Mutation Breeding Of Streptomyces Albospinus CT205 And The Application In Preventing Celery Root Rot

High-yield production level of biocontrol bacterium is an important indicator for the development of new soil-borne disease biological agents.Streptomyces albospinus CT205 is an important biocontrol strain.Its metabolized antibiotic is Cycloheximide.At present,it is used as a protein.inhibitor in medical and applied to the prevention and control of agricultural diseases and insect pests.The purpose of this study was to improve the production performance of wild strain CT205 by mutagenic screening and optimization of fermentation conditions,compare the difference of volatile organic compounds(VOCs)produced by mutant strains and wild strain,discusses the CT205 metabolites on resistance of plant system,and preparation of mutant strain biological inoculants for prevention and control of celery root rot.The main results were summarized as follows:Total 124 mutant strains were obtained through four mutation breeding methods of acridine orange,microwave,UV and UV-LiCl.After initial screening of yeast antagonistic circle,re-screening of liquid shake flask fermentation,and content stability test,a high-yield Cycloheximide mutant strain U-L-4 was obtained.The content of Cycloheximide was increased from 27.0 mg/L to 42.0 mg/L.In order to further increase the content of Cycloheximide produced by mutant strain,the single factor experiment was used to optimize the liquid fermentation conditions of the mutant strain U-L-4.The optimized U-L-4 liquid fermentation formula(g/L)was:mannitol 50,peptone 2,CaCO3 3.0,NaCl 2.0,MgSO4.7H2O 0.5,K2HPO4 0.5.The initial pH of the medium was 7.5,the liquid volume was 50/500 mL,the inoculation volume was 10%,and the content of Cycloheximide in the fermentation broth was 64.22 mg/L at 28 ℃ for 5 days,which was 140.74%higher than that from CT205.The methods of determining Cycloheximide were compared,and it was found that High Performance Liquid Chromatography(HPLC),biological activity and Spectrophotometry all showed a great linear relationship with Cycloheximide standard,of which the correlation coefficient was R2HPLC method(0.9992)>R2 biological activity method(0.9937)≈R2 spectrophotometry(0.9965).The methods of HPLC and biological activity weresuitable for determination of the active substances in CT205 fermentation broth and crude extracts,and Spectrophotometric method is not suitable for more samples to detect the contents of impurities such as fermented liquid.The inhibition zone against Yeast of mutant strain AO-9 obtained by acridine orange mutation-induced mutation was larger than that of CT205,but the content of Cycloheximide was lower than CT205,and it was speculated that VOCs of the mutated strain might be different.The inhibition effect on pathogens of VOCs produced by the biocontrol strain was studied using flat-plate buckling test,it was found that the VOCs produced by mutant strains U-L-4 and AO-9 could significantly inhibit the growth and spore formation of F.oxysporum cucumber compared with the wild strain CT205.U-L-4 was more effective than AO-9.The VOCs produced from mutant strains U-L-4,AO-9 and wild strain CT205 were detected and contrasted by GC-MS.It was found that the amount of alcohols(2-methylisoborneol,etc.)and olefin substances(2-methyl2-borneol,etc.)produced from the mutant strains AO-9 and U-L-4 was significantly increased compared with the wild strain CT205,among which,the olefins produced from the mutant strains AO-9 and U-L-4 increased by 40.71%and 32.47%respectively compared with the wild strain CT205;alcohol substances compared with the wild strain CT205,it increased by 1.10%and 0.63%respectively.The Eremophilene and(-)-β-pinene had strong antibacterial activity,and the yield of the mutant strains AO-9 and U-L-4 increased by 6.75%and 0.25%compared with the CT205.Further,it was confirmed that the mutant strain AO-9 had a large inhibition circle for the Yeast,and the amount of Cycloheximide was lower than that of the wild-type strain,which was caused by the production of volatile substances with high antibacterial activity had increased.To explore the effects of active substance Cycloheximide and VOCs produced by S.albospinus CT205 on cucumber plant defense system.The results showed that the activity of Phenylalanine ammonia lyase(PAL)and Peroxidase(POD)enzymes in treatments such as,25mg/L Cycloheximide,Compound VOCs,25mg/L Cycloheximide+Compound VOCs and BTH were significantly higher than the control,which increased by 179.56%,35.35%,95.57%,83.14%;and 86.16%,77.57%,97.10%,99.49%respectively.At 24 h,the activity of Polyphenol oxidase(PPO)in each treatment was significantly higher than the control.which increased by 28.82%,33.09%,17.28%and 79.19%respectively.At 96 h,the activity of Superoxide dismutase(SOD)in each treatment was significantly higher than that in the control,which increased by 11.72%,412.25%,122.97%and 57.42%respectively.The content of salicylic acid content in cucumber leaves increased significantly compared with the control.Among them,the maximum value was 0.3729 μg/g in 25 mg/L Cycloheximide+Compound VOCs treatment for 96 h,which was 9.8 times higher than the control.It was preliminarily explained that the active substance produced by strain CT205 could induce the systemic resistance of cucumber.U-L-4 biology agent was prepared by solid-state fermentation(soil and sand:wheat bran:peanut cake:coconut bran:millet:meat and bone powder=4:2:1:1:1:1:1),and The number of spore was up to 8.0×108 CFU·g-1 after cultured for 14 days.In the fieldtext of celery,it was found that the number of pathogenic fungi in rhizosphere soil of plants was significantly reduced,and the yield of celery per mu was increased by 75%.The biology agent had a certain role in promoting growth while preventing and controlling diseases.