Mechanisms Of Biological Control Agaist Peanut Stem Rot By Pseudomonas Chlororaphis ZM-1

Peanut stem rot is a serious plant disease caused by the pathogen Sclerotium rolfsii,which brings serious losses to agricultural development.At present,the traditional chemical control methods for this disease have disadvantages such as environmental pollution and increased drug resistance of pathogenic bacteria.As an important plant root biocontrol bacteria,Pseudomonas has been proven to effectively inhibit pathogenic bacteria and promote plant growth.Therefore,research on the physiology and function of this type of biocontrol bacteria is important for the development of new biological control of plant diseases means are important.On the other hand,biocontrol bacteria are subject to a variety of stresses from the external environment during their survival,and bacteria have also evolved corresponding mechanisms to deal with them.Therefore,the research on the coping mechanism of exogenous stress is more conducive to the exertion of the biocontrol effect of bacteria.A strain zm-1 with bacteriostatic effect against various pathogenic fungi was isolated in our laboratory before,and it was identified as a subspecies of Pseudomonas chlororaphis.After analysis,the strain has phenazine antibacterial substances and genes encoding superoxide dismutase,so it is speculated that it has the ability to secrete antibacterial antibiotics and cope with exogenous oxidative stress.In order to determine whether it can effectively inhibit Peanut stem rot,we analyzed the functions and roles of related genes through various research methods such as bioinformatics,molecular biology and microbiology.The specific results are as follows:(1)Pseudomonas chlororaphis zm-1 can produce extracellular antifungal substances.Through antagonism experiments,it was found that both the cells and extracellular extracts of wild-type zm-1 could inhibit the growth of S.rolfsii.The components of extracellular antibacterial compounds were identified by ultra-high liquid chromatography-mass spectrometry as 1-hydroxyphenazine,phenazine-1-carboxylic acid and core phenazine.(2)Genes phzE,phzH encode the production of related phenazines.According to NCBI analysis,the phenazine biosynthesis proteins in zm-1 are highly homologous to the anthranilate synthase family proteins from Pseudomonas aeruginosa.This family of proteins has been reported to be associated with the phenazine-1-carboxylic acid-producing Pseudomonas chlororaphis.The phenazine-specific anthranilate synthase in zm-1 is highly homologous to the phenazine-type biosynthetic protein of phzE from Pseudomonas chlororaphis.Therefore,two mutants,ΔphzH and ΔphzE,were constructed,and the extracellular compounds of the two mutants were analyzed.It was found that ΔphzE did not produce any phenazine compounds,and ΔphzH no longer produced 1-hydroxyphenazine,but still produced phenazine-1-carboxylic acid and core phenazine.(3)Genes phzE and phzH are involved in the bacteriostatic and biocontrol processes of Pseudomonas chlororaphis.The antagonistic capacity of ΔphzH decreased,while of ΔphzE almost completely disappeared.The results of pot experiment and field experiment under greenhouse conditions showed that the biocontrol effect of ΔphzH was significantly lower than that of wild-type Pseudomonas chlororaphis zm-1(75.63%)to 47.21%.In addition,ΔphzE almost completely lost its ability to inhibit S.rolfsii(its biocontrol effect was reduced to 6.19%).(4)The genes sodA,sodB encode different superoxide dismutases in zm-1.Superoxide dismutase has been shown to be highly conserved and present in almost all aerobic organisms.Bioinformatics analysis showed that Sod A and Sod B belonged to Mn SOD and Fe SDO.Both proteins were determined to have superoxide dismutase activity by activity assays.(5)The genes sodA and sodB are involved in the regulation of zm-1’s physiology and response to oxidative stress,and the gene sodB plays a major role in this process.To verify the function of the two encoding genes,two single knockout mutants,ΔsodA and ΔsodB,were constructed and their various phenotypes were evaluated.The results showed that the genes sodA and sodB affected the movement ability,protease production ability and biofilm formation of zm-1,which indicated that both genes were involved in the physiological process of zm-1.During the experiment of the bacterial response to oxidative stress,it was found that the ability of ΔsodA to cope with oxidative stress when the gene sodA was deleted was only slightly different from that of the wild-type zm-1,while when the gene sodB was deleted,the mutant was very sensitive to oxidants.In the presence of VK3,both ΔsodB solid spot plates and liquid shake bacteria exhibited growth defects.Thus,it was shown that the gene sodB plays a key role in the antioxidant effect of zm-1,which was further confirmed by subsequent Quantitative Real-Time PCR(q RT-PCR).(6)The genes sodA and sodB further affected the biocontrol ability of the bacteria by regulating zm-1 in response to oxidative stress.Oxidative stress is one of the important environmental stresses that biocontrol bacteria need to deal with when they play a biocontrol role.Combining the roles of the two genes in oxidative stress,we speculate that they may maintain the homeostasis in bacteria by removing superoxide anions,thereby ensuring the The normal performance of subsequent biological defense capabilities.During the biological control experiment of Peanut stem rot under greenhouse conditions using two superoxide dismutase mutants and wild-type zm-1,it was found that wild-type zm-1 inhibited the growth of S.rolfsii growth,while ΔsodA,ΔsodB aligned with S.rolfsii.Therefore,it is speculated that the superoxide dismutase gene can ensure the normal function of the bacteria by regulating the bacteria’s response to oxidative stress,and provide a guarantee for the biological control of Peanut stem rot disease.These results help to further understand the biocontrol properties of Pseudomonas chlororaphis zm-1 and lay a theoretical foundation for further research.Based on the above research results,we concluded that Pseudomonas chlororaphis zm-1has the potential to control Peanut stem rot disease.The phenazine derivatives produced and secreted by zm-1 play a key role in the control of peanut scabies caused by S.rolfsii.In addition,superoxide dismutase,a key enzyme in the antioxidant system of bacteria,also provides a guarantee for maintaining the normal function of the bacteria and the exertion of biocontrol capabilities.These findings provide new ideas for the effective control of Peanut stem rot disease.