Microplot Design and Plant and Soil Sample Preparation for ¹⁵Nitrogen Analysis

This methodology can help researchers quantify the impacts of fertilizer drive nitrogen on the soil crop system and answer questions about nitrogen use efficiency. The main advantage of this technique is that it allows for multiple in seasons soil and plant sampling events over two consecutive growing seasons. This methodology could help us better understand the nitrogen cycle processes of mineralization and mobilization to improve nitrogen fertilizer management guidelines.

To set up a field plot, plant six cornrows with 76 centimeters of spacing with a final 15.2 by 4.6 meter plot dimension. Establish 1.5 meter border areas from each end of the lengthwise dimension and an additional 1.5 meter long border area adjoining the sampling and harvest areas. Designate rows two and three as the in season plant and soil sampling area and rows four and five as the harvest area for corn grain yield.

Establish a 2.4 by 3.8 meter micro plot area centered on the width dimension for collection of all the nitrogen enriched plant and soil samples, leaving 38 meters of unsampled border on the length and width dimensions to minimize any edge effects. Then delineate the treatment plot and micro plot corners with different colored flags. Wear shoe coverings when accessing the micro plots and minimize micro plot foot traffic to prevent contamination of the unenriched sampling areas, removing the foot covers when exiting the micro plot area.

To apply the Nitrogen-15 enriched fertilizer, dilute 10 atom percent Nitrogen-15 enriched urea in conventional urea to five atom percent nitrogen enriched urea and dissolve the urea in two liters of deionized water to ensure uniform enrichment of the urea fertilizer. Use a calibrated backpack carbon dioxide sprayer to evenly apply the Nitrogen-15 enriched urea solution to the micro plots. Then incorporate urea containing fertilizers with light tillage, hand rakes or 64 centimeters of irrigation within 24 hours of application to minimize the volatilization loss potential.

At each sampling stage, collect a six above ground, Nitrogen-15 unenriched corn plant composite sample from within the sampling area and a six above ground corn plant composite sample from the Nitrogen-15 enriched micro plot. Chop VH and R1 above ground biomasses and place the chopped biomass in labeled bags or drying and a forced air oven at 60 degrees Celsius until constant mass. Record the biomass dry weight and thoroughly mix and grind 100 to 200 grams of dried plant material until it can pass through a two millimeter sieve.

Then thoroughly mix the ground material and store the sub sample in a labeled coin envelope for further processing. For soil sample processing, within eight days of fertilizer application, use a hand probe to collect a four core 1.8 centimeter diameter composite soil sample from the unenriched sampling area at VH and R1 concurrent with the plant sampling and use a separate hand probe to collect a 15 core 1.8 centimeter diameter composite soil sample from the micro plot. Homogenize each composite soil sample in a bucket and place the samples in pre labeled paper bags.

Then drY the soil samples at 35 degrees Celsius in a forced air oven until constant mass, before grinding each sample until it can pass through a two millimeter sieve. For sample processing in the laboratory dry the ground plan samples overnight in the 60 degrees Celsius oven, before individually grinding the dried plant and soil samples in roller jars at four times g for six to 24 hours until the samples obtain a fine flour like consistency. Then transfer the finely ground material into clean labeled 20 milliliters silylation vials.

To determine the total and Nitrogen-15 concentration in each sample, wearing nitrile gloves, first use laboratory wipes and ethanol to clean the micro scale, work surfaces, spatula and forceps. Place the clean utensils on a lab wipe on the lab bench and use forceps to gently flare out the opening of the sample capsule. Oven and release the modified capsule one to two millimeters above the micro scale weigh pan and tear the capsule.

Use forceps to return the capsule to the clean work surface and use the spatula to carefully add the required mass of finely ground sample material to the capsule. Use the forceps to slowly crimp the top third of the loaded capsule and fold over to seal. Continue folding and compressing the capsule, taking care not to puncture or tear the tin until a spherical shape has been obtained.

Use the forceps to drop the wrapped capsule several times from a one centimeter height onto a clean dark surface to check for leaks. If no dust appears, weigh the sample as just demonstrated and place the capsule in one well of a 96 well plate, recording the well placement. Between each sample encapsulation, clean each of the utensils and surfaces with ethanol and laboratory wipes, paying special attention to the spatula and forceps edges.

In this representative analysis, the fertilizer-derived nitrogen concentration in the above ground corn biomass sample was greatest earlier in the growing season and decreased with each successive sampling period. The soil-derived nitrogen however, was consistently the greatest fraction of above ground biomass nitrogen, illustrating the importance of the soil nitrogen supply for optimal corn growth. At physiological maturity in the first year, approximately 27%of the above ground biomass nitrogen was fertilizer-derived with similar proportions observed in the grain, stover and cob fractions.

At physiological maturity in the second year, only 2%of first year fertilizer-derived nitrogen was recovered in the above ground biomass. With approximately 1.6 kilograms per hectare of first year fertilizer derived nitrogen exported in the grain. Within eight days of fertilizer application, the majority of the fertilizer derived nitrogen was in the top 15 centimeters of the soil profile as expected.

However approximately 22 kilograms of nitrogen per hectare had already moved into the deeper depths, while four to 10%of the fertilizer-derived nitrogen was unaccounted for. Indeed, by the end of the first and second years, less than 50%of the fertilizer-derived nitrogen was accounted for within the soil corn system, while the remainder was either lost to the environment or leached below the 90 centimeter soil sample depth. When attempting this procedure, extreme care should be taken to avoid cross contamination which can invalidate the results.

Inorganic and organic fractions of the soil may be further analyzed to improve our understanding of nitrogen cycling dynamics. Plant samples analyzed by plant parts may be used to inform our understanding of nitrogen uptake and translocation over time.