Cultivating the Bacterial Microbiota of Populus Roots

There are currently limited bacterial isolates from the Populus root microbiome for use as reference genomes for metagenomic comparisons, comparative bacterial genomics, and synthetic community construction. To increase these resources, we isolated a large (3,211) bacterial culture collection from fine roots (<2 mm) of two Populus species. We sampled the rhizosphere, which includes soil adhered tightly to the roots, rhizoplane organisms, the root endosphere, and macerated surface-sterilized roots, in an effort to capture as much diversity as possible. Dilution plating and subsequent colony isolation resulted in 3,208 pure bacterial cultures (see Table S1 in the supplemental material) from Populus deltoides and Populus trichocarpa roots and rhizosphere, sampled across two Populus common garden sites (39) and natural environments in Tennessee, Georgia, Oregon, and North Carolina between 2011 and 2018. In addition, three isolates were obtained by single-cell flow cytometry sorting on agar medium in order to begin to access less abundant members of the Populus microbiome. The majority of the isolates (87%) were cultured using R2A media (Table S1) (40), although various other media and root extracts were also used to increase strain diversity. The diversity of the microbial isolates was assessed based on 16S rRNA gene sequence and determined to include bacterial isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla: Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia (Fig. 1, Table 1; see also Table S1). The greatest number of isolates are from 5 genera, Pseudomonas (18.2%, 584), Bacillus (13.7%, 439), Rhizobium (13.4%, 430), Streptomyces (11.8%, 377), and Variovorax (5.9%, 190). The large number of bacteria from these groups is likely the result of their abundance within the microbiome, their presence in the majority of samples, and culture conditions that favor their growth, as many of these genera are known to be more readily cultivated in a laboratory setting. At 97% sequence similarity, we estimate that 464 distinct species exist within the culture collection, with the most species coming from the genera Paenibacillus (9.7%, 45), followed by Bacillus (7.8%, 36), Pseudomonas (6.7%, 31), Streptomyces (5.8%, 27), and Rhizobium (5.6%, 26). The majority of the isolates were cultured from the surface-sterilized root endosphere (50%) followed by rhizosphere (37.3%) and unsterilized root tissues (12.7%) of Populus (Fig. 2). Although more individual isolates were cultured from the root endosphere, a greater number of unique genera was isolated from the rhizosphere (91 genera) versus root endosphere (83 genera). The genera with the greatest number of isolates (Pseudomonas, Bacillus, Rhizobium, Streptomyces, and Variovorax) were present across both host species (P. deltoides and P. trichocarpa) and across all sampled trees, while other genera showed host species specificity (Fig. 2). Strains from the genera Collimonas (Betaproteobacteria) and Arthrobacter (Actinobacteria), while not exclusive, were primarily and more frequently isolated from P. trichocarpa (Collimonas, 1 strain from P. deltoides versus 106 strains from six out of 11 P. trichocarpa trees; Arthrobacter, 3 strains from 3 of 41 P. deltoides trees versus 71 strains from 9 out of 11 P. trichocarpa trees). The classes Cytophagia, Verrucomicrobiae, and Acidobacteria were isolated only from P. deltoides trees, although only a few strains from Cytophagia and 1 strain each from Verrucomicrobiae (41) and Acidobacteria (42) are present within the collection. Host species may be only one contributing factor, as climate and soil type could also influence the distribution of isolates. Some genera were more abundant in either the root endosphere or rhizosphere. Streptomyces, although present in both the root endosphere and rhizosphere, had the most strains isolated from the non-surface-sterilized root samples (Fig. 2). A few genera (Microbacterium, Tardiphaga, and Bosea) were cultured only from the root endosphere and unsterilized root samples, but only a few strains are present within the collection (Fig. 2).

For two genera, Rhizobium and Variovorax, more isolates were cultured from the root endosphere samples compared to rhizosphere (Fig. S1). Based on RDP classification, the Variovorax isolates, when assigned against the RDP database, are found to belong to 5 different species, V. boronicumulans, V. ginsengisoli, V. guangxiensis, V. paradoxus, and V. soli (Table S2). Even though more isolates were cultured from the root endosphere, four of the species listed above were isolated from both root endosphere and rhizosphere. The only notable exceptions are the isolates identified as Variovorax soli, which were exclusively found in the rhizosphere or non-surface-sterilized root samples, suggesting that these isolates remain on the outer surfaces of the plants. Similar to Variovorax, the Rhizobium isolates had many species shared across both root endosphere and rhizosphere (Table S3). This suggests that isolates within a species have specific adaptations for survival in the different root regions. One species of Rhizobium identified in the isolate collection as being in the rhizosphere was R. paranaense. This species is commonly found in beans as a nitrogen-fixing symbiont (43). A species only identified in the endosphere, R. cellulosilyticum, was previously isolated from sawdust of Populus alba and generally has polysaccharide-hydrolyzing ability, suggesting a mechanism for its entry into the endosphere compartment (44); however, further studies are needed to confirm root endosphere colonization mechanisms.

To explore to what extent the bacterial isolates reflect the natural diversity of the Populus microbiota, we compared 16S gene sequences of culture collection isolates with rRNA gene survey data from root endosphere, rhizosphere, and bulk soil samples. We used two gene survey studies. The first study contained samples collected from Populus trichocarpa trees grown in common gardens at Corvallis, OR, and Clatskanie, OR (designated the common garden study). These samples were also utilized for isolation in the collection. The second study was a previously published data set available for P. deltoides grown at a site in Knoxville, TN (designated the Atlas study) (15). Overall, 50% of the sequences found in the amplicon data (root endosphere and rhizosphere) had a representative within the culture collection compared at 97% identity levels (Fig. 3). The common garden study had less cultured representatives in the root endosphere (44.2% ± 19.7%) compared to the rhizosphere data (58.8% ± 25.3%). The Atlas study had slightly more cultured representatives in the root endosphere (51% ± 30.3%) than the rhizosphere (49% ± 15.2%). Very little of the culture collection was matched in the bulk soil samples from either the Atlas (4.7% ± 2.1%) or the common garden (7.5% ± 2.6%) studies, highlighting the selectivity and recruitment by plants for organisms in the environment. The top sequence variants in the root endosphere and rhizosphere were identified as Pseudomonas species (Fig. 4). In studies and both rhizosphere and root endosphere, the majority of the top sequence variants had a match to the culture collection, with one notable exception. In the common garden study root endosphere samples, the top sequence variant that was present in almost all samples was identified as an Acinetobacter species. Although the culture collection does contain an Acinetobacter species, the sequence variant was only 95% genetically similar based on 16S rRNA. The sequence variant from the common garden sample had 100% sequence identity to Acinetobacter johnsonii species based on a BLAST search. A phylogenetic analysis of Acinetobacter species, including the sequence variant and the isolated strain from the culture collection, show the sequence variant grouped with Acinetobacter johnsonii species while the culture collection isolate grouped with Acinetobacter calcoaceticus (Fig. S2).

To investigate if isolates from the collection could establish a reproducible community, a simplified 10-member community was created using isolates from Populus deltoides. The community was created to resemble a natural poplar bacterial community at the phylum taxonomic rank (15), consisting of genome-sequenced organisms to allow us to uniquely assign genetic material to organisms to test hypotheses about functionality of individuals in a community; this community will be referenced as PD10. The 10 members included Streptomyces mirabilis YR139 (Actinobacteria), Bacillus sp. strain BC15 (Firmicutes), Sphingobium sp. strain AP49 (Alphaproteobacteria), Caulobacter sp. strain AP07 (Alphaproteobacteria), Rhizobium sp. strain CF142 (Alphaproteobacteria), Paraburkholderia sp. strain BT03 (Betaproteobacteria), Variovorax sp. strain CF313 (Betaproteobacteria), Duganella sp. strain CF402 (Betaproteobacteria), Pseudomonas sp. strain GM17 (Gammaproteobacteria), and Pantoea sp. strain YR343 (Gammaproteobacteria). Based on KEGG orthology (KO), which looks at the functional potential contained in genomes, this phylogenetically and functionally diverse community represents the majority of the functional potential present in the genome-sequenced strains from our culture collection. Specifically, of the 9,886 KO terms detected in the P. deltoides root endosphere metagenome (45), the PD10 community represented 4,621 (47%) of the KO terms. Interestingly, we detected 121 KO terms unique in the PD10 genomes, likely due to the ability to resolve low-abundance genes in microbiome samples (Fig. S3). The PD10 community was used in three experiments on axenic Populus roots. The first experiment examined the PD10 members over four time points after inoculation. Initial poplar roots were dominated by a Microbacterium species that was not part of the PD10 community, suggesting endogenous origin (Fig. 5A). The Microbacterium species was also present in the uninoculated control samples (Fig. S4). The poplar root environment supported a final community of 8 of the 10 members, although the community was dominated by Pantoea sp. strain YR343 (80% relative abundance).

The second experiment was single inoculations of each of the PD10 members onto axenic Populus roots, and colonization of roots was assessed by counting CFU for each strain individually. The number of CFU ranged from 10^2.5 CFU/g of root by Caulobacter AP07 to 10^7.7 CFU/g of root by Paraburkholderia sp. strain BT03 (Fig. S5). Both Paraburkholderia sp. strain BT03 and Pantoea sp. strain YR343 had greater colonization rates than all of the other strains. In the third experiment, the PD10 community was inoculated onto axenic Populus deltoides and Populus trichocarpa roots, and the final community was evaluated using quantitative PCR (qPCR). Similarly, to the single inoculations, when inoculated in a community, Paraburkholderia sp. strain BT03 and Pantoea sp. strain YR343 had the highest colonization rate assessed through qPCR independent of the host species (Fig. 5B). Three strains, Caulobacter sp. strain AP07, Bacillus sp. strain BC15, and Pseudomonas sp. strain GM17, decreased in colonization potential when inoculated in a community compared to single-strain inoculations.

Here, we present a culture collection of 3,211 bacterial isolates cultured from the Populus root endosphere and rhizosphere. This is the most comprehensive collection of Populus isolates reported. It represents a large diversity of the natural Populus bacterial microbiome and will allow for in-depth analysis of plant-microbe interactions. With such an expansive collection, it will be possible to further elucidate the genetic potential within a microbiome. This collection could lead to a better understanding of the bacterial genetics involved in host-microbe interactions and elucidate the mechanisms by which microbes can assist plant hosts. Having a diverse collection of cultures will allow researchers to create larger synthetic communities with a known set of functional genes within controlled experimental manipulations to elucidate individual microbial roles and community adaptations. Overall, this collection will expand knowledge of plant-microbe interactions.

The goal was to establish a diverse collection of bacterial isolates from the Populus root endosphere and rhizosphere microbiota. To capture as much diversity as possible, we utilized a wide array of root samples from natural and agricultural systems and different geographic locations, which are further detailed below. Bacterial strains were isolated through dilution plating on designated agar media (see Table S1 in the supplemental material). Roots from Populus deltoides or P. trichocarpa were sampled between 2009 and 2018 from Tennessee (29 trees), Georgia (12 trees), Oregon (33 trees), and North Carolina (11 trees) (Table S1). The genome-wide association study (GWAS) was of common gardens located near Corvallis, OR, and Clatskine, OR. These plantations consist of ∼1,100 individual genotypes of Populus trichocarpa replicated in three blocks, as described by Evans et al. (39). The Corvallis GWAS site lies on a sandy to gravelly loam mollisols in the Camas and Newberg series. The Clatskanie site lies on a silt loam entisol in the Wauna series. The Bellville, Georgia, testbed sites consist of plantations of wild-type and RNA knockdown lines of Populus deltoides and lies on loamy ultisols in the Irvington series. The Caney Fork and Yadkin Rivers populations consist of wild Populus deltoides that were sampled at 10 to 15 locations along the watershed of each river in Dekalb and Smith counties in Tennessee (Caney Fork) and Davie and Yadkin counties in North Carolina. Soil characteristics at these sites vary and were described previously (16, 18). The root samples were packed on wet ice in the field and sent to Oak Ridge National Laboratory for processing. Fine roots (<2 mm) were excised and processed as described previously (16, 84). To collect the rhizosphere bacteria, the roots were washed 5 times with sterile water, and the rhizosphere samples were collected from serial dilution of the washes. To collect root endosphere bacteria, the roots were then surface sterilized by a 30-s incubation in 95% ethanol, 3-min incubation in 5% NaOCl, and then 6 washes with sterile water (16). Following sterilization, roots were macerated in 10 ml of MgSO₄ (10 mM) and serially diluted to cultivate endosphere microbes. Nonsterilized roots of some samples were macerated and plated and are referred to as root (Table S1). Cultures were isolated through 3 rounds of restreaking on agar medium. For all strains, 16S rRNA Sanger sequencing was carried out using universal primers (27f-1492r) (Table S1). Strains for genome sequencing were first picked based on phylogenetic diversity, followed by tree species and root region (endosphere and rhizosphere), with emphasis on strains from the root endosphere. Genome sequencing of strains (Table S2) was primarily carried out at the Joint Genome Institute (JGI; Walnut Creek, CA, USA) (https://jgi.doe.gov/), and annotations were carried out using the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4) (85), as previously described (45, 46). The 16S rRNA sequences were input into the CD-HIT (86) web server and clustered at 97% sequence similarity to estimate the number of sequences.

The above-described method of direct plating resulted in a large amount of diversity from the Populus microbiome; however, this method likely favors fast-growing and abundant strains. To cultivate slow-growing or rare members, a cell-sorting approach was used. Three strains in the collection, Terriglobus albidus strain ORNL, Roseimicrobium sp. strain ORNL1, and Starkeya sp. strain ORNL1, were isolated through cell-sorting methods. Surface roots (approximately 5 inches deep) and associated soil (20 g total) were collected from a mature, wild Populus deltoides on the ORNL reservation, and 50 ml liquid R2A medium was added and the slurry was rocked at room temperature for 1 h. The suspension was filtered through sterile miracloth, and the liquid was centrifuged at 500 × g for 5 min to remove large particles and then passed through a 70-μm sieve. The filtrate was then centrifuged at 10,000 × g, 18°C, for 10 min to pellet the microbial cells. The pellet was gently resuspended in 4 ml R2A medium, overlaid on top of a Histodenz (Sigma-Aldrich, St. Louis, MO) solution (4 g Histodenz dissolved in 5 ml R2A) in 10-ml ultraclear centrifuge tubes (Beckman, Palo Alto, CA), and centrifuged at 10,000 × g for 40 min at 18°C (Optima LE-80K; Beckman Coulter, Brea, CA), as we previously described (74). The microbial white fraction that formed at the two-phase interface was recovered with a pipette and diluted in R2A medium for flow cytometric cell separation. The microbial sample was stained with 5 μM Syto 9 nucleic acid stain (Life Technologies, Grand Island, NY). The stained samples were sorted on a Cytopeia Influx cell sorter (BD, Franklin Lakes, NJ), with sorting gates selected based on relative particle size (forward scatter) and fluorescence intensity. Single particles were arrayed on petri plates containing R2A agar or DSMZ medium 1426 at a density of 100 particles per plate. Following incubation at 28°C for 5 to 10 days, colonies were screened by amplification of small subunit rRNA genes with bacterial universal primers (27F-1492R), followed by Sanger sequencing. A colony of Terriglobus albidus was identified on R2A agar with cells sorted from a low-fluorescence, small-particle-size gate (P1; Fig. S6). A colony of Roseimicrobium strain ORNL1 was identified on DSMZ medium 1426 plates sorted with large, high-fluorescence cells (gate P12; Fig. S6). A more detailed description of the microbial cultivation results using cell sorting will be published elsewhere. The genomes of Terriglobus albidus strain ORNL, Roseimicrobium strain ORNL1, and Starkeya sp. strain ORNL1 have been completed and published (41, 42, 53).

A 16S rRNA alignment was created from all the isolates using SINA (87) with the SILVA 132 alignment (88) as a reference. A cyanobacteria (Prochlorococcus marinus NR_125480.1) was used as the outgroup, since it resides outside all the isolates phylogenetically (89). Maximum likelihood phylogenetic inference was performed using IQ-TREE multicore (v1.6.8) (90). The best-fit model (TIMe+R16) for phylogenetic inference was selected based on Bayesian information criterion using ModelFinder (91) packaged with IQ-TREE. Alignment sites containing only gaps and/or Ns were removed before model selection and phylogenetic inference using FAST (92). The final tree was imported into ggtree (93) to add taxonomic ranks and metadata.

To ascertain how much of the natural poplar microbiome has been successfully cultivated, we obtained 16S rRNA sequencing data from two previous poplar studies, referred to as the common garden (NCBI SRA BioProject no. PRJNA666202) and Atlas (15). The Atlas study was conducted at a site in Blount County, Tennessee, with transitions from horizon silt loams to horizon silt clay loams. Five clonal individuals of Populus deltoides and P. trichocarpa x deltoides hybrid were sampled in August 2014. Further site and sampling description are detailed in reference 15. The common garden was set up as follows. Bulk soil, rhizosphere, and endosphere samples were collected from a diverse set of 32 genotypes of P. trichocarpa from two common gardens (Clatskanie, OR, and Corvalis, OR). These genotypes were selected to represent a wide range of phenotypic diversity (e.g., lignin%, S:G ratio, growth rate, lectin content, etc.). Sample preparation and DNA extraction were processed as described previously (18). DNA extracts were sent to JGI for sequencing. We trimmed the raw sequences to remove primers using cutadapt (v.1.18) (94). The sequences were imported into QIIME2 (v. 2019.1) (95) for further processing. Sequence variants were assigned using DADA2 (96) implemented in the QIIME2 plugin. Since multiple sequencing runs were performed, sequence variants were determined for each sequencing run and then combined per the DADA2 software recommendations. Taxonomy was assigned to the sequence variants using a naive Bayesian classifier trained on the SILVA 132 database (88) via the q2-feature-classifier (97). Sequence variants identified as chloroplast, mitochondria, or unassigned were removed before further analysis. The remaining sequence variants were reassigned a consensus taxonomy using the vsearch (98) plugin in QIIME2 with the culture collection 16S rRNA as the reference database. To determine if the top sequence variants were represented in the culture collection, we graphed the top 100 sequence variants using their relative abundance and relative frequency. Relative frequency was defined as the number of plant samples the sequence variant was present in divided by the total number of plant samples. All figures were created using ggplot2 (v 3.0.0) (99) in R (100).

The 16S rRNA gene sequences from Acinetobacter species were downloaded from NCBI, and Psychrobacter arcticus (NR_042907.1) was used as an outgroup. The Acinetobacter 16 rRNA sequences, including the top sequence variant from the common garden and the isolate strain, were aligned using SINA (87) with the SILVA 132 alignment (88) as a reference.

Maximum likelihood phylogenetic inference was performed using IQ-TREE multicore (v1.6.8) (90). The best-fit model (TIMe+R16) for phylogenetic inference was selected based on Bayesian information criterion using ModelFinder (91) packaged with IQ-TREE. Alignment sites containing only gaps and/or Ns were removed before model selection and phylogenetic inference using FAST (92). The final tree was imported into ggtree (93).

One 10-strain bacterial community was created from the culture collection from Populus deltoides. The 10-member community was made at the phylum taxonomic rank to resemble the distribution seen in the natural community of wild poplar. The 10 members included Streptomyces mirabilis YR139 (Actinobacteria), Bacilius sp. strain BC15 (Firmicutes), Sphingobium sp. strain AP49 (Alphaproteobacteria), Caulobacter sp. strain AP07 (Alphaproteobacteria), Rhizobium sp. strain CF142 (Alphaproteobacteria), Paraburkholderia sp. strain BT03 (Betaproteobacteria), Variovorax sp. strain CF313 (Betaproteobacteria), Duganella sp. strain CF402 (Betaproteobacteria), Pseudomonas sp. strain GM17 (Gammaproteobacteria), and Pantoea sp. strain YR343 (Gammaproteobacteria). Genome annotations using KEGG orthology were used to compare to P. deltoides microbiome metagenome (IMG accession number 3300006177) (45). Specifically, KO terms represented in the 10-member community were compared directly to the list of KO terms identified in the representative metagenome for presence/absence. The 10-member community from Populus deltoides will furthermore be referenced as PD10. Three experiments were performed using the PD10 community. For all experiments on Populus, the initial inoculum was prepared as described below. To prepare the community for inoculation, strains were grown in 5 ml of R2A medium overnight at 25°C and 200-rpm shaking. Bacterial suspensions were washed with 10 mM MgSO₄ and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.01. We combined 1 ml of each strain, and 10 ml total of inoculum was added to the plant soil immediately prior to planting. Plant culturing and inoculation were previously described (37).

The first experiment examined the PD10 community assemblage over time in P. trichocarpa root tissue. The PD10 community was grown as described above and inoculated into the soil before planting of P. trichocarpa axenic plants. Ten plants were used for each time point, 5 uninoculated controls and 5 inoculated plants. Root tissue was harvested at 1, 7, 14, and 21 days postinoculation. DNA was extracted as described above. The 16S rRNA gene was amplified for both the cultures and root tissues and sequenced using the Illumina protocol described in reference 15. Raw amplicon reads were processed as described above through QIIME 2. Taxonomy was also assigned using the consensus vsearch (98) option in QIIME2 against a database of 16S sequences from the PD10 community members. The resulting sequence variant table, mapping file, and taxonomy file were imported into Phyloseq (version 1.22.3) (101) in R (version 3.4.4) (100) for visualization. We corrected for 16S rRNA copy number using a custom R script (available at https://github.com/dlcarper/CopyNumberCorrection) and the number of copies obtained from the isolate genomes. Raw sequences were deposited in the NCBI SRA database under BioProject number PRJNA659670.

The second experiment was single inoculations of each of the PD10 members onto axenic Populus roots, and colonization of roots was assessed by counting the number of CFU for each strain individually. Plants were removed from the microcosm. Roots were submerged in sterile distilled water to remove the clay from the root system. The wet weight of plant root tissue was recorded, and root tissue was macerated with 10 mM MgSO₄ at 1 ml per g of root tissue. Macerated sample was serially diluted (10-fold) with 10 mM MgSO₄. Each sample was plated onto R2A medium plates and allowed to grow for 48 h at 25°C, after which the colonies were counted.

Finally, in the third experiment, the PD10 community was inoculated onto axenic Populus delotides and Populus trichocarpa roots, and the final community was evaluated using qPCR. Two plant species were used for inoculation, P. deltoides (genotype WV94) and P. trichocarpa (genotype BESC-819) to test for differences in host selectivity. Five plants from P. deltoides and P. trichocarpa were inoculated with PD10 communities. Plants were grown for 21 days in growth chambers under 16 h light, 8 h dark per day with ∼50% humidity. The plants were harvested by removing the entire plant from the soil and rinsing the roots with sterile water to remove loose soil. Root material was flash frozen in liquid nitrogen and stored at −80°C. Tissue lysis was obtained using three rounds of alternating bead beating (1 min) and liquid nitrogen freezing, followed by extraction using the PowerPlant kit according to the manufacturer’s instructions. DNA was used for microbial quantification by qPCR. qPCR for bacterial quantification was performed using the iTAQ kit (Bio-Rad, Hercules, CA, USA) on a CFX96 system according to manufacturer’s instructions using bacterial genomic DNA as a standard for quantification, as described previously (37).

Raw sequences for the common garden data set were deposited in the NCBI SRA database under BioProject number PRJNA666202. Raw sequences for the constructed communities were deposited in the NCBI SRA database under BioProject number PRJNA659670 for poplar data.