The Research On Formulation Application Technology Of Bacillus Subtilis Strain TR21and Its Active Substances Purification

It was found that Bacillus subtilis strain TR21had good control effect on banana fusarium wilt in pot and field experiments. In order to get basic date and scientific guidance of TR21preparations for field production, the effect of some popular bactericides, fungicide, fertilizers and plant growth regulator inbanana planting on the survival of TR21spores in wettable powder were tested. Then based on the fact that TR21had the capacity to induce banana producing induced systemic resistance, a new formulation-suppository had been produced with the raw powder of TR21, and its control effect were carried out by pot experiment. Finally, antagonistic protein and lipopeptid produced by TR21were isolated and purified to provide theoretic support for the improvement of the TR21formulations. The results as follows:1. The influences of popular bactericides, fungicide, fertilizers and plant growth regulator used in banana planting on the survival of TR21spores in wettable powder(WP). The results showed that the plant growth regulator which contained0.136%gibberellin, indoleacetic acid and brassinolide WP had no influence on the survival of TR21spores. TR21spores’survival was not influenced after they had been treated for7days by two kinds of bactericides which was80%oxytetracyclini hydrochloridum WP and20%thiodiazole-copper SC but it was significantly influenced when the spores had been treated for15days by other bactericides which contained2%kasugamycin and45%copper oxychloride WP. Fungicieds which main component was20%carbendazim and30%thiram WP had significant influence on the survival of TR21spores after treated by1days and7days; three kinds of fertilizers have no influence on survival of TR21spores.2. The suppository preparation of strain TR21and its pot experimental control effect. Suppository of TR21spores was made by mixed4000D polyethylene glycol and TR21raw powder, but its potted control effect was not very good. The results showed that60days after inoculation of the pathogen, the control effects of raw powders and suppository of TR21were9.68%and-14.52%respectively.3. The antagonistic protein of TR21. The crude antagonistic protein was precipitated by20%ammonium sulfate and had antagonistic function on Fusarium oxysporum f. sp. cubense Race4strain FOC009. Its antifungal activities was persistent when heated at100℃for30min, or treated with UV radiation for30min. Furthermore, its antagonistic capacity remained strong in a wide pH range from6.0to10.0or after treated by trypsase, pepsin and protease K. Then the protein was isolated and purified by ion exchange and hydrophobic role chromatography, and SDS-PAGE showed the molecular weight of the purified protein was about36kDa.4. The lipopeptid produced by TR21. Lipopeptid was crude extracted from TR21culture and was detected by AB4000Q TRAP mass spectrometer. The results showed that the lipopeptid material contained surfactin, which was a kind of systemic induce elicitor produced by Bacillus. Surfactins produced by TR21included three homologues with molecular weight of1008D,1022D and1036D.