A Workflow for the Quantitative Assessment of the Endophytic and Epiphytic Bacterial Microbiomes of the Bark of Populus trichocarpa

[Presenter] A workflow for the quantitative assessment of the endophytic and epithetic bacterial microbiomes of the bark of Populus trichocarpa. Here we designed and tested a detailed workflow to study the microbiome colonizing the internal tissues or the surfaces of the bark of Populus trichocarpa. This workflow describes in detail the sampling of the endophytic and epithetic microbiomes of the bark of Populus trichocarpa, the processing of collected materials, the extraction of genomic DNA, the PCR amplification of target regions, the preparation of DNA libraries and the sequencing of the endophytic and epithetic bacterial microbiomes via next generation sequencing technology. We also provide a brief analytical procedure for the obtained sequencing data. This proven workflow will provide an effective approach for empirically investigating the endophytic and epithetic microbiome communities of the bark of Populus trichocarpa and other forestry species. Grow three month old Populus trichocarpa saplings in a growth chamber, cut the 10 centimeter long stem segments of Populus trichocarpa from two to 12 centimeters above the sterilized soil surface using a sterilized pruner. Place the stem segments in a sterilized square Petri dish. Repeatedly wipe the epidermis of the stem on the sterilized square Petri dish using sterile cotton with 0.1%. Tween 20. Store each piece of cotton containing microorganisms, epithetic samples in a sterile 1.5 milliliter centrifuge tube at minus 80 degrees centigrade. After wiping the stem surface, cut open the bark with a sterile scalpel, peel off the bark and place it in a square Petri dish. Cut the bark stripped from the stem into approximately one centimeter segments with a sterile scalpel. Sterilize the segment surfaces by soaking in 70% alcohol for two minutes, and then soak the segments in sodium hypochlorite with 0.1%, Tween 80 for five minutes. Finally, sterilize the segments in 70% alcohol for 30 seconds. Then wash all the samples three times with sterile distilled water and place them on a clean bench for air drying. Store each sterilized stem bark endophytic samples in a sterile two milliliter centrifuge tube at minus 80 degrees centigrade. Place the stem bark sections in sterile five milliliter hard tubes and add three sterilized steel beads into each tube. Freeze the hard tubes containing the samples in liquid nitrogen for 10 minutes. Crush the bark samples for five minutes with a bead beater. Then freeze the hard tube again in liquid nitrogen for one minute and repeat the bead beating step for five minutes. Add the crushed stem bark of Populus trichocarpa and the cotton adhered epithetic bacterial communities, 0.25 grams each, to each of the bead tubes and then add 60 microliters of solution C1 supplied by a commercial kit, and gently mixed by inversion. Next, homogenize the endophytic and epithetic samples using a bead beater in four one minute runs. After each run, place the samples on ice for 30 seconds. Centrifuge the tubes at 10,000 times gravity for one minute, extract the endophytic and epithetic samples using a kit following the manufacturer's instructions, add 30 microliters of solution C6 to the center of the white filter membrane and incubate at 25 degrees centigrade for five minutes. Centrifuge at 10,000 times gravity at room temperature for one minute. Measure the concentrations of the genomic DNA extracted from epithetic and endophytic samples using a micro volume spectrophotometer. Dilute each DNA sample with sterile ddH2O to two nanograms per microliter to obtain the DNA template for PCR amplification. Amplify the V5 to V7 region of the bacterial 16S rRNA gene using the forward primer 799F and the reverse primer 1193R. Meanwhile, amplify the V4 region of the bacterial 16S rRNA gene using the forward primer 5115F and the reverse primer 806R. Mix every sample thoroughly and centrifuge to eliminate bubbles. Perform amplification of the V5 to V7 or V4 region of the bacterial 16S rRNA genes on a thermal cycler. Load five microliter liters of each sample Amplicon on a 1% Agarose gel and perform electrophoresis at 120 volts for 30 minutes. View the gel under UV lights to validate successful PCR amplification. For each sample, mix three replicates of the PCR amplification products in a PCR tube. Determine the amplified products' concentration using a commercial kit according to the manufacturer's instructions. Measure fluorescence using a microplate reader. Plot a standard DNA curve and use it to calculate the DNA concentrations of the amplified products. According to their measured concentration, mix the obtained PCR products of the endophytic or epiphytic samples from the bark of Populus trichocarpa in equal concentration to build the Amplicon library. Conduct Agarose gel electrophoresis using electrophoresis apparatus. Briefly prepare a 1.3% Agarose gel, 1.3 grams of Agarose powder in 100 milliliters of one times TAE buffer. Remove the combs after gel solidification and place the gel in an electrophoresis tank with one times TAE buffer. Next, add one volume unit of six times orange loading buffer to five volume units of each PCR product with 100 microliters of the ladder loaded into each well. Add 30 microliters of the DNA ladder to one well. After connecting the positive and negative wires, perform electrophoresis 80 volts for 50 minutes. Excise DNA fragments from the Agarose gel using a clean, sharp scalpel. Weigh the gel slice and transfer it to a colorless tube. Add buffer to immerse the gel slice. Perform gel extraction using the gel extraction kit following the manufacturer's instructions. Determine the purified Amplicon library's concentration on a fluorimeter using a commercial kit following the manufacturer's protocol for sequencing profiling. Read standard and sample assay tubes using a fluorimeter and confirm the quality of the library by visualizing a single band. Analyze and annotate the 16S rRNA gene sequences using the CHAM II. Make taxonomic annotations using the Silver Database according to reference sequences. Visualize these results using the ggplot2 package in R. High quality genomic DNA of the endophytic and epithetic bacterial microbiomes of the bark of Populus trichocarpa was obtained. The PCR amplification of all 12 samples yielded single strong bands with the correct size. The Amplicon library was further built with a single peak which fulfilled the pre-sequencing requirements. For the endophytic microbiomes, high quality reads were obtained using the primer pairs 799F/1193R and 515F/806R respectively. The primer pair 799F/1193R yielded significantly fewer amplified plant reads. The number of ASVs from epithetic microbiome of the bark of Populus trichocarpa was significantly higher when using the 799F/1193R compared to 515F/806R primers. Shannon Index was estimated to be lower in the epiphytic bacterial community of the bark than in the endophytic bacterial community using the primer pair 799F/1193R. Taxonomic characteristics of bacterial communities in the endophytic and epiphytic bark portions of Populus trichocarpa were further explored using the primer pair 799F/1193R. A reliable and efficient workflow from sample collection to data analysis was built to quantify the composition and diversity of the endophytic and epithetic bacterial microbiomes of the bark of Populus trichocarpa. Our protocol may serve as a path for studying the bark microbiome of woody plant species, allowing a better understanding of the characteristics of bacterial communities inhabiting other woody plants.