Studies On Antimicrobial Activity Of Endophytes From Platycladus Orientalis

Platycladus orientalis is a traditional Chinese medicine for relieving cough, dispelling phlegm, detoxification and detumescence. This thesis focuses on isolation, fermentation and strain identification of endophytes isolated from Platycladus orientalis, and the purification and structural identification of antimicrobial ingredients. The primary results are as follows:1. 25 strains of endophytic fungi were obtained from PDA medium, and 69 strains of endophytic actinomyces were obtained from GAUZE’s Medium NO.1, starch-casein medium and Modified HV medium.2. The results indicated that nine strains of endophytes exhibited antimicrobial activity. Finally, strain ZH32 and F3 were preferred for further investigation on their active ingredients.3. The endophytic fungus strain ZH32 was identified as Chaetomium globosum, based on the analysis of its ITS sequences. An antimicrobial metabolite, WH-01, was isolated and purified by means of macroporous adsorption resin chromatography, silica gel column chromatography and high performance liquid chromatography(HPLC) in sequence. It was identified as Chaetoglobosin A by HRMS and NMR spectroscopy analysis. Its antimicrobial activity against plant pathogenic microorganisms was comprehensively evaluated for the first time. The minimum inhibitory concentrations(MICs) against Ralstonia solanacearum, Pseudomonas syringae pv.actinidiae and Xanthomonas oryzae pv. oryzae were 12.5 μg/m L. WH-01 also showed significant inhibitory effects on mycelial growth of eight plant pathogenic fungi, and its EC50 values against Alternaria alternate and Curvularia lunata were 1.66 and 4.58 μg/m L, respectively. Besides, the EC50 value of WH-01 against spore germination of Botrytis cinerea was 8.82 μg/m L.4. The endophytic actinomycete strain F3 was identified as Streptomyces ambofaciens, according to the analysis of its 16 S r DNA sequences. An antibacterial metabolite, WH-02, was isolated and purified by means of macroporous adsorption resin chromatography, silica gel column chromatography, gel column chromatography and high performance liquid chromatography(HPLC) in sequence. Based on the HRMS and NMR data, WH-02 was elucidated as a Telomycin analogue, and its constituent amino aicds and the sequence were identical with those of Telomycin. The absolute configuration of its constituent amino aicds will be further investigated. WH-02 showed potent antibacterial activitives against Ralstonia solanacearum, Pseudomonas syringae pv.actinidiae, Xanthomonas oryzae pv. oryzae, Bacillus subtilis, Bacillus cereus, Staphyloccocus aureus and Escherichia coli, and their MICs were 6.25, 6.25, 6.25, 3.13, 25, 25 and 6.25 μg/m L, respectively.