SOIL MICROBIOLOGY Impacts of Sampling Design on Estimates of Microbial Community Diversity and Composition in Agricultural Soils Sarah C. Castle 1 & Deborah A. Samac 1,2 & Michael J. Sadowsky 3,4 & Carl J. Rosen 3 & Jessica L. M. Gutknecht 3 & Linda L. Kinkel 1,4 Received: 7 May 2018 /Accepted: 3 January 2019 # This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2019 Abstract Soil microbiota play important and diverse roles in agricultural crop nutrition and productivity. Yet, despite increasing efforts to characterize soil bacterial and fungal assemblages, it is challenging to disentangle the influences of sampling design on assessments of communities. Here, we sought to determine whether composite samples—often analyzed as a low cost and effort alternative to replicated individual samples—provide representative summary estimates of microbial communities. At three Minnesota agricultural research sites planted with an oat cover crop, we conducted amplicon sequencing for soil bacterial and fungal communities (16S V4 and ITS2) of replicated individual or homogenized composite soil samples. We compared soil microbiota from within and among plots and then among agricultural sites using both sampling strategies. Results indicated that single or multiple replicated individual samples, or a composite sample from each plot, were sufficient for distinguishing broad site-level macroecological differences among bacterial and fungal communities. Analysis of a single sample per plot captured only a small fraction of the distinct OTUs, diversity, and compositional variability detected in the analysis of multiple individual samples or a single composite sample. Likewise, composite samples captured only a fraction of the diversity represented by the six individual samples from which they were formed, and, on average, analysis of two or three individual samples offered greater compositional coverage (i.e., greater number of OTUs) than a single composite sample. We conclude that sampling design significantly impacts estimates of bacterial and fungal communities even in homogeneously managed agricultural soils, and our findings indicate that while either strategy may be sufficient for broad macroecological investigations, composites may be a poor substitute for replicated samples at finer spatial scales. Keywords Agriculture . Soil . Microbiota . Spatial sampling . Composite sampling . Amplicon sequencing . Bacteria . Fungi . ITS2 . 16S-V4 Introduction Microbiota are critical to crop nutrition and productivity, and hold promise for improving yields and resilience in agricul- tural production systems. For instance, the composition and structure of plant and soil microbial communities can be im- portant drivers of plant nutrient availability [42], stress response [37, 46], and disease incidence and suppression [30, 43–45]. Efforts to collect data on plant and soil microbi- ota in agricultural systems are rapidly expanding, and yet re- gardless of its potential significance, a theoretical framework connecting microbial diversity and composition with agricul- tural production is lacking. In part, this challenge arises due to the high spatial and temporal heterogeneity of microbial com- munities (e.g., [12, 19, 24, 31]), which can be locally patchy Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00248-019-01318-6) contains supplementary material, which is available to authorized users. * Sarah C. Castle sccastle@umn.edu 1 Department of Plant Pathology, University of Minnesota, Minneapolis, USA 2 USDA-ARS, Plant Science Research Unit, Saint Paul, MN, USA 3 Department of Soil, Water, and Climate, University of Minnesota, Minneapolis, MN, USA 4 Biotechnology Institute, University of Minnesota, Minneapolis, MN, USA Microbial Ecology https://doi.org/10.1007/s00248-019-01318-6