Name: microplot design and plant and soil sample preparation for 15nitrogen analysis
Uploaded: 2020-05-10T14:00:00
Description: Watch this Scientific Journal Video about Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis at JoVE.com

The authors acknowledge the support of the Minnesota Corn Research &#38; Promotion Council, the Hueg-Harrison Fellowship, and the Minnesota&#39;s Discovery, Research and InnoVation Economy (MnDRIVE) Fellowship.

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.
<ol>
	<li>Describe the experimental field site includin...

<ol>
	<li>Sharp, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Stable Isotope Geochemistry. , (2017).</li><li>Van Cleemput, O., Zapata, F., Vanlauwe, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+on+Nitrogen+Management+in+Agricultural+Systems.">Guidelines on Nitrogen Management in Agricultural Systems.</a> Guidelines on Nitrogen Management in Agricultural Systems. 29 (29), 19 (2008).</li><li>Hauck, R. D., Meisinger, J. J., Mulvaney, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+considerations+in+the+use+of+nitrogen+tracers+in+agricultural+and+environmental+research.">Practical considerations in the use of nitrogen tracers in agricultural and environmental research.</a> Methods of Soil Analysis: Part 2-Microbiological and Biochemical Properties. , 907-950 (1994).</li><li>Bedard-Haughn, A., Van Groenigen, J. W., Van Kessel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracing+15N+through+landscapes:+Potential+uses+and+precautions.">Tracing 15N through landscapes: Potential uses and precautions.</a> Journal of Hydrology. 272 (1-4), 175-190 (2003).</li><li>Peterson, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Agricultural Field Experiments: Design and Analysis. , (1994).</li><li>Follett, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innovative+15N+microplot+research+techniques+to+study+nitrogen+use+efficiency+under+different+ecosystems.">Innovative 15N microplot research techniques to study nitrogen use efficiency under different ecosystems.</a> Communications in Soil Science and Plant Analysis. 32 (7/8), 951-979 (2001).</li><li>Russelle, M. P., Deibert, E. J., Hauck, R. D., Stevanovic, M., Olson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+water+and+nitrogen+management+on+yield+and+15N-depleted+fertilizer+use+efficiency+of+irrigated+corn.">Effects of water and nitrogen management on yield and 15N-depleted fertilizer use efficiency of irrigated corn.</a> Soil Science Society of America Journal. 45 (3), 553-558 (1981).</li><li>Schindler, F. V., Knighton, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+Fertilizer+Nitrogen+Applied+to+Corn+as+Estimated+by+the+Isotopic+and+Difference+Methods.">Fate of Fertilizer Nitrogen Applied to Corn as Estimated by the Isotopic and Difference Methods.</a> Soil Science Society of America Journal. 63, 1734 (1999).</li><li>Stevens, W. B., Hoeft, R. G., Mulvaney, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+Nitrogen-15+in+a+Long-Term+Nitrogen+Rate+Study.">Fate of Nitrogen-15 in a Long-Term Nitrogen Rate Study.</a> Agronomy Journal. 97 (4), 1037 (2005).</li><li>Recous, S., Fresneau, C., Faurie, G., Mary, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+labelled+15N+urea+and+ammonium+nitrate+applied+to+a+winter+wheat+crop.">The fate of labelled 15N urea and ammonium nitrate applied to a winter wheat crop.</a> Plant and Soil. 112 (2), 205-214 (1988).</li><li>Abendroth, L. J., Elmore, R. W., Boyer, M. J., Marlay, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Corn Growth and Development. , (2011).</li><li>Gomez, K. A., Gomez, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Statistical Procedures for Agricultural Research. , (1984).</li><li>Khan, S. A., Mulvaney, R. L., Brooks, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diffusion+Methods+for+Automated+Nitrogen-15+Analysis+using+Acidified+Disks.">Diffusion Methods for Automated Nitrogen-15 Analysis using Acidified Disks.</a> Soil Science Society of America Journal. 62 (2), 406 (1998).</li><li>Horneck, D. A., Miller, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+Total+Nitrogen+in+Plant+Tissue.">Determination of Total Nitrogen in Plant Tissue.</a> Handbook of Reference Methods for Plant Analysis. , 75-84 (1998).</li><li>. Carbon (13C) and Nitrogen (15N) Analysis of Solids by EA-IRMS Available from: <a target="_blank" href="https://stableisotopefacility.ucdavis.edu/13cand15n.html">https://stableisotopefacility.ucdavis.edu/13cand15n.html</a> (2019)</li><li>Stevens, W. B., Hoeft, R. G., Mulvaney, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+Nitrogen-15+in+a+Long-Term+Nitrogen+Rate+Study:+II.+Nitrogen+Uptake+Efficiency.">Fate of Nitrogen-15 in a Long-Term Nitrogen Rate Study: II. Nitrogen Uptake Efficiency.</a> Agronomy Journal. 97 (4), 1046 (2005).</li><li>. Fertilizing Corn in Minnesota Available from: <a target="_blank" href="https://extension.umn.edu/crop-specific-needs/fertilizing-corn-minnesota">https://extension.umn.edu/crop-specific-needs/fertilizing-corn-minnesota</a> (2018)</li><li>Blake, G. R., Hartge, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bulk+Density.">Bulk Density.</a> Methods of Soil Analysis: Part 1 Physical and Mineralogical Methods. , 363-375 (1986).</li><li>Jokela, W. E., Randall, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+Fertilizer+Nitrogen+as+Affected+by+Time+and+Rate+of+Application+on+Corn.">Fate of Fertilizer Nitrogen as Affected by Time and Rate of Application on Corn.</a> Soil Science Society of America Journal. 61 (6), 1695 (2010).</li><li>Hart, S. C., Stark, J. M., Davidson, E. A., Firestone, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitrogen+Mineralization,+Immobilization,+and+Nitrification.">Nitrogen Mineralization, Immobilization, and Nitrification.</a> Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. (5), 985-1018 (1994).</li><li>Olson, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+tagged+nitrogen+fertilizer+applied+to+irrigated+corn.">Fate of tagged nitrogen fertilizer applied to irrigated corn.</a> Soil Science Society of America Journal. 44 (3), 514-517 (1980).</li><li>Follett, R. F., Porter, L. K., Halvorson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Border+Effects+on+Nitrogen-15+Fertilized+Winter+Wheat+Microplots+Grown+in+the+Great+Plains.">Border Effects on Nitrogen-15 Fertilized Winter Wheat Microplots Grown in the Great Plains.</a> Agronomy Journal. 83 (3), 608-612 (1991).</li><li>Balabane, M., Balesdent, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Input+of+fertilizer-derived+labelled+n+to+soil+organic+matter+during+a+growing+season+of+maize+in+the+field.">Input of fertilizer-derived labelled n to soil organic matter during a growing season of maize in the field.</a> Soil Biology and Biochemistry. 24 (2), 89-96 (1992).</li><li>Recous, S., Machet, J. M., Mary, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+partitioning+of+fertilizer-N+between+soil+and+crop:+Comparison+of+ammonium+and+nitrate+applications.">The partitioning of fertilizer-N between soil and crop: Comparison of ammonium and nitrate applications.</a> Plant and Soil. 144 (1), 101-111 (1992).</li><li>Bigeriego, M., Hauck, R. D., Olson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uptake,+Translocation+and+Utilization+of+15N-Depleted+Fertilizer+in+Irrigated+Corn.">Uptake, Translocation and Utilization of 15N-Depleted Fertilizer in Irrigated Corn.</a> Soil Science Society of America Journal. 43 (3), 528 (1979).</li><li>Glendining, M. J., Poulton, P. R., Powlson, D. S., Jenkinson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of15N-labelled+fertilizer+applied+to+spring+barley+grown+on+soils+of+contrasting+nutrient+status.">Fate of15N-labelled fertilizer applied to spring barley grown on soils of contrasting nutrient status.</a> Plant and Soil. 195 (1), 83-98 (1997).</li><li>Khanif, Y. M., Cleemput, O., Baert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field+study+of+the+fate+of+labelled+fertilizer+nitrate+applied+to+barley+and+maize+in+sandy+soils.">Field study of the fate of labelled fertilizer nitrate applied to barley and maize in sandy soils.</a> Fertilizer Research. 5 (3), 289-294 (1984).</li></ol>

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...

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&#160;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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 mL scintillation vial</td>
			<td>ANY; Fisher Scientific is one example</td>
			<td>0334172C</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL borosilicate glass bottle</td>
			<td>QORPAK</td>
			<td>264047</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well plate</td>
			<td>EA Consumables</td>
			<td>E2063</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>EA Consumables</td>
			<td>E2079</td>
			<td></td>
		</tr>
		<tr>
			<td>Cloth parts bag (30x50 cm)</td>
			<td>ANY</td>
			<td>NA</td>
			<td>For corn ears</td>
		</tr>
		<tr>
			<td>CO2 Backpack Sprayer</td>
			<td>ANY; Bellspray Inc is one example</td>
			<td>Model T</td>
			<td></td>
		</tr>
		<tr>
			<td>Coin envelop (6.4x10.8 cm)</td>
			<td>ANY; ULINE is one example</td>
			<td>S-6285</td>
			<td>For 2-mm ground plant samples</td>
		</tr>
		<tr>
			<td>Corn chipper</td>
			<td>ANY; DR Chipper Shredder is one example</td>
			<td>SKU:CS23030BMN0</td>
			<td>For chipping corn biomass</td>
		</tr>
		<tr>
			<td>Corn seed</td>
			<td>ANY</td>
			<td>NA</td>
			<td>Hybrid appropriate to the region</td>
		</tr>
		<tr>
			<td>Disposable shoe cover</td>
			<td>ANY; Boardwalk is one example</td>
			<td>BWK00031L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 Proof</td>
			<td>ANY; Decon Laboratories Inc. is one example</td>
			<td>2701TP</td>
			<td></td>
		</tr>
		<tr>
			<td>Fabric bags with drawstring (90x60 cm)</td>
			<td>ANY</td>
			<td>NA</td>
			<td>For plant sample collection</td>
		</tr>
		<tr>
			<td>Fertilizer Urea (46-0-0)</td>
			<td>ANY</td>
			<td>NA</td>
			<td>~0.366 atom % 15N</td>
		</tr>
		<tr>
			<td>Hand rake</td>
			<td>ANY; Fastenal Company is one example</td>
			<td>5098-63-107</td>
			<td></td>
		</tr>
		<tr>
			<td>Hand sickle</td>
			<td>ANY; Home Depot is one example</td>
			<td>NJP150</td>
			<td>For plant sample collection</td>
		</tr>
		<tr>
			<td>Hand-held soil probe</td>
			<td>ANY; AMS is one example</td>
			<td>401.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydraulic soil probe</td>
			<td>ANY; Giddings is one example</td>
			<td>GSPS</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid, 12N</td>
			<td>Ricca Chemical</td>
			<td>R37800001A</td>
			<td></td>
		</tr>
		<tr>
			<td>Jar mill</td>
			<td>ANY; Cole-Parmer is one example</td>
			<td>SI-04172-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Mill</td>
			<td>Perten</td>
			<td>3610</td>
			<td>For grinding grain</td>
		</tr>
		<tr>
			<td>Microbalance accurate to four decimal places</td>
			<td>ANY; Mettler Toledo is one example</td>
			<td>XPR2</td>
			<td></td>
		</tr>
		<tr>
			<td>N95 Particulate Filtering Facepiece Respirator</td>
			<td>ANY, ULINE is one example</td>
			<td>S-9632</td>
			<td></td>
		</tr>
		<tr>
			<td>Neoprene or butyl rubber gloves</td>
			<td>ANY</td>
			<td>NA</td>
			<td>For working in HCl acid bath</td>
		</tr>
		<tr>
			<td>Paper hardware bags (13.3x8.7x27.8 cm)</td>
			<td>ANY; ULINE is one example</td>
			<td>S-8530</td>
			<td>For soil samples and corn grain</td>
		</tr>
		<tr>
			<td>Plant grinder</td>
			<td>ANY; Thomas Wiley Model 4 Mill is one example</td>
			<td>1188Y47-TS</td>
			<td>For grinding chipped corn biomass to 2-mm particles</td>
		</tr>
		<tr>
			<td>Plastic tags</td>
			<td>ULINE</td>
			<td>S-5544Y-PW</td>
			<td>For labeling fabric bags and microplot stalk bundles</td>
		</tr>
		<tr>
			<td>Sodium hydroxide pellets, ACS</td>
			<td>Spectrum Chemical</td>
			<td>SPCM-S1295-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Soil grinder</td>
			<td>ANY; AGVISE stainless steel grinder with motor is one example</td>
			<td>NA</td>
			<td>For grinding soil to pass through a 2-mm sieve</td>
		</tr>
		<tr>
			<td>Tin capsule 5x9 mm</td>
			<td>Costech Analytical Technologies Inc.</td>
			<td>041061</td>
			<td></td>
		</tr>
		<tr>
			<td>Tin capsule 9x10 mm</td>
			<td>Costech Analytical Technologies Inc.</td>
			<td>041073</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea (46-0-0)</td>
			<td>MilliporeSigma</td>
			<td>490970</td>
			<td>10 atom % 15N</td>
		</tr>
	</tbody>
</table>

microplot design and plant and soil sample preparation for 15nitrogen analysis

Many&#160;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&#160;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&#8729;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.

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...

Watch this Scientific Journal Video about Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis at JoVE.com

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

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.

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

Many&#160;nitrogen fertilizer studies evaluate the overall effect of a treatment on end-of-season measurements such as grain yield or cumulative N losses. ...

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.

Research

JoVE Journal

Environment

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

Design and Construction of an Urban Runoff Research Facility

As the urban population increases, so does the area of irrigated urban landscape. Summer water use in urban areas can be 2-3x winter base line water use ...

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Protocol for Microplastics Sampling on the Sea Surface and Sample Analysis

Microplastic pollution in the marine environment is a scientific topic that has received increasing attention over the last decade. The majority of ...

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...