Isolation, Purification, Identification And Activity Research Of Antibiotic Peptaibols From The Fermentation Broth Of Trichoderma Harzianum

Peptaibols are a group of linear polypeptide antibiotics with the a-helix tructure characterized by the presence of a high proportion of the nonprotein-aminoisobutyric acid, the C-terminal-bonded amino alcohols acetyl N-acylgroup, They are mainly producted by fungi strain of Trichoderma spp.and have attracted particular attention as a result of their antibiotic activities and membrane-modifying properties. The purpose of this study is to further understand the mechanism of the Trichoderma harzianum antagonistic antibacterial mechanism and lay the foundation to expand its application through isolation, purification, identification and activity research of antibiotic peptaibols from the fermentation broth of Trichoderma harzianum.In this paper:Peptaibol antibiotics were isolated using a bioassay-guided fractionation against R.solani. To purify the peptaibols, the fermentation broth was fractionated by polystyrene resin XAD-2, sephadex LH-20, High Performance Thin-layer Chromatography (HTLC) and High performance liquid chromatography (HPLC). A specific chromogenic reaction with4,4’-Methylenebis (N,N-dimethylaniline)(TDM) was used for the preliminary identification of peptaibols. The sequence of the peptaibol antibiotics were determined by LC-ESI-MS2analysis. The influence of peptaibols on the mycelial growth, the sclerotium formation and germination, mycelial morphology, cell wall integrity and the permeability of membrane system, as well as the effect on mycelium growth of the pathogenic fungus when combined with Trichoderma were finally determined. We got the results as follows:(1) Twenty peptaibol antibiotics include Alamethicin F-50, Alamethicin II, Atroviridin B, Atroviridin C, Stilboflavins B1, Stilboflavins B2, Stilboflavins B3, Stilboflavins B4, Trichokindin la, Trichokindin Ila, Trichokindin Ⅲa, Trichokindin_Ⅳ, Trichocompactins Ⅱa, Trichocompactins Ⅲa, Trichocompactins IV, Peptaibol FC1-5, were identified from the fermentation broth of T. harzianum and five of the peptaibols were novel. Nine peptaibol antibiotics include Alamethicin F-50, Alamethicin II, Atroviridin B, Atroviridin C, Peptaibol FC1-5were Finally prepared by HPLC.(2) Peptaibols Alamethicin F-50, Alamethicin II, Atroviridin B, Atroviridin C could significantly inhibit the hyphal growth of T.Cucmeris, R.Solani, M.oryzae, F. cucumerinum and F.niveum with EC50values of3.00,3.51,3.39,3.89μg·ml-1;1.32,1.78,1.53,1.91μg·ml-1;2.54,3.20,2.75,3.30μg·ml-1;8.22,10.0,8.71,10.79μg·ml-1;17.7,20.89,18.6,22.5μg·ml-1.(3) Peptaibols Alamethicin F-50, Alamethicin II, Atroviridin B, Atroviridin C could significantly inhibit the germination of the spores of F. cucumerinum, F.niveum and M.oryzae with EC50values of5.74,6.75,6.10,6.55μg·ml-1;12.9,16.8,13.5,18.5μg·ml-1;24.3,28.7,26.5,30.2μg·ml-1(4) Peptaibols FC could destroy the cell wall integrity and increase the permeability of cell membrane system of pathogenic fungus, and the hyphae of the pathogenic fungi treated with peptaibols Alamethicin F-50and peptaibols FC showed deformity. Furthermore, peptaibols FC could effectively inhibit sclerotium formation of R.Solani.(5) Peptaibols exerted synergistic antibiotic effect against R.solani with EC50values of1.03ug·ml-1, and M.oryzae with EC50values of4.67μg·ml-1, when applied with trichodermin.