Endophytic Actinomycetes From Tea Plants(Camellia Sinensis) In Fujian Province:Isolation,Identification And Analysis Of Bioactive Metabolites

Endophytic actinomycetes are a promising source of novel bioactive metabolites,especially antibiotics,with abuntant species diversity and functional diversity.Tea plants(Camellia sinensis)produce arsenals of phytochemicals,which are linked to a number of medicinal and nutritional properties.There are rich tea plant resources in Fujian province,China,which provides a good condition for collecting endophytic actinobacterial resources.However,a systematic investigation into the abundance and diversity of cultivated actinomycetes residing in tea plants in Fujian and their activities has not been performed.Therefore,with seven isolation media,endophytic actinomycetes were isolated from roots,stems and leaves of 16 tea cultivars in Fujian.Their diversity,anti-microbial and plant-growth promoting activites were explored.One such bioactive strain was chosen to further characterize the antifungal metabolites.The results were as follows:Preliminary screening of strains was based on their colony morphology.By 16 S rRNA sequencing,a total of 46 strains were identified as actinomycetes from nine tea cultivars.Their abundance and diversity were shown to be influenced by both the genotypes and tissue types of tea plants.Among nine tea cultivars,most actinobacteria were recoved from Rougui,Tieguanyin and Fuyun 6.With regard to tissue types,actinomycetes were more enriched in tea roots and leaves.Based on 16 S rRNA phylogeny,these isolates were taxonomically grouped into 11 families and 13 genera,including Kribbella,Mobilicoccus,Piscicoccus,Streptomyces,Actinomadura,Nocardia,Kytococcus,Leifsonia,Microbacterium,Micromonospora,Mycobacterium,Nocardiopsis and Pseudonocardia.The genus Streptomyces was most prevalent whereas rare genera,Mobilicoccus and Piscicoccus,were reported for the first time to occur as plant endophytes.Besides,six strains had homology less than 98.6% compared with type strains,speculating that they may represent new species.To study the biosynthetic potentials for secondary metabolites in endophytic actinomycetes from tea plants,PCR screening of polyketide synthase genes(pks-I and pks-II)and nonribosomal peptide synthetase genes(nrps)were performed.A total of 37 strains showed positive to at least one of these genes.Antimicrobial assays against a set of bacterial and fungal pathogens uncovered that 16 of 37 strains had antimicrobial activities,of which XY006 showed a good broad-spectrum of antifungal activity.And XY191,XY192 and XY208 exhibited strong inhibitory activities against both Staphylococcus aureus and Bacillus subtilis.The results demomstrated that endophytic actinomycetes associated with tea plants have a high potential for producing antimicrobial metabolites.Given that actinobacteria play an important role in promoting plant growth,indole acetic acid(IAA)production and 1-aminocyclopropane-1-carboxylic acid deaminase(ACCD)activities were determined and recorded in 93.5% and 21.7% of all isolates,respectively.The IAA production ranged between 2.2 and 43.1 ?g/mL.More importantly,eight strains not only produced IAA,but also showed ACC deaminase activities.Overall,these results indicate that in general,endophytic actinomycetes isolated from tea plants possess promising antimicrobial activities and plant-growth promoting properties.Further,XY006 was selected to study the antifungal compound(s).Guided by the antifungal bioassay,bioactive metabolites were isolated and purified through various purification methods,including organic solvent extraction,ODS reversed phase column chromatography,high performance liquid chromatography(HPLC),and Oasis HLB column chromatography.Ultimately,three antifungal compounds were purified.Compound 1 may be soyasaponins based on the MS/MS fragmentations in comparison to known metabolites in public databases,which should be further detected by NMR to determine chemical structure.The amount of compound 2 and 3 were too small to identity the structure by NMR.Nevertheless,deduced from MS/MS fragmentations,they were tentatively assigned as lipopeptin A and lipopeptin B,both of which are cyclic lipopeptides.Their structures only differ by one-CH2-group.