Isolation,Identification And Control Of Fusarium Solani From Micropterus Salmoides And Transcriptome Analysis Of Micropterus Salmoides After Infection With Fusarium Solani

Micropterus salmoides,Largemouth black bass,is one of the important culture species China due to its fresh meat,fast growth and wide adaptability.However,disease has become one of the factors restricting the development of its industry.Fusarium is widely distributed and can infect various plants,humans and aquatic animals.Fish are susceptible to infection with Fusarium when their body surface is damaged or in a bad breeding environment,with rapid onset and high fatality rate and causing great economic losses.However,there is no effective prevention and control method for Fusarium disease.Therefore,it is necessary to carry out research on it.In July 2019,the head,pectoral,dorsal and caudal fins of M.salmoides reared in the circulating water chamber showed congestion and inflamation,then ulcerated white filaments and finally died.In this study,a pathogenic Fusarium strain was isolated and purified from the diseased part,and its biological characteristics,chemical and biological control methods were studied.Transcriptome analysis on spleen tissues of M.salmoides after infected with Fusarium.The main research results are as follows:1.The dominant bacterium MY1 was isolated from the nidus site,which was confirmed to be pathogenic by artificial infection.The median lethal dose(LD₅₀)for M.salmoides was 1.03×10⁴cfu/m L.MY1 was identified as F.solani by morphological characteristics and r DNA-ITS sequence analysis.2.F.solani could grow in the range of 10℃～35℃and p H=4.32～11.44.The optimal temperature was 25℃～30℃and the optimal p H was 7.85.When Na Cl≥10%,the growth stopped.PDA was the most suitable medium for growth.The lactose was the most suitable carbon source and the optimal nitrogen source was sodium nitrate.Light had no significant effect on its growth.Spores could be produced in the range of10℃～35℃,p H=4.32～11.44,and the optimal temperature was 25℃and p H 7.85.When Na Cl≥8%,the spores nonproduction.The optimal medium for sporulation was PDA.The most suitable carbon sources were starch and D-fructose and the optimal nitrogen source was peptone.Light was conducive to sporulation.3.The hyphae of F.solani was sensitive to preconazole,garlicand and metalaxyl,the MIC values was 4μg/m L,0.1 g/m L and 25 mg/L,respectively.The spores were sensitive to amphotericin B,clotrimazole,conazole,nystatin and metalaxyl,with MIC of 8,16,8,32 ug/m L and 50 mg/L,respectively.4.A total of 42 strains of bacteria were isolated from the body surface and aquaculture water of M.salmoides.And one strain of antagonist JK10 was screened out by using F.solani as the indicator bacterium.Combined with morphological,biochemical and 16S r RNA sequence analysis,JK10 was identified as Bacillus subtilis.It was resistance to aminotronam and other 9 antibiotics.The strain was safe and the antibacterial activity could be stably inherited to the next generation.5.Through single factor test and orthogonal test,the optimal fermentation conditions of B.subtilis was determined:temperature 30℃,p H value 8,rotation speed180 r/min,inoculation amount 0.5%,liquid loading amount 50 m L,and the optimal medium composition was 14 g/L lactose,7 g/L yeast powder,10 g/L potassium chloride.Compared with before optimization,the strain logarithm end was 2 h earlier,the OD_600nmvalue was 1.15 times of the previous value,and the inhibition rate was increased by 9.5%.6.B.subtilis produced protease,amylase and volatile substances,which had a certain inhibitory effect on the growth of F.solani.Antibacterial substances mainly existed in sediments,which had good resistance to heat,acid and alkali and ultraviolet light,and was suitable for storage at room temperature.7.Transcriptome analysis on spleen tissues of M.salmoides before and after infection with F.solani.43.72 Gb of clean reads were obtained and 89458 unigenes expressions were detected.2195 differentially expressed genes were detected,among which 1519 were up-regulated and 676 were down-regulated.GO item analysis showed that the differential genes were mainly involved in small molecule metabolism,immune process,ketoacid metabolism process,etc.Differential gene of KEGG pathway was enriched in rheumatoid arthritis,cytokine receptor interaction,NF-k B signaling pathway,etc.In addition,7 immune genes were randomly screened for q RT-PCR validation,the expression trend was consistent with that of RNA-seq,which confirmed the reliability of transcriptome data.Enriched the database of M.salmoides,providing reference information for the study of Fusarium pathogenicity and immune mechanism.In this paper,the biological characteristics and control methods of the pathogenic fungi were studied and the transcriptome analysis was conducted on the spleen tissues before and after infection with the F.solani,which provided a basis for the prevention and control of disease,and also laid a foundation for the study of immune mechanism of the M.salmoides against fungal infection.