Biological Charcteristics And Purification Of Antimicrobial Substance (S) Of Bacillus Strain B₂₁

Bacillus strain B₂₁, which isolated from tomato plants, could inhibit the growth ofRalstonia solanacearum. The biological characteristic and the purification ofantimicrobial substance(s) of Bacillus strain B₂₁ were studied in this dissertation.On the basis of morphologycal, physiological and biochemical characteristics aswell as 16S rDNA sequence, strain B21 was identified as Bacillus subtilis. Strain B₂₁ couldgrow in pH of 4.0-10.0 and at temperature of 10℃-49℃, the optimum pH and temp-erature were 6.0-8.0 and 34℃respectively. It could use many carbons as carbon source,such as sucrose, glucose hydrate, lactose, raffinose maltose, and it could also use manyorgonic nitrogen as nitrogen source, such as peptone, beef extract, yeast extract andcasein. The suitable culture conditions for strain B₂₁ producing antibiotic substance(s)were that the sucrose was used as carbon source, yeast extract and casein was used asnitrogen source, the temperature was 34-37℃, the initial pH was 7.0-7.5, and theculture time was 7d.Antimicrobial substance(s) produced by Bacillus subtiliss train B₂₁ was purifiedsuccessive through steps of ammonium sulfate, DEAE-52 anion exchange chromatog-ramphy and Sephadex G-75 filtration chromatography. The purified substance(s)showed only one spot on silicon thin layer chromatography (TLC). The results of ultafil-tration showed that the substance(s) which molecular weight was smalller than 30 kD orbigger than 50 kD has not inhibitory activity against Ralstonia solanacearum. Determin-ing by SDS-PAGE, the molecular weight of the purified substance(s) was about 40 000D. The inhibitant activity of the crude antimicrobial substance(s) against Ralstoniasolanacearum was depressed partially evidently when it was treated with 100℃for 20minutes, and its inhibitant activity lossed at 120℃for 20 minutes. All those indicatedthat the antimicrobial substances could not endure high temperature. The antimicrobialsubstance(s) was partially sensitive to proteinase K, and was insensitive to trypsin andpronase. The inhibitant active pH range of the antimicrobial substance(s) was wide,from 4.0 to 10.0.The pictures of scan microscope showed that the spores of phytopathogenic fungigerminated abnormally after treated with antimicrobiai substance(s), and the myceliaappeared distortion and the leak of the contents in cells.The antimicrobial substance(s) has inhibitory activity against many plant pathog-enic bacteria and fungi, such as Ralstonia solanacearum E.F.Smith., Xanthomonasaxonopodis pv.Citri, Xanthomonas oryaze pv. oryaze, Xanthomonas oryaze pv.oryaze(Fang et al) Swings et al., Magnaporthe grisea (Hebert) Barr., Exserohilum turcicum(Pass.) Lenonard & Suggs, Bipolaris maydis (Nishik. & Miyabe) shoem., Alternariabrassicae (Berk.) Sacc., Curvularia iunata (Walker) Boedijin, Alternaria musae Bour.etBat., Cercospora musae Zimm., Helminthosporium torulosum (Syd) Ashby, Colletotrich-um coccdes (Wall) Hughes & Colletotrichum sp., Fusarium moniliforme Sheid., Rhizoc-tonia solani Kuhn and Sclerotium rolfsii Sacc..