Abstract
Many actinobacteria produce secondary metabolites that include antimicrobial compounds. Since most of the actinobacteria cannot be cultivated, their antimicrobial potential awaits to be revealed. We hypothesized that the actinobacterial endophyte communities inside Melia toosendan (Chinaberry) tree are diverse, include strains with antimicrobial activity, and that antimicrobial activity can be detected using a cultivation independent approach and co-occurrence analysis. We isolated and identified actinobacteria from Chinaberry, tested their antimicrobial activities, and characterized the communities using amplicon sequencing and denaturing gradient gel electrophoresis as cultivation independent methods. Most of the isolates were identified as Streptomyces spp., whereas based on amplicon sequencing the most abundant OTU was assigned to Rhodococcus, and Tomitella was the most diverse genus. Out of the 135 isolates, 113 inhibited the growth of at least one indicator organism. Six out of the 7577 operational taxonomic units (OTUs) matched 46 cultivated isolates. Only three OTUs, Streptomyces OTU4, OTU11, and OTU26, and their corresponding isolate groups were available for comparing co-occurrences and antimicrobial activity. Streptomyces OTU4 correlated negatively with a high number of OTUs, and the isolates corresponding to Streptomyces OTU4 had high antimicrobial activity. However, for the other two OTUs and their corresponding isolate groups there was no clear relation between the numbers of negative correlations and antimicrobial activity. Thus, the applicability of co-occurrence analysis in detecting antimicrobially active actinobacteria could not be proven.
Highlights
Free-living and endophytic actinobacteria are common in terrestrial and marine environments
Actinobacteria were identified by amplicon sequencing, denaturing gradient gel electrophoresis (DGGE) and Sanger sequencing, all targeting the 16S rRNA gene using actinobacteria specific primers
Sequences from amplicon sequencing were grouped to 7577 operational taxonomic units (OTUs) at 97% similarity level
Summary
Free-living and endophytic actinobacteria are common in terrestrial and marine environments. Culture-independent methods, for example amplicon sequencing targeting the bacterial 16S rRNA gene, have shown that Streptomyces species form only a small fraction of actinobacterial communities in soil, marine sediments, and inside a plant[1,2], and that revealing the diversity of actinobacteria necessitates a focused approach. Actinobacterial endophyte communities that include producers of antimicrobial compounds are expected to display social and asocial interactions[10]. We hypothesized that the actinobacterial endophyte communities inside traditional medicinal plant Melia toosendan (Chinaberry) are diverse, and include strains with antimicrobial activity. We (i) isolated strains from Chinaberry, identified the strains using 16S rRNA gene sequencing, and tested their antimicrobial activities, and (ii) applied denaturing gradient gel electrophoresis and amplicon sequencing targeting the 16S rRNA gene as cultivation independent approaches. Co-occurrences of the OTUs were analyzed, and the results from co-occurrence and antimicrobial activity analyses were compared
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