Application Research On Two Strains Of Pseudoalteromonas Spp. With Antagonistic Activity Against Vibrio And Survey On Microbial Composition In Aquaculture Environment In Guangxi Zhuang Autonomous Region

1.Biosecurity evaluation of Pseudoalteromonas piscicida 2515 as probiotic for shrimp aquacultureIn order to evaluate the biosafety of a strain Pseudoalteromonas piscicida 2515（strain 2515 for short）,having antagonism against Vibrio spp.,as a disease-resistant probiotic for shrimp aquaculture,the strain was added into the nursery water at concentration of 10⁵,10⁶,10⁷and 10⁸ CFU/m L respectively and Bacillus licheniformis used as positive control to test the effect of bacterial virulence on the survival rate of larvae development of Fenneropenaeus chinensis from stage of fertilized egg to naupliiⅢ,naupliiⅢto ZoeaⅠ,ZoeaⅡto mysisⅠand mysisⅡto postlarvaⅠ.Meanwhile the effect of strain 2515 on the survival rate of Penaeus vannamei and the density of vibrio at grow out stage was investigated by adding the strain at concentration of 10⁶CFU/m L into the high-density shrimp aquaculture water.The hemolytic activity of strain 2515 on sheep and fish blood was tested on blood plate,also the hemolytic activity of strain 2515 on the blood cells of shrimp was observed by microscope;Furthermore,the effect of heating treatment on the antibacterial activity of strain 2515was studied.The results showed that the survival rates of P.chinensis larvae in all the developing stages at all concentration of strain 2515 in experimental groups were not lower than that in positive and blank control,except in the groups from naupliiⅢto ZoeaⅠat the concentration of 10⁶ and10⁷CFU/m L,which were significant lower than that in positive and blank control.The addition of strain 2515 to the aquaculture water of P.vannamei also had no significant effect on the survival rate compared with groups of B.licheniformis and the blank control,however the density of Vibrio spp.in the experimental group was significantly reduced.Strain 2515 had hemolytic activity on red blood cells of sheep and fish,but had no hemolytic activity on blood cells of shrimp.Heating at 55℃for 1h can inactivate the strain 2515,while the activity of antibacterial substances in the strain could be increased by 55.8%.The results not only provide reference data for the evaluating of biosafety of strain 2515,but also provide a method for the evaluation of biosafety of aquaculture probiotics.2.Colonization simulation of strain 2515 in aquaculture sea water and effect of culture conditions on its antibacterial activityIn order to test the colonization ability of strain 2515（initial concentration 10⁶CFU/m L）in aquaculture sea water,shrimp feed and environmental strains including Bacillus Amyloliquefaciens,Shewanella sp.,B.licheniformis and Vibrio parahaemolyticus were added into the water to simulate the shrimp aquaculture environment and the colonization effect was assessed by testing the density of strain in30 days.The results showed that The concentration of strain 2515 began to decrease and maintained at the order of 10⁵CFU/m L after inoculation 24 hours in group of no feed adding into the aquaculture water under the conditions of mono-culture,while in the group with feed adding the strain density could be stabilized above 10⁶CFU/m L after inoculation 120h.Under the mixed culture conditions of multi-bacteria,the strain2515 in the no feed adding group can be maintained at the order of 10⁴⁶CFU/m L within0-120h after inoculation,and then began to decrease and maintain at the order of10⁴CFU/m L,however in the feed-adding group it can maintain above 10⁶CFU/m L.To investigate the effect of fermentation time strain 2515 in different culture medium and metal ion on the antibacterial effect,the strain were inoculated in the medium 2216E and LB and antibacterial effect were analyzed at 12h,24h,36h and 48h respectively,also magnesium chloride and ferric citrate at concentrations of 0、5、5、11、22 and0、0.05、0.1、0.2g/L were add into the LB medium respectively to analyze the effect of different concentrations of Mg²⁺and Fe³⁺on the antibacterial effect of strain 2515.The results show that,under the same fermentation time,strain 2515 had a better antibacterial effect against V.anguillarum in medium LB than in 2216E,the fermentation time with the highest antibacterial effect for both medium were 36h.Iron ions could promote the antibacterial activity of strain 2515.Adding ferric citrate to the LB liquid medium at 0.2g/L could obtain the highest antibacterial effect with the diameter of inhibitory circle（14.1±0.60）mm,11.1 percent higher than that in the control group.3.Isolation,identification and application of an Antagonism against pathogen in aquacultureA strain 2018082905（strain 2905 for short）with antagonism against vibrio was isolated from the sea cucumber pond,and the antibacterial effect was evaluated by the tested strain Aeromonas hydrophila,A.salmonicida,V.parahaemolysis,V.splendidus,V.anguillarum,V.harveyi and V.fischeri.The results showed that the strain had strong antibacterial activity against A.salmonicida and V.fischeri,but no antibacterial effect against A.hydrophila.The hemolytic effect on blood cells of sheep,fish and shrimp was used to study by microscopic observation and blood plate method with the bacteria concentration at 10⁸CFU/m L.The results showed that strain 2905 had hemolytic effect on fish red blood cells and no hemolytic effect on sheep blood and shrimp blood cells.Drug resistance of strain 2905 was analyzed using the drug sensitive test paper.The results indicated that strain 2905 contained multiple resistance genes.Also strain 2905was preliminary identified by the 16S r RNA gene sequencing and show that it was one strain of Pseudoalteromonas sp.close to P.piscicida.The results can provide candidate strain and basic data for the screening and application of antagonism against pathogenic bacteria in marine aquaculture.4.Investigation on environmental microbes of aquaculture animals in GuangxiIn order to clarify the composition of environmental microbes in aquaculture animals in Guangxi and to identify the main pathogenic bacteria that harmful to farmed animals,the environmental microorganisms from marine and freshwater cultured animals in Guangxi were collect and separate using culture medium of 2216E,LB,and BHI in September 2018 and July 2019 respectively,moreover identification of the bacterial isolates were carried out by 16S r RNA gene sequencing.A total of 177samples were collected in the two surveys,and 260 strains of bacteria were isolated.Among of them,244 strains of bacteria were isolated from fresh water aquaculture animals and their pond water,belonging to 56 genera,which contained pathogens A.hydrophila,A.caviae,A.dhakensis,A.jandaei,Streptococcus agalactiae,Photobacterium damselae,V.alginolyticus,V.cholerae,V.harveyi,V.parahaemolyticus and V.vulnificus etc.The farmed animals with symptomatic symptoms are mainly tilapia Oreochromis mossambicus.Among the 7 strains of S.agalactiae isolated from Tilapia,there are 2 strains are far from other strains in Gene-tree.There are 16 strains of bacteria isolated from Siganus guttatus,Epinephelus awoara,P.monodon,P.vannamei and their pond environment,belonging to 6 genera,which contained the main pathogens V.alginolyticus,V.cholerae,V.vulnificus,V.fluvialis etc.The results could provide basic information on the composition of pathogens bacteria of aquaculture animals and strain materials used for the prevention and control of bacterial diseases in aquaculture animals in Guangxi.