Screening, Identification And Fermentation Conditions Of Antagonistic Organisms Against Pseudomonas Solanacarum

Ginger bacterial wilt is a destructive disease caused by Pseudomonas solanacarum (Smith) Smith. The prevention against the disease was always through agricultural and chemical measures since long before, but the effect of prevention was not good. Application of chemical pesticides brought entironment pollution. Biological control by natural antagonistic organisms is a potential measure against plant diseases.In our test an antagonistic actinomyces was isolated from ginger soil and was studied.168 strains of actinomyces were isolated by tube dilution assay from the soil of ginger in Chengdu, Jianyang, Yaan and Hongya. SR-11 strain was screened from these strains of actinomycetes though the first screened and rescreened double with deck agar method and aperture-strikeing method.In the antimicrobial spectrum test, Adopting the method of aperture-strikeing and hyphal extension inhibition assay of plate to test the inhibitive effect on Escherichia coli, Bacillus subtilis, Staphylococcus aureus and some kinds of pathogenic fungi such as Curuvularia Lunata (Walk) Boed, Rhizoctonia solani, Cochlibolus Heterostrophus, Exserohilum Turcicum, Cochliobolus carbonum, Pestalotiopsis foedans, Altalaria altanata, Fusarium graminearum etc.The result showed that it haddistinctively inhibitive effect on gram-positive bacterium, gram-negative bacterium and some kinds of pathogenic fungi. The stability of antagonistic substance produced by SR-11 was studied. The activity of the inhibitive substance was not influenced when in 100℃ for 10 minutes. In 100℃ for 10 minutes,the inhibitive zone diminished distinctly,but the substance still had inhibitive effect on the pathgen. In pH 2.0~11.0 for 30 minutes or treated by Trypsinase and Proteinase K,the inhibitive zone is not changed. Results indicate that the antagonistic substance is stable against heat and acid and alkali and enzyme.Traditional phenotype classification, chemotaxonology and modern molecular taxonology were used to study the strain SR-11.It's morphological, cultural physiological, biochemical characteristics, chemotaxonomy and 16S rDNA sequences analysis were studied. The substrate myceliums have no partition, the aerial myceliums are ramose; The spore-bearing filaments are spiral, the spores are oval and the surface are smooth. Cell wall type I , Sugar type C. When SR-11 is matured, the aerial myceliums are gray and the strain can give out an earthy smell. A phylogenetic tree was constructed by comparing with the published 16S rDNA sequences of the related bacteria species. In the phylogenetic tree the overall similarity value between strain SR-11 and 12 type Streptomyces sp are 96.5-98.3%.The effects of a number of factors on the production of Streptomyces SR-11 were studied, including pH, seed volume, volume of culture media, ultural temperature and time, carbon source, nitrogen source. The results showed that the optimal carbon source and nitrogen source were glucose, soybean powder and peptone, respectively. The optimization pH7.0~7.4,cultural temperature:28℃, and Streptomyces SR-11 was cultivated for 96h on 140r/ min shaker in flask with 8%~12% volume medium. On the basis of these, the recipe of fermentation medium was optimized through uniform -design. A suitable fermentation condition was found for antibacterial production of strain SR-11. The optimal conditions were as follows: peptone0.3%, soybean powderO. 8%, glucose 2. 8 % , NaCl 0. 50 %.