Comparative analysis of three next-generation sequencing techniques to measure nosZ gene abundance in Missouri claypan soils.

Quantitative next-generation sequencing techniques have been critical in gaining a better understanding of microbial ecosystems. In soils, denitrifying microorganisms are responsible for dinitrogen (N2) production. The nosZ gene codes for nitrous oxide reductase, the enzyme facilitating the reduction of nitrous oxide (N2O) to N2. The objectives of this research were to: 1) understand how soil depth influences RNA concentration and nosZ gene abundance; 2) assess the spatial dependence of nosZ gene abundance in two claypan soil fields; and 3) compare and evaluate multiple RNA-based sequencing methods for quantifying nosZ gene abundance in soils in relation to dinitrogen (N2) production. Research sites consisted of two intensively studied claypan soil fields in Central Missouri, USA. Soil cores were collected from two landscape transects across both fields and analyzed for extractable soil RNA at two depths (0-15 cm and 15-30 cm). Measurements of nosZ gene abundance were obtained using real-time quantitative polymerase chain reaction (RT-qPCR), droplet digital polymerase chain reaction (ddPCR), and nanostring sequencing (NS). In both fields, soil RNA concentrations were significantly greater at 0-15 cm depth compared to 15-30 cm. These data indicated low overall soil microbial activity below 15 cm. Due to low quantities of extractable soil RNA in the subsoil, nosZ gene abundance was only determined in the 0-15 cm depth. Sequencing method comparisons of average nosZ gene abundance showed that NS results were constrained to a narrow range and were 10-20-fold lower than ddPCR and RT-qPCR at each landscape position within each field. Droplet digital PCR appears to be the most promising method, as it reflected changes in N2 production across landscape position.

Economically optimal nitrogen rate reduces soil residual nitrate.

Post-harvest residual soil NO(3)-N (RSN) is susceptible to transfer to water resources. Practices that minimize RSN levels can reduce N loss to the environment. Our objectives were (i) to determine if the RSN after corn (Zea mays L.) harvest can be reduced if N fertilizer is applied at the economically optimal N rate (EONR) as compared to current producer practices in the midwestern USA and (ii) to compare RSN levels for N fertilizer rates below, at, and above the EONR. Six experiments were conducted in producer fields in three major soil areas (Mississippi Delta alluvial, deep loess, claypan) in Missouri over 2 yr. Predominant soil great groups were Albaqualfs, Argiudolls, Haplaquolls, and Fluvaquents. At four transects in each field, six treatment N rates from 0 to 280 kg N ha(-1) were applied, the EONR was determined, and the RSN was measured to a 0.9-m depth from five treatment plots. The EONR at sampling sites varied from 49 to 228 kg N ha(-1) depending on site and year. Estimated average RSN at the EONR was 33 kg N ha(-1) in the 0.9-m profile. This was at least 12 kg N ha(-1) lower than RSN at the producers' N rates. The RSN increased with increasing Delta EONR (total N applied - EONR). This relationship was best modeled by a plateau-linear function, with a low RSN plateau at N rates well below the EONR. A linear increase in RSN began anywhere from 65 kg N ha(-1) below the EONR to 20 kg N ha(-1) above the EONR at the three sites with good data resolution near the EONR. Applying N rates in excess of the EONR produced elevated RSN values in all six experiments. Our results suggest that applying the EONR will produce environmental benefits in an economically sound manner, and that continued attempts to develop methods for accurately predicting EONR are justified.