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Microbiomes play critical roles in host functioning and therefore there is increasing interest in the microbiome assembly of plants. However, sampling strategies for long-lived perennial trees need to be standardised to produce robust data that accurately represents the microbiome over time. This issue is currently unresolved because there is little evidence indicating which portion of perennial tree species (e.g., root region or surrounding soil) is the best to sample to produce the most accurate measure of microbiome communities. Our aim was to sample different compartments of a plant’s belowground microbiome to identify the optimal sampling strategy to account for the microbial community present. We found that the structure of the microbial community depends most strongly on the environment (site) and compartment of sample collected (bulk soil, rhizosphere, or rhizoplane), rather than the depth or cardinal direction of the sample. We also found that the microbial community increased in diversity with increased distance from the tree within the rhizoplane and rhizosphere. The data presented here provides systematic evidence for a pragmatic and robust sampling regime that was tested and validated across different environments and soil types while controlling for host genotype. This sampling regime will enable effective partitioning of root compartments when studying the microbiome associated with perennial tree species, allowing targeted questions about the microbiome to be explored with greater accuracy.


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