Screening Of Marine Antagonistic Bacteria And Its Inhibition Of Sclerotinia Sclerotiorum

Sclerotinia sclerotiorum is an important plant pathogenic fungi that cause a variety of plants such as rapeseed, soybean diseases, and can survive in the soil for a long time as Sclerotia. It’s incidence is difficult to prevent. Marine Bacillus amyloliquefaciens GM-1strain, which with a strong inhibition of Sclerotinia sclerotiorum and other plant pathogens, is islotated from marine water of Lianyungang. Antibacterial mechanism and stability of the fermentation broth were systematically studied. And liquid flask fermentation conditions was optimized. The main contents and conclusions are as follows:1. Isolation of biocontrol marine microorganisms to Sclerotinia sclerotiorum. The220marine microorganisms were isolated from the marine samples collected from the sea area of Lianyungang, consisting of171bacteria,32actinomycetes, and17fungi.34strains(about15.45%of the total isolates) of them had antimicrobial activity by means of antagonistic effect, and the isolation quantity of antimicrobial active strains from bacteria was highest (27strains). After duplicate sieves,8strains(consisting of6bacteria,2actinomycetes) were observed inhibiting Sclerotinia sclerotiorum and8other plant pathogenic fungi obviously. Finally, the GM-1strain has the strongest antifungal activities to9plant pathogenic fungi and broad-spectrum antibacterial.2. This study aimed to identify GM-1strain’s species. The isolates were belong to Bacillus sp. by means of phenotypic characteristics observation, physiological and biochemical indexes determination. In the16S rDNA sequence and gyrB sequence analysis, these sequences fragment of16S rDNA sequence and gyrB amplified from total DNA of strain GM-1by PCR. Then these sequences analysis were down by the application of BLAST on the websites of NCBI and the phylogenetic tree was drawn.The results showed that GM-1strain shared99%homology with Bacillus amyloliquefaciens(AB014097.1). Combined with the results of phenotypic characteristics observation, physiological and biochemical, GM-1strain was identified as a Bacillus amyloliquefaciens. 3. Inhibitory spectrum and stability of Marine Bacillus amyloliquefaciens GM-1strain fermentation broth were studied. Oxford cup method was used to measure inhibitory spectrum on10plant pathogens and Growth rate method was used to determine stability of Bacillus amyloliquefaciens GM-1strain on Sclerotinia sclerotiorum. The results showed that GM-1strain had better activity to10plant pathogens and its width of inhibition zone to Coniell a diplodiella and Bipolar is sorokiniana was both more than3.40cm. It was stable to heat, light and ultraviolet rays. It was steady at pH6-8and insensitive to protease. GM-1strain has broad antimicrobial spectrum and strong stability.4. Inhibition and disease prevention effect of GM-1strain were determined in this study. Antibacterial effect of GM-1strain was measured by dual culture method and Oxford Cup method. The damaging effect to mycelium of Sclerotinia sclerotiorum was observed by microscopic, and enzyme production of GM-1strain was measured by flat transparent circle method. Disease prevention effect of GM-1strain was determined by pot experiment. The results indicated that fermentations and culture filtrate of GM-1strain had obvious antimicrobial effects on Sclerotinia sclerotiorum. The inhibition zones got30mm and16mm, respectively, after7days culture. GM-1strain induced alterations on hyphae morphology of S. sclerotiorum, such as distortion and deformation. Moreover, its fermentations and culture filtrate inhibited the germination of sclerotia, and the inhibition of fermentations compared with culture filtrate is more significant. Research on enzyme production capacity of GM-1strain found that it has cellulase and protease activity. Pot experiments indicated the strain suppressed Rape sclertiniose, the control efficiency reached68%. It could promoted the germination of seed, These demonstrated that strain GM-1should be useful in biological control of Sclerotinia sclerotiorum.5. The optimal nitro gen source, carbon source and salt for the cell growth and the production of antimicrobial substance by strain GM-1were tested. The medium composition and fermentation conditions were optimized by orthogonal experiments.The results showed that the best medium was sucrose lOg/L, beef extract16g/L, yeast extract3g/L, FeSO40.4g/L. The optimal fermentation conditionswere the combination of temperature28℃, initial pH7.0, incubation time60h,10%inoculum volume percentage and medium volume70mL/250mL 6. Isolation and purification of active ingredient produced by strain GM-1. Through bioassay-guided purification, two antifungal compounds,2and Al, were separated from fermentation liquid of marine Bacillus amyloliquefaciens strain GM-1via silica gel column chromatography. Sephadex LH-20, thin layer chromatography and HPLC.