Screening Of Antagonistic Bacterium Strain Against Fish Saprolegnia Sp. And Antagonism Analysis

Saprolegniasis is a common disease,has caused huge damage to aquaculture.There is no effective chemical drugs so far,but using the antagonism between biological to control diseases,has been used in fishry production.The purpose of this study is to obtain pathogens of Saprolegniasis for species identification;explore the conditions of its growth;the strong pathogenic strains as the indicator bacteria,screening of antagonistic bacterium strain against fish Saprolegnia sp. for species identification;optimization the culture conditions of antagonistic bacteria;explore the mechanism of antagonism,purification of the active subsrances,lay the foundation for saprolegniasis' s biocontrol.The results are as follows:(1) Saprolegnia LF04 was isolated from the nidus of tilapia with saprolegniasis,was identified as Saprolegniaceae,Saprolegmia,very likely is a new species,named Saprolegnia LF04;the challenge assays of Saprolegnia LF04 showed that the infection rate of Carp eggs was 90%, the infection rate of Perca fluviatilis eggs was 52%;the optimal temperature was 25?,optimal pH was 7.0,more sensitive to sodium chloride,when the concentration reached to 2%,the growth of Saprolegnia LF04 completely inhibited.(2) The two antagonistic bacterium:strain JL04 and strain JL50 were screened, Saprolegnia LF04 as the indicator bacteria;identified by morphological characteristics and molecular,JL04 was bacillus subtilis,JL50 was bacillus pubtilis.(3) The challenge and prevention assays of strain JL04 on fish showed that the two antagonistic bacterium have no pathogenicity to fish,fish reduced the probability of infection by 45.0%(JL04) and 38.4%(JL50);passage experiments showed that the antagonistic properties of the two antagonistic bacterium could be stabilized passed to offspring;the antibacterial spectrum of JL04 was more widely than JL50;strain JL50 just has antagonistic to a specific Saprolegnia(LF04 and LF01), JL04 can better adapt to the actual needs of production.(4) Antagonistic mechanism analysis of strain JL04 showed that the inhibition rate of fermentation broth of the strain JL04 on saprolegnia hyphal growth was 70%,the bacteria-free culture supernatants was 40%,both can inhibite cysts germination,therefore,it's believed that strain JL04 secretes antimicrobial substances in the culture medium; the bacteria-free culture supernatants was sensitive to temperature,pH, protease K,so,it is believed that the antimicrobial substances was protein;through the elution by Sephadex G-75 gel filtration and SDS-PAGE test,suggested a molecular mass of the protein about 2.7×104Da.We can know that the antagonistic mechanism of strain JL04 was secreting antimicrobial substances to the extracellular, inhibition of mycelial growth and spore germination.(5) Optimization the culture conditions of strain JL04: The results of fermentation conditions showed that strain JL04 was sensitive to temperature,pH, the optimal temperature was 32?, optimal pH was 5.0-6.0,effect of inoculation was not significant;through single factor experiment and orthogonal experiment, initially obtained the optimized formula of fermentation medium of JL04 strain in laboratory:6g/L tryptone,6g/L glucose,3.5g/L yeast extract,3.5g/L NH4NO3,2g/L MgSO4,3g/L CaCl2,1g/L MnSO. The optimal medium,the time of reached to stable phase shortens 2 hours,and the inhibition rate of bacteria-free culture supernatants improved 12.5%.