Study On Optimization Of Fermentation Conditions And Biocontrol Mechanisms Of The Actinomycetes Strain LG-9 To Cotton Verticillium Wilt

Verticillium wilt of cotton,causedbyVerticillium dahlia Kleb.,which seriously restricts the development of cotton industry.At present,there is no ideal chemical agent to control Verticillium wilt of cotton.Our laboratory screened a strain of actinomycetes LG-9 with inhibitory effect against Verticillium dahlia.It was proved that the strain LG-9 could significantly inhibit Verticillium dahlia.Objectives:In order to provide theoretical and practical basis for biological control of cotton Verticillium wilt,the work was done from 4 aspects:Optimization fermentation conditions of actinomycetes LG-9;determination of antimicrobial activity of fermentation liquid toward Verticillium dahlia;determination of effects of fermentation liquid on cotton seedling promotion and the inducement of cotton seedling resistance;preliminary analysis on the properties of antimicrobial substances.Methods:Single factor,Plackett-Burman design,steepest grade and response surface design were chosen to select the optimal fermentation medium for LG-9 and to optimize fermentation conditions;The inhibitory effect of fermentation liquid on Verticillium dahlia was determined by mycelial growth inhibition method,spore germination inhibition method,biomass inhibition method;Evaluating the effection of LG-9 againsting cotton Verticillium wilt by pot and field ferment filtrate root-drenching test;The effect of fermentation liquid on the germination of cotton seed and the growth of cotton seedling was studied by soaking seed and seeding root-drenching,and measuring the length of root and hypocotyl of cotton seed and the biomass of cotton plant on the ground and underground;The dynamic of peroxidase?POD?,polyphenol oxidase?PPO?,phenylalanine ammonia-lyase?PAL?,catalase?CAT?and H₂O₂ content in cotton seedlings were determined after ferment filtrate root-drenching;The antibacterial activity substance properties of LG-9 fermentation liquid were studied by solvent extraction and ammonium sulfate precipitation method.Results:The most suitable fermentation medium of LG-9 was selected from 10 mediums,that was millet medium,and the the diameter of the inhibition zone was the largest,21.0 mm.The optimal fermentation conditions of the strains LG-9 were loading volume of liquid medium 101.19 mL in 250 mL triangle bottle,pH7.2,inoculation amount of 7.5%,temperature 31.32?,rotate 160 r/min,time 69.02 h,the diameter of the inhibition zone was 22.45 mm under these conditions.The inhibitory rate of 5 times-fold dilution of antagonistic LG-9 fermentation liquid on the mycelial growth of Verticillium dahlia was the largest?76.77%?,the inhibition rate of spore yield was 84.35%,the inhibition rate of spore germination was80.00%,and the inhibition rate of biomass was 88.22%.The activity of superoxide dismutase?SOD?,CAT,PPO decreased and malonaldehyde?MDA?content was increased of Verticillium dahlia by 5 times-fold dilution of LG-9 fermentation liquid.The pot trials control efficiency of two inoculation methods of LG-9pro-inoculation and mixed inoculation with Verticillium dahlia were 42.86%and 47.17%,respectively.Field experiment showed that the ferment filtrate of LG-9 exhibited a protective efficacy of 49.07%against cotton Verticillium wilt after the 3 times of root-drenching.Cotton seeds soaking in the 5,25,100,and250-fold dilutions of LG-9 fermented liquid promoted the germination of seeds and the growth of radicles and hypocotyl.At 72 h,the germination rate of cotton seeds ranged from 28.3%to 51.7%,the radicle length of cotton seedlings ranged from 4.15 to 4.74 cm and the hypocotyl length ranged from 4.08 to 4.61cm.The plant height of cotton,the fresh weight of aboveground and underground parts,the dry weight of ground and underground and the length of main root were increased by 5 times dilution solution of LG-9ferment filtrate root-drenching alone and mixed inoculation with Verticillium dahlia.The treatment of the root of the ferment filtrate could induce the increase of POD,PPO,PAL and CAT enzyme activity,and induce the active oxygen outbreak in a certain period of time.The fermentation liquid was sensitive to high temperature,insensitive to trypsin and pepsin,and slightly sensitive to proteinase K.The antibacterial activity of fermentation liquid under-20 and 4 degrees Celsius cannot be kept for a long time.Antagonistic substances can dissolve in methanol and acetonitrile,which belong to polar substances.The active substance in LG-9 fermentation liquid could be precipitated by ammonium sulfate;the strongest inhibition activity was under the ammonium sulfate condition of 80%.Conclusion:The actinomycetes strains LG-9 fermentation liquid had significant antibacterial activity in vitro for Verticillium dahlia.The most suitable medium for producing antibacterial substance from LG-9was millet medium.The LG-9 fermentation conditions were optimized by the response surface methodo logy,and the antibacterial activity of the fermentation liquid increased by 10.42%before the optimization.The fermentation liquid could promote the germination of cotton seed and the growth of cotton seedling,induced the increase of resistance enzymes of cotton seedling and the content of reactive oxygen,and had better control effect on cotton Verticillium wilt.The results showed that actinomycetes LG-9 had good potentiality in preventing and controlling cotton Verticillium wilt of cotton.