Functionally discrete fine roots differ in microbial assembly, microbial functional potential, and produced metabolites.

Traditionally, fine roots were grouped using arbitrary size categories, rarely capturing the heterogeneity in physiology, morphology and functionality among different fine root orders. Fine roots with different functional roles are rarely separated in microbiome-focused studies and may result in confounding microbial signals and host-filtering across different root microbiome compartments. Using a 26-year-old common garden, we sampled fine roots from four temperate tree species that varied in root morphology and sorted them into absorptive and transportive fine roots. The rhizoplane and rhizosphere were characterized using 16S rRNA gene and internal transcribed spacer region amplicon sequencing and shotgun metagenomics for the rhizoplane to identify potential microbial functions. Fine roots were subject to metabolomics to spatially characterize resource availability. Both fungi and bacteria differed according to root functional type. We observed additional differences between the bacterial rhizoplane and rhizosphere compartments for absorptive but not transportive fine roots. Rhizoplane bacteria, as well as the root metabolome and potential microbial functions, differed between absorptive and transportive fine roots, but not the rhizosphere bacteria. Functional differences were driven by sugar transport, peptidases and urea transport. Our data highlights the importance of root function when examining root-microbial relationships, emphasizing different host selective pressures imparted on different root microbiome compartments.

Functionally-explicit sampling can answer key questions about the specificity of plant-microbe interactions.

The rhizosphere is a nexus for plant-microbe interactions and, as a host-structured environment, a location of high activity for distinct microbes and plant species. Although our insights into this habitat have exploded in recent years, we are still limited in our ability to answer key questions about the specificity of these root-microbial relationships. In particular, it can be difficult to confirm or reject microbiome heritability in many plant systems and to pinpoint which microbial taxa are key to plant functioning. Like other host-structured environments, the rhizosphere is structurally, chemically, and biologically complex, driven largely by differences in root anatomy, location, and function. In this Correspondence, we describe a review of 377 "rhizosphere microbiome" research papers and demonstrate how matching a sampling method to the biological question can advance our understanding of host-microbe interactions in a functionally heterogeneous environment. We found that the vast majority of studies (92%) pool all roots from a root system during sampling, ignoring variation in microbial composition between roots of different function and limiting insight into key root-microbial relationships. Furthermore, approaches for removing root-associated microbes are highly variable and non-standard, complicating multi-study analyses. Our understanding of the strength and nature of host-microbe relationships in heterogenous host-microbiome environments can be clarified by targeting sampling to locations of high interaction. While the high complexity of the rhizosphere creates logistical challenges, we suggest that unambiguous language and refined approaches will improve our ability to match methods to research questions and advance our understanding of the specificity of plant-microbial interactions.

The hierarchy of root branching order determines bacterial composition, microbial carrying capacity and microbial filtering.

Fine roots vary dramatically in their functions, which range from resource absorption to within-plant resource transport. These differences should alter resource availability to root-associated microorganisms, yet most root microbiome studies involve fine root homogenization. We hypothesized that microbial filtering would be greatest in the most distal roots. To test this, we sampled roots of six temperate tree species from a 23-year-old common garden planting, separating by branching order. Rhizoplane bacterial composition was characterized with 16S rRNA gene sequencing, while bacterial abundance was determined on a subset of trees through flow cytometry. Root order strongly impacted composition across tree species, with absorptive lower order roots exerting the greatest selective pressure. Microbial carrying capacity was higher in absorptive roots in two of three tested tree species. This study indicates lower order roots as the main point of microbial interaction with fine roots, suggesting that root homogenization could mask microbial recruitment signatures.