Distinct Primary vs. Lateral Root-Associated Bacterial and Fungal Communities Are Masked by Composite Root Sampling

Root systems comprise distinct primary and lateral roots, yet conventional microbiome studies often rely on composite root samples, potentially obscuring root-type-specific profiles. We hypothesized that 1) primary and lateral roots harbor distinct microbial communities that may be masked by composite sampling, 2) soil phosphorus (P) acts as an environmental filter with root-type-dependent effects on microbial diversity and composition, and 3) composite sampling obscures root-type-enriched, functionally relevant taxa. We investigated rhizosphere soil and root-associated bacterial and fungal communities of field-grown canola under P-fertilized (+P; 18.7 kg P ha⁻¹ as monoammonium phosphate) and P-omitted (-P) conditions using segmented (primary and lateral roots separately) or composite root sampling. In the rhizosphere, P fertilization reduced bacterial alpha diversity and significantly shifted both bacterial and fungal community structure, with an additional contribution of root type for bacteria. In roots, alpha diversity patterns were primarily associated with root type, while community structure was microbial-domain-specific: bacterial communities exhibited root-type-dependent responses to P, whereas fungal communities differed mainly between root types. Consistent with our hypotheses, segmented sampling revealed root-type-associated taxonomic enrichments and P-responsive shifts that were diminished or lost in composite samples, underrepresenting potentially important plant-associated taxa. Collectively, this study demonstrates that composite sampling can mask ecologically and agronomically relevant microbial associations across root architectures, highlighting the importance of root-type resolution in plant-microbiome research.