Name: A Workflow for the Quantitative Assessment of the Endophytic and Epiphytic Bacterial Microbiomes of the Bark of Populus trichocarpa (Video) | JoVE Journal
Uploaded: 2025-06-27T14:20:00.000+00:00
Duration: 12 min 5 s
Description: Learn a workflow for quantitative assessment of endophytic and epiphytic bacterial microbiomes in Populus trichocarpa bark using next-generation sequencing. This method enables comprehensive profiling of plant-microbe interactions.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

We thank Mr. David Anthony Atherton for providing the video narration and members of the B.N. laboratory for valuable advice. This work was supported by the National Science Foundation of China (grant number 32071741 [to BN]), the Key R&#38;D Plan Projects in Xinjiang Uygur Autonomous Region (grant number 2022B02014), the &#34;Tianchi Talents&#34; Introduction Plan (to BN), the National Key R&#38;D Program of China (grant number 2021YFD2200203 [to GQ]), and the Innovation Project of State Key Laboratory of Tree Genetics and Breeding (Northeast Forestry University) (grant number 2019A01 [to BN]).

1.Sample collection
<ol>
	<li>Grow 3-month-old P. trichocarpa saplings in a growth chamber (21&#8211;25&#176;C; 16-h light/8-h dark cycle with supplemental light of approximately 300 &#956;Em&#8722;2 s&#8722;1 from a three-band linear fluorescent lamp [T5 28 W 6400 K]; and 60&#8211;80% humidity)26.</li>
	<li>Cut the 10 cm long stem segments of P. trichocarpa from 2&#8211;12 cm above the sterilized soil surface (Figure 1A) using a sterilized pruner. Place the stem segments in a sterilized square Petri dish (13 cm x 13 cm). 
	NOTE: Six trees of the same size were selected for this study, of which three were used for the PCR amplification with each primer set.</li>
</ol>
2. Sample processing
<ol>
	<li>Repeatedly wipe the epidermis of the stem on the sterilized square Petri dish using a sterile cotton swab dipped in 0.1% Tween 20 to more efficiently collect the microorganisms colonizing on the bark surfaces (Figure 1B). 
	NOTE: Sub-humid sterile cottons are used to collect microorganisms adhering to the stem surface. Do not use cotton that is too dry or too wet when wiping the stem surface. Tween 20 is a surfactant capable of reducing surface tension, a property beneficial for obtaining more microbial samples from plant surfaces27,28.</li>
	<li>Cut the cotton tip from the swab using a sterile blade, and place each cotton tip containing epiphytic microbial samples in a sterile 1.5 mL centrifuge tube. Store the samples at &#8722;80 &#176;C.</li>
	<li>After wiping the stem surface, cut open the bark from which the epiphytic microorganisms were sampled using a sterile scalpel, peel off the bark, and place it in a square Petri dish.</li>
	<li>Cut the bark stripped from the stem in step 2.3 into approximately 1 cm segments (Figure 1C) with a sterile scalpel.</li>
	<li>Sterilize the segments by soaking them in 70% (v/v) alcohol for 2 min. Next, soak the segments in sodium hypochlorite (2.5% effective chlorine content) supplemented with 0.1% Tween 80 for 5 min.</li>
	<li>Sterilize the segments obtained in step 2.5 for 30 s in 70% (v/v) alcohol29. Then, wash all the samples three times with sterile distilled water and place them on a clean bench for air-drying. 
	NOTE: The bark samples are stirred back and forth using sterile tweezers to ensure sufficient surface sterilization.</li>
	<li>Store each surface-sterilized stem bark segment (endophytic samples) in a sterile 2 mL centrifuge tube at &#8722;80 &#176;C.</li>
</ol>
3. Genomic DNA extraction
<ol>
	<li>Perform the following steps to pretreat the samples for DNA extraction.
	<ol>
		<li>Place the surface-sterilized stem bark segments obtained in step 2.7 in sterile 5 mL hard tubes and add three sterilized steel beads (4 mm) into each tube. 
		NOTE: Perform this step on a clean bench to prevent microbial contamination. The cotton balls prepared in step 2.2 (epiphytic samples) do not need to be pre-processed and are directly used for genomic DNA extraction.</li>
		<li>Freeze the hard tubes containing the samples in liquid nitrogen for 10 min.</li>
		<li>Crush the bark samples for 5 min with a bead beater. Then, freeze the hard tube again in liquid nitrogen for 1 min and repeat the bead-beating step for 5 min.</li>
	</ol>
	</li>
	<li>Extract genomic DNA. 
	NOTE: Use a commercial DNA extraction kit to isolate genomic DNA from the endophytic and epiphytic samples.
	<ol>
		<li>Add the crushed stem bark of P. trichocarpa and the cotton-adhered epiphytic bacterial communities (0.25 g each) to each of the bead tubes. Then add 60 &#181;L of solution C1 supplied by a commercial kit and gently mix by inversion.</li>
		<li>Next, homogenize the endophytic and epiphytic samples using a bead beater in four 1-min runs. After each run, place the samples on ice for 30 s.</li>
		<li>Centrifuge the tubes at 10,000 x&#160;g for 1 min.</li>
		<li>Extract the endophytic and epiphytic samples using a kit (see Table of Materials) following the manufacturer&#8217;s instructions.</li>
		<li>Add 30 &#181;L of solution C6 to the center of the white filter membrane and incubate at 25 &#176;C for 5 min.</li>
		<li>Centrifuge at 10,000 x&#160;g at room temperature for 1 min.</li>
	</ol>
	</li>
</ol>
4. PCR amplification
<ol>
	<li>Standardize the extracted DNA concentration.
	<ol>
		<li>Measure the concentrations of the genomic DNA extracted from epiphytic and endophytic samples using a microvolume spectrophotometer.</li>
		<li>Dilute each DNA sample with sterile ddH2O to 2 ng/&#956;L to obtain the DNA template for PCR amplification.</li>
	</ol>
	</li>
	<li>Amplify these DNA samples.
	<ol>
		<li>Amplify the V5&#8211;V7 region of the bacterial 16S rRNA gene using the forward primer 799F (5&#8217;-AACMGGATTAGATACCCKG-3&#8217;) and the reverse primer 1193R (5&#8217;-ACGTCATCCCCACCTTCC-3&#8217;) 10. Amplify the V4 region of the bacterial 16S rRNA gene using the forward primer 515F (5&#8217;-GTGCCAGCMGCCGCGGTAA-3&#8217;) and the reverse primer 806R (5&#8217;-GGACTACHVGGGTWTCTAAT-3&#8217;) 30. 
		NOTE: The genomic DNA extracted from each sample is utilized as template for each of the triplicated PCR amplification reactions, and an additional reaction containing no DNA is set as a negative control. For every sample, the PCR reaction mixture volume is 25 &#956;L. For the primer pair 799F/1193R, each reaction contains 12 &#956;L of PCR water, 1 &#956;L of each of the F/R primers (10 &#956;M), 1 &#956;L of DNA template, and 10 &#956;L of mix solution. For the primer pair 515F/806R, each reaction contains 12 &#956;L of PCR water, 1 &#956;L of each of the F/R primers (10 &#956;M), 1 &#956;L of DNA template, and 10 &#956;L of mix solution. PCR reactions with the primer pair 799F/1193R are performed with the following cycle conditions: initial denaturation at 98 &#176;C for 5 min, 25 cycles of 98 &#176;C for 30 s, 53 &#176;C for 30 s, and 72 &#176;C for 45 s, and a final extension at 72 &#176;C for 5 min. The PCR reactions with the primer pair 515F/806R are conducted with the following cycle conditions: initial denaturation at 94 &#176;C for 3 min, 35 cycles of 94&#176;C for 45 s, 50 &#176;C for 60 s, and 72 &#176;C for 90 s, and a final extension for 10 min at 72 &#176;C.</li>
		<li>Mix every sample thoroughly and centrifuge to eliminate bubbles.</li>
		<li>Perform amplification of the V5&#8211;V7 or V4 region of the bacterial 16S rRNA genes on a thermal cycler.</li>
	</ol>
	</li>
	<li>Detect the amplified products.
	<ol>
		<li>Load 5 &#181;L of each amplicon on a 1% agarose gel and perform electrophoresis at 120 V for 30 min.</li>
		<li>View the gel under UV light to validate successful PCR amplification. The sizes of the PCR products obtained with the primer pairs 799F/1193R and 515F/806R should be approximately 430 bp and 400 bp, respectively.</li>
	</ol>
	</li>
</ol>
5. Preparation of amplicon sequencing Library
<ol>
	<li>Mix the amplified products and determine their concentrations.
	<ol>
		<li>Mix the products generated from the three replicates of the PCR amplification for each sample in a PCR tube.</li>
		<li>Add 5 &#956;L of the PCR products and 195 &#956;L of working solution into a well of a 96-well microplate according to the manufacturer&#8217;s instructions for the kit (see Table of Materials). Next, add 10 &#956;L of the standard DNA (DNA reagents with concentrations of 0, 5, 10, 20, 40, 60, 80 and 100 ng/&#956;L from the kit) and 190 &#956;L of working solution into a well of the same microplate. Following this step, determine the fluorescence intensity of the PCR products and standard DNA.</li>
		<li>Measure fluorescence using a microplate reader. 
		NOTE: The excitation/emission of standard fluorescein wavelengths are set at 480/530 nm to measure the quantity of the double-stranded DNA in each sample.</li>
		<li>Establish the standard curve of DNA concentrations and fluorescence intensities using the standard DNA concentrations of the standard DNA solutions (0, 5, 10, 20, 40, 60, 80 and 100 ng/&#956;L) and the fluorescence intensity of each of standard DNA solution. Substitute the fluorescence intensity value of each of the PCR products into the standard curve to calculate the DNA concentrations of the PCR products.</li>
	</ol>
	</li>
	<li>Build amplicon sequencing libraries.
	<ol>
		<li>According to the concentrations detected in step 5.1.4, mix the remaining PCR products amplified by the same primer pair in equal concentrations to build the amplicon sequencing library. 
		NOTE: The fraction of the PCR products of each sample used for quantifying the concentration of the double-stranded DNA by measuring the fluorescence intensity is not included in the amplicon sequencing library, which is built from the remaining PCR products of the same sample.</li>
	</ol>
	</li>
	<li>Perform gel extraction.
	<ol>
		<li>Conduct agarose gel electrophoresis using electrophoresis apparatus. Briefly, prepare a 1.3% agarose gel (i.e., 1.3 g of agarose powder in 100 mL of 1 x&#160;TAE buffer). 
		NOTE: Care is taken to ensure that the quantity of the wells in the gel is sufficient to hold each PCR product and DNA ladder; in this study, 1.3% agarose gel is prepared by dissolving 1.3 g of agarose powder in 100 mL of 1 x&#160;TAE buffer.</li>
		<li>Remove the combs after gel solidification and place the gel in an electrophoresis tank with 1 x&#160;TAE buffer. Next, add 1 volume unit of 6 x&#160;orange loading buffer to 5 volume units of each PCR product, with 100 &#181;L of the latter loaded into each well. Add 30 &#181;L of the DNA ladder to one well. After connecting the positive and negative wires, perform electrophoresis at 80 V for 50 min. 
		NOTE: In this study, gel extraction is employed to purify the amplicon sequencing library represented by the band of 400 bp or 430 bp amplified with the primer pair 515F/806R or 799F/1193R, respectively.</li>
		<li>Excise DNA fragments from the agarose gel using a clean, sharp scalpel.</li>
		<li>Weigh the gel slice and transfer it to a colorless tube. Add buffer to immerse the gel slice. 
		NOTE: The maximum amount of gel per spin column is 400 mg. Three volume units of buffer are added to one volume unit of a given gel slice.</li>
		<li>Perform gel extraction using the Gel Extraction Kit (see Table of Materials) following the manufacturer&#8217;s instructions. 
		NOTE: The amplicon library constructed from the products of the PCR targeting the bacterial 16S rRNA gene used for sequencing the endophytic and epiphytic bacterial communities is obtained using this method.</li>
	</ol>
	</li>
	<li>Confirm the purified amplicon sequencing library&#8217;s quality.
	<ol>
		<li>Determine the concentration of double-stranded DNA in the purified amplicon sequencing library obtained in step 5.3.5 by a fluorometer using a commercial kit (see Table of Materials) following the manufacturer&#8217;s protocol (Figure 2).</li>
		<li>Determine the target DNA fragment size of the amplicon sequencing library obtained in step 5.3.5 by using a nucleic acid quality control analysis system with commercial chips and kits (see Table of Materials) following the manufacturer&#8217;s protocol. 
		NOTE: Make sure that the fragment size is within the expected range and there are no primer dimers in the amplicon sequencing library.</li>
		<li>Determine the effective concentration of the sequencing library obtained in step 5.3.5 by using the quantitative real time polymerase chain reaction (qRT-PCR) with a commercial kit (see Table of Materials) following the manufacturer&#8217;s protocol.</li>
		<li>Dilute the amplicon sequencing library to meet the loading concentration recommended for the sequencing platform.</li>
	</ol>
	</li>
</ol>
6. Sequence processing and statistical analysis
<ol>
	<li>After sequencing the amplicon library obtained in step 5.4.4 by using a sequencing platform (see Table of Materials)31. Create a folder named emp-paired-end-sequences in the working directory, and then save the compressed sequencing file in fastq format in this folder. Then open Ubuntu and activate the conda environment and Quantitative Insights into Microbial Ecology 2 (QIIME2)(v2021.2)32.</li>
	<li>Write a mapping file and save it as a text file. The file content is shown in Table 2.</li>
	<li>Split the sequencing data obtained with the amplicon sequencing library and remove primers from the sequencing data using the mapping file.
	<ol>
		<li>Use the following command to split the sequencing data: time qiime demux emp-paired --m-barcodes-file Mapping_file.txt --m-barcodes-column BarcodeSequence --p-rev-comp-mapping-barcodes --i-seqs emp-paired-end-sequences.qza --o-per-sample-sequences demux.qza --o-error-correction-details demux-details.qza</li>
		<li>Use the following command to remove primers: time qiime cutadapt trim-paired --i-demultiplexed-sequences paired-demux.qza --p-front-f 799F or 515F --p-front-r 1193R or 806R --o-trimmed-sequences paired-end-demux.qza</li>
	</ol>
	</li>
	<li>Screen and remove any chimera using the DADA2 pipeline33. Cluster the filtered sequences into amplicon sequence variants (ASVs) at 99% similarity.
	<ol>
		<li>Use the following command to screen and remove chimera and cluster sequences into ASVs: time qiime dada2 denoise-paired --i-demultiplexed-seqs paired-end-demux.qza --p-trim-left-f 0 --p-trim-left-r 0 --p-trunc-len-f 220 --p-trunc-len-r 220 --o-table table.qza -o-representative-sequences rep-seqs.qza --o-denoising-stats denoising-stats.qza --p-n-threads 0</li>
	</ol>
	</li>
	<li>Make taxonomic annotations using the SILVA database (v138) based on the reference sequences34.
	<ol>
		<li>Use the following command to annotate the ASVs obtained in step 6.4: time qiime feature-classifier classify-sklearn --i-classifier gg-13-8-99-515-806-nb-classifier.qza --i-reads rep-seqs.qza --o-classification taxonomy.qza</li>
	</ol>
	</li>
	<li>Create an ASV table in QIIME2 (v2021.2) by using the following commands: 
	qiime tools export --input-path table.qza --output-path exported-feature-table 
	qiime tools export --input-path taxonomy.qza --output-path exported-feature_table 
	biom add-metadata -i feature-table.biom -o table.womd.biom --observation-metadata-fp taxonomy.tsv --observation-header OTUID, taxonomy --sc-separated taxonomy 
	biom convert -i table.womd.biom -o ASV-table.txt --to-tsv --header-key taxonomy 
	NOTE: These commands generate a table containing the number of reads of all ASVs for each sample.</li>
	<li>Calculate the number of ASVs and reads of the endophytic and epiphytic bacterial communities generated with the primer pairs 799F/1193R and 515F/806R, respectively.
	<ol>
		<li>Calculate the total number of ASVs having &#8805; 1 reads at the levels of phylum, class, order, family and genus, respectively, in each sample in the ASV table obtained in step 6.6 by counting.</li>
		<li>Calculate the total number of reads in each sample based on the sum of the number of reads of all ASVs in the ASV table obtained in step 6.6 by counting.</li>
		<li>Calculate the number of plant reads in each sample using the number of reads of ASVs annotated as chloroplasts or mitochondria in the ASV table obtained in step 6.6 by counting.</li>
		<li>Calculate the number of bacterial reads in each sample using the following formula: 
		Number of bacterial reads = Number of total reads &#8722; Number of plant reads.</li>
		<li>Calculate and visualize the average number of ASVs (at the levels of phylum, class, order, family and genus, respectively) or reads (in total or of plant and bacteria, respectively) of the endophytic and epiphytic bacterial communities generated by the primer pairs 799F/1193R and 515F/806R by using Excel (2019) and Prism (v8.0) software.</li>
		<li>Evaluate the significance of the differences between the numbers of ASVs (at the levels of phylum, class, order, family and genus, respectively) or reads (in total or of plant and bacteria, respectively) generated with the primer pairs 799F/1193R and 515F/806R by using the Student&#8217;s t-test in the Prism (v8.0) software.</li>
	</ol>
	</li>
	<li>Filter the reads annotated as chloroplasts and mitochondria by using the following command: time qiime taxa filter-table --i-table table.qza --i-taxonomy taxonomy.qza --p-exclude mitochondria, chloroplast --o-filtered-table ASV-table-no-mitochondria-no-chlorplast.qza</li>
	<li>Create phylogenetic tree by using the following command: time qiime phylogeny align-to-tree-mafft-fasttree --i-sequences rep-seqs.qza --o-alignment aligned-rep-seqs.qza --o-masked-alignment masked-aligned-rep-seqs.qza --o-tree unrooted-tree.qza --o-rooted-tree rooted-tree.qza</li>
	<li>Calculate and visualize the alpha diversities represented by Shannon index of the endophytic and epiphytic bacterial communities by using the QIIME2 (v2021.2) and the &#8216;ggplot2&#8217; package in R (v4.0.5)35.
	<ol>
		<li>Use the following command to calculate the alpha diversities: time qiime diversity core-metrics-phylogenetic --i-phylogeny rooted-tree.qza --i-table ASV-table-no-mitochondria-no-chlorplast.qza --p-sampling-depth the lowest read value among all samples --m-metadata-file Mapping_file.txt --output-dir core-metrics-result</li>
	</ol>
	</li>
	<li>Calculate the relative abundances of the populations at the phylum level of the endophytic and epiphytic bacterial communities. Generate a file in qzv format named as &#8220;taxa-bar-plots&#8221; by using the following command of QIIME2 (v2021.2): time qiime taxa barplot --i-table ASVs-table-no-mitochondria-no-chlorplast.qza --i-taxonomy taxonomy.qza --m-metadata-file Mapping_file.txt --o-visualization taxa-bar-plots.qzv
	<ol>
		<li>Open the file taxa-bar-plots.qzv online at https://view.qiime2.org/. Download and save the newly generated file from level2. Calculate the relative abundance by the following formula with R (v4.0.5): 
		Relative abundance of each bacterial phylum (%) = (number of reads of each bacterial phylum in each sample / number of total reads of each sample) x&#160;100 %. 
		Visualize the data by using the ggplot2, RColorBrewer, reshape and ggalluvial packages in R (v4.0.5).</li>
	</ol>
	</li>
	<li>Analyze the significance of the difference between the relative abundance of each of the dominant phyla (defined as a phylum possessing a relative abundance of &#62; 0.5%) in the endophytic and epiphytic bacterial communities sequenced with the primer pair 799F/1193R by using the one-way analysis of variance (ANOVA) with Tukey&#8217;s post-hoc test at a significance level of 0.05 in STAMP software36.</li>
</ol>

<ol><li>Aguirre-Von-Wobeser, E., Alonso-Sanchez, A., Mendez-Bravo, A., Villanueva Espino, L. A., Reverchon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Barks+from+avocado+trees+of+different+geographic+locations+have+consistent+microbial+communities%5BTitle%5D)+AND+%22Archs+Microbiol%22%5BJournal%5D)">Barks from avocado trees of different geographic locations have consistent microbial communities.</a> Archs Microbiol. 203 (7), 4593-4607 (2021).</li>
<li>Konopka, B., Pajtik, J., Seben, V., Merganicova, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+bark+thickness+and+bark+biomass+on+stems+of+four+broadleaved+tree+species%5BTitle%5D)+AND+%22Plants+%28Basel%29%22%5BJournal%5D)">Modeling bark thickness and bark biomass on stems of four broadleaved tree species.</a> Plants (Basel). 11 (9), 1148(2022).</li>
<li>Rosell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bark+in+woody+plants%3A+Understanding+the+diversity+of+a+multifunctional+structure%5BTitle%5D)+AND+%22Integr+Comp+Biol%22%5BJournal%5D)">Bark in woody plants: Understanding the diversity of a multifunctional structure.</a> Integr Comp Biol. 59 (3), 535-547 (2019).</li>
<li>Kobayashi, K., Aoyagi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microbial+community+structure+analysis+inacer+palmatumbark+and+isolation+of+novel+bacteria+IAD-21+of+the+candidate+division+FBP%5BTitle%5D)+AND+%22PeerJ%22%5BJournal%5D)">Microbial community structure analysis inacer palmatumbark and isolation of novel bacteria IAD-21 of the candidate division FBP.</a> PeerJ. 7, e7876(2019).</li>
<li>Pellitier, P. T., Zak, D. R., Salley, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+filtering+structures+fungal+endophyte+communities+in+tree+bark%5BTitle%5D)+AND+%22Mol+Ecol%22%5BJournal%5D)">Environmental filtering structures fungal endophyte communities in tree bark.</a> Mol Ecol. 28 (23), 5188-5198 (2019).</li>
<li>Dreyling, L., Schmitt, I., Dal Grande, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tree+size+drives+diversity+and+community+structure+of+microbial+communities+on+the+bark+of+beech+%28fagus+sylvatica%29%5BTitle%5D)+AND+%22Front+For+Glob+Change%22%5BJournal%5D)">Tree size drives diversity and community structure of microbial communities on the bark of beech (fagus sylvatica).</a> Front For Glob Change. 5, 858382(2022).</li>
<li>Jo, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Changes+in+microbial+community+structure+in+response+to+gummosis+in+peach+tree+bark%5BTitle%5D)+AND+%22Plants+%28Basel%29%22%5BJournal%5D)">Changes in microbial community structure in response to gummosis in peach tree bark.</a> Plants (Basel). 11 (21), 2834(2022).</li>
<li>Dong, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((core+microbiota%2C+and+function+of+the+rhizosphere+soil+and+bark+microbiota+in+Eucommia+ulmoides%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">core microbiota, and function of the rhizosphere soil and bark microbiota in Eucommia ulmoides.</a> Front Microbiol. 13, 855317(2022).</li>
<li>Bodenhausen, N., Bortfeld-Miller, M., Ackermann, M., Vorholt, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+synthetic+community+approach+reveals+plant+genotypes+affecting+the+phyllosphere+microbiota%5BTitle%5D)+AND+%22PLoS+Genet%22%5BJournal%5D)">A synthetic community approach reveals plant genotypes affecting the phyllosphere microbiota.</a> PLoS Genet. 10 (4), e1004283(2014).</li>
<li>Bodenhausen, N., Horton, M. W., Bergelson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+communities+associated+with+the+leaves+and+the+roots+of+Arabidopsis+thaliana%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Bacterial communities associated with the leaves and the roots of Arabidopsis thaliana.</a> PLoS One. 8 (2), e52369(2013).</li>
<li>Stone, B. W. G., Weingarten, E. A., Jackson, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Role+of+the+phyllosphere+microbiome+in+plant+health+and+function%5BTitle%5D)+AND+%22Annual+plant+reviews%22%5BJournal%5D)">The Role of the phyllosphere microbiome in plant health and function.</a> Annual plant reviews. 1, 1-24 (2018).</li>
<li>Lambais, M. R., Lucheta, A. R., Crowley, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+community+assemblages+associated+with+the+phyllosphere%2C+dermosphere%2C+and+rhizosphere+of+tree+species+of+the+atlantic+forest+are+host+taxon+dependent%5BTitle%5D)+AND+%22Microb.+Ecol%22%5BJournal%5D)">Bacterial community assemblages associated with the phyllosphere, dermosphere, and rhizosphere of tree species of the atlantic forest are host taxon dependent.</a> Microb. Ecol. 68 (3), 567-574 (2014).</li>
<li>Arrigoni, E., Antonielli, L., Pindo, M., Pertot, I., Perazzolli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissue+age+and+plant+genotype+affect+the+microbiota+of+apple+and+pear+bark%5BTitle%5D)+AND+%22Microbiol+Res%22%5BJournal%5D)">Tissue age and plant genotype affect the microbiota of apple and pear bark.</a> Microbiol Res. 211, 57-68 (2018).</li>
<li>Germain, H., Séguin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Innate+immunity%3A+Has+poplar+made+its+bed%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">Innate immunity: Has poplar made its bed.</a> New Phytol. 189 (3), 678-687 (2011).</li>
<li>Varnagirytė-Kabašinskienė, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluation+of+shoot+collection+timing+and+hormonal+treatment+on+seedling+rooting+and+growth+in+four+poplar+genomic+groups%5BTitle%5D)+AND+%22Forests%22%5BJournal%5D)">Evaluation of shoot collection timing and hormonal treatment on seedling rooting and growth in four poplar genomic groups.</a> Forests. 15 (9), (2024).</li>
<li>Lu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ptr-mir397a+is+a+negative+regulator+of+laccase+genes+affecting+lignin+content+in+Populus+trichocarpa%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Ptr-mir397a is a negative regulator of laccase genes affecting lignin content in Populus trichocarpa.</a> Proc Natl Acad Sci USA. 110 (26), 10848-10853 (2013).</li>
<li>Krabel, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+root+and+aboveground+biomass+development+of+hybrid+poplars+%28populus+spp.%29+under+drought+conditions%5BTitle%5D)+AND+%22Can+J+For+Res%22%5BJournal%5D)">Early root and aboveground biomass development of hybrid poplars (populus spp.) under drought conditions.</a> Can J For Res. 45 (10), 1289-1298 (2015).</li>
<li>Vitulo, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bark+and+grape+microbiome+of+vitis+vinifera%3A+Influence+of+geographic+patterns+and+agronomic+management+on+bacterial+diversity%5BTitle%5D)+AND+%22Front+Microbiol%22%5BJournal%5D)">Bark and grape microbiome of vitis vinifera: Influence of geographic patterns and agronomic management on bacterial diversity.</a> Front Microbiol. 9, 3203(2018).</li>
<li>Aschenbrenner, I. A., Cernava, T., Erlacher, A., Berg, G., Grube, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+sharing+and+distinct+co-occurrence+networks+among+spatially+close+bacterial+microbiota+of+bark%2C+mosses+and+lichens%5BTitle%5D)+AND+%22Mol+Ecol%22%5BJournal%5D)">Differential sharing and distinct co-occurrence networks among spatially close bacterial microbiota of bark, mosses and lichens.</a> Mol Ecol. 26 (10), 2826-2838 (2017).</li>
<li>Liang, X., Wan, D., Tan, L., Liu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamic+changes+of+endophytic+bacteria+in+the+bark+and+leaves+of+medicinal+plant+eucommia+ulmoides+in+different+seasons%5BTitle%5D)+AND+%22Microbiol+Res%22%5BJournal%5D)">Dynamic changes of endophytic bacteria in the bark and leaves of medicinal plant eucommia ulmoides in different seasons.</a> Microbiol Res. 280, 127567(2024).</li>
<li>De Muinck, E. J., Trosvik, P., Gilfillan, G. D., Hov, J. R., Sundaram, A. Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+ultra+high-throughput+16s+rRNA+gene+amplicon+sequencing+library+preparation+method+for+the+illumina+Hiseq+platform%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">A novel ultra high-throughput 16s rRNA gene amplicon sequencing library preparation method for the illumina Hiseq platform.</a> Microbiome. 5 (1), 68(2017).</li>
<li>Anguita-Maeso, M., Haro, C., Navas-Cortes, J. A., Landa, B. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primer+choice+and+xylem-microbiome-extraction+method+are+important+determinants+in+assessing+xylem+bacterial+community+in+olive+trees%5BTitle%5D)+AND+%22Plants+%28Basel%29%22%5BJournal%5D)">Primer choice and xylem-microbiome-extraction method are important determinants in assessing xylem bacterial community in olive trees.</a> Plants (Basel). 11 (10), 1320(2022).</li>
<li>Wang, C., Liu, D. W., Bai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreasing+soil+microbial+diversity+is+associated+with+decreasing+microbial+biomass+under+nitrogen+addition%5BTitle%5D)+AND+%22Soil+Biol+Biochem%22%5BJournal%5D)">Decreasing soil microbial diversity is associated with decreasing microbial biomass under nitrogen addition.</a> Soil Biol Biochem. 120, 126-133 (2018).</li>
<li>Almeida, A., Mitchell, A. L., Tarkowska, A., Finn, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Benchmarking+taxonomic+assignments+based+on+16s+rRNA+gene+profiling+of+the+microbiota+from+commonly+sampled+environments%5BTitle%5D)+AND+%22GigaScience%22%5BJournal%5D)">Benchmarking taxonomic assignments based on 16s rRNA gene profiling of the microbiota from commonly sampled environments.</a> GigaScience. 7 (5), giy54(2018).</li>
<li>Liland, K. H., Vinje, H., Snipen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microclass%3A+An+R-package+for+16s+taxonomy+classification%5BTitle%5D)+AND+%22BMC+Bioinform%22%5BJournal%5D)">Microclass: An R-package for 16s taxonomy classification.</a> BMC Bioinform. 18 (1), 172(2017).</li>
<li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+AREb1+transcription+factor+influences+histone+acetylation+to+regulate+drought+responses+and+tolerance+in+Populus+trichocarpa%5BTitle%5D)+AND+%22Plant+Cell%22%5BJournal%5D)">The AREb1 transcription factor influences histone acetylation to regulate drought responses and tolerance in Populus trichocarpa.</a> Plant Cell. 31 (3), 663-686 (2019).</li>
<li>Cregger, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Populus+holobiont%3A+Dissecting+the+effects+of+plant+niches+and+genotype+on+the+microbiome%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">The Populus holobiont: Dissecting the effects of plant niches and genotype on the microbiome.</a> Microbiome. 6 (1), 31(2018).</li>
<li>Maria Rodríguez, J. O., Ziani, K., Maté, J. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+effect+of+plasticizers+and+surfactants+on+the+physical+properties+of+starch+based+edible+films%5BTitle%5D)+AND+%22Food+Res+Int%22%5BJournal%5D)">Combined effect of plasticizers and surfactants on the physical properties of starch based edible films.</a> Food Res Int. 39 (8), 840-846 (2006).</li>
<li>Beckers, B., De Beeck, M. O., Weyens, N., Boerjan, W., Vangronsveld, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+variability+and+niche+differentiation+in+the+rhizosphere+and+endosphere+bacterial+microbiome+of+field-grown+poplar+trees%5BTitle%5D)+AND+%22Microbiome%22%5BJournal%5D)">Structural variability and niche differentiation in the rhizosphere and endosphere bacterial microbiome of field-grown poplar trees.</a> Microbiome. 5 (1), 25(2017).</li>
<li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Global+patterns+of+16s+rrna+diversity+at+a+depth+of+millions+of+sequences+per+sample%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+USA%22%5BJournal%5D)">Global patterns of 16s rrna diversity at a depth of millions of sequences per sample.</a> Proc Natl Acad Sci USA. 108, 4516-4522 (2011).</li>
<li>Mcpherson, M. R., Wang, P., Marsh, E. L., Mitchell, R. B., Schachtman, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+analysis+of+microbial+communities+in+soil%2C+rhizosphere%2C+and+roots+in+perennial+grass+experiments%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and analysis of microbial communities in soil, rhizosphere, and roots in perennial grass experiments.</a> J Vis Exp. 24 (137), e57932(2018).</li>
<li>Caporaso, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((QIIME+allows+analysis+of+high-throughput+community+sequencing+data%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">QIIME allows analysis of high-throughput community sequencing data.</a> Nat Methods. 7 (5), 335-336 (2010).</li>
<li>Callahan, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dada2%3A+High-resolution+sample+inference+from+illumina+amplicon+data%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">Dada2: High-resolution sample inference from illumina amplicon data.</a> Nat Methods. 13 (7), 581-583 (2016).</li>
<li>Pruesse, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+comprehensive+online+resource+for+quality+checked+and+aligned+ribosomal+RNA+sequence+data+compatible+with+ARB%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB.</a> Nucleic Acids Res. 35 (21), 7188-7196 (2007).</li>
<li>Ginestet, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ggplot2%3A+Elegant+graphics+for+data+analysis%5BTitle%5D)+AND+%22J+R+Stat+Soc+Ser+A+Stat+Soc%22%5BJournal%5D)">Ggplot2: Elegant graphics for data analysis.</a> J R Stat Soc Ser A Stat Soc. 174, 245-245 (2011).</li>
<li>Parks, D. H., Tyson, G. W., Hugenholtz, P., Beiko, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stamp%3A+Statistical+analysis+of+taxonomic+and+functional+profiles%5BTitle%5D)+AND+%22Bioinform%22%5BJournal%5D)">Stamp: Statistical analysis of taxonomic and functional profiles.</a> Bioinform. 30 (21), 3123-3124 (2014).</li>
<li>Holmes, S. P., McMurdie, P. J., Callahan, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exact+sequence+variants+should+replace+operational+taxonomic+units+in+marker-gene+data+analysis%5BTitle%5D)+AND+%22ISME+J%22%5BJournal%5D)">Exact sequence variants should replace operational taxonomic units in marker-gene data analysis.</a> ISME J. 11, 2639-2643 (2017).</li>
<li>Chernov, T. I., Tkhakakhova, A. K., Kutovaya, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Assessment+of+diversity+indices+for+the+characterization+of+the+soil+prokaryotic+community+by+metagenomic+analysis%5BTitle%5D)+AND+%22Eurasian+Soil+Sci%22%5BJournal%5D)">Assessment of diversity indices for the characterization of the soil prokaryotic community by metagenomic analysis.</a> Eurasian Soil Sci. 48 (4), 410-415 (2015).</li>
<li>Li Yu, X., Hu, X. Y., Wang, X. X., Zhang, X. Y., Du, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+protocol+specialized+for+microbial+DNA+extraction+from+living+poplar+wood%5BTitle%5D)+AND+%22Not+Bot+Horti+Agrobot+Cluj+Napoca%22%5BJournal%5D)">A protocol specialized for microbial DNA extraction from living poplar wood.</a> Not Bot Horti Agrobot Cluj Napoca. 50 (4), (2022).</li>
<li>Jeffrey, L. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bark-dwelling+methanotrophic+bacteria+decrease+methane+emissions+from+trees%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Bark-dwelling methanotrophic bacteria decrease methane emissions from trees.</a> Nat Commun. 12 (1), 2127(2021).</li>
<li>Chelius, M. K., Triplett, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+diversity+of+archaea+and+bacteria+in+association+with+the+roots+of+zea+mays+l%5BTitle%5D)+AND+%22Microb+Ecol%22%5BJournal%5D)">The diversity of archaea and bacteria in association with the roots of zea mays l.</a> Microb Ecol. 41 (3), 252-263 (2001).</li>
</ol>

The authors have nothing to disclose.

Developed from the published methodology for examining bark microbiota7,18, the workflow proposed by us provides general guidance for identifying and characterizing endophytic and epiphytic microbiomes of the bark of woody plants. This method comprises protocols for sample collection and processing, genomic DNA extraction, PCR amplification, preparation of amplicon sequencing libraries, and sequencing data analysis, serving as a potential blueprint for designing ...

Bark is the outermost layer of the stems of woody plants, referring to all the tissues outside the vascular cambium, including the phloem and the periderm1,2. Extensive research has demonstrated that bark plays pivotal roles in the translocation of water and carbohydrates, the protection of trees from fire and mechanical damage3, and adaptation to abiotic4and biotic stresses5. More importantly, as the interface between a tree and the surrounding air, bark also serves as a unique habitat for microorganisms6. Notably, bark-associated microbes have been confirmed to possess the functions of promoting host growth7, assisting trees in nutrient acquisition8, enhancing plant hormone synthesis9, and protecting hosts from phytopathogen infection5. However, far less attention has been paid to the microbial communities colonizing bark compared to the phyllosphere and rhizopshere microbiota10,11. Limited knowledge exists regarding the biological features of the microbiome of tree bark. The objective of the present methodology paper is to provide investigators with a set of protocols for describing the diversity and composition of the endophytic and epiphytic bacterial microbiome of woody plant bark.
On the basis of the published methods for investigating bark microbiota12,13in this work, we designed and tested an elaborate workflow based on 16S rRNA gene amplicon sequencing analysis to explore the bacterial microbiome settling the internal tissues or the surfaces of the stem bark of Populus trichocarpa, an important model organism used in woody plant biology14. P. trichocarpa has the characteristics of rapid growth15, relative ease of experimental manipulation16, and easy genetic transformation, and also, known for its wide application in timber production17. Specifically, the method put forward in this work begins with a detailed description of the sampling of the endophytic and epiphytic microbiota of the stem bark of P. trichocarpa. With the inclusion of a step for surface-sterilizing the bark with alcohol and sodium hypochlorite after harvesting the microbiome colonizing the surface, this study established a procedure for simultaneously and separately collecting microorganisms residing in the inner structure of the bark (endophytes) and those living on the external surface of the bark (epiphytes). To the best of our knowledge, among the published studies on the woody plant bark microbiome, no assays have been performed on the endophytic and epiphytic microbial communities gathered from the same bark sample based on a clear distinction made between the interior and exterior niches of the bark18,19,20. Thus, the method developed in this research will enable researchers to examine the diversity and the composition of the bacterial communities inhabiting bark in a precise and comprehensive manner. In addition, this study provides detailed descriptions of the procedures for genomic DNA extraction, polymerase chain reaction (PCR) amplification, the preparation of the 16S rRNA gene amplicon sequencing library21, and sequencing data analysis. In particular, this study employed a pair of primers targeting the hypervariable V5-V7 regions of the bacterial 16S rRNA gene to reduce the unspecific amplification of the mitochondrial and chloroplast sequences of host plants22, which yielded unbiased profiles of the bark-associated microbiome. The &#945;-diversities were evaluated using the Shannon index23,a popular diversity index widely used in the research of ecology, and the taxonomic composition at the phylum level was calculated and visualized in the Quantitative Insights into Microbial Ecology (QIIME) 24and R software25. Together with the methods for exploring microbiota colonizing barks established in the previous study 1,6, our methodology may serve as a blueprint for designing protocols for studying the bark microbiome of other woody plant species, particularly economically important trees of forest.

<table><tbody><tr><td>Equipment:</td><td></td><td></td><td></td></tr><tr><td>Bead beater</td><td>DHS Life Science</td><td>401265</td><td></td></tr><tr><td>Centrifuge for 1.5 mL tubes</td><td>Thermo Scientific</td><td>75002440</td><td></td></tr><tr><td>Centrifuge for PCR tubes</td><td></td><td></td><td></td></tr><tr><td>Microplate reader</td><td>BioTek</td><td>8040528</td><td></td></tr><tr><td>Nanodrop</td><td>DHS Life Science &amp;amp; Technology Co., Ltd.</td><td>2010/2020&amp;nbsp;</td><td>microvolume spectrophotometer</td></tr><tr><td>Qubit 3.0 Fluorometer&amp;nbsp;</td><td>invitrogen</td><td>Q33216</td><td>fluorometer&amp;nbsp;</td></tr><tr><td>Refrigerator(-80)</td><td>Panasonic Healthcare Co., Ltd</td><td>17060107</td><td></td></tr><tr><td>T100 Thermal Cycler</td><td>Bio-Rad</td><td>1861096</td><td>thermal cycler</td></tr><tr><td>Tanon EPS-600&amp;nbsp;</td><td>Tanon</td><td>A-179047-2313</td><td>For electrophoresis</td></tr><tr><td>LabChip GX Touch HT</td><td>PerkinElmer</td><td>CLS138625</td><td>Library DNA fragment distribution detection, nucleic acid quality control analysis</td></tr><tr><td>Applied Biosystems QuantStudio 12K</td><td>Applied Biosystems</td><td>4485694</td><td>Quantification library</td></tr><tr><td>Illumina Novaseq6000</td><td>Illumina&amp;nbsp;</td><td></td><td>Library sequencing</td></tr><tr><td>Materials:</td><td></td><td></td><td></td></tr><tr><td>Gloves</td><td>Any</td><td>NA</td><td></td></tr><tr><td>aerosol barrier tip 10 &amp;mu;L</td><td>Axygen</td><td>TF-300-R-S</td><td></td></tr><tr><td>aerosol barrier tip 20&amp;mu;L</td><td>Axygen</td><td>TF-20-R-S</td><td></td></tr><tr><td>aerosol barrier tip 200&amp;mu;L</td><td>Axygen</td><td>TF-200-R-S</td><td></td></tr><tr><td>aerosol barrier tipr 1000&amp;mu;L</td><td>Axygen</td><td>TF-1000-R-S</td><td></td></tr><tr><td>centrifuge tube 1.5 ml</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>Corning 96-well Black/Clear Flat Bottom Microplate</td><td>Corning</td><td>3631</td><td></td></tr><tr><td>cotton stick</td><td>ZHENDE MEDICAL</td><td>76606</td><td></td></tr><tr><td>DNeasy PowerSoil Kit</td><td>QIAGEN&amp;nbsp;</td><td>12888-100</td><td>For DNA extraction</td></tr><tr><td>enzyme-free centrifuge tube 1.5 ml</td><td>USA SCIENTIFIC</td><td>1415-2600</td><td></td></tr><tr><td>ethanol</td><td>Tianjin Yongda Chemical Reagent Company Limited</td><td>64-17-5</td><td></td></tr><tr><td>hard tubes(5 ml)</td><td>DHS Life Science</td><td>0401261-17</td><td></td></tr><tr><td>Hot Master Mix&amp;nbsp;</td><td>Quanta Bio</td><td>2200400</td><td></td></tr><tr><td>microcentrifuge tube 2.0 ml</td><td>Axygen</td><td>MCT-200-C-S</td><td></td></tr><tr><td>PCR Water</td><td>QIAGEN&amp;nbsp;</td><td>17000-10</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit&amp;nbsp;</td><td>QIAGEN</td><td>28704</td><td>For gel extraction</td></tr><tr><td>Quant-iT dsDNA Assay Kits,Broad Range</td><td>Invitrogen</td><td>Q33130</td><td>For concentration determination of the amplified products and the purified amplicon libraries</td></tr><tr><td>square petri dish</td><td>Changde Bkman Biotechnology Co.,Ltd</td><td>110301014</td><td></td></tr><tr><td>steel beads</td><td>Shangyu Yixin Ball Industry Co., Ltd.</td><td>YXB36985</td><td></td></tr><tr><td>sodium hypochlorite solution</td><td>Tianjin Beilian Fine Chemicals Development Co., Ltd&amp;nbsp;</td><td>7681-52-9</td><td></td></tr><tr><td>Tween 20</td><td>Nachuan Biotechnology Studio</td><td>9005-64-5</td><td></td></tr><tr><td>Dual Protocol DNA High Sens Reagent Kit</td><td>PerkinElmer</td><td>CLS760672</td><td>DNA library detection</td></tr><tr><td>VAHTS Library Quantification Kit for Illumina</td><td>Vazyme Biotech Co., Ltd</td><td>NQ104</td><td>Quantification library</td></tr></tbody></table>

a workflow for the quantitative assessment of the endophytic and epiphytic bacterial microbiomes of the bark of populus trichocarpa

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

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.

High-quality genomic DNA of the endophytic and epiphytic bacterial microbiomes of the bark of P. trichocarpa was obtained using the optimized protocol (Table 1). The PCR amplification of all 12 samples yielded single strong bands with the correct size (Figure 3). Both primer pairs 799F/1193R and 515F/806R successfully amplified the target products from endophytic or epiphytic bark tissue samples at around 400 and 430 bp, respective...

This protocol presents a reliable and efficient workflow from sample collection to data analysis for profiling the endophytic and epiphytic bacterial microbiomes present in the bark of Populus trichocarpa.

Microorganisms colonizing plant surfaces and internal tissues may possess beneficial functions in promoting plant growth and health. However, information ...

a-workflow-for-quantitative-assessment-endophytic-epiphytic-bacterial

Research

JoVE Journal

Biology

We provide a technique to preserve intact tree phloem and prepare it for observation.  We create an apparatus called a phloem sandwich that allows for the introduction and observation of arthropods, microbes, and other organisms that inhabit phloem tissues.

Technique for Studying Arthropod and Microbial Communities within Tree Tissues

A whole cell bioreporter assay with Burkholderia sartisoli RP037-mChe was developed to detect fractions of an organic contaminant (i.e., fluorene) available for bacterial degradation after active transport by mycelia bridging air-filled pores in a water unsaturated model system.

A Whole Cell Bioreporter Approach to Assess Transport and Bioavailability of Organic Contaminants in Water Unsaturated Systems

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

We describe how to quantify leaf dwelling arthropods by sealing the leaves and end of branches in a bag, clipping and freezing the bagged material, and rinsing the previously frozen material in water to separate arthropods from the substrate for quantification.

A Method for Quantifying Foliage-Dwelling Arthropods

We describe a semi-quantitative approach of measuring characteristics of corticolous (bark-dwelling) arthropod communities. We placed commercially manufactured sticky traps on tree boles to estimate abundance, total length (a surrogate to biomass), richness, and Shannon diversity for comparison among tree species.

Quantifying Corticolous Arthropods Using Sticky Traps

Glyphosate-based products (GBP) are the most common broad-spectrum herbicides worldwide. In this article, we introduce general guidelines to quantify the effect of GBP on microbiomes, from field experiments to bioinformatics analyses.

Quantification of the Potential Impact of Glyphosate-Based Products on Microbiomes

The protocol here describes the methods for the assessment of the arbuscular mycorrhizal colonization patterns and strategy in roots for two species: Zea mays and Festuca rubra. The use of the MycoPatt method permits the calculation of parameters, the conversion of mycorrhizal structures into digital data, and the mapping of their real position in roots.

Mycorrhizal Maps as a Tool to Explore Colonization Patterns and Fungal Strategies in the Roots of Festuca rubra and Zea mays

We propose an optimized Scanning Electron Microscopy protocol for visualizing highly heterogeneous and delicate samples containing plant and fungal biomass, together with microbiota and biofilm. This protocol allows describing the spatial dimensions of the microbiota organization.

Microbiota of Attine Ants' Gardens: Visualizing a Microbial Landscape by Scanning Electron Microscopy

This protocol delivers a modular, BSL-2 workflow combining crystal-violet biomass assays, time-lapse phase-contrast kinetics, confocal 3-D/matrix mapping, SEM ultrastructure, and an in vivo hamster infection module to cultivate, quantify, characterize and investigate Leptospira biofilm functional role, enabling standardized evaluation of mutants and anti-biofilm interventions across laboratories.

A Modular Workflow for Quantitative, Structural and Functional Analysis of Leptospira Biofilms

Learn a workflow for quantitative assessment of endophytic and epiphytic bacterial microbiomes in Populus trichocarpa bark using next-generation sequencing. This method enables comprehensive profiling of plant-microbe interactions.

A Workflow for the Quantitative Assessment of the Endophytic and Epiphytic Bacterial Microbiomes of the Bark of Populus trichocarpa (Video) | JoVE Journal

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

Explore More Videos