Crop rotational diversity can improve crop productivity and soil health and boost soil microbial diversity. This research hypothesized that a three-year rotation of corn-soybean-wheat (CSW), compared to a two-year corn-soybean (CS) rotation, would result in a more diverse and more complex soil bacterial community, together with a greater abundance of beneficial bacteria. This was evaluated in a replicated experiment established in 2013 at two locations in Ohio (USA). The soil bacterial communities under soybean were compared between CS and CSW, at both studied sites, in 2018 and 2019, through 16S rDNA amplicon metabarcoding. 

A crop rotation field experiment was established in 2013 at two Ohio State University research locations, Northwest Agricultural Research Stations (NWARS) and Western Agricultural Research Stations (WARS). The experimental treatments were a two-year corn-soybean (CS) rotation compared to a three-year corn-soybean-wheat (CSW). The experiment was managed as a no-till system, with herbicide and fungicide applications as needed. The complete root and associated soil were sampled with a shovel at an approximate depth of 10-15 cm. Eight samples were collected within each plot and the shovels were cleaned with 70% ethanol between plots. Each sample was placed in a plastic bag and kept cool in a cold box while it was transferred from the field to the laboratory, and at 8°C until processing. Soils were vacuum dried at 60°C for 30 minutes. The total soil DNA was extracted from 0.25g of dried soils using the SurePrepTM Soil DNA Isolation Kit (Fisher BioReagents, NJ, USA), according to manufacturer recommendations, and followed by a ribonuclease A (RNAse A) treatment. Bacterial community characterization, through amplicon sequencing, was performed using a two-step amplification protocol. In the first PCR, the primers 515F and 806R (Apprill et al., 2015; Parada et al., 2016) targeting the V3-V4 region of the 16S rDNA were used, followed by a double-indexing PCR approach. Samples were pooled into a single sequencing library based on equimolar concentrations and sequenced using an Illumina MiSeq (Illumina, San Diego, CA, USA) sequencing platform with the 300PE chemistry, with two separate runs for samples collected in 2018 and 2019, respectively. A total of 256 DNA samples from soybean-associated soils, corresponding to two treatments, 4 plots/treatment, and 8 samples/plot for two locations in Ohio, and two growing (2018 and 2019) seasons were processed. In addition, positive controls, including the commercial ZymoBIOMICS Microbial Community DNA Standard (Zymo Research, Irvine, CA, USA) and a mixture of DNA of known soil isolates and two types of negative controls were included in sequencing runs and analysis for both years of analysis.