Screening, Identiifcation And Fermentation Conditions Of Antagonistic Germ Against Setosphaeira Turcica

Northern corn leaf blight which caused by Setosphaeria turcica is an importantfungal disease and severely damaged to corn production. The prevention against thedisease was always through agricultural and chemical measures since long before, butthe effect of prevention was not good. Application of chemical pesticides broughtentironment pollution. Biological control by natural antagonistic organisms is apotential measure against plant diseases. Therebefore, the main of sudy was to getantagonistic bacterias isolated from soil and carried on preliminary research.445bacteria strains were isolated from Jingyu county and Changbai Mountainand23of them showed strong antagonistic effect against the pathogen. CB113wasscreened from these strains through the first screened and rescreened by Oxford cupdiffusion. The strain was further rescreened by using filter paper method and thefermentation and thallus were extracted respectively by ethyl acetate and methanol.Morphological, cultural physiological, biochemical characteristics and16S rDNAsequences analysis were studied. The result showed that it can utilize fructose, sucrose,maltose and cannot utilize D-xylose, D-glucose and mannitol rhamnose etc. Catalase,gelatin liquefaction and citric acid were positive, and V-P, starch hydrolysis, indoletest, phenylalanine dehydrogenase and nitrate reduction were negative. Milk litmustest showed that the strain produced alkaline and proteases, which could becompletely peptonization. Strain CB11316S rDNA sequences was submitted to theNCBI data, the landing No.JN007069. A phylogenetic tree was constructed bycomparing with the published16S rDNA sequences of the related bacteria species.The strain of Pseudomonas syringae pv. Syringae B728and Pseudomonas fluorescensPf-5homology were respectively98.11%and98.11%. CB113was preliminarilyidentified as Pseudomonas fluorescens and was named Pseudomonas fluorescensCB113.The main reason of fluorscenct Pseudomonas can produce multiple antibiotics including phenoxazine, pyoluteorin, pyrrolnitrin, and2,4-diacetylphloroglucinol.Through the petri dish and biochemical properties, the results indicated that the strainproduced siderophore, phosphatase, protease and IAA,and produced pyrrolnitrin, andNPRS,but not produced phenoxazine, PKS,2,4-diacetylphloroglucinol andpyoluteorin.The effects of a number of factors on the production of CB113were studied,including pH, volume of culture media, ultural temperature and time, carbon sourceand nitrogen source. The optimization pH7.0, cultural temperature28℃, and CB113was cultured for60h in flask with1%volume medium. The results showed that theoptimal carbon source and nitrogen source were fructose and peptone. The optimalconditons were as follows: fructose2.5%, peptone1%, K₂HPO₄0.2%, MgSO₄.7H₂O0.1%.The stability of antagonistic substance produced by CB113was studied. WhenpH5-9, the antagonist on the pathogen indicator bacteria of the zone of inhibition didnot change significantly. When increasing temperature, the inhibition zone diameterwas diminished, but the substance still had inhibitive effect on the pathgon. The timeof ultraviolet radition was not significantly the inhibition zone. Results indicate thatthe antagonistic substance is stable against heat, acid and ultraviolet radition.