A Workflow for the Quantitative Assessment of the Endophytic and Epiphytic Bacterial Microbiomes of the Bark of <em>Populus trichocarpa</em>

Microorganisms colonizing plant surfaces and internal tissues may possess beneficial functions in promoting plant growth and health. However, information on the microbiome of bark tissues of woody plants remains limited, especially regarding the endophytic and epiphytic bacterial microbiota of the bark of Populus. To overcome this limitation, we established a workflow to quantify the composition and diversity of the endophytic and epiphytic bacterial microbiota colonizing the bark of Populus trichocarpa. Briefly, the epidermis of the stem of P. trichocarpa was repeatedly wiped with a cotton ball dipped in 0.1% Tween 20 to acquire epiphytic bacterial samples. The stripped epidermis was sterilized and then repeatedly frozen and crushed using liquid nitrogen and a bead beater, respectively, to collect endophytic bacterial samples. Genomic DNA was extracted from the cotton-adhered epiphytic bacterial communities and the crushed bark of P. trichocarpa, and underwent ploymerase chain reaction (PCR) amplification with primers targeting the hypervariable V5-V7 and V4 regions of the bacterial 16S rRNA gene. Three replicates of the obtained PCR products of each endophytic or epiphytic sample were mixed in equal concentrations to build the amplicon library, which was then sequenced. The obtained sequences were analyzed by sequence splicing, quality filtering, chimera removal, and taxonomic annotations. In summary, we established a reliable and efficient workflow from sample collection to data analysis for determining the endophytic and epiphytic bacterial microbiomes of the bark of P. trichocarpa through 16S rRNA gene profiling. Together with the methods for exploring microbiota colonizing barks established in the previous study, our methodology may serve as a blueprint for designing protocols for investigating the bark microbiome of other woody plant species, particularly economically important trees of forests.