Inhibition Mechanism Of Endophytic Bacillus Subtilis Itb57 On Tobacco Phytophthora Nicotianae

Tobacco black shank is a typical soil-borne disease caused by tobacco P. nicotianae, Pathogens can produce zoospores, breed fast and large, it can infect and damage tobacco of the whole growth period. The main methods of controlling tobacco black shank have agricultural control, chemical control and biological control, biological control of plant disease control is the hotspot for international research. It is significance for tobacco yield and farmer’s income to develop effective bio-control agents antagonistic tobacco black shank.Bacillus subtilis Itb57 is isolated from healthy tobacco plants, previous studies have showed that the strain effects on tobacco black shank disease prevention and control of the greenhouse effect reaches 69.3%, and the field control effects for two consecutive annual average above 60.0%, it indicates that the control effect is stable, therefore, this paper further explore the mechanism of inhibit the growth of the fungus from cell morphological, physiological and biochemical level of P. nicotianae and antagonism material types of Itb57 strain and so on, provide a theoretical basis for the use of biocontrol bacteria. The conclusions are as follows:l.The effect of Itb57 strain fermentation filtrate on P. nicotianae cell morphologyAfter treatment with Itb57 fermentation filtrate, the P. nicotianae mycelial growth was significantly inhibited, the inhibition rate was 39.6%; With different concentration of fermentation filtrate of the P. nicotianae mycelial, the results showed that with the increase of concentration of fermentation filtrate, sporangium formation inhibition rate, sporangium germination inhibition rate and resting spore germination inhibition rate increased gradually, When the concentration of fermentation filtrate was 50%, sporangium formation inhibition rate reached 79.7%, the sporangia germination inhibition rate was 70.8%, the concentration of 50%fermentation filtrate cultivate resting spore 8 h, its germination inhibition rate was 59.6%, and the microscopic observation found Itb57 strains fermentation filtrate smaller influence on the morphology of the fungus sporangium.2.The effect of Itb57 strain fermentation filtrate on P. nicotianae physiology and biochemistryAfter treatment with Itb57 fermentation filtrate,The main component of the cell wall of P. nicotianae mycelial β-1,3- glucan content was significantly lower than the control, when the culture filtrate concentration of 5%, the alkali-soluble β-1,3- glucan, alkali insoluble β-1,3- glucan soluble β-1,3- glucan content was 7.2%,16.7%,12.3%, compared with the control, decreased by 51.2%,42.5%,39.4%; With ferment filtrate concentration increases, P. nicotianae β-1,3- glucan synthase activity decreased, while the cultivation of the fungus silk solution conductivity value and malondialdehyde content increased gradually, the membrane showed that the cell membrane integrity was damaged, membrane lipid peroxide was serious; With concentration of 5% fermentation filtrate, hyphae DNA content compared to was reduced by 14.6%. P. nicotianae mitochondrial complex enzyme I, complex enzyme II, complex enzyme III, IV activity was significantly lower than the control; hypha protein compared with no significant difference.3.Research on Itb57 strain of antibioticsItb57 strain fermentation filtrate by the concentration of 20%,30%,40%,50%, 60%,70% ammonium sulfate precipitation after collecting crude extract, antagonism activity test results found that 40% of the ammonium sulfate from crude extract on the growth of P. nicotianae had inhibitory effect, when the concentration of 70% ammonium,the inhibition effect was the best, the bacteriostatic circle diameter of 1.5 cm; With the increase of the concentration of sulfuric acid, and the resulting crude extract protein content also increased; crude protein SDS-PAGE electrophoresis results showed that the size of the relative molecular mass of approximately 45 kDa protein antagonist activity may be associated with related; Molecule detection had found Itb57 strains produce spore binding protein (TasA) gene.Itb57 strains fermentation filtrate through acid precipitation collecting fat peptides crude extract, with P. nicotianae as indicator, the bacteriostatic circle diameter of 1.2 cm, lipopeptide crude extracts by HPLC analysis, preliminary determined Itb57 strains fermentation filtrate of surface active element; And molecular detection has found Itb57 strains produce surface active element (srfA) gene.