The plant microbiota forms complex associations with its host to support health in its natural environment. Root-associated bacteria (RAB) colonize root compartments and can influence plant functions. The goal of this study was to characterize the microbiome connected to the rhizosphere of the orchid Dactylorhiza traunsteineri. Our community analysis, based on sequencing of 16S ribosomal DNA (rDNA), found that Proteobacteria, Actinobacteria, Myxococcota, Bacteroidota, and Acidobacteria are the main types of bacteria in the D. traunsteineri rhizosphere. Using deep shotgun metagenomics and new metagenome assembly, we identified 47 MAGs, revealing important metabolic and biosynthetic abilities of the orchid's RAB.
Overall design: D. traunsteineri individuals (from the turfosa subspecies) were taken from the Kitzbuehel alpine town region in the western Austrian province of Tyrol, potted, and kept at the Botanical Garden of the University of Vienna under conditions similar to those of the sampling region. Metagenomic DNA was isolated from the rhizosphere soil and root wash samples, which were dried in a sterile environment. For bacterial community analysis, the V3-V4 fragment of the 16S rDNA gene was amplified from the genomic DNA extracted from the soil and root samples using 341F and 805R primers with adapters, in duplicates, using Q5 high-fidelity DNA polymerase. The 16S rRNA gene copy concentrations in DNA extracts were measured with quantitative PCR and normalized to the same 16S rRNA fragment copy number to improve comparability and reduce PCR bias, and purified using the Agencourt AMPure XP purification system. Sequencing indexes were added in a second amplification reaction with sample-specific barcodes using Q5 high-fidelity DNA polymerase. Amplicon libraries were purified using the Agencourt AMPure XP purification system and measured using the PicoGreen kit. The libraries were combined and diluted to 4 nM before being sequenced for 300 cycles pair-end on the Illumina MiSeq system using the MiSeq Reagent Kit v2. Shotgun metagenomics sequencing was done on the genomic DNA from the soil and root samples. The libraries were prepared using 1000 ng of genomic DNA, which was broken up using the Bioruptor sonication device. Fragments were cleaned up using the GeneJet PCR Purification Kit and selected for a 300 bp size using Agencourt AMPure XP beads. Sequencing libraries were prepared following the standard NEBNext Ultra II DNA Library Prep with Sample Purification Beads procedure. The final library concentrations were measured using the Qubit dsDNA HS Assay Kit and the average size of the library was measured using the Agilent 5200 Fragment Analyzer System. The libraries were combined and diluted to 4 nM before being sequenced for 600 cycles pair-end on the Illumina MiSeq system using the MiSeq Reagent Kit v3. Less...