Investigation On Endophytes Of Euonymus Japonicas And Its Bioactive Metabolites

Plant endophytes are one of the microbial population, which widely reside in the healthy tissue of their host and can produce a variety of secondary metabolites. They have been reported to be the most important sources of bioactive natural products with novel structures. Euonymus japonicas is a high anti-pollution plant, and it has been used in traditional medicine to treat traumatic injury, painful urination and other diseases.The paper is about the research work on the bioactive endophytes, which were isolated from Euonymus japonicus, including antimicrobial activity of fermentation substance, strain identification, the purification and structural identification and optimization of fermentation medium. The results are as follows:1. 42 strains of endophytic actinomyces and 21 strains of endophytic fungi were obtained from Euonymus japonicus by the tissue homogenate and slices of sample respectively. Antimicrobial activity of fermentation substance indicated that five strains of endophytes exhibited biological activities, and strain G1 and W1 were preferred for further research by the biological activities, fermentation status and other factors.2. According to the results of 16 S rDNA sequence analysis, the endophytic actinomycete strain G1 was basically identified as Streptomyces flavofuscus G1. Two antimicrobial compounds, G13 and G19, were isolated from cultures, which were identified as phencomycin and 4’-deacetyl-(-)-griseusin A respectively. The bioassay results showed that 4’-deacetyl-(-)-griseusin A exhibited strong antibacterial activity against Xanthomonas oryzae pv. oryzae,Pseudomonas aeruginosa,Bacillus cereus, and Staphylococcus aureuse, with minimum inhibitory concentration(MIC) value of 1.56 μg/mL. In addition, 4’-deacetyl-(-)-griseusin A could also effectively inhibit the mycelial growth of plant pathogenic fungi Valsa mali and Fusarium graminearum, and the IC50 values were 14.70 and 24.35 μg/mL, respectively. Phencomycin exhibited weak antibacterial activity against the tested bacteria, and it exhibited strong antifungal activity against the mycelial growth of Sclerotinia sclerotiorum and V. mali, with IC50 values of 5.21 and 4.82 μg/mL, respectively.3. According to the results of ITS sequence analysis, the endophytic fungus strain W1 was basically identified as Cylindrocarpon olidum W1. One high yield antimicrobial compound, W1-4, were isolated from the mycelia, which were identified as ascochlorin. Its antimicrobial activity against plant pathogenic microorganisms was evaluated for the first time. The bioassay results showed that it exhibited weak antibacterial activity against the tested bacteria, and it could inhibit the mycelial growth of ten plant pathogenic fungi, and inhibition rate against V. mali is 82.61% with the concentration of 100 μg/mL.4. The central composite design and response surface methodology was used for medium optimization of Cylindrocarpon olidum W1 to improve the production of ascochlorin. The best formula is: corn flour 30 g/L, fish meal 8.098 g/L, KCl 6.928 g/L. In the optimal culture medium, the content of ascochlorin(1015.32 μg/mL) is 4.81 times of the original content(211.08 μg/mL).