Structure Analysis And Bioactivity Determination Of Two Kinds Of Lipopeptides From Deep-sea Bacteria

Plant diseases caused by plant pathogenic fungi bring great economic losses to food production of our country.Because of the threat of chemical pesticides to the ecological environment and the survival of human beings,people have been focused on safer biological pesticides.Biological pesticides from microorganisms had been widely applied because of their wide sources,simple process and difficulty in producing drug resistance.This study plans to screen marine bacterial strains with strong antifungal activity against plant pathogenic fungi,and carries out related research on them.The contents are as following:In this research,a large number of marine bacterial strains were isolated from the deep-sea sediments,and two strains with high antifungal activity against Magnaporthe grisea were screened,which were named as Bacillus sp.wsm-1 and Bacillus sp.CS30 according to 16S rRNA sequencing,respectively.The corresponding antifungal agents were purified by sequential acidic precipitation,methanol extraction,silica gel column chromatography,and reversed phase high-performance liquid chromatography（RP-HPLC）.The antifungal agents produced by Bacillus sp.wsm-1 were identified as two iturin-like cyclic lipopeptide homologs with a difference of-CH₂ by analysis of amino acid composition and tandem mass spectrometry.The amino acid sequence of the purified antifungal agents wasβ-amino fatty acid-Asn-Ser-Asn-Pro-Tyr-Asn-Gln,which were different from previous reportedlipopeptides,then these two cyclic lipopeptides were deduced as two novel lipopeptides and designated as C14 iturin W and C15 iturin W,respectively.The antifungal agents produced by Bacillus sp.CS30 belonged to the family of surfactin and the two homologues were named as surfactin CS30-1 and surfactin CS30-2,respectively.In order to detect the differences of the antifungal activity between two lipopeptide homologues and corresponding antifungal mechanism,the bacteriostatic concentrations of C14 iturin W and C15 iturin W against M.grisea and their effects on the sub-microstructure of M.grisea hyphae were determined in this study.It was found that the antifungal activity of C15 iturin W was significantly higher than that of C14 iturin W and they inhibited the growth of M.grisea by destroying the cellular structure,thus leading to cytoplasmic leakage and cell death.In addition,due to the strong anti-tumor inhibitory activity of surfactin CS30-2,the corresponding submicroscopic structure of its action mechanism was observed.The result showed that the cancer cells almost lost the filiform structures and shifted to a round shape and appeared serious cytoplasmic leakage after treated with surfactin CS30-2.In view of the large difference of the antifungal activity between the homologues C14iturin W and C15 iturin W,different carbon sources,nitrogen sources and amino acids were added,and their effects on the production of C14 iturin W and C15 iturin W were measured separately on the basis of optimal medium（NB medium）when optimizing the fermentation conditions of strain Bacillus sp.wsm-1 in this study.The results turned out that most of the added carbon sources could increase the production of C14 iturin W,but had a significant inhibitory effect on the C15 iturin W;in the addition of nitrogen sources,tryptone had a significant promotion effect on both homologs,which could be considered as the best nitrogen source;the addition of six detected amino acids could promote the production of two homologues to a certain extent.In summary,the two marine bacterial strains screened in this study had a strong antifungal activity against plant pathogenic fungi,and had good potential application prospects in the prevention and control of plant diseases.Determination of antibacterial activity,exploration of the mechanism and the optimization of fermentation conditions can provide the basis for its future development and utilization.