the positive mutant strains 30s-36,40s-22,and 60s-60 have similar phosphorus metabolism capacity at 10h.Although they are stronger than the original strain,the difference is not significant?p>0.05?.The ability of Bacillus to metabolize phosphorus was basically the same,and ARTP mutagenesis did not have much effect on phosphorus metabolism.4.The results of environmental tolerance showed that when the temperature exceeds 35°C,the p H exceeds 7,and the Na Cl concentration exceeds 1%,the bacteriostatic rate of the original strain and the positive mutant strains 30s-36,40s-22,60s-60 begin to decrease.In the environment of heavy metal Ba⁺,it has higher biomass and bacteriostasis rate.In the environment of heavy metal Ag⁺,the biomass and bacteriostatic ability of each strain is low,and each strain cannot grow in the environment of heavy metal Pb⁺,Cu⁺,Zn⁺.The positive mutant strain showed stronger antibacterial ability to R-67 than the original strain in different environments.5.The results of phenylacetic acid production of Bacillus megaterium showed that the content of phenylacetic acid in the culture medium of the original strain was200.25 ng/m L;the positive mutants 30s-36,40s-22,and 60s-60 were 203.51 ng/m L and 430.50 ng/m L,623.47 ng/m L,respectively.The ability of each mutant strain to produce phenylacetic acid was improved compared to the starting strain,of which40s-22 and 60s-60 were increased by 114.98%and 211.35%respectively compared to the starting strain.6.The bacteriostatic mechanism of R-67 by phenylacetic acid,a monomeric compound of Bacillus megaterium,the EC₅₀of phenylacetic acid to R-67 was 0.475mg/m L.R-67 was found to have smaller colony diameters,irregular edges,and looser hyphae than the blank control after 5 days.After 15 days of treatment,the number of nodules decreased,which was only 25.96%of the blank control?p<0.05?.The effect of phenylacetic acid on R-67 increased the content of extracellular electrolytes and soluble sugar,and the mycelium appeared wrinkles,deformities,dissolution,protoplast coagulation,swollen apex.and the content of malondialdehyde and ROS in the bacteria increased,speculating that phenylacetic acid can stimulate Excessive oxygen free radicals produced by R-67 trigger membrane lipid peroxidation,destroy cell membrane integrity,and leak intracellular electrolytes and macromolecular carbohydrates,resulting in cell death.Moreover,it was found that the content of soluble protein and key enzymes of tricarboxylic acid cycle?SDH,MDH?in R-67bacteria decreased,speculating that phenylacetic acid can also affect R-67 protein synthesis and energy metabolism to exert bacteriostatic effects.