Study On The Inhibition Characteristics Of Macroalgae Ulva Fasciata Against Vibrio

Vibrio is a common pathogen in marine aquaculture environment.It is not only an important factor restricting the development of marine aquaculture,but also a foodborne pathogen that threatens human health.Although a large number of antibiotics additions can inhibit the proliferation of Vibrio to a cetain extent,it also induces the production of some resistant Vibrio.In recent years,most studies have shown that not merely macroalgae has the function of purifying water quality,but also the substances extracted from them have strong antibacterial properties.The WHO has also recommended that extracted antibiotic substance from macroalgae can replace antibiotics to control the problem of aquaculture pathogen.In order to further expand the usage of macroalgae for aquaculture water quality rehabilitation,it is necessary to study the inhibition characteristics of macroalgae viable tissue against Vibrio,and related research reports are still less.Thus,the effect of typical macroalgae of Ulva fasciata on three vibrio species growth activity(Vibrio parahaemolyticus strain ATCC17802,isolated Vibrio parahaemolyticus strain Vp1 and Vibrio alginolyticus strain Va2)was examined in this study.Three indicators of cultivability,motility and biofilm formation were used to reveal the vibrio growth activity.In addition,a comparative transcriptomic analysis of Vibrio parahaemolyticus and Vibrio alginolyticus in response to Ulva fasciata was conducted to reveal the differential genes expression of vibrio species via the inhibition of Ulva fasciata.The main results of this study are presented as follows:1)A comparative cultability of Vibrio in response to Ulva fasciata.Experimental results showed that the viable tissue of the Ulva fasciata significantly inhibited the culturability of the Vibrio parahaemolyticus strain ATCC17802,Vp1 and Va2 via colony counting method of TCBS.When Ulva fasciata was continuously cultured for more than 3d at the density of 12 g L-1,the culturability inhibition efficiency reached 99%.A higher density(12g L-1)of Ulva fasciata achieved a significantly higher inhibition rate than that of a lower density(3g L-1).In addition,a higher concentration of Vibrio suspension,longer time was required to detect no culturable vibrio.2)A comparative motility and biofilm formation study of Vibrio in response to Ulva fasciata.The motility of Vibrio was measured by the diameter of bacterial circles formed by inoculating the bacteria on the swimming plate,and the biofilm formation of vibrio was measured by the crystal violet assay.Experimental results showed that vibrio motility and biofilm formation under different conditions(algae density,nitrogen and phosphorus levels,salinity and bacterial suspension concentration)were all significantly inhibited by Ulva fasciata.After 3-days' co-culture with Ulva fasciata,the maximum biofilm formation ability was inhibited,with the highest inhibition rate of 67.10%,94.69% and 88.50% for strain ATCC17802,Vp1 and Va2,respectively.The highest inhibition efficiencies of 30.29 %,20.26 % and 39.49 % of swimming behavior were found for the strain ATCC17802,Vp1 and Va2,respectively.Correspondingly,the inhibition efficiencies of twitching behavior were 47.68 %,39.49 %,and 31.8 %,respectively.Thus,the live culture of Ulva fasciata could turn vibrio into an uncultivable state,and reduce the motility ability and biofilm formation of vibrio.3)A comparative transcriptomic analysis of Vibrio in response to Ulva fasciata.The RNA transcriptome of Vibrio parahaemolyticus Vp1 and Vibrio alginolyticus Va2 in response to Ulva fasciata were analyzed by Illumina Hi Seq XTM Ten sequencing platform.Following Ulva fasciata 24 h-treatment,many down-regulated genes and up-regulated genes were found on vibrio.Thereinto,differential expressed genes(DEGs)screening of Vibrio parahaemolyticus Vp1 samples yielded 210 significant differences,including 90 up-regulated genes and 120 down-regulated genes.GO function enrichment and KEGG pathway analysis showed that,DEGs of Vibrio parahaemolyticus Vp1 were mainly enriched in flagella assembly,bacterial chemotaxis and biofilm pathway.The down-regulated genes mainly involved flagellin Fli C,flagellar motor switch protein Fli G and Fli N,methyl acceptor chemotactic protein MCP,chemotaxis protein Che Y,etc.The DEGs of Vibrio alginolyticus Va2 were mainly enriched in pyrimidine metabolism,bacterial chemotaxis and flagella assembly,and the down-regulated genes included uridine phosphorylase and flagellin motor protein Fli G.Thus,the down-regulation of these genes were relative with the results of vibrio motility and biofilm formation due to the inhibition of Ulva fasciata.