Identification Of A Antagonistic Bacterium Strain And Preliminary Study On Its Antagonistic Substances

Both the planting area and totle yield of potato in China are at the first place in the world. Now the potato crop is the fourth major crops,fowling wheat,rice and corn.It mainly grows on drouthy and semiarid areas with cool climate in Ninmeng,Yunnan,Guizhou provinces and northwest,northeast of China. Since potato in these areas as the traditional food are nutritious and high-yield. Jixi county is "Potatoes' country in China" in Ningxia province. But dry rot of potato tube disease on potato wildly spead in recent years,which influence potatoes' postharvest storage and production.At present, using medicament to prevent and cure the pathogen is the main means towards postharvest disease. But pesticide usage do harm to human health conditions.The main aim of the experiment is to isolate and select antagonistic bacteria against fungicidal disease on dry rot of potato. Exploit the bacteria as biologic pesticide which is safe to people and high efficacity to dry rot of potato tube disease.Some "Non-clear organism" were discovered since 1992 in China.Although its essence is still unknown, many antagonistic organisms are found in it. Thus, the "Non-clear organism" is a finer experimental material to isolate and select antagonistic bacteria against dry rot of potato tube disease. A total number of 48 bacterial strains were isolated from the "Non-clear organism",within which 19 strains are Bacillus. As Bacillus strains propagate quietly and produce spores which is easy to storage and transport, they are advantageous to industrial produce. Six strains had antagonistic activity against the potato dry-rot disease by plate test. According to the result of the suppression assay, strain B₁₃ was selected as the one of the best biocontrol effect with 16mm diameter of inhibition zone. Through indoor experiment, it had a strong antagonistic action to tested pathogens and bacteria, especially to dry-rot of potato and tobacco wildfire disease. Also its biocontrol ability is very steady during its propagation.After knowing the useful strain's systematic position,then we can know the strain's inherent performance,which is benefit for exploiting product. In terms of the traditional identification way, according to its morphological, cultural, and physiological biochemical characteristics of B₁₃, plus its 16SrDNA sequence, it was identified as Bacillus subtilis.Generally,natural microorganism strains evenly metabolize and unable to accumulate overmuch production.When altering their growth condition, some production can be possibly excessively accumulated. The fermented conditions for strain Bo's growth and producing fungi-antagonistic active substance were determined and the results are as follow. The compositions of fermentation medium are: beaf extract 0.3%, Tryptone 0.5%, glucose 1%, NaCl 0.5%, yeast extract 0.1%, pH7.0. When the fermention time is 48h,the optimal original culture pH is 7.2 and cultivation temperature is 37°C, and the optimal load is 500mL flask contains 100mL medium when the shaking speed is 180r/min and the optimal volume of inoculum is 7%. Under these conditions the strain can grow better and gain the highest antifungus activity with 22mm diameter of inhibition zone.The property of the antagonistic metarial is also studied in this experiment.By centrifugation B₁₃'s fermentation fluid and removing the bacterium, (NH4)₂SO₄ which end concentration achieves 70% degree of saturation was joined into the upper liquid. Over night, collecting precipitates and dissolving with water solution. Its UV scanning spectrum shows that the precipitates have absorption at point 280nm. Two apexes appear by Sephadex G 75 gel filtration chromatography. The antifungus activity of apex I is lower than apex II. The precipitates were purified by SDS-PAGE which showed three trips with each molecular weight of 175kDa, 62kDa and 28kDa.But the three proteins are not identified in limited time and condition which is a pity to construct better strain by geen engineering methods. So the main task to continue the research is to identify the proteins.Knowing the proteins' synthetical pass way and their regulative mechanism to construct antibiotics high-expression Bacillus subtilis B₁₃ ,which has significant sense to the strain's industrial application.