Comparison Of Gut Microbiota In Freshwater Fishes From Different Regions And Screening For Antagonistic Bacteria Against Fish Pathogens

The Qinling-Huaihe line is the North-South boundary of China.There are obvious differences in environment and climate between Wangcheng District（in Hunan Province belonging to southern China）and Yucheng City（in Shandong Province belonging to northern China）,and the intestinal flora abundance and structure of freshwater fish are also different.In this study,we selected grass carps（Ctenopharyngodon idella）,crucian carps（Carassius auratus）and carps（Cyprinus carpio）in the two areas.The 16S rRNA gene V4 hypervariable regions of intestinal flora in fish were amplified and sequenced,and then the sequencing data were analyzed.The results showed that the difference between Shandong grass carp group and Hunan grass carp group was significantly greater than that within groups（R=0.307,P<0.05）.The relative abundance of intestinal microflora in each group was not consistent,but the core microfloraes were Proteobacteria,Fusobacteria,Bacteroidetes and Firmicutes.（LDA>2.0,P<0.05）.This study also compared the relative abundance of Streptomyces、Bacillus、Lactobacillus、Bifidobacterium、Aeromonas、Vibrio、Shewanella、Edwardsiella and Flavobacterium.The results showed that the abundance of Aeromonas in Hunan grass carp group and Hunan carp group was higher than that in Shandong grass carp group and Shandong carp group,Vibrio were enriched in Shandong grass carp group and Hunan carp group,while the relative abundance of Bifidobacterium in Shandong grass carp group and Hunan grass carp group was higher than that in Hunan grass carp group and Hunan carp group.With the rapid development of aquaculture in China,fish diseases have become more and more serious problems,and the probiotics can reduce the incidence of diseases without damaging the fish pond environment.In this study,Aeromonas hydrophila,Edwardsiella tarda and Staphylococcus aureus were used as indicator bacteria to screen a actinomycete with obvious bacteriostatic activity from the soil samples of Yucheng Wetland Park.After 16S rRNA sequencing,it was identified as S.phaeochromogenes XA1.The morphological characteristics,growth and propagation and the utilization of carbon sources of the strain in the medium were observed by experiments.In order to further study the effect of the strain on the growth of grass carp,the fermentation supernatant and bacteria of strain XA1 were added to the feed of grass carp respectively for feeding test.The results showed that compared with the CK（basic feed）of the control group,the test group added supernatant promoted the growth of grass carp to a certain extent（P<0.05）.After the treatment of Aeromonas hydrophila,the survival rate of grass carp in group B（basic feed+0.5%fermentation supernatant）was higher than that in the control group（P<0.05）.This study showed that the secondary metabolites of strain XA1 played an important role in promoting fish growth and reducing mortality.Then we used methanol to extract the fermentation products of strain XA1,and the thermostability test showed that there were high temperature resistant antibacterial substances in the metabolites of strain XA1.The crude extract was separated by silica gel column chromatography and purified by semi preparative HPLC.The compound Z1 was identified by mass spectrometry and nuclear magnetic resonance.The result showed that the compound Z1 was4’,5,7-trihydroxyisoflavone with molecular weight of 270 and molecular formula of C₁₅H₁₀O₅.In conclusion,we compared the intestinal microflora differences of freshwater fish in the two areas,and screened an antagonistic strain of fish pathogenic bacteria in this study,which laid a foundation for further research on fish health cultivation and disease control.