Cultivating the Bacterial Microbiota of Populus Roots

Abstract

The integral role of microbial communities in plant growth and health is now widely recognized, and, increasingly, the constituents of the microbiome are being defined. While phylogenetic surveys have revealed the taxa present in a microbiome and show that this composition can depend on, and respond to, environmental perturbations, the challenge shifts to determining why particular microbes are selected and how they collectively function in concert with their host. In this study, we targeted the isolation of representative bacterial strains from environmental samples of Populus roots using a direct plating approach and compared them to amplicon-based sequencing analysis of root samples. The resulting culture collection contains 3,211 unique isolates representing 10 classes, 18 orders, 45 families, and 120 genera from 6 phyla, based on 16S rRNA gene sequence analysis. The collection accounts for ∼50% of the natural community of plant-associated bacteria as determined by phylogenetic analysis. Additionally, a representative set of 553 had their genomes sequenced to facilitate functional analyses. The top sequence variants in the amplicon data, identified as Pseudomonas, had multiple representatives within the culture collection. We then explore a simplified microbiome, comprised of 10 strains representing abundant taxa from environmental samples, and tested for their ability to reproducibly colonize Populus root tissue. The 10-member simplified community was able to reproducibly colonize on Populus roots after 21 days, with some taxa found in surface-sterilized aboveground tissue. This study presents a comprehensive collection of bacteria isolated from Populus for use in exploring microbial function and community inoculation experiments to understand basic concepts of plant and environmental selection. IMPORTANCE Microbial communities play an integral role in the health and survival of their plant hosts. Many studies have identified key members in these communities and led to the use of synthetic communities for elucidating their function; however, these studies are limited by the available cultured bacterial representatives. Here, we present a bacterial culture collection comprising 3,211 isolates that is representative of the root community of Populus. We then demonstrate the ability to examine underlying microbe-microbe interactions using a synthetic community approach. This culture collection will allow for the greater exploration of the microbial community function through targeted experimentation and manipulation.

Keywords: 16S rRNA gene sequencing; bacterial isolation; culture collection; isolation; plant microbiome; poplar.

FIG 1

Phylogenetic tree created from 16S rRNA sequencing of all isolates consolidated into genera. Tip points are colored by the bacterial phylum/class (Proteobacteria) to which they belong. The bar graph corresponds to the number of isolates in the collection in that genus.

FIG 2

Heatmap showing the number of isolates from root endosphere and rhizosphere. The genus to which the isolates belong is listed along the y axis. The larger groupings are the tree species across the top and the bacterial class along the right side.

FIG 3

Proportion of 16S sequencing data, from previous poplar studies, that is present within the culture collection from the rhizosphere, endosphere, and soil.

FIG 4

Relative abundance of top 100 sequence variants from previous poplar microbiome studies plotted against their relative frequency (how many trees they were found in). Color indicates the phylogenetic class to which the sequence variant was identified.

FIG 5

Quantification of 10-member bacterial community strains on Populus root tissue. (A) Taxonomic plots of strain relative abundance based on 16S rRNA gene amplicon sequence of 10-member bacterial community on Populus trichocarpa root tissues and monitored over time. (B) Strain-specific qPCR results from each of the 10-member strains inoculated in a community on Populus trichocarpa and Populus deltoides roots with significant differences of colonization between species noted. Ns, not significant.