Antagonistic Substances And Screening Of Rhizospheric Spore-producing Bacilli Against Tobacco Brown Spot

669 rhizospheric spore-producing bacilli were obtained from healthy tobacco roots. By dual culture on PDA plates and determining inhibition activity of antibiotic substances, it was found that 9 potential biocontrol isolates showed good antagonistic effects to Alternaria alternata, the pathogen of tobacco brown spot, and the diameter of inhibition zone of bacteria and crude antibiotic substances was up to 10 mm. Inoculation of detached leaves of tobacco variety K326 with bacterial suspension and crude antibiotic substances of B6 and B75 resulted in control effect reaching 75.6%, 76.9% and 62.5%, 64.7%, respectively. Results in field experiment showed that preparation of B6 and B75 reduced the incidence of tobacco brown spot to 70.3%, 75.8% and 60.3%, 64.4%, respectively. Crude antibiotic substances of B75 could inhibit mycelium growth, sporulation and spore germination of tobacco brown spot pathogen.Classical physiological and biochemical methods, Biolog, and 16s rDNA analysis were used to identify the strain B75 which could control tobacco brown spot disease. Physiological and biochemical identification showed that it belonged to Bacillus subtilis, Biolog also showed it was Bacillus subtilis. 16s rDNA analysis was used for further identification, which showed the 16s rDNA sequences of B75 shared 99.8% homologic with published sequence of B subtilis from Genbank, and both sequences constituted a branch in Phylogenetic tree. Based on these results, it is considered that the strain B75 belongs to B subtilis.An antagonistic strain B75, isolated from tobacco rhizospheric soil, began to produce antagonistic substance (AS) at logarithmic growth phase, and reached a peak before growth stopped in NB medium. In 50-250ml NB medium of 500ml flask with pH 4.0-8.5, B75 could produce AS at 25-50℃, while 100ml medium in 500ml flask, pH 6.0 and 30℃were the best conditions. Among 17 carbon and 6 nitrogen source tested, mannitol and yeast extract were demonstrated to be more suitable for production of B75 AS.With 40% (NH₄) ₂SO₄ fractional precipitation, after 8,000r/min centrifugation, the crude antagonistic substance of Bacillus subtilis B75 was obtained. The crude extract was thermostable, moreover it inhibitory activity was also stable at 120℃30 min. The more pH up-graded, the more solubility of the crude extract increased, and its activity decreased at pH 8 in room temperature. Enzymic hydrolysated by protease, pepsin and proteinase K, its activity not decreased. The dissolved matter with PBS (pH6.8) was separated by gel filtration on sephadex G25 column, and the crude extract was further purified with source 15 phenyl column on FPLC and RPC18 chromatography on HPLC,. The molecular weight of the purified matter is 1463.61 and 1477.82 according to Q-TOF2. And the purified matter was identified as fengycin by Q-TOF2 and ApexQ FT-MS analysis.Antagonistic substances were precipitated from liquid culture filtrates of Bacillus subtilis B75 with ammonium sulfate and hydrochloric acid, and exacted from its whole cells with phosphate buffer (50 mmol/L, pH 6.8) , methanol, ethanol, acetonitrile, acetone, dichloromethane, trichloromethane and ethyl acetate. The precipitation and exacts were detected of biological activities, and analyzed with RP-HPLC. The results showed that the biological activity of the precipitation of ammonium sulfate was stronger than of hydrochloric acid, the exacts of methanol, ethanol, acetonitrile and acetone had biological activities, and their activities were lower than of the precipitation of ammonium sulfate and hydrochloric acid. At the identical conditions of RP-HPLC, the impurities of the precipitation of ammonium sulfate was more than of hydrochloric acid, activity peaks of the precipitation of ammonium sulfate and hydrochloric acid existed fine distinction, activity peaks of liquid culture filtrates of Bacillus subtilis B75 were distinctly different from its whole cells, and activity peaks of the exacts of methanol, ethanol, acetonitrile and acetone could superimpose completely. These results illustrated that antagonistic substances existed in liquid culture filtrates and whole cells of Bacillus subtilis B75, and antagonistic substances of liquid culture filtrates were distinctly different from whole cells.