Isolation Of Endophytic Actinobacteria And Chemistry Of Antimicrobial Compound From Zostera Marina

Actinobacteria are the main source of bioactive substances.Bacterial resistance to antibiotics has become a major clinical problem exacerbated by the widespread and sometimes inappropriate use of antibiotics.It becomes increasingly important to find new microbial resources and new lead compounds.Zostera marina is the main component of seagrass beds.Because of the unique living environment of seagrass bed,Zostera marina contains abundant microorganism resources.At present,the research on Zostera marina mainly focuses on ecological restoration,but there are few reports on actinobacteria and their active compounds from Zostera marina.Therefore,the study on endophytic actinobacteria from Zostera marina in the area of Weihai will enrich the resources of endophytic actinobacteria and provide a good way to discover new natural products.Culture-dependent method with 7 kinds of culture media was used to isolate endophytic actinobacteria from Zostera marina collected from Shuangdao Bay in Weihai of Shandong province.The filter paper method was performed to determine the potential antimicrobial activities of the crude extracts of the isolated actinobacterial strains against 7 indicator bacteria.The secondary metabolites from the actinobacterial strains DYZ076 with good activities were accumulated by solid fermentation,and separation techniques of natural products were applied to isolate compounds from the crude extracts of their fermentation broth.Compound structures were elucidated by comprehensive analysis of modern spectral technology and mass spectrometry.In this study,134 strains of endophytic actinobacteria were isolated from the samples of Zostera marina by using 7 kinds of culture media,such as ISP2,of which83 were from roots and 51 were from leaves.The isolation effects of different culture media were different.Of 134 isolates,100 strains(74.63% of the total)displayed antimicrobial activity against at least one indicator strains.27 strains against at least two indicator strains,19 strains had antimicrobial activities against three indicator strains,and 16 strains against four indicator strains,and one strains against five indicator strains.Most of the active strains showed inhibition against Gram-positive indicator bacteria,and fewer strains expressed antimicrobial activity against Gramnegative bacteria.The solid culture medium of rice was used for the accumulation of secondary metabolites of actinobacteria DYZ076.TLC analysis results showed that the metabolites of this strain were rich.The silica gel column chromatography was applied to isolate 10 secondary metabolites,and the component 8 had antimicrobial activities.Component 8 was selected for semi-preparative high performance liquid chromatography(HPLC)analysis,and the component 8-4 had the strongest inhibitory effects.Then the structure of component 8-4 was determined for Actinomycin D.Our results suggested that the endophytic actinobacteria from Zostera marina are rich and have good antimicrobial activities.