Advancements in molecular technology have reduced the constraints that the grain of observation, or the spatial resolution and volume of the sampling unit, have on the characterization of plant associated microbiomes. With discrete ecological sampling and massive parallel sequencing, we can more precisely portray microbiome community assembly and microbial recruitment to host tissue over space and time. Here, we differentiate rarefied community richness and relative abundance in bacterial microbiomes of Salvia lyrata dependent on three spatial depths, or physical distances from the soil surface within the rhizosphere microhabitat as a proxy for the root system zones. To assess the impact of sampling grain on rarefied community richness and relative abundance, we evaluated the variation of these metrics between samples pooled prior to DNA extraction and samples pooled after sequencing. The V3-V4 fragment of the 16S rRNA gene was used to describe and compare bacterial communities in the rhizosphere. All samples were loaded into a MiSeq instrument on a V3 flow cell set to read a 2x275, or paired end, cycle at the University of Tennessee Genomics Core, Knoxville, Tennessee, USA. Processing of the 16S amplicon data were completed with the open source pipeline DADA2 version 1.8. All merged sequences were matched to SILVA version 132. Lastly, the effective number of bacterial species associated with each rhizosphere depth and each pooled sample was described using a Hill numbers approach.
Results/Conclusions:
A distance-based redundancy analysis on a quantitative Jaccard distance and weighted UniFrac distance revealed that rhizosphere microbiomes vary in richness between rhizosphere soil depths. At all orders of diversity, rarefied microbial richness was consistently lowest nearest the root tips (approximately 4cm from soil surface) in comparison to other rhizosphere soil depths. We additionally show that fine grain sampling recovers greater microbial richness through 16S rRNA gene sequencing. In summary, to further elucidate the extent host specific microbiomes assemble within the rhizosphere, the grain at which bacterial communities are sampled should reflect and encompass fine scale heterogeneity of the system.