| Presently, diseases caused by a variety of pathogens are increasingly endangering the health and life of human beings while the chemical drug abuse contributed to the resistence of some pathogens. Since the new century, the population is increasing rapidly and the demand for food is becoming more and more urgent. The crop yield reduction caused by plant pathogens exacerbating the food crisis. Therefore, seeking new, safe and efficient natural antimicrobial drugs becomes the urgent matter. Endophytes are a series of special microorganisms residing in living plants which are relatively unstudied for a long time. However, more and more researches have proven that endophytes are valuable sources of novel antimicrobial products. Presently a number of antimicrobial natural products belong to diverse structural classes have been obtained from endophytes. Thus, endophytes possess broad development prospects. Endophytic Brevibacillus brevis 011 was used in this study to investigate the antifungal activity and mechanism of crude extracts produced by 011 strain. The effects of post-harvest treatment with crude extracts on the fresh-keeping of tomatoes were investigated. We also separated, purified and identified the antifungal compounds derived from crude extracts.1. In order to investigate the utilization potential of crude extracts produced by endophyte 011,14 pathogenic funguses were selected to evaluate their antagonistic activity using plate-perforation method. Results indicated that the crude extracts of 011 strain showed the weakest antagonistic activity on Colletotrichum gloeosporioides Penz while displayed the strongest inhibitory effect on Alternaria sonali. The treatment of crude extracts resulted in the pathogen mycelia grew in abnormal shape and the germination of spores was inhibited. The crude extracts also caused damage to cytoplasmic membrane accompanied with induction of lipid peroxidation, increasement of hydrogen peroxide content in the cell and the decline of protein synthesis in mycelia membrane. When the concentration of crude extracts was at 50 mg.mL-1, it also expressed obvious inhibitive effect on ergosterol synthesis. The activities of pectinase and cellulose were both significantly lower than that of control after treatment.2. The effects of post-harvest treatment with endophytic strain 011 crude extract on the fresh-keeping of tomatoes (Xiangfenyihao of Hunan province) were investigated. Results indicated that after 25 d storage, the decay rate treated with crude extracts significantly decreased by 52.17% compared to sterile water group. In the whole storage period, crude extracts treatment inhibited the mass loss rate, significantly delayed the reduction of Vc content, reduced the loss of reducing sugar, soluble protein and the content of titratable acid. It significantly inhibited the accumulation of O2-·and MDA levels and maintained the integrity of the cell membrane. The crude extracts treatment could significantly enhance anti-reversibility enzyme activity of POD, CAT, SOD and PAL.3. Six bioactive gradients 12-1-12-6 were obtained from crude extracts after separated by HPLC. Two bioactive components 12-1-1 and 12-1-2 were obtained from gradient 12-1 after separated by HPLC. The weights of componends 12-1-1 and 12-1-2 were 1.6 and 0.7mg, respectively, and their minimal inhibitory concentrations (MIC) were 11.25 and 8.75 μg/mL. Three bioactive components 12-2-1,12-2-2 and 12-2-3 were obtained from gradient 12-1 after separated by HPLC. Their weights were 0.6,0.8 and 7.1 mg, respectively, and their MIC values were 30,10 and 15.6μg/mL, respectively. Nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HI-MS) showed that the compound was cyclic lipopeptide with a molecular mass of 1042 Da. Its molecular formula was C48H74N12O14. The hrdrophilic part was composed of 7 a-amino acid residues including Tyr, Ser, Gln, Asn and Pro. The amino acid residues connected into ring by amido linkage. The hydrophobic portion was (3-amino fatty acid which was made up of 14 carbon atoms. The primary structure was Pro-Ser-Asn-βAFA-Asn-Tyr-Asn-Gln. Compound 12-2-3 belongs to Iturin family and has the similar structure with Mycosubtilin. It is the homolog of Mycosubtilin. |
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