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Endophytic microbial communities of lichen are emerging as novel microbial resources and for exploration of potential biotechnological applications. Here, we focused on a medicinal lichen Usnea longissima, and investigated its bacterial and fungal endophytes. Using PacBio 16S rRNA and ITS amplicon sequencing, we explored the diversity and composition of endophytic bacteria and fungi in U. longissima collected from Tibet at five altitudes ranging from 2,989 to 4,048 m. A total of 6 phyla, 12 classes, 44 genera, and 13 species of the bacterial community have been identified in U. longissima. Most members belong to Alphaproteobacteria (42.59%), Betaproteobacteria (33.84%), Clostridia (13.59%), Acidobacteria (7%), and Bacilli (1.69%). As for the fungal community, excluding the obligate fungus sequences, we identified 2 phyla, 15 classes, 65 genera, and 19 species. Lichen-related fungi of U. longissima mainly came from Ascomycota (95%), Basidiomycota (2.69%), and unidentified phyla (2.5%). The presence of the sequences that have not been characterized before suggests the novelty of the microbiota. Of particular interest is the detection of sequences related to lactic acid bacteria and budding yeast. In addition, the possible existence of harmful bacteria was also discussed. To our best knowledge, this is the first relatively detailed study on the endophytic microbiota associated with U. longissima. The results here provide the basis for further exploration of the microbial diversity in lichen and promote biotechnological applications of lichen-associated microbial strains.

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BACKGROUND: Antimicrobial resistance (AMR) is a major threat to public health. Microorganisms equipped with AMR genes are suggested to have partially emerged from natural habitats; however, this hypothesis remains inconclusive so far. To understand the consequences of the introduction of exogenic antimicrobials into natural environments, we exposed lichen thalli of Peltigera polydactylon, which represent defined, highly diverse miniature ecosystems, to clinical (colistin, tetracycline), and non-clinical (glyphosate, alkylpyrazine) antimicrobials. We studied microbiome responses by analysing DNA- and RNA-based amplicon libraries and metagenomic datasets. RESULTS: The analyzed samples consisted of the thallus-forming fungus that is associated with cyanobacteria as well as other diverse and abundant bacterial communities (up to 108 16S rRNA gene copies ng-1 DNA) dominated by Alphaproteobacteria and Bacteroidetes. Moreover, the natural resistome of this meta-community encompassed 728 AMR genes spanning 30 antimicrobial classes. Following 10 days of exposure to the selected antimicrobials at four different concentrations (full therapeutic dosage and a gradient of sub-therapeutic dosages), we observed statistically significant, antimicrobial-specific shifts in the structure and function but not in bacterial abundances within the microbiota. We observed a relatively lower response after the exposure to the non-clinical compared to the clinical antimicrobial compounds. Furthermore, we observed specific bacterial responders, e.g., Pseudomonas and Burkholderia to clinical antimicrobials. Interestingly, the main positive responders naturally occur in low proportions in the lichen holobiont. Moreover, metagenomic recovery of the responders' genomes suggested that they are all naturally equipped with specific genetic repertoires that allow them to thrive and bloom when exposed to antimicrobials. Of the responders, Sphingomonas, Pseudomonas, and Methylobacterium showed the highest potential. CONCLUSIONS: Antimicrobial exposure resulted in a microbial dysbiosis due to a bloom of naturally low abundant taxa (positive responders) with specific AMR features. Overall, this study provides mechanistic insights into community-level responses of a native microbiota to antimicrobials and suggests novel strategies for AMR prediction and management. Video Abstract.

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Lichens are a symbiotic association between a fungus and a green alga or a cyanobacterium, or both. They can grow in practically any terrestrial environment and play crucial roles in ecosystems, such as assisting in soil formation and degrading soil organic matter. In their thalli, they can host a wide diversity of non-photoautotrophic microorganisms, including bacteria, which play important functions and are considered key components of the lichens. In this work, using the BioLog® EcoPlate system, we studied the consumption kinetics of different carbon-sources by microbial communities associated with the thallus and the substrate of Peltigera lichens growing in a Chilean temperate rain forest dominated by Nothofagus pumilio. Based on the similarity of the consumption of 31 carbon-sources, three groups were formed. Among them, one group clustered the microbial metabolic profiles of almost all the substrates from one of the sampling sites, which exhibited the highest levels of consumption of the carbon-sources, and another group gathered the microbial metabolic profiles from the lichen thalli with the most abundant mycobiont haplotypes. These results suggest that the lichen thallus has a higher impact on the metabolism of its microbiome than on the microbial community of its substrate, with the latter being more diverse in terms of the metabolized sources and whose activity level is probably related to the availability of soil nutrients. However, although significant differences were detected in the microbial consumption of several carbon-sources when comparing the lichen thallus and the underlying substrate, d-mannitol, l-asparagine, and l-serine were intensively metabolized by both communities, suggesting that they share some microbial groups. Likewise, some communities showed high consumption of 2-hydroxybenzoic acid, d-galacturonic acid, and itaconic acid; these could serve as suitable sources of microorganisms as bioresources of novel bioactive compounds with biotechnological applications.

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