| Fungus disease is the main factor of crop yield loss. Many strategies have beeninvestigated to manage the disease. Among them, though high effect, chemical control iscost high and causing environment pollution. Biological control, by using low molecularfungicide pepotide may not cause environment pollution, chemical substance resistanceand also has stable control effect.Endophytic Bacillus subtillis BY-2 was isolated from oilseed rape, and hadbiocontrol effect to fungi disease. After return inoculation treatment to ilseed rape, thesame endophtie B. subtilis as BY-2 obtained. In 10 days after inoculation, the number ofendophytic bacterium could reach up to 10~3cfu/g fresh plant, and after 30 days, thenumber of bacteria was maintained 10~3cfu/g fresh plant. It was also found colonizinginside other crops, including soybean, corn and wheat. BY-2 was plant endophyticbacterium and was also the main colony of plant rhizosphere, it could reproduce aroundoilseed rape rhizoshpere, and enter plants by natural hole, wound and meristem. Thebacterium number of BY-2 could reach to 10~9/g soil in the rape rhizosphere. Comparing toanother PGPR Pseudomonus alcaligenes A9, its number adhering to root surface was alsohigher.BY-2 was antagonist to 15 main crop fungi pathogen, including Scleorotiniasclerotioum of oilseed rape, Thanatephorous cucumeris (Frank) Donk of flee, Fsariumoxysporum f.sp. vasinfectum of cotton and so on. In the confronting incubation againstS.sclrotioum on PDA plates, the inhibiton zone reached to 3.7cm. Meantime, myceliummalformation of S.sclerotiorum caused by BY-2 was observed, such as short growth,cytoplasm condensation, cell wall break and protoplasm leak. Even germination ofsclerotia was also inhibited. BY-2 could also induce increase of plant resistance-relatedenzymes such as PPO, POD and PAL.In LB borth, cultured under 28℃, 150rpm/min, in 48hrs, BY-2 reach logarithmicphase, and bacterium number could reach 10~10/ml. The antiobitic substance yield gothighest after 72 hour's culture. Though the bacterium grew fastest at 37℃, yield andactivity of antibiotic substance got the highest at 28℃. BY-2 could grow normal in pH5-9,it grew slowly in pH3 and better in pH6-7. The best growth condition for BY-2 wascultured under 150rpm/min, 120ml broth in 250ml flask.BY-2 could produce two kinds of antibiotic substance, one of which was a volatilecomponent that could effectively inhibit growth of S. sclerotiorum and the other wassecreted into the culture medium. The extracellular secreted substance was the mainantibiotic substance of BY-2. This kind of substance could be collected by adjust theculture supernant pH to 3 to precipitate it, and be extracted by methanol. The extracted antibiotic substance had same size of inhibition zone against S. scleotiorum as BY-2culture supernant.The extracellular antibiotic substance could be purified by methods of acidicprecipitation, methanol extract, Sephadex G25 chromatography column separation. Thepurified antibiotic substance formed a peak at 1.969min in HPLC when methanol andwater used as the fluent phase. When tfifluoroacetate-methanol was used as the fluentphase, peak at 1.969min in the fuent phase of methanol and water was separate to 6 peak,their molecular mass were 995,1009,1023,1037,1051 and 1087Da, respectively. Itconsisted of Aln, Gln, Thr and Lle, and the ratio was Aln:Gln:Thr:Lle=1.1:1:1.5.According to the amino acid constitute analysis and molecular mass analysis, theantibiotic substance fell into the analogous of lipopeptied compound. Their molecularstructure were similar with surfactin's in lipopeptide though the rate of amino acid wasdifferent from the other members of surfactin and the higher molecular homologous1051Da and 1087Da were not reported before. The antibiotic substance was resistant tohigh temperature and ultraviolation, active in broad pH range, stable in organic solventand could keep long time.BY-2 and its antibiotic substance could effectively control a lot of fungi diseases.The control efficacy against S. scletotiorum was 100% on detached leaves in vitro. Itcould reduce 86.4% of the incidence ofS. scletotiorum on oilseed rape in the pot test, andaslo reduced incidence of stern wilt in the field. When used in the same concentration, thecontrol efficacy of the antibiotic substance was better than BY-2 culture after theconservation of several weeks. Besides oilseed rape S. scletotiorum, BY-2 and itsantiobiotic substance could also control S. scletotiorum of sunflower and soybean,Thanatephorous cucumeris (Frank) Donk office, Bipolaris maydis. |
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