Preparation Of Endophytic Fungal SQGX-6 Metabolite From Panax Ginseng And Its Treatment Of Aeromonas Hydrophila Infection In Black Carp

Aeromonas hydrophila as a human-animal-fish commensal conditional pathogen has been widely spread worldwide,leading to high mortality rates and huge economic losses,which seriously threaten the healthy development of the farming industry.Although antibiotics have reduced the mortality of Aeromonas hydrophila infections to some extent,with the emergence of antibiotic residue problems and multi-drug resistant strains,the application of antibiotics has been limited in many ways.Therefore,there is an urgent need to develop novel drugs to fight against Aeromonas hydrophila.With the continuous research on Chinese herbal medicines,the functions of medicinal plants and their secondary metabolites in antitumor,antibacterial and antioxidant have been highlighted,which have good prospects for the screening of natural active compounds and the development of novel drugs.In order to determine the pathogen and pathogenicity of cyprinid hemorrhagic disease caused in a farm in Jingzhou,Hubei,a dominant strain of QAB5 was isolated from the diseased cyprinid using traditional pathogen isolation methods,and was subjected to morphological characteristics,physiological and biochemical properties,16S rRNA gene sequence,MLST multiple sequence locus typing,extracellular enzyme activity analysis,virulence gene detection,artificial infection test,biofilm formation ability determination and The results showed that strain QAB5 was a Aeromonas hydrophila with strong pathogenicity,MLST sequence type was ST251,containing hemolytic activity,protease and lipase activity,and virulence genotype was aer⁺hly⁺ahp⁺ast⁺alt⁺act⁺;artificially infected healthy cyprinid showed similar symptoms to natural disease cyprinid such as unresponsiveness,abdominal bleeding,anal redness and intestinal bleeding.The LD₅₀ was determined to be 4.4×10⁶ CFU/m L,which was a highly pathogenic and virulent strain;the isolated strain QAB5 was sensitive to 13antibiotics such as kanamycin and chloramphenicol,and resistant to 4 antibiotics such as ampicillin and rifampicin;the Chinese herbal medicines whole scorpion,umeboshi and clove had obvious in vitro inhibitory effects on strain QAB5.The results of this study provide evidence to reveal the pathogenic mechanism of Aeromonas hydrophila and provide practical reference for the prevention and control of hemorrhagic diseases caused by Aeromonas hydrophila.In order to screen for endophytic fungal isolates of Panax ginseng with significant inhibitory effect on Aeromonas hydrophila,the secondary metabolites of 19 strains were extracted with ethyl acetate,screened for antibacterial activity using Oxford cup plate method,and their chemical diversity was determined by thin-layer chromatography to screen one strain and determine its optimal culture conditions,and their antioxidant activity and antibacterial spectrum were determined by combining DPPH method and Oxford cup plate method The antioxidant activity and antibacterial spectrum were determined by DPPH and Oxford cup plate methods.The results showed that the secondary metabolite extracts of Panax ginseng endophytes G3,G5,G7,G8,GX-2,GX-4,GX-5 and SQGX-6 had significant in vitro inhibitory effects on Aeromonas hydrophila,among which strains G5,GX-5 and SQGX-6 had the best inhibitory effects.The secondary metabolite extract of strain SQGX-6 showed richer chemical diversity by thin layer chromatography and was identified as the subject of study;strain SQGX-6 showed strong inhibition activity of its secondary metabolite at 50%culture volume and 7 days;in addition,the secondary metabolite extract[DPPH·]of strain SQGX-6 showed 94%clearance and had a wide range of pathogenic bacteria such as Aeromonas hydrophila and Streptococcus lactis It has a broad-spectrum bacterial inhibitory effect.The results of this study provide a reference for the screening of biocontrol strains of Aeromonas hydrophila.In order to clarify the chemical composition of the secondary metabolite extract of SQGX-6 and its practical application in the treatment of aquatic diseases caused by Aeromonas hydrophila,this study used the endophytic fungus SQGX-6 from Panax notoginseng as the object of study,and analyzed the composition of the secondary metabolite extract of SQGX-6 and its main compound structure type by ultra performance liquid chromatography（UPLC-MS）.On the basis of determining the relative safe doses of SQGX-6 secondary metabolite extracts in cyprinid fish,the secondary metabolites of SQGX-6 were used to treat cyprinid fish infected with Aeromonas hydrophila,and the antibacterial and therapeutic effects of the secondary metabolites of SQGX-6 in vivo were comprehensively evaluated by statistical analysis of tissue bacterial load,organ index and liver antioxidant capacity.The results showed that the secondary metabolite extracts of SQGX-6 mainly contained 618 chemical components such as gentianic acid,adenine,7-ethyl-10-hydroxycamptothecin,aflatoxin,oxidized prebiotics and salicylic acid;among them,flavonoids were the most abundant substance types,accounting for 16.019%of the total,followed by alkaloids,accounting for 15.21%.In vitro antibacterial experiments showed that the minimum inhibitory concentration of SQGX-6 secondary metabolite extracts against Aeromonas hydrophila was 0.78125 mg/m L and the minimum bactericidal concentration was 1.5625 mg/m L;the relative safe drug concentrations for in vivo treatment were 64,16 and 4μg/g concentration groups.The results of in vivo treatment experiments showed that the liver and spleen bacterial loads of cyprinid in the SQGX-6 extract treatment group were significantly lower than those in the attacking and DMSO groups（P<0.05）;the results of organ index showed that the treatment with enrofloxacin and SQGX-6 secondary metabolite extract showed significant differences compared with the attacking group（P<0.05）.The antioxidant capacity assay revealed that the liver T-SOD activity of cyprinid fish was significantly decreased（P<0.05）,and the corresponding MDA content was significantly increased（P<0.05）and the liver GSH content was significantly decreased（P<0.05）after the infection by Aeromonas hydrophila.The administration of enrofloxacin and SQGX-6 secondary metabolite extracts significantly increased T-SOD activity（P<0.05）,significantly decreased MDA content（P<0.05）and significantly increased GSH content（P<0.05）in the liver of cyprinid fish.In the above results,none of the three concentration groups of the SQGX-6 secondary metabolite extract group showed significant differences between them（P>0.05）.