Plant and soil measurements

Plants were sampled in late September when plants reached reproductive maturity (physiological maturity, R6) to assess aboveground biomass and N uptake. This sampling time reflects the plant stage when all physiological activity (of importance here: N uptake and translocation) has essentially stopped, and plants begin to dry. The collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. Five plants, located in a row with no skips or doubles, were cut at ground level in each plot, separated into stalks, leaves, and ears. Row lengths were measured to determine the ground area. Leaves were scanned with a leaf area meter (LI-3100C; LI-COR, Lincoln, Nebraska, USA). All plant components were oven-dried at 65 °C and weighed. Aboveground vegetative biomass included all aboveground parts (e.g., stalks, cobs, husks) except grain. Representative sub-samples of each oven-dried component were ground for subsequent elemental analyses. Grain yield was determined at plant maturity (R6) by collecting ears by hand from an area within the center of the plots, two rows wide and 5 m long. Grain was separated from the ears, weighed separately, and adjusted to 15.5% moisture content for standardization.

End-of-season soil cores (6.35 cm diameter) were collected in October 2018 to determine root biomass (kg ha⁻¹) and available soil N at four depths (0–30, 30–60, 60–90, 90–120 cm). Four soil cores were collected with a Giddings probe in each plot: two in-row cores and two inter-row cores. Visible roots from each depth increment were removed by hand-picking from samples. Collected roots were then oven-dried, weighed, and sub-replicates averaged for each plot on a ground area basis. Row and between-row soil samples from each plot were composited within each depth, air-dried, and sieved to 2 mm. Stalks, leaves, grain, and root subsamples were analyzed for total C and N using a combustion analyzer (LECO Tru-SPEC, St. Joseph, MI). To determine soil NO₃⁻ concentration at each depth, a 5 g of subsample homogenized field moist soil was extracted with 25 mL of 1 M KCl solution and measured with colorimetry²⁷ with a UV–Vis Spectrophotometer (Shimadzu, Kyoto, Japan).