Bacterial Community Characterization Of Grass Carp Aquaculture System And Screening Antagonistic Bacteria Of Fish Pathogenic Strain Aeromonas Veronii

In China,grass carp is one of the major carps cultured in freshwater ponds.In recent years,with the increasing density of grass carp culture,it leads to deterioration of water quality and disease outbreak.The study of microbial community characteristics of aquaculture water could promote the healthy culturing of fish.This study was aimed to reveal the microbial community characteristics of aquaculture system for grass carp and compare the differences between microbial community structure of aquaculture water with fish disease outbreak and healthy aquaculture water.Lesion tissue of fish was collected from a farm in Hunan Province,then the isolated strains were identified as a fish pathogen,and screening its antagonistic bacteria.1.Analysis of the microbial community characteristics of pond for grass carp.The high-throughput sequencing and Bioinformatics technique was used,and the microbial community structure and a-diversity indices between the rearing water of healthy and diseased groups were studied and compared.Microbial metagenomic sequencing analysis showed that different water samples were total identified to 38 phyla,70 classes,133 orders,269 families,423 genera.There was no significant difference between the α-diversities of healthy and diseased aquaculture water,however,the results of principal coordinates analysis(PCoA)and the sample clustering of heatmap showed that the microbial structure of healthy and diseased aquaculture water were quite different.Ordination via canonical correspondence analysis(CCA)on the high-throughput sequencing data and environmental parameters indicated that the composition of microbial community was significantly influenced by COD.2.Isolation and identification of fish pathogen strain.In this study,we isolated and purified a set of suspected strains of Aeromonas veronii from lesion tissue of fish.We selected a strain which has the typical characteristic.According to physiological and biochemical characteristics,and the analysis of 16S rDNA sequences,the isolated fish pathogenic bacterium was identified as a novel Aeromonas veronii strain and named as AvX005(GenBank:KU641161).This bacteria can make grass carp pathogenic by artificial infection experiments,and we studied its antibiotic susceptibility and growth characteristics.3.Screening antagonistic bacteria of Aeromonas veronii and analysis of their active substances.This study found that Bacillus amyloliquefaciens has a broad-spectrum antagonistic activity against fish pathogenic bacterias and has obvious antagonistic activity against Aeromonas spp.Bacillus amyloliquefaciens and Aeromonas veronii coaggregation rate reached 36.98%.Animal protection tests showed that Bacillus amyloliquefaciens has a 80%immune protection rate for grass carp.The antibacterial activity of Bacillus amyloliquefaciens was identified by physical and chemical properties.The results showed that the antibacterial substances has relatively strong thermal stability,acid resistance,but it is very sensitive to strong alkaliand.At the same time,it can be extracted by organic solvents and acid precipitation.It is speculated that the antimicrobial substances was not a kind of protein material.4.Isolation and identification of antibacterial substances.The antibacterial substances of Bacillus amyloliquefaciens were isolated and purified by UHPLC and identified by mass spectrometry.It was found that the antimicrobial substance was a kind of light yellow substance,easily soluble in methanol,ethanol and other organic solvents,and its molecular weight was 1045.29.According to the previous studies,we speculate that the antibacterial activity of compounds is likely to be lipopeptide.It is expected to be developed into microecological preparations for the prevention and treatment of aquaculture diseases.