Antifungal Actinomycetes Screening, Identification Of Its Active Metabolite, A Preliminary Study

This thesis studies on the screening of 20 strains of actinomycetes which were supplied by the Cell Engineering Group of Microbial Laboratory of Shen Yang Institute of Ecology of Chinese Academy of Sciences.In the subsequent screening program of the fungicidal activity against Alternaria longipes,strains which have the better fungicidal activity were exhibited.Meanwhile, we did the corn seed soaking experiment with the fermentation broth of the 7 strains which had good effect on growth promotion.The results showed that the strain F-9 had the excellent antagonism and growth promotion,so we selected F-9 as the objective strain.After the purification of strain F-9,we chose the F-9-3 which had the strongest function on bacteriostasis activeness and growth promotion,determined its antibacterial spectrum and expanded the soaks seeds experiment,the results indicated that the indoor sterilizing effect of the strain was very good,the strain had the inhibitory action to the partial cause of disease fungus,the fermentation broth of the strain F-9-3 also had strongly promotion for the growth of oil bean,hot pepper and cabbage.By means of observation on substrate mycelium,aerial mycelium,spore and sporothrix, cultural characteristics,biochemical and physiological characteristics,and the 16SrRNA sequence determination,F-9 was primarily identified as a variety for which the name Streptomyces olivaceoviridis.The single factor and orthogonal experiment indicated that the fermentation medium was corn meal 70g,MgSO₄ 0.5g,KH₂PO₄ 3g,KCl 2.5g,and Na₂HPO₄ lg in one liter.The optimal cultivation temperature was 28℃and optimal pH was 6.5;the most suitable shake rate was 220r/min;the optimal fermentation time was 96h;and the most suitable volume of cultivated liquid in 250ml conic bottle was 50mL.The research results which were obtain at the storage condition of room temperature and generation culture showed that the product of the strain has high stability.Stability test of fermentation products of F-9 had been carried out to acid,alkali,temperature and transfer of culture.The fungicidal activity of the fermentation products of F-9 was no marked decrease after heated at 60℃for one hours,however,at 100℃,the fungicidal activity was great changed along with the time expand,one hour later,there was no activity.The results show that in acidic conditions,the activity was easily inactivated,and the relative stability in the neutral or alkaline conditions.The bioactive substance was mainly isolated from fermentation broth of F-9-3 by solvent extraction,silica gel column chromatography and HPLC.The bioactive component was eluted under the condition of mobile phase(CH₃OH:CHCl₃=10:3/2:1)by silica gel column chromatography;then,we merged the same bioactive substances which were identified by the means of thin layer chromatography,and determined by High Performance Liquid Chromatography with the C₁₈column.The mobile phase was acetonitrile:KH₂PO₄ buffer (0.02mol/L)=30:70.The flow rate was 1ml/min,when the retention time came to 15.543,we got the chromatographic peak which had the high chromatographic resolution and the peak shape is good.Under such chromatographic condition,the sample was obtained by the means of preparative liquid chromatography,and the molecular weight(MW)of active substance was conceived as 495 with the method of ESI-MS.