| Spirogyra is harmful filamentous algae in aquaculture water,which can affect the growth of Eriocheir sinensis.The traditional physical and chemical methods of preventing and removing Spirogyra are costly and easy to produce secondary pollution.The use of algae-lysing bacteria for ecological prevention and control has become a hot spot in Spirogyra control research due to its advantages of safety and pollution-free.In this study,Bacillus subtilis A4 with high spirogyra-inhibiting activity was isolated,screened and identified from the aquaculture environment with S.gracilis FACHB-354as an indicator algae species.It was confirmed that it had good safety from the aspects of virulence genes,toxic substance production ability,drug resistance and virulence to zebrafish,Daphnia magna and E.sinensis.On this basis,its excellent preservation protectant was further developed,and its mechanism of action was further analyzed,to lay a theoretical and strain resource foundation for the construction of Spirogyra ecological control technology in E.sinensis aquaculture water.The specific research results are as follows:1.The isolate A4 with good algicidal effect on Spirogyra was screened from the aquaculture environment by using S.gracilis FACHB-354 as an indicator.The algicidal rate of isolate A4 against S.gracilis FACHB-354 was 65.46%.The isolate A4 was identified as B.subtilis by molecular identification and physiological and biochemical identification.In addition,B.subtilis A4 had a wide algicidal spectrum against Spirogyra.It exhibited significant algicidal effects on S.gracilis FACHB-354 with algicidal ratios above 50%under the conditions of 3.0×10~6 to 3.0×10~9 CFU/m L,15℃to 35℃,and photoperiods of 14h:10h,24h:0h and 0h:24h(light/dark).2.B.subtilis A4 had no hemolytic activity,did not produce harmful metabolites such as biogenic amines,indole,ammonia,and hydrogen sulfide,and did not carry virulent nhe A,bce T,ces,cyt K,ent FM,hbl A,hbl C,and hbl D genes.In addition,B.subtilis A4 was highly sensitive to 30 antibiotics such as cephalosporins,peptides,amide alcohols,aminoglycosides,β-lactams,macrolides,quinolones,sulfonamides,tetracyclines,and nitrofurans,and did not possess neither antibiotic resistant genes like MCR-1,flo R,aaac(6’)-Ib,bla CTX,erm A,erm C,qnr A,qnr B,sul1,sul2,tet A and tet M nor resistance plasmid.Its median lethal concentrations to zebrafish,D.magna and E.sinensis was tested to be above 1.5×10~9 CFU/m L.3.By analyzing the preservation effect of 14 protectants on B.subtilis A4 at 25°C,4°C and-20°C,it was found that the preservation effect of B.subtilis A4 at-20°C was the best.Three kinds of protectants,skim milk powder,sodium glutamate and sorbitol,were screened out,which had good protective effects on B.subtilis A4 during frozen storage(-20°C).On this basis,a high-efficiency freeze-storage compound protectant for B.subtilis A4 was developed.The best compound ratio of skim milk powder,sorbitol and sodium glutamate was 5:5:2,the best effective content was 20%,and the best mixed ratio with B.subtilis A4 bacterial solution was 2:1.Under this condition,the survival rate of B.subtilis A4 was 89.50%after 60 days of frozen storage at-20°C.In addition,the compound protectant also had good protective effect on probiotics such as Bacillus licheniformis C1,B.subtilis JK-2 and B.licheniformis AQ19-11,the survival rates of strains C1,JK-2 and AQ19-11 were 90.73%,81.50%and 88.15%,respectively,after 60days of frozen storage at-20°C.4.The B.subtilis A4 showed an algicidal mode of indirect attack on Spirogyra,and its extracellular products can cause oxidative damage to S.gracilis FACHB-354 cells by significantly enhancing the the malondialdehyde content and the activities of peroxidase,catalase and superoxide dismutase.In addition,the non-targeted metabolomics comparative analysis of B.subtilis A4 and GT5(with low algicidal activity)was carried out.In the positive ion mode,a total of 796 metabolites were detected and annotated,including 230 metabolites were identified as differential metabolites,of which 108metabolites were significantly up-regulated expression and 122 metabolites were significantly down-regulated expression.In the negative ion mode,a total of 671metabolites were detected and annotated,including 227 differential metabolites,of which176 metabolites with significantly up-regulated expression and 51 metabolites with significantly down-regulated expression.The differential metabolites annotated to the KEGG functional pathway included 70 metabolites such as urocanic acid,formiminoglutamic acid,prostaglandin I2,phenylacetylglycine,5’-guanylic acid,3α,7α,12α-trihydroxy-5β-cholestanate,and pyrocatechol,mainly steroids,peptides,organic acids,nucleic acids,lipids,hormones and transmitters,carbohydrates,and antibiotics.It is mainly involved in arginine biosynthesis,lysine degradation,histidine metabolism,arginine and proline metabolism,glycerophospholipid metabolism,arachidonic acid metabolism,glycine,serine and threonine metabolism,two-component system,cysteine and methionine metabolism,alanine,aspartate and glutamate metabolism,biotin metabolism,benzoate degradation,lysine biosynthesis,ABC transporters,and other metabolic pathways,which may be related to the excellent Spirogyra inhibition activity of B.subtilis A4. |
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