The Diversity Of Endophytic Actinobacteria From Peganum Harmala And Primary Investigation Of Their Secondary Metabolites

With the rapid development of drug-resistant bacteria,it is very important to find novel antibacterial active compounds.The separation ratio of new active compounds derived from endophytic actinomycetes is higher than that of actinomycetes derived from soil environment.Peganum harmala L.,one perennial medicinal plant in the desert area,has a wide range of biological activities.Therefore,in this experiment,we tried to obtain bioactive natural,by isolating endophytic actinomycetes of Peganum harmala L.,performing diversity analysis and subsequent mining of secondary metabolites.Nine kinds of separation media under p H 7 and p H 9 were used to separate and identify actinomycetes from rhizosphere,root,stem,leaf,seed of Peganum harmala L.and analyze its diversity.A total of 449 strains were identified,distributed in 8 orders,9 families,and 14 genera.Streptomyces,Amycolatopsis,and Nocardiopsis were the predominant groups,and other rare Actinomycetes also included Streptomonospora,Nocardia,Pseudonocardia,Actinokineospora,Saccharothrix,Microbacterium,Myceligenerans,Promicromonospora,Nocardioides,Micromonospora,and Glycomyces.Among them,the maximum similarity between the 16 S r RNA gene sequence of 6 isolates and the effectively published strain sequence in the database is less than 98.65%,which are potential novel species.And p H value of 9.0 was favorable for the isolation of potential new species of rare actinomycetes.The diversity results showed that the species diversity of endophytic actinomycetes associated with the gobi Peganum harmala L.was the most abundant and the most of Amycolatopsis were isolated from the samples in this habitat.A multi-phase taxonomic study was conducted on one of the potential new species of actinomycetes TRM 65233.Through genotypic characteristics,morphological characteristics,physiological biochemical characteristics and chemical classification characteristics,TRM 65233 was identified as novel species of genus Actinokineospora.The new species is named Actinokineospora pegani.A total of 10 pathogens included multi-drug-resistant pathogenic drug-sensitive strains for instance Enterococcus faecalis,Staphylococcus aureus,Klebsiella pneumoniae,Acinetobacter baumannii,Pseudomonas aeruginosa,Escherichia coli,Salmonella,Shigella Castellani,Candida albicans,and Cryptococcus neoformans were used as targets by agar diffusion method and microplate half Quantitative method for antibacterial activity screening.88 active actinomycetes were screened out of 449 strains as endophytic actinobacteria associated with Peganum harmala L.,including 77 strains of Streptomyces,6 strains of Amycolatopsis,3 strains of Nocardiopsis,1 strains of Micromonospora,1 strains of Glycomyces.S.rochei TRM 65215 had an inhibitory effect on 10 pathogens,and the inhibition rate was generally greater than 80%.It is suggested that the endophytic actinomycetes from Peganum harmala L.had a broad spectrum of antibacterial activity,and was an antagonistic actinomycetes resource for Staphylococcus aureus,Pseudomonas aeruginosa,Candida albicans and other pathogens.Amycolatopsis lurida TRM 64558 has antibacterial activity against methicillin-resistant Staphylococcus aureus,therefore,the secondary metabolites of fermentation broth from TRM 64558 were preliminary explored.The effective components in the broth were enriched and purified by macroporous resin adsorption chromatography,silica gel column chromatography and preparative liquid chromatography,and a monomer compound was obtained.The structure was identified as 4-Phenyl-3-Butenoic Acid.The resources of endophytic actinomycetes from Peganum harmala L.are abundant and diverse,including a variety of supposed novel species of rare actinomycetes,which provides the species support for the development and utilization of rare actinomycetes.Among the various active strains screened,the biosynthetic gene cluster of secondary metabolites of TRM 64558 was predicted to contain the gene cluster of glycopeptide antibiotics for the treatment of drug-resistant bacterial infections,which has the potential to exploit the active compounds and is worth further studying.