微图设计及植物和土壤样品制备，进行15次氮气分析
Summary
介绍了用于15N 示踪剂研究的微图设计，以适应多个季节内植物和土壤采样事件。提出了用于15N分析的土壤和植物样品收集和处理程序，包括研磨和称重方案。
Abstract
许多氮肥研究评估处理对季末测量（如谷物产量或累计 N 损失）的总体影响。需要采用稳定的同位素方法，跟踪和量化通过土壤作物系统获得N（FDN）的肥料的命运。本文的目的是描述一种小图研究设计，利用非封闭15个N富集微图，在两个生长季节进行多个土壤和植物采样事件，并提供样品收集、处理和处理方案，用于总共15个N分析。这些方法通过一项从明尼苏达州中南部种植到玉米的复制研究进行演示（Zea Mays L.）。每次处理包括六排玉米（76厘米的行间距）15.2米长，一端嵌入一个微图（2.4米 x 3.8米）。肥料级尿素在种植时以135公斤N+ha-1施用，而微图在种植过程中多次采集了富集量至5原子%15N的尿素，并注意使用单独的工具，并在所有过程中使用物理分离未富集和浓缩的样品，尽量减少交叉污染。土壤和植物样品干燥，地面通过2毫米的屏幕，然后地面到面粉一样一致性使用辊罐磨。跟踪器研究需要额外的规划、样品处理时间和人工，并且比传统的 N 研究产生更高的 15N 浓缩材料和样品分析成本。然而，使用质量平衡方法，具有多个季节采样事件的跟踪研究使研究人员能够通过土壤作物系统估计 FDN 分布，并估计系统中对 FDN 的下落不明。
Introduction
化肥氮（N）的使用对于满足全球不断增长的人口的粮食、纤维、饲料和燃料需求至关重要，但农业领域的氮气损失可能会对环境质量产生负面影响。由于N在土壤作物系统中经历了许多转变，因此，要改进提高N使用效率并最大限度地减少环境损失的管理方法，必须更好地了解N循环、作物利用率和肥料N的总体命运。传统的 N 肥料研究主要侧重于处理对季末测量的影响，如作物产量、作物 N 吸收相对于 N 速率（明显的肥料使用效率）和残留土壤 N。虽然这些研究量化了整个系统N投入、产出和效率，但它们无法识别或量化土壤作物系统中来自肥料来源或土壤的N。必须使用使用稳定同位素的不同方法来跟踪和量化土壤作物系统中衍生的N（FDN）肥料的命运。
氮有两种稳定的同位素，14N和15N，在自然界中相对恒定比为272：1，为14N/15N151（浓度为0.366原子% 15N或3600 ppm 15N2，3）。增加15N富肥增加了土壤系统15N含量。由于15N富肥与未富土N混合，14N/15N比率的测量变化15使研究人员能够跟踪FDN在土壤轮廓和作物,3，4。质量平衡可以通过测量系统中15N 示踪器的总量及其每个部件2来计算。由于15种N富肥比传统肥料贵得多，15个N富肥通常嵌入到处理地块中。本文的目的是描述利用微图对玉米（Zea mays L.）的多个季节土壤和植物取样事件进行小图研究设计，并提出为总共15个N分析准备植物和土壤样本的协议。然后，这些结果可用于估算 N 肥料的使用效率，并在散装土壤和作物中为 FDN 创建部分 N 预算核算。
Protocol
1. 现场现场描述 注：在进行15N示踪场试验时，选定地点应尽量减少因土壤、地形和物理特征而变化5。由于坡度、风或水迁移或耕种，横向土壤移动后可能发生交叉污染，而土壤N的垂直分布可能受到地下水流和瓦片排水的影响。 描述实验场址，包括过去的管理（如以前的作物和耕种）、经纬度、土壤物理和化学性质（例如?…
Representative Results
本文提供的结果来自2015年位于明尼苏达州瓦塞卡附近的明尼苏达大学南方外展研究中心的一个现场。该基地在 2015 年以前被管理为玉米大豆 +甘氨酸最大值 （L.） Merr] 旋转，但在 2015 和 2016 生长季节作为玉米玉米轮作进行管理。土壤是一个Nicollet粘土土（细洛米，混合，超活性，梅西阿基克哈普卢多斯）-韦伯斯特粘土土（细洛米，混合，超活性，中西泰皮克恩多阿…
Discussion
稳定的同位素研究是通过土壤作物系统跟踪和量化FDN的有用工具。然而，有三个主要假设与N示踪器研究有关，如果违反，可能会使使用这种方法得出的结论无效。它们是 1） 示踪剂在整个系统中均匀分布，2） 研究下的进程以相同的速率发生，3） N 离开15N 富集池不返回3。由于本研究对整个土壤作物系统中FDN总量分布感兴趣，假设2和假设3是关注最少的21。<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者感谢明尼苏达州玉米研究与促进委员会、休格-哈里森研究金和明尼苏达州发现、研究和无病毒经济 （MnDRIVE） 研究金的支持。
Materials
| 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 (30×50 cm) | ANY | NA | For corn ears |
| CO2 Backpack Sprayer | ANY; Bellspray Inc is one example | Model T | |
| Coin envelop (6.4×10.8 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 (90×60 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 (13.3×8.7×27.8 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 5×9 mm | Costech Analytical Technologies Inc. | 041061 | |
| Tin capsule 9×10 mm | Costech Analytical Technologies Inc. | 041073 | |
| Urea (46-0-0) | MilliporeSigma | 490970 | 10 atom % 15N |
References
- Sharp, Z. . Principles of Stable Isotope Geochemistry. , (2017).
- Van Cleemput, O., Zapata, F., Vanlauwe, B. Guidelines on Nitrogen Management in Agricultural Systems. Guidelines on Nitrogen Management in Agricultural Systems. 29 (29), 19 (2008).
- Hauck, R. D., Meisinger, J. J., Mulvaney, R. L. Practical considerations in the use of nitrogen tracers in agricultural and environmental research. Methods of Soil Analysis: Part 2-Microbiological and Biochemical Properties. , 907-950 (1994).
- Bedard-Haughn, A., Van Groenigen, J. W., Van Kessel, C. Tracing 15N through landscapes: Potential uses and precautions. Journal of Hydrology. 272 (1-4), 175-190 (2003).
- Peterson, R. G. . Agricultural Field Experiments: Design and Analysis. , (1994).
- Follett, R. F. Innovative 15N microplot research techniques to study nitrogen use efficiency under different ecosystems. Communications in Soil Science and Plant Analysis. 32 (7/8), 951-979 (2001).
- Russelle, M. P., Deibert, E. J., Hauck, R. D., Stevanovic, M., Olson, R. A. Effects of water and nitrogen management on yield and 15N-depleted fertilizer use efficiency of irrigated corn. Soil Science Society of America Journal. 45 (3), 553-558 (1981).
- Schindler, F. V., Knighton, R. E. Fate of Fertilizer Nitrogen Applied to Corn as Estimated by the Isotopic and Difference Methods. Soil Science Society of America Journal. 63, 1734 (1999).
- Stevens, W. B., Hoeft, R. G., Mulvaney, R. L. Fate of Nitrogen-15 in a Long-Term Nitrogen Rate Study. Agronomy Journal. 97 (4), 1037 (2005).
- Recous, S., Fresneau, C., Faurie, G., Mary, B. The fate of labelled 15N urea and ammonium nitrate applied to a winter wheat crop. Plant and Soil. 112 (2), 205-214 (1988).
- Abendroth, L. J., Elmore, R. W., Boyer, M. J., Marlay, S. K. . Corn Growth and Development. , (2011).
- Gomez, K. A., Gomez, A. A. . Statistical Procedures for Agricultural Research. , (1984).
- Khan, S. A., Mulvaney, R. L., Brooks, P. D. Diffusion Methods for Automated Nitrogen-15 Analysis using Acidified Disks. Soil Science Society of America Journal. 62 (2), 406 (1998).
- Horneck, D. A., Miller, R. O. Determination of Total Nitrogen in Plant Tissue. Handbook of Reference Methods for Plant Analysis. , 75-84 (1998).
- . Carbon (13C) and Nitrogen (15N) Analysis of Solids by EA-IRMS Available from: https://stableisotopefacility.ucdavis.edu/13cand15n.html (2019)
- Stevens, W. B., Hoeft, R. G., Mulvaney, R. L. Fate of Nitrogen-15 in a Long-Term Nitrogen Rate Study: II. Nitrogen Uptake Efficiency. Agronomy Journal. 97 (4), 1046 (2005).
- . Fertilizing Corn in Minnesota Available from: https://extension.umn.edu/crop-specific-needs/fertilizing-corn-minnesota (2018)
- Blake, G. R., Hartge, K. H. Bulk Density. Methods of Soil Analysis: Part 1 Physical and Mineralogical Methods. , 363-375 (1986).
- Jokela, W. E., Randall, G. W. Fate of Fertilizer Nitrogen as Affected by Time and Rate of Application on Corn. Soil Science Society of America Journal. 61 (6), 1695 (2010).
- Hart, S. C., Stark, J. M., Davidson, E. A., Firestone, M. K. Nitrogen Mineralization, Immobilization, and Nitrification. Methods of Soil Analysis, Part 2. Microbiological and Biochemical Properties. (5), 985-1018 (1994).
- Olson, R. V. Fate of tagged nitrogen fertilizer applied to irrigated corn. Soil Science Society of America Journal. 44 (3), 514-517 (1980).
- Follett, R. F., Porter, L. K., Halvorson, A. D. Border Effects on Nitrogen-15 Fertilized Winter Wheat Microplots Grown in the Great Plains. Agronomy Journal. 83 (3), 608-612 (1991).
- Balabane, M., Balesdent, J. Input of fertilizer-derived labelled n to soil organic matter during a growing season of maize in the field. Soil Biology and Biochemistry. 24 (2), 89-96 (1992).
- Recous, S., Machet, J. M., Mary, B. The partitioning of fertilizer-N between soil and crop: Comparison of ammonium and nitrate applications. Plant and Soil. 144 (1), 101-111 (1992).
- Bigeriego, M., Hauck, R. D., Olson, R. A. Uptake, Translocation and Utilization of 15N-Depleted Fertilizer in Irrigated Corn. Soil Science Society of America Journal. 43 (3), 528 (1979).
- Glendining, M. J., Poulton, P. R., Powlson, D. S., Jenkinson, D. S. Fate of15N-labelled fertilizer applied to spring barley grown on soils of contrasting nutrient status. Plant and Soil. 195 (1), 83-98 (1997).
- Khanif, Y. M., Cleemput, O., Baert, L. Field study of the fate of labelled fertilizer nitrate applied to barley and maize in sandy soils. Fertilizer Research. 5 (3), 289-294 (1984).
Citer Cet Article
Spackman, J. A., Fernandez, F. G. Microplot Design and Plant and Soil Sample Preparation for 15Nitrogen Analysis. J. Vis. Exp. (159), e61191, doi:10.3791/61191 (2020).