Study On Isolation And Antimicrobial Activity Of Endophytes From Acorus Calamus Linn

As Traditional Chinese medicine,Acorus calamus Linn.grows in sullage and has a strong antimicrobial activity.Some endophytic fungi could produce large new natural products,some metabolite produced by endophytic fungi were identical or semblable with host plant.It now appears that endophytes is an enormous,relatively untapped source of diversity the microbial.In this paper the endophytes from Acorus calamus Linn.were isolated for the first time.With high throughput microdilution method,the endophytes of antifungal,antimitotic and antimicrobial activity were preliminary screened by model of PyricuLaria oryzae P-2b and bacterium,through detecting deformation of mycelia germinated from conidia of P. Oryzae P-2b.S9 strain possessed strongest antimicrobial activity to broad-spectrum bacterium,yeast and mycelial fungi.The further research was made for the activity substance,classification,stability, Optimization of fermentation conditions and the plant for antimicrobial activity.This research is for the purpose of exploring for endophytes from Acorus calamus Linn.to produce antimicrobial activity constituent possibility,providing the idea for studies the new resource to search new antimicrobial compounds.Endophyte is a new resource of antimicrobial activity and potential for application.Main contents of this thesis as follows:1.The plant materials are thoroughly surface treated with 75%ethanol,sodium hypochlorite solution and sterilizated water.Controlled experiments that detecting of biologic safe cabinet,rinse water and tissue blotting were set simultaneously were extremely essential.There are a great variety of endophytes in Acorus calamus Linn. 113 strains were isolated from stem and leaf.2.With high throughput microdilution method and screening model of P.Oryzae P-2b and bacteria,50 strains against P.Oryzae P-2b,46 strains against bacteria were screened from 113 strains.S9,S15,S24 and S70 had intense anti-plant pathogenic fungi and anti-human pathogenic fungi ability,and S9 have a broad-spectrum antimicrobial activity.3.Growth characteristic test have been examine by the changes of saccharide value,biomass,pH value and minimum inhibitory concentration.we find Logarithmic phase are in the first to third day and Stable phase are in the fourth to sixth day and from the seventh day is Decline phase.During the whole Growth cycle,there are big changes in saccharide value,biomass,pH value and minimum inhibitory concentration.4.The stability of S9 have been studied by temperature,illumination,pH and existing from generation to generation.S9 have a good stability on temperature,illumination and generation.However,pH has great effect on antimicrobial activity of S9,which has greater antimicrobial activity in acidic environment than in alkaline environment.The activity components are outside cell but not the Macromolecules such as Polysaccharide and Protein.5.Optimize fermentation conditions by Single factor test,Plackett-Burman and Response Surface Method,the optimization of fermentation conditions for S9 is 28℃, 140 r/min,saccharose 26 g·L^-1,KH₂PO₄ 1mmol·L^-1,potato 250 g·L^-1,Yeast extract 20 g·L^-1.6.Active systematic separation portions of six strains of S9 were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The activity components are analyzed by GC-MS.Strain identification by ITS sequence analysis and Colony morphology,S9 belong to Trichoderma sp.and is similar with Hypocrea lixii sp.The key activity components analyzed by GC-MS are 2-(2,2-dimethylpropylidene)cyclohexane-1,3-dione.7.Active systematic separation portions of six strains of Acorus calamus Linn. were traced in order to determined active intensity with indicator and found the Petroleum ether has the strongest antimicrobial activity.The key activity components analyzed by GC-MS are 1-methoxy-4-phenylethynyl-Benzene.