Screen A Stain Which Antagonism The Pathogen Vibrio Splendidus In Apostichopus Japonicus And The Study On The Antagonism Biology

Owing the nutritional and biological value of Apostichopus japonicus,the mariculture of sea cucumber has been developed quickly in Shandong,Liaoning and Fujian.But the bacterial diseases were serious occurrence repeatedly in aquaculture.Recently,the antibiotics were used to prevent and treat the bacterial diseases to the A.japonicus,such as skin ulceration disease.But the antibiotics caused many problems such as environmental pollution and raise of bacterial drug resistance.Due to the merits of easy storage,no side effect,environmental friendliness and enforcing the immunity,the probiotics which has be used as an alternative strategy to control pathogens in aquaculture of immunized animals has garnered much attention.Accordingly,screening some bacteria which have strong antagonism activity as potential probiotics have importantly potential research and social value.In our experiment,the Vibrio splendidus isolated from the focal lesions of A.japonicus was the pathogenic bacteria of skin ulceration disease and used as the indicator.One antagonistic strain was screened from the intestine of A.japonicus.And the strain which could inhibit the V.splendidus were screened by disk diffusion method and fermentation method.Meanwhile,the antagonism mechanism of the strain for V.splendidus was preliminary studied.The main results as follows:①Screening and identification of antagonistic bacteria: The cell free supernatant of strain LS2 which could strongly against the pathogen V.splendidus had been determined.And the antagonistic activity of stain LS2 was calculated to be 86.83 ± 1.47%.It was identified as Bacillus cereus by 16 S rDNAand morphology.②Screening and identification of the antagonistic substance: The antagonistic substance of the supernatant of B.cereus LS2 was identified by bioassay-guided method,the antagonistic substance was secreted into the supernatant.The molecular weights of antagonistic suatance is under 3 kDa.And the antibacterial rate was 64 ± 7.9% and 38.9 ± 3.79% after heat and alkali treated.And antagonistic substance which is amphiphilic could be precipitated by concentrated hydrochloric acid.The antagonistic activity of B.cereus LS2 may be lipopeptide.③Preliminary study on the mechanism: Study on the expression of m RNA and proteins in the V.splendidus which was treated by LS2.The different expression of proteins had been screened in the SDS-PAGE and identified by MAl DI-Tof/Tof MS.Then the expression of DLDH1 and EF-Tu had been increased with the proteins and m RNA.Meanwhile,the formation of biomembrane had been reduced with the proteins increasing.We had structured the DLDH1over-expression of V.splendidus strain which displayed lower growth rate compared with the control V.splendidus.In this experiment,we screened a strain,Bacillus cereus LS2,which has strong antagonistic activity against V.splendidus.The LS2 provides a choice and thinking to alternative antibiotics,and provides a certain theoretical basis for biological control of sea cucumber rotten skin syndrome.