Screening And Identifying Of Antagonistic Actinomycetes Against Ralstonia Solancearum And Optimizing Of Fermentation Conditions

Tobacco bacterial wilt caused by Ralatonia solanacearum is a bacterial disease. It is widely occurred in Guangdong, Fujian and other southern areas of China. Recently, it has the tendency that expanded into north of China. It has happened and damaged severely in Shandong, Henan and Shaanxi province. It has become one of the major diseases on tobacco. Traditional methods of chemical control is not only has severe impacts on ecological environment as well as high costs, serious pollution and soil ecology, but also affects the quality of agricultural products caused by exceeded hazardous substances. Therefore, it has great significance to screen biological strains which can effectively prevent and cure tobacco bacterial wilt. In order to provide the theoretical basis of biological control of tobacco bacterial wilt, this dissertation focused particularly on studies on the strain identification, optimization of fermentation condition, stability of fermentation products and investigation of growth-promoting effect, the main results are as follows:1. Eighty-seven actinomycetes strains were isolated from different soil samples using dilution method. Plate resist method combined with agar diffusion method were applied to screen antagonists. After primary selection by plate resist method, 6 strains were acquired, among which 3 strains behaved stronger inhibition activities. In the following screen, one strain named Y23 showed the strongest inhibition activity with inhibition zone of 14.8mm.The strain Y23 showed different inhibition activity against many kinds of tobacco pathogenic fungi such as T. basicola, C. nicotianae and A. alternate etc. besides R. solancearum.2. It's morphological, cultural physiological, biochemical characteristics, chemotaxonomy and 16S rRNA sequences analysis were studied. The substrate mycelium have no partition, the aerial mycelium are ramose; the spores are rod-shaped and the surface are smooth. The cell wall typeⅠand sugar type C showed the strain with streptomyces character. The phylogenetic tree analysis from almost complete 16S rRNA showed that the strain S. lavendulae was very similar with 9 typical Streptomyces for 97.9～99.3 % identity. The 16S rRNA sequence data confirmed the generic assignment. In the phylogenetic tree strain Y23 was most closely related to S. lavendulae, sharing 16S rRNA similarity value of 99.3 %. However, the morphological and physiological characteristic between strain Y23 and S. lavendulae revealed a little differences. Based on the polyphase taxonomical data, strain Y23 was primarily identified as S. lavendulae.3. The fermentation conditions of S. lavendulae were studied.The single factor and orthogonal experiment indicated that the optimum fermentation condition of strain Y23 for producing the most effective ferment filtrate were cultured in 1.0 % starch soluble, 0.4 % powdered yeast, 0.025 % K2HP04, 0.05 % MgS04, 0.05 % NaCl, 0.001 % FeS04, the initial pH of 7.0, at 26℃and shaked at 200 r/min for 96 h. These results are valuable to strain application, antibiotics purification and its industrialization.4. Bioassay of the stability of fermented broth of strain Y23 against R. solanacearum. The antibacterial substances from strain Y23 showed stability to high temperature, and it does not lose its antagonistic activity under 100℃in 30 min; There was no significant drop in activity at pH 3, but unstability to high alkali condition and the inhibition rate decreased by 11.25 % at the pH of 10; Proteinase K have little impact on antimicrobial substances. We were also found that the fermentation supernatante at 4℃has better stability.