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A microplot design for 15N tracer research is described to accommodate multiple in-season plant and soil sampling events. Soil and plant sample collection and processing procedures, including grinding and weighing protocols, for 15N analysis are put forth.
Many nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. A stable isotope approach is necessary to follow and quantify the fate of fertilizer derived N (FDN) through the soil-crop system. The purpose of this paper is to describe a small-plot research design utilizing non-confined 15N enriched microplots for multiple soil and plant sampling events over two growing seasons and provide sample collection, handling, and processing protocols for total 15N analysis. The methods were demonstrated using a replicated study from south-central Minnesota planted to corn (Zea mays L.). Each treatment consisted of six corn rows (76 cm row-spacing) 15.2 m long with a microplot (2.4 m by 3.8 m) embedded at one end. Fertilizer-grade urea was applied at 135 kg N∙ha-1 at planting, while the microplot received urea enriched to 5 atom % 15N. Soil and plant samples were taken several times throughout the growing season, taking care to minimize cross-contamination by using separate tools and physically separating unenriched and enriched samples during all procedures. Soil and plant samples were dried, ground to pass through a 2 mm screen, and then ground to a flour-like consistency using a roller jar mill. Tracer studies require additional planning, sample processing time and manual labor, and incur higher costs for 15N enriched materials and sample analysis than traditional N studies. However, using the mass balance approach, tracer studies with multiple in-season sampling events allow the researcher to estimate FDN distribution through the soil-crop system and estimate unaccounted-for FDN from the system.
Fertilizer nitrogen (N) use is essential in agriculture to meet the food, fiber, feed, and fuel demands of a growing global population, but N losses from agricultural fields can negatively impact environmental quality. Because N undergoes many transformations in the soil-crop system, a better understanding of N cycling, crop utilization, and the overall fate of fertilizer N are necessary to improve management practices that promote N use efficiency and minimize environmental losses. Traditional N fertilizer studies primarily focus on the effect of a treatment on end-of-season measurements such as crop yield, crop N uptake relative to the N rate applied (apparent fertilizer use efficiency), and residual soil N. While these studies quantify the overall system N inputs, outputs, and efficiencies, they cannot identify nor quantify N in the soil-crop system derived from fertilizer sources or the soil. A different approach using stable isotopes must be used to track and quantify the fate of fertilizer derived N (FDN) in the soil-crop system.
Nitrogen has two stable isotopes, 14N and 15N, that occur in nature at a relatively constant ratio of 272:1 for 14N/15N1 (concentration of 0.366 atom % 15N or 3600 ppm 15N2,3). The addition of 15N enriched fertilizer increases the total 15N content of the soil system. As 15N enriched fertilizer mixes with unenriched soil N, the measured change of 14N/15N ratio allows researchers to trace FDN in the soil profile and into the crop3,4. A mass balance can be calculated by measuring the total amount of 15N tracer in the system and each of its parts2. Because 15N enriched fertilizers are significantly more expensive than conventional fertilizers, 15N enriched microplots are often embedded within the treatment plots. The purpose of this methods paper is to describe a small-plot research design utilizing microplots for multiple in-season soil and plant sampling events for corn (Zea mays L.) and to present protocols for preparing plant and soil samples for total 15N analysis. These results can then be used to estimate N fertilizer use efficiency and create a partial N budget accounting for FDN in the bulk soil and the crop.
1. Field site description
NOTE: When performing 15N tracer field trials, selected sites should minimize variation due to soil, topography, and physical features5. Cross-contamination may occur following lateral soil movement due to slope, wind or water translocation, or tillage while the vertical distribution of soil N may be impacted by subsurface water flow and tile-drainage6.
2. Plot design
3. Soil and plant sample precautions
4. 15N enriched fertilizer preparation and application
5. Field sample processing: aboveground corn biomass
6. Field sample processing: soil
7. Lab sample processing: grind soil and plant samples
8. Weigh ground plant and soil samples for total N and 15N analysis
9. Calculations
The results presented in this paper come from a field site established in 2015 at the University of Minnesota Southern Outreach and Research Center located near Waseca, MN. The site was managed as a corn-soybean [Glycine max (L.) Merr] rotation prior to 2015 but was managed as a corn-corn rotation during the 2015 and 2016 growing seasons. The soil was a Nicollet clay loam (fine-loamy, mixed, superactive, mesic Aquic Hapludolls)-Webster clay loam (fine-loamy, mixed, superactive, m...
Stable isotope research is a useful tool for tracking and quantifying FDN through the soil-crop system. However, there are three main assumptions associated with N tracer studies that if violated may invalidate conclusions drawn from using this methodology. They are 1) the tracer is uniformly distributed throughout the system, 2) processes under the study occur at the same rates, and 3) N leaving the 15N enriched pool does not return3. Because this study is interested in the distributio...
The authors have nothing to disclose.
The authors acknowledge the support of the Minnesota Corn Research & Promotion Council, the Hueg-Harrison Fellowship, and the Minnesota's Discovery, Research and InnoVation Economy (MnDRIVE) Fellowship.
Name | Company | Catalog Number | Comments |
20 mL scintillation vial | ANY; Fisher Scientific is one example | 0334172C | |
250 mL borosilicate glass bottle | QORPAK | 264047 | |
48-well plate | EA Consumables | E2063 | |
96-well plate | EA Consumables | E2079 | |
Cloth parts bag (30x50 cm) | ANY | NA | For corn ears |
CO2 Backpack Sprayer | ANY; Bellspray Inc is one example | Model T | |
Coin envelop (6.4x10.8 cm) | ANY; ULINE is one example | S-6285 | For 2-mm ground plant samples |
Corn chipper | ANY; DR Chipper Shredder is one example | SKU:CS23030BMN0 | For chipping corn biomass |
Corn seed | ANY | NA | Hybrid appropriate to the region |
Disposable shoe cover | ANY; Boardwalk is one example | BWK00031L | |
Ethanol 200 Proof | ANY; Decon Laboratories Inc. is one example | 2701TP | |
Fabric bags with drawstring (90x60 cm) | ANY | NA | For plant sample collection |
Fertilizer Urea (46-0-0) | ANY | NA | ~0.366 atom % 15N |
Hand rake | ANY; Fastenal Company is one example | 5098-63-107 | |
Hand sickle | ANY; Home Depot is one example | NJP150 | For plant sample collection |
Hand-held soil probe | ANY; AMS is one example | 401.01 | |
Hydraulic soil probe | ANY; Giddings is one example | GSPS | |
Hydrochloric acid, 12N | Ricca Chemical | R37800001A | |
Jar mill | ANY; Cole-Parmer is one example | SI-04172-50 | |
Laboratory Mill | Perten | 3610 | For grinding grain |
Microbalance accurate to four decimal places | ANY; Mettler Toledo is one example | XPR2 | |
N95 Particulate Filtering Facepiece Respirator | ANY, ULINE is one example | S-9632 | |
Neoprene or butyl rubber gloves | ANY | NA | For working in HCl acid bath |
Paper hardware bags (13.3x8.7x27.8 cm) | ANY; ULINE is one example | S-8530 | For soil samples and corn grain |
Plant grinder | ANY; Thomas Wiley Model 4 Mill is one example | 1188Y47-TS | For grinding chipped corn biomass to 2-mm particles |
Plastic tags | ULINE | S-5544Y-PW | For labeling fabric bags and microplot stalk bundles |
Sodium hydroxide pellets, ACS | Spectrum Chemical | SPCM-S1295-07 | |
Soil grinder | ANY; AGVISE stainless steel grinder with motor is one example | NA | For grinding soil to pass through a 2-mm sieve |
Tin capsule 5x9 mm | Costech Analytical Technologies Inc. | 041061 | |
Tin capsule 9x10 mm | Costech Analytical Technologies Inc. | 041073 | |
Urea (46-0-0) | MilliporeSigma | 490970 | 10 atom % 15N |
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