Isolation And Identification Of Main Antimicrobial Substance Produced By Endophytic Bacteria Y13 In The Camellia Oleifera

Camellia olifera is one of the most important woody oil crops in china that has big economic value.As the tea industry is growing,there are more and more diseases happening to Camellia olifera.Camellia oleifera anthracnose is one of these serious diseases,it leads to serious drop in buds and leaves in Camellia olifera so that inhibit the quantity and quality of tea industry.Recently,using massive chemical pesticide leads to the strains Camellia olifera have got resistance to chemical pesticide and pollute the environment seriously as well.So the biocontrol for Camellia olifera is paid more and more attentions.Strain Y13 isolated from Camellia belongs to Bacillus.The strain has been shown strong activity against Colletotrichum gloeosporioides.In order to improve the effectivity and stability,we isolate,purify and identify the inhibitory compounds through methyl alcohol precipitation,solid phase extraction,reversed phase high performance liquid chromatography and ITMS.The main results are as following:(1)Isolation of inhibitory compounds produced from endophytic bacteria Y13.First of all,we get the fermentation broth through the liquid fermentation and isolate inhibitory compounds from Y13 fermentation broth by ethanol precipitation and ammonium sulfate precipitation.The result shows that the extracts showed more strong inhibition against Colletotrichum gloeosporioides by ethanol precipitation than by ammonium sulfate precipitation.So we chosen the ethanol precipitation as the extract method we used.40%?60%?70%?80%?90%these five concentrations of ethanol are designed and compared.Finally,the result showed that the extract from 80%ethanol showed the biggest inhibition zone against the mycelium growth of C.Gloeosporioides.The diameter of inhibition zone up to 19.50mm,so 80%are screened as the optimum concentration.(2)Purification of inhibitory compounds produced from endophytic bacteria Y13.Firstly,Inhibitory compounds were concentrated by solid phase extraction from Y13 fermentation broth.We chosen methyl alcohol as extractant and the concentrations are 0?45%?65%?95%respectively.The inhibition results showed that 65%methyl alcohol can concentrate most of inhibitory compounds.Secondly,these inhibitory compounds are purified better through reversed phase high performance liquid chromatography.20 main peak fractions are assayed for their antifungal activity in an agar plate assay,separately.The result indicated that 16 of 20 compounds showed activity.20 main peak fractions can be divided in two groups.One group at retention time from 10.5 to 26.0 min showed inhibition.The peak fraction at retention time 11.0min showed strongest activity.The other group at retention time from 27.5-39.5min dark the mycelial of C.gloeosporioides.The compound at retention time 37.5 showed strongest activity.Optimized the condition of chromatography again and again.Finally,we chosen preparative C18(5um 10×250mm)as the chromatographic column and the mobile phase are acetonitrile and ultrapure water,the concentration of acetonitrile is from 44%to 94%,the speed of flow is 0.7ml/min,the wavelengh is 210nm.After that,we test the pureness of inhibitory compounds by analysis HPLC.The result showed that all of compounds have pure enough to satisfy the demand of ion trap mass spectra.(3)Structural identification of inhibitory compounds produced from endophytic bacteria Y13.The structures of main inhibitory compounds were identified by ion trap mass spectra.Their structures were analysed by combining collision induced dissociation with Pulsed-Q Dissociation.The result showed that the fraction at retention time 11.0min is homologs of Iturin whose primary chemical structure is Cyclo(C14?AA-Asn-Tyr-Asn-Gln-Pro-Asn-Ser),at the same time,all of fractions at retention time 10.5-25.0min are homologs of Iturin as well.There are at least 40 categories in these compounds.The fraction at retention time 37.5min is the homologs of Fengycin whose primary chemical structure are R'(C15)-Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Val hat belongs to FengycinA2 and R'(C14)-Glu-Orn-Tyr-Thr-Glu-Ala-Pro-Gln-Tyr-Ile that belongs to FengycinAl.Other compounds at retention time 27.5-39.5min were identified as well.They are more than 60 categories homologs of Fengycin too.Finally,we identified and analysed the rest of fractions which have no inhibitory activity against C.Gloeosporioides,they are the homologs of Surfactin and the categories up to 30.