Isolation And Identification Of Sebastes Schlegeli Vibrio And Screening Of Antagonistic Bacteria

In the process of Sebastes schlegeli(S.schlegelii)cage breeding,due to high density of cage breeding,organic matter accumulation,resulting in a large number of bacteria,Bacterial infection of S.schlegelii will cause disease and even death.Four different vibrio species were isolated from three ulcerated fish,including Vibrio scophthalmi(V.scophthalmi),V.harveyi,V.alginolyticus and V.parahaemolyticus.V.alginolyticus and V.parahaemolyticus were isolated from the same fish.V.scophthalmi was isolated from S.schlegelii for the first time.By detecting extracellular enzymes,V.harveyi,V.alginolyticus and V.parahaemolyticus all contained extracellular protease,lecithin enzyme,lipase and gelatinase,while no extracellular enzymes were detected in V.scophthalmi.Virulence genes were detected by PCR.V.harveyi,V alginolyticus and V.parahaemolyticus all had Flab.V alginolyticus and V.parahaemolyticus all carried TcpA,while V.scophthalmi,V.alginolyticus and V.parahaemolyticus all had ToxS.Other virulence genes Tdh,Trh,Tlh,ToxR and Zot were not detected.According to the above characteristics,these four pathogens were identified as having potential pathogenic effects on S.schlegelii.After antibiotic detection of four vibrio bacteria,it was found that four pathogens were resistant to a variety of antibiotics,only moderately sensitive to erythromycin,multi-drug resistance was obvious.The results of drug sensitivity test showed that all the four vibrio strains had relatively strong resistance.A more healthy and green method for disease prevention and control is needed.Therefore,the four pathogenic vibrio antagonists were screened,and 17 strains with inhibitory effect on pathogenic bacteria were screened.It is mainly divided into V.campbellii,Pseudoalteromonas sp.and Shewanella sp.Among them,Pseudoalteromonas sp.can be used as probiotics for marine fish,and subsequent experiments are mainly carried out around Pseudoalteromonas sp..The concentration standard curve and growth curve of Pseudoalteromonas sp.were measured.The standard curve and growth curve showed that it had strong adaptability to the environment,vigorous vitality,and could reach high cell concentration in a short time.According to the preliminary investigation of its bacteriostatic mechanism,its extracellular products only had an inhibitory effect on V.splendid preserved in the laboratory,and the fermentation bacteria suspension and cell had an inhibitory effect on V.alginolyticus,V parahaemolyticus and V.splendid,and the bacteriostatic effect was stronger with the participation of Pseudoalteromonas sp..In order to explore the influence of culture conditions on Pseudoalteromonas sp.,the single factor experiment showed that Pseudoalteromonas sp.J-13 had significant differences in cell concentration under different treatment factors,and had significant differences in inhibitory effects on V.alginolyticus and V.parahaemolyticus.In addition,under different treatment factors,the optimal inhibition conditions of J-13 against two different bacteria were also different.Only under the influence of temperature and liquid loading,the inhibition trend of J-13 on vibrio was basically the same.Under the condition of low liquid loading and 33℃ culture,Pseudoalteromonas sp.J-13 had the strongest inhibition effect on the two kinds of vibrio.The results of orthogonal experiment showed that the optimal conditions for cell culture of Pseudoalteromonas sp.J-13 were 33 ℃,pH6 and 1%salinity.The optimal bacteriostatic culture conditions for J-13 against SF-5 were 33℃,pH8 and 3%salinity.The optimal bacteriostatic culture conditions for J13 against SF-6 were 33℃,pH6 and 3%salinity.The optimal cell culture conditions and the optimal bacteriostatic conditions of J-13 were different,and the culture conditions should be changed according to different needs.These studies will lay a solid foundation for the prevention and control of S.schlegelii.