| Apple tree canker disease(caused by Valsa mali) commonly occurred in China, in the resent past severely restricted the development of the apple industry. A number of effective strategies have been developed to control the disease of apple tree canker, including the use of chemical fungicides. However, they have series of problems including environment and food safety concerns. Thus, it is urgent to develop a novel, effective, and safe bio-control agent to control apple tree canker disease. The Bacillus amyloliquefaciens strain TS-1203 was identified as a possible biocontrol agent for Valsa mali because in invitro experiments showed that has an inhibitory effect on the pathogen of apple tree canker. Therefore, the objectives of the present study were to develop and analyze the stability of and active antimicrobial based on Bacillus amyloliquefaciens TS-1203. The main results were as follows:1) Stability of active antimicrobial substance and antibacterial spectrum determination:Different physical and chemical factors had different impact on the stability of an active antimicrobial substance based on B. amyloliquefaciens TS-1203. Physical factors, such as natural light had no significant effect on the stability of the active antimicrobial substance. However, the inhibitory effect of the biocontrol agent on Valsa mali was decreased significant significantly when exposed to 100℃ or irradiated for 180 s with UV light. Among all the chemical factors, the inhibitory effect on Valsa mali was not decreased when the p H ranged from 6 to 10, or when treated with metal ions including Mg2+ and Fe3+. On the other hand, treatment with 80 % ammonium sulfate had significant impact on the inhibitory rate of TS-1203 crude extraction decreasing it by 7.23 %. Also, ethyl acetate and acetone significantly decreased the inhibitory effect of TS-1203 crude extraction on Valsa mali. Moreover, the crude extraction of TS-1203 had higher inhibitory effect on some species of plant pathogens, especially the inhibitory rate on the plant pathogens of Rhizoctonia solani and Alternaria alternata were more than 80 %.2) Active antimicrobial substance isolation, purification and identification:The optimal extracting agent for isolating the active antimicrobial substances from the TS-1203 strain of B. amyloliquefaciens was ethyl acetate, and the optimal separating solvent for purifying the active antimicrobial substances was selected as petroleum, acetone, and chloroform at a ratio of 24: 9: 1. Lipopeptides and more than ten species of volatile substances were isolated and identified by GC-MS technology; some included hydrocarbons, benzene, alcohols, amines, sulfides and esters. Three species of volatile substances were selected from the volatile substances to determine their inhibitory effect on Valsa mali. The results showed that xylene had the highest inhibitory effect on Valsa mali growth, and the inhibitory effect was 100 %, while the isopropanol and phenylacetamide had lower inhibitory effect in comparison with xylene, and also the inhibitory effect were 62.67 % and 19.00 %, respectively.3) Optimization of bio-control agent:0.50 % twain-80, 0.10 % sodium citrate and 0.50 % lignosulfonate were selected as the surfactants, preservatives and UV protectant respectively to develop the bio-control agents. The inhibitory rate of the bio-control agent prepared in this way was more than 80 %. Meanwhile, central composite design combined with response surface methodology was applied to optimize the best adjuvant condition to develop the bio-control agents of TS-1203. The predicted and the actual results had a good match, and resulted in the best combination to be in 65 m L fermentation of TS-1203, which contained 650 μL of twain-80, 165 mg sodium citrate, 308 mg lignosulfonate; the inhibitory rate was verified as 83.98 % under optimal combination condition. In addition, sodium alginate has significantly inhibitory effect on the number of colonies growth in comparison with control, while the inhibitory effect of polyvinyl alcohol and sodium carboxymethylcellulose were lower than sodium alginate. Thus, combining both agents and active antimicrobial substances, and including organic bentonite, polyvinyl alcohol and sodium carboxymethyl cellulose as carrier, was possible to develop and effective bio-control.4) Development of bio-control agent:All the index for the bio-control agent of TS-1203 wettable powder were in accordance with the quality indices of national regulation, which include the index of p H at 6.86, and the suspension percentage of 75.82 %, wetting time of 84 s, fineness of 99 %, and the active ingredient contents at 1.43×108 cfu/g, which was slightly lower than national regulation. Compared with bentonite, the film-forming properties of polyvinyl alcohol andcarboxymethyl cellulose sodium were better judging by visual analysis, which was consistent with the quality assessment of smearing the preparation. In addition, compared with room temperature, there has no significant difference between the two preparations of wettable powder and smearing preparation when stored at 52(±2) ℃ and 4(±2) ℃ for 14 d. |
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