# NITROGEN LEACHING AFTER SOLID MANURE APPLICATION IN AUTUMN BEFORE SPRING SOWING

DELIN, S.¹

¹ Swedish University of Agricultural Sciences, Sweden

## INTRODUCTION

Regulation of manure application in autumn due to risk for leaching can be problematic for manure rich in straw that could be unbeneficial for the crop to apply close to sowing and on clay soils that cannot be ploughed in spring. The risk for leaching is likely to differ between different on soil texture and manure characteristics.

## MATERIAL AND METHODS

In 80 cm deep lysimeters (Persson & Bergström, 1991) with loamy sand or silty clay, manure was applied in October, November or March during three consecutive years before sowing spring oats (2014 and 2016) or spring barley (2015). Nitrogen (N) leaching measurements started before the first manure application in October 2013 and continued until the end of the year 2017. On sandy loam, manure with high or low Carbon:Nitrogen ratio (C:N=18 or 10) was compared for all times of application. On silty clay, application of manure with low C:N ratio in October and November was compared. There was also one control treatment with no manure application for each soil type. The experiments were conducted in an outdoor facility in Sweden. All treatments had three replicates with in total nine lysimeters with silty clay and 21 lysimeters with loamy sand. The treatments were kept in the same lysimeters for all three years. In parallel field plot experiments, effects on ammonia emissions and grain yield were measured. In addition, soil samples were taken in autumn and early spring in the layers 0-30 cm, 30-60 cm and 60-90 cm and analysed for ammonium and nitrate nitrogen. The field experiments were located at the same sites as where soil was collected for the lysimeters and had the same treatments in three replicates plus two additional treatments with different mineral nitrogen fertiliser additions. In parallel incubations, net nitrogen mineralisation depending on manure C:N ratio was studied for fifteen different solid manures in loamy sand and for the two manures used in lysimeter and field experiment on the silty clay. The incubations were performed in the laboratory at 10°C.

## RESULTS AND DISCUSSION

Nitrogen leaching was not affected by timing of manure application, when manure C:N ratio was 18. However, when C:N ratio was 10, nitrogen leaching was elevated with around 10 kg N per ha after manure application in October compared to November and March (Figure 1). One year after manure application, no significant differences in nitrogen concentration between treatments were observed, which indicates no significant residual effects of manure application on N leaching during the following year. The grain yield in the parallel field experiments was similar between application times of manure, but tended to be lower after application of manure with high C:N ratio in March and after application of manure in November on clay soil. Ammonia emissions did not differ between manure types, but were on average higher after application in October when air temperature was on average 12°C compared to in November and March when air temperature was on average 5°C. Incubation results indicated that only manure with C:N ratio below 14 tended to release enough mineral N to be considered as risk for leaching during the first months after application, which is in accordance with other similar investigations (Qian & Schoenau, 2002). The effects of manure application on leaching were similar between the two soil types in the lysimeter study. However, the soil analyses from the field experiments indicated no effects of manure application on leaching on the silty clay, only on the loamy sand. This difference in results between field and lysimeters for the silty clay could be explained by moister soil conditions in the field, leading to larger losses with denitrification rather than by leaching.
<figure><img src="image_80.png" /><figcaption>Figure 1. Yearly nitrogen leaching depending on time for application of solid manure before spring sowing on the loamy sand.</figcaption></figure>

## CONCLUSION

Nitrogen leaching after application of manure was unaffected by time for application for manure with high (>14) C:N ratio, whereas for manure with low C:N ratio (<14) application in October caused higher N leaching than application in November and March.

**Acknowledgements:** This project was financed by Formas. The author wish to thank all of those who helped performing the experiments and all who gave valuable advice, including Ingrid K Thomsen and Lars Bergström. Special thanks to Rolf Tunared who built the lysimeter facility.

## REFERENCES

Qian, P. Schoenau, J.J. 2002. Availability of nitrogen in solid manure amendments with different C:N ratios. Canadian Journal of Soil Science 82, 219-225.

Persson, L. Bergström, L. 1991 Drilling method for collection of undisturbed soil monoliths. Soil Sci Soc Am J 55, 285-287.
# HUMAN URINE AS A NITROGEN FERTILIZER: A GREENHOUSE EXPERIMENT

MARTIN, T.M.P.¹,², LEVAVASSEUR, F.¹, ESCULIER, F.²,³, HOUOT, S.¹

¹ ECOSYS, INRA, AgroParisTech, Université Paris-Saclay, 78850, Thiverval-Grignon, France; ² LEESU, ENPC, UPEC, AgroParisTech, Université Paris-Est, 75455 Marne-la-Vallée Cedex 2, France; ³ METIS, Sorbonne Universités, CNRS, EPHE, 75005 Paris, France

## INTRODUCTION

Urine source separation is a promising paradigm for the development of a sustainable urban metabolism. Today, recycling of nutrients from wastewaters on crops is low. Only 4 % of the nitrogen and 41 % of the phosphorus present in the wastewater of Paris urban area are recycled according to Esculier et al., 2018. On the other hand, N injected into agricultural systems comes mostly from highly energy-consuming processes and phosphorus is a fossil resource for which the peak production may be reached in the next decades (Cordell et al., 2009). N fertilizers consumption and almost all the P consumption in the Ile-de-France region could be covered by the excretions of the Paris urban area inhabitants (Esculier et al., 2018). This valorization is especially possible via the urines, which contains most of these nutrients (about 75% of the N and 50% of the P loads in domestic wastewater according to Eme and Boutin, 2015) in a small volume. Treatments can be done on urine in order to obtain different types of products: liquid, solid, concentrated ... (e.g. nitrification permits to avoid ammonia volatilization and allows concentration of nutrients by distillation). Few studies exist on urine based fertilizer, although some authors have found a higher fertilizing potential than organic fertilizer and similar to mineral fertilizer (Kirchmann and Pettersson, 1995). The aim of this work was thus to characterize the N use efficiency of such products.

## MATERIAL AND METHODS

A greenhouse pot experiment was carried out during 56 days with English ryegrass (Lolium Perenne). The experiment included 6 treatments corresponding to 5 organic products and a mineral N treatment: urine (U), nitrified concentrated urine (NCU), cattle slurry (CS), compost (C), compost mixed with urine (C+U) and mineral nitrogen solution (MNS). The cattle slurry was used as a reference for classical organic fertilizer used by farmers. We tested a mix between urine and compost as it could be done easily on composting platform and could increase the mineral nitrogen content in compost. Mix was done just before the beginning of the experimentation using 20 g of compost for 16.7 g of urine. The products were mixed with 1.3 kg of loam soil just before the seeding of ryegrass. Each product was tested for two doses (X and Y, only Y for compost because of the low nitrogen availability), plus an unfertilized control treatment.

<table><caption>Table 1. Product characteristics (mean value and [standard deviation]): pH, dry matter content (DM), total (Tot-N) and mineral (Min-N) nitrogen concentrations, phosphorus (P₂O₅) and potassium (K₂O) concentrations. Treatment input in soil: dose, total (Tot-N input) and mineral nitrogen input (Min-N input) for the different products: urine (U), nitrified concentrated urine (NCU), cattle slurry (CS), compost (C), compost mixed with urine (C+U) and mineral nitrogen solution (MNS).</caption><thead><tr><th rowspan="2">Product</th><th rowspan="2">pH</th><th rowspan="2">DM %</th><th colspan="4">Product characteristics</th><th colspan="2">Treatment input in soil</th></tr><tr><th>Tot-N g.L<sup>-1</sup>/g.kg<sup>-1</sup>*</th><th>Min-N g.L<sup>-1</sup>/g.kg<sup>-1</sup>*</th><th>P<sub>2</sub>O<sub>5</sub> g.L<sup>-1</sup>/g.kg<sup>-1</sup>*</th><th>K<sub>2</sub>O g.L<sup>-1</sup>/g.kg<sup>-1</sup>*</th><th>Dose</th><th>Tot-N input mg.kgMS<sup>-1</sup></th><th>Min-N input mg.kgMS<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="2">U</td><td>9.1</td><td>1.6</td><td>6.6</td><td>5.5</td><td>0.4</td><td>2.3</td><td>X</td><td>131.4</td><td>108.5</td></tr><tr><td>[0.0]</td><td>[0.5]</td><td>[0.0]</td><td>[0.1]</td><td>[0.0]</td><td>[0.1]</td><td>Y</td><td>262.7</td><td>216.9</td></tr><tr><td rowspan="2">NCU</td><td>3.9</td><td>X</td><td>66.2<sup>1</sup></td><td>66.2</td><td>10.7</td><td>50.6</td><td>X</td><td>120.6</td><td>120.6</td></tr><tr><td>[0.0]</td><td></td><td></td><td>[5.5]</td><td>[0.2]</td><td>[1.8]</td><td>Y</td><td>241.4</td><td>241.4</td></tr><tr><td rowspan="2">CS</td><td>8.0</td><td>12.0</td><td>6.0*</td><td>1.4*</td><td>3.1*</td><td>3.8*</td><td>X</td><td>184.0</td><td>42.2</td></tr><tr><td>[0.2]</td><td>[0.1]</td><td>[0.1]</td><td>[0.0]</td><td>[0.1]</td><td>[0.1]</td><td>Y</td><td>368.1</td><td>84.5</td></tr><tr><td rowspan="2">C</td><td>8.8</td><td>74.1</td><td>14.3*</td><td>0.2*</td><td>5.2*</td><td>9.8*</td><td rowspan="2">Y</td><td rowspan="2">684.9</td><td rowspan="2">10.0</td></tr><tr><td>[0.1]</td><td>[0.8]</td><td>[0.4]</td><td>[0.0]</td><td>[0.2]</td><td>[0.1]</td></tr><tr><td>C+U</td><td>X</td><td>41.1<sup>1</sup></td><td>10.8<sup>1*</sup></td><td>3.1<sup>1*</sup></td><td>3.3*</td><td>7.3*</td><td>X</td><td>473.5 (131.4 from U)</td><td>113.5 (108.5 from U)</td></tr></tbody></table>
<table><thead><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th></tr><tr><th></th><th></th><th></th><th></th><th>[0.5]</th><th>[1.0]</th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>MNS</td><td>4.7</td><td>0</td><td>222</td><td>222</td><td>0</td><td>0</td><td>X</td><td>947.1 (262.7 from U)</td><td>226.9 (216.9 from U)</td></tr><tr><td></td><td>[0.0]</td><td></td><td>[6.2]</td><td>[6.2]</td><td></td><td></td><td>Y</td><td>77.8</td><td>77.8</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>155.6</td><td>155.6</td></tr></tbody></table>

¹Estimated values. Org N was neglected for NCU. For C+U, DM, Tot-N, Min-N were estimated with the ratio mix of U and C.

The experiment included 52 pots: 13 treatments and 4 replicates. The pots were randomized and moved every week to avoid heterogeneous radiation. Temperature (17 °C to 23 °C) and soil humidity (90 % to 100 % of field capacity) were controlled. When leaching occurred, the leachates were recovered and put back on the corresponding pots to avoid mineral nitrogen losses. The above ground biomass was cut 21, 40 and 56 days after the beginning of the experiment. Fresh and dried biomasses were weighed and nitrogen content was analyzed by Dumas method. The nitrogen use efficiency (NUE) was calculated as the slope of the relationship between plant nitrogen uptake and applied nitrogen doses including the control. NUE for C+U was calculated using only nitrogen from U as applied nitrogen dose. An ANOVA was performed using R to determine significant differences among treatments: ANOVA followed by Tukey HSD or Kruskal-Wallis test followed by Willcoxon-Mann-Whitney test.

## RESULTS AND DISCUSSION

Under the conditions of the experiment, the NUE of the raw urine (U) or treated urine (NCU) were close to, or even higher than that of the mineral fertilizer (MNS). The larger proportion of mineral N in these products (83% for U, 100% [estimated] for NCU) compared to cattle slurry (23% of mineral nitrogen) permitted a higher assimilation of nitrogen than with this conventional organic fertilizer. The higher NUE for U and NCU than for mineral fertilizer could be explained by the supply of other nutriments with these products (e.g., 0.6 for U to 1.6 for NCU grams of phosphorus and 3.5 for U to 7.6 for NCU grams of potassium for 10 grams of nitrogen), and by a buffering capacity which helped to maintain a higher soil pH. When applied with compost, the NUE of urine was slightly reduced due to mineral nitrogen organization of some of the mineral nitrogen in the urine, but remained good (about 85% of that of urine alone) in addition to being an amendment.

<figure><img src="image_81.png" /><figcaption>Figure 1. Nitrogen use efficiency (NUE) according to the product and calculated for dose X and Y (only Y for compost): urine (U), nitrified concentrated urine (NCU), cattle slurry (CS), compost (C), compost mixed with urine (C+U), mineral nitrogen solution (MNS).</figcaption></figure>

## CONCLUSION

Urine based products are a promising new source of nitrogen fertilization in agriculture. The implementation of alternative sanitation systems can also lead to great environmental and social benefits: decreases in energy consumption, reductions in greenhouse gases emissions, improvement in surface water quality, sustainable nutrient source and reconnection of urban and rural territories. The work is going on with complementary experiments to study the innocuousness of such new source of nitrogen and the potential environmental impacts through gas emissions after application.

**Acknowledgements:** We thank the OCAPI project and the Ile-de-France Federation for Research on the Environment (FIRE) for their financial support.
REFERENCES

Cordell, D., Drangert, J.-O., and White, S., 2009. The story of phosphorus: Global food security and food for thought. Glob. Environ. Change 19, 292–305.

Eme, C. and Boutin, C., 2015. Composition des eaux usées domestiques par source d'émission à l'échelle de l'habitation. IRSTEA, ONEMA, CréaPure Éd.

Esculier, F., Le Noë, J., Barles, S., Billen, G., Creno, B., Garnier, J., Lesavre, J., Petit, L., Tabuchi, JP., 2018. The biogeochemical imprint of human metabolism in Paris Megacity: a regionalized analysis of a water-agro-food system. J. Hydrol.

Kirchmann, H., and Pettersson, S., 1995. Human urine-chemical composition and fertilizer use efficiency. Fertil. Res. 40, 149–154.
# NUTRIENT CYCLING IN GRASSLAND SYSTEMS; N, P AND C CYCLING IN A PLOT EXPERIMENT ON THE NORTH WYKE FARM PLATFORM

LE COCQ, K.¹, MCAULIFFE, G.¹,², HORROCKS, C.¹, DARCH, T.¹, TAKAHASHI, T.¹,², BLACKWELL, M.¹, MISSELBROOK, T.¹, LEE, M.R.F.¹,², CARDENAS, L.¹

1 Rothamsted Research, North Wyke, Okehampton, Devon, EX20 2SB, United Kingdom; 2 University of Bristol, Bristol Veterinary School, Langford House, Langford, Somerset, BS40 5DU.

## INTRODUCTION

While our understanding of N cycle contribution to greenhouse gas (GHG) emissions from grasslands is increasing, datasets which elucidate the influence of urine and manure deposits from grazing cattle on N cycling, microbial populations and GHG emissions are less complete. Nutrient inputs to soil from grazing cattle are spatially variable and therefore it is difficult to capture information about the specific effect of urine and dung deposits on the soil chemistry and microbial community from these events over different periods of time. The North Wyke Farm Platform (NWFP) provides the opportunity to study GHG emissions concomitantly with soil nutrient cycling under three different grassland management scenarios (farmlets); permanent pasture (PP), white clover + perennial ryegrass mix with no N fertiliser added (WC) and high sugar perennial ryegrass monoculture (HS). Here, we report the results of a plot experiment assessing the impact of bovine dung and urine deposits on N, P and C cycling in grassland on the three aforementioned grassland management systems, with a focus on N.

## MATERIAL AND METHODS

### Experimental design

Plots measuring 2 x 1 m on three representative fields of the NWFP farmlets were treated with: synthetic urine (standard composition across all fields), cattle urine, dung, a fertiliser N control (as ammonium nitrate) and a zero N control. Cattle urine and dung treatments were collected and bulked within NWFP farmlets and only reapplied to their farmlet of origin. No fertiliser N was applied to the plots on the WC farmlet. Each plot was replicated three times on each field in a randomised block design. The same volume of each treatment was applied and samples were taken to determine the N content of each of the treatments on the day of application. Baseline data for gas emissions, soil chemistry, nucleic acid and herbage were taken before application of the treatments in June 2017. Following application of treatments, GHG emissions (N₂O, CO₂ and CH₄) were measured weekly. Soil samples were collected seven times following application of the treatments from each plot until the final sampling on 10/10/2017.

### Sample collection and analysis

Greenhouse gas emissions were measured using a static chamber technique, as described in Cardenas et al. (2016), using 2 chambers per plot. For soils, 10 cm deep cores from three points randomly selected from a grid within each plot were combined to make one composite sample per plot. The samples were stored at 4°C until processing upon which any plant material was removed, and the soils were crumbled and homogenised. 50 g were subsampled for potassium chloride (KCl) extractions and subsequent analysis of reactive N, and the remaining soil was frozen. Wet weight of the samples was recorded before freeze drying. Dried weights were taken and the % moisture was determined gravimetrically. A sterile cork borer was used to take 8 cores for microbial analysis from each plot and immediately flash frozen in liquid N on site and subsequently stored at -80 °C prior to analysis.

Soil and dung samples were analysed for total N, P, C as well as iodine, selenium, potassium, molybdenum and manganese following standard laboratory procedures. Herbage was sampled twice during the experiment following treatment and analysed for yield, micronutrient content and total N, P and C. Data were analysed using
analysis of variance (ANOVA) to test for statistical differences between experimental treatments. GenStat (VSNI, 2017) was used to carry out all statistical tests. Microbial genes involved in N cycling will be quantified using qPCR. Cumulative values for N₂O fluxes were log transformed and analysed for probability of statistical significance with ANOVA.

## RESULTS AND DISCUSSION

Preliminary results showed a significant difference in total N₂O fluxes between NWFP farmlets (P=0.022). Preliminary soil N analysis results showed that soil TON content followed the same trend across NWFP farmlets but differences were observed between the experimental treatments. Full results of soil C, P and N content will be presented following complete analysis.

## CONCLUSION

Conclusions based on preliminary results are that the forage diet of grazing cattle have a significant influence on the GHG emissions and N cycling following dung and urine deposition. Further conclusions will be drawn following full analysis of the experimental results.

**Acknowledgements:** This work was supported as part of Rothamsted Research's Institute Strategic Programme – Soil to Nutrition (BBS/E/C/00010320) funded by the UK Biotechnology and Biological Sciences Research Council. The North Wyke Farm Platform is a UK National Capability supported by the Biotechnology and Biological Sciences Research Council (BBSRC BB/J004308/1).

## REFERENCES

Cardenas, L.M., Misselbrook, T.M., Hodgson, C., Donovan, N., Gilhespy, S., Smith, K.A., Dhanoa, M.S. and Chadwick, D., 2016. Effect of the application of cattle urine with or without the nitrification inhibitor DCD, on dung on greenhouse gas emissions from a UK grassland soil. *Agriculture, ecosystems & environment*, 235, pp.229-241.

Orr, R.J., Murray, P.J., Eyles, C.J., Blackwell, M.S.A., Cardenas, L.M., Collins, A.L., Dungait, J.A.J., Goulding, K.W.T., Griffith, B.A., Gurr, S.J. and Harris, P., 2016. The North Wyke farm platform: effect of temperate grassland farming systems on soil moisture contents, runoff and associated water quality dynamics. *European journal of soil science*, 67(4), pp.374-385.

VSN International (2017). GenStat For Windows 19th Edition. VSN International, Hemel Hempstead, UK. Web page: genstat.co.uk
# THE IMPACTS OF SOIL INCORPORATION OF CONVENTIONAL AND NOVEL ORGANIC FERTILIZERS ON N AVAILABILITY AND MICROBIAL PARAMETERS

GEBREMIKAEL, M.¹, RANASINGHE, A², SALEHI, P.¹, PIPAN, M³, DE NEVE, S.¹

¹ Ghent university, Belgium; ² University of Kelaniya, Sri Lanka; ³ Entomics Biosystems Ltd, UK

## INTRODUCTION

In addition to the conventional organic inputs into the soil, numerous new materials have become available during the last decades following novel agricultural industries, applications and processes. A rapid increase of such agricultural waste co-products and by-products (AWCB) is posing a threat to the environment. The need for sustainability and increasing agricultural productivity to feed the growing world population calls for treating, recycling and valorising AWCB in a sustainable way. **Agrocycle**, an EU funded project under H2020 research and innovation program, aims to provide mechanisms to achieve a 10% increase of the recycling and valorisation of agricultural waste by 2020, and to maximize the use of by-products and co-products via the creation of new sustainable value chains.

One of the mechanisms in this regard is the use of AWCB as a soil amendment and biofertilizer. Accordingly, we (together with other Agrocycle partners) develop and test the potential and sustainability of existing AWCB and new biofertilizers (BF) as soil organic matter amendment and N sources for crop production.

For a sustainable use of these organic materials, knowledge on their agricultural value and environmental risk is needed for farmers and policy makers. We conducted two incubation experiments using 11 conventional and novel organic fertilizers (OF) spanning a wide range of C:N ratio (6.5:1-60:1), consistency (liquid and solid) and sources (digestates, crop residues, phytoremediation plants and oil industry and food wastes). This abstract presents the N availability from the AWCB and BF, and shows an overview of impacts of these materials on microbial biomass and enzyme activity in the soil.

## MATERIAL AND METHODS

Soil samples were collected from 0-15cm of an organically managed maize field at Rumbeke, Belgium. After drying and sieving, samples were preincubated for 10 days, and filled into PVC tubes (h=10 cm, r=5 cm). Because of the large differences in C:N, it was not practical to apply a rate based on the same C or N content. The list of AWCB applied at field rates are: Anaerobic digestates of various feedstocks such as cattle manure only (AD_Man), AD from cattle slurry activated with crop residues (ActivatedAD), crop residues (AD_Crop), and pig slurry-crop residue mixture (AD_PigCrop). Other OF include: Composts from rice bran (RB compost), olive prunings (OliveW Compost), municipal wastes (MW Compost); Phytoremediation plant biomass (PhytoR), fresh crop residues of wheat (W-straw), sugar beet (SugarbtR), and a novel biofertilizer frass (insect poo and leftover collected during the larval growth of an insect on mixed food waste). Two separate incubation experiments were conducted at constant temperature and moisture content. Samples were destructively removed from each treatment at six sampling points during 120-147 days long incubation. Mineral N and other biological parameters including enzymes, microbial biomass C and PLFA were measured at each sampling time.

## RESULTS AND DISCUSSION

The percentage of N mineralized from the AWCB and BF varied over time (Figure 1). Based on the results of the incubation experiment, the AWCB and BF can generally be categorized into three groups in relation to their potential to N mineralization. The first group, particularly wheat straw, municipal waste compost and olive waste composts resulted in no net N mineralisation during the entire period of the incubation. The second group such as sugar beet residue and the general food waste frass did not result in net N mineralisation until 30 and 90 days
of the incubation, respectively. A significant percent of N ranging from 9% to 68% was mineralized from the remaining AWCB and BF during the entire period of the incubation.

<figure><img src="image_82.png" /><figcaption>Figure 1. The evolution of total mineral N expressed as % of total N added in the amendment.</figcaption></figure>

The application of these materials significantly enhanced biological quality parameters, particularly microbial biomass C and enzymatic activities (both dehydrogenase and betaglucosidase activities). AWCB and BF with relatively higher C:N ratio such as wheat straw, frass and the three composts (MWC, OliveWC, Ricebran compost) resulted in higher dehydrogenase enzyme activities compared to the one with lower C:N ratio such as digestates during the initial period of the experiment. The differences amongst the AWCB and BF became less pronounced towards the end of the incubation experiment suggesting that the impacts of these materials on intracellular biological soil quality parameters are only short-term.

## CONCLUSION

The data on the evolution of N mineralization-immobilization dynamics has important implication for synchronization of plant N need with the release of N from the AWCB and BF to maximize crop production. The data can also be used to categorize these materials as a soil amendment, immobilizing materials or biofertilizer. Frass has a potential to be used as biofertilizer and probably as immobilizing material in a similar way that catches crops are applied.

**Acknowledgements:** The materials investigated are provided by partners of the Agrocycle project (CREA-Italy; Demeter-Greece; AGRii-UK and INAGRO-Belgium. This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement N° 690142.
20th Nitrogen Workshop

Coupling C-N-P-S cycles

June 25-27, 2018

Le Couvent des Jacobins, Rennes – France

**Session III: Local process studies – Posters**
# SUBSTITUTION OF UREA BY CALCIUM AMMONIUM NITRATE IN A RAINFED SEMI-ARID CROP: EFFECT ON N OXIDES EMISSIONS, YIELD AND QUALITY

GUARDIA, G.<fnref n="1" />; SÁNCHEZ-MARTÍN, L.<fnref n="1" />; SANZ-COBENA, A.<fnref n="1" />; RODRÍGUEZ DE QUIJANO, M.<fnref n="1" />; FUERTES-MENDIZÁBAL, T.², GONZÁLEZ-MURUA, C.², VALLEJO, A.<fnref n="1" />

## INTRODUCTION

The use of calcium ammonium nitrate (CAN) instead of urea (U) can increase the emission of nitrous oxide (N₂O), a powerful greenhouse gas (IPCC, 2014), in areas with predominance of denitrification (e.g. grasslands) (Roche et al., 2016). However, contrasting results have been found in semi-arid arid cropping systems (e.g. Mediterranean) with low organic C contents in soil and predominance of nitrification, even under irrigated conditions (Guardia et al., 2017; 2018). This experiment aimed to assess the effect of CAN and U on N₂O and nitric oxide (NO) emissions, nitrogen (N) use efficiency (NUE), yield and breadmaking quality in a non-irrigated winter wheat crop (Triticum aestivum L.).

## MATERIAL AND METHODS

### Experimental design and management

A field experiment was set up in central Spain (latitude 40°25'N, longitude 3°29'W) in an alkaline silty loam soil with a 2.1% of oxidizable organic matter (Walkley-Black). A randomized complete block design with three replicates was established. The treatments were U, CAN (which were applied in at a rate of 120 kg N ha⁻<fnref n="1" />) and control (C) which did not receive synthetic N fertilization. The N fertilizers were applied at top-dressing (26th February, tillering stage).

### N oxides sampling and analyses

Gas samples were taken from February to July by the closed chamber technique and N₂O was analyzed by Gas Chromatography. Nitric oxide was measured using a gas flow-through system by using a closed chamber and a chemiluminescence detector (Guardia et al., 2017).

### N efficiency, yield and breadmaking quality

Wheat was harvested on 21st June with a research plot combine. Previously, the plants of one row were harvested to determine the total N content of grain and straw, which was analyzed using a TruMac CN Leco elemental analyzer. In addition, the SDS-sedimentation (SDSS) volume, which is highly correlated with water deformation energy and therefore with gluten strength, was determined as described by Callejo et al. (2016). Grain reserve proteins (gliadins and glutenins) were determined as described in Fuertes-Mendizábal et al. (2013). The NUE was calculated as the ratio between aboveground N uptake (subtracting that of C) and the amount of synthetic N applied. The N surplus was calculated as the N application minus the aboveground N uptake.

## RESULTS AND DISCUSSION

### N oxides emissions

Both N₂O and NO emissions peaked after N fertilization and were low from two months after fertilization to the end of wheat cropping cycle (and also before N fertilization). CAN decreased significantly both cumulative N₂O (by 58%, Fig. 1a) and NO (by 69%, Fig. 1b) emissions. Our results were in agreement with Guardia et al. (2018), who

<footnote n="1">¹ ETSI Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid (UPM), Spain, ² Department of Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Bilbao, Spain.</footnote>
found that most N₂O has been demonstrated to come from ammonium (NH₄⁺) oxidation rather from nitrate (NO₃⁻) reduction in a Mediterranean maize crop.

<figure><img src="image_83.png" /><figcaption>Figure 1. Cumulative N₂O (a) and NO (b) emissions from fertilization to harvesting. Different letters within columns indicate significant differences by applying the Least Significant Difference (LSD) test at P< 0.05. Vertical bars indicate standard errors.</figcaption></figure>

## Yield and breadmaking quality

We observed a positive response of wheat yield, protein content and SDSS volume to N fertilization. However, no significant differences were observed between U and CAN (which tended to increase grain N content and gluten strength), except for the gliadin to glutenin ratio. In comparison to U, CAN significantly increased NUE (by 29%) and decreased N surplus (by 49%).

## CONCLUSION

The substitution of U by CAN can be recommended to achieve an optimum balance between N oxides mitigation, yields, breadmaking quality and N efficiency in rainfed semi-arid cereal crops.

**Acknowledgements:** The authors are grateful to the Spanish Ministry of Economy and Innovation for their economic support through Project AGL2015-64582-C3-3-R (MINECO/FEDER). Funding for this research was also provided by EuroChem Agro GmbH.

## REFERENCES

- Callejo, M.J., Benavente, E., Ezpeleta, J.I., Laguna, M.J., Carrillo, J.M., Rodríguez-Quijano, M., 2016. Influence of teff variety and wheat flour strength on breadmaking properties of healthier teff-based breads. J. Cereal Sci., 68, 38-45.

- Fuertes-Mendizábal, T., González-Torralba, J., Arregui, L.M., González-Murua, C., González-Moro, M.B., Estavillo, J.M., 2013. Ammonium as sole N source improves grain quality in wheat. J. Sci. Food Agr. 93, 2162-2171.

- Guardia, G., Cangani, M.T., Andreu, G., Sanz-Cobena, A., García-Marco, S., Álvarez, J.M., Recio-Huetos, J., Vallejo, A., 2017. Effect of inhibitors and fertigation strategies on GHG emissions, NO fluxes and yield in irrigated maize. Field Crop. Res. 204, 135-145.

- Guardia, G., Vallejo, A., Cardenas, L.M., Dixon, E.R., García-Marco, S., 2018. Fate of 15N-labelled ammonium nitrate with or without the new nitrification inhibitor DMPSA in an irrigated maize crop. Soil Biol. Biochem. 116, 193-202.

- IPCC, 2014. Climate Change 2014: Synthesis Report. Contribution of Working Groups I, II and III to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change [Core Writing Team, R.K. Pachauri and L.A. Meyer (eds.)]. IPCC, Geneva, Switzerland, 151 pp.

- Roche, L., Forrestal, P.J., Lanigan, G.J., Richards, K.G.; Shaw, L.J., Wall, D.P., 2016. Impact of fertiliser nitrogen formulation, and N stabilisers on nitrous oxide emissions in spring barley. Agr. Ecosyst. Environ. 233, 229-237.
# NITROGEN AND PHOSPHOROUS AVAILABILITY OF ORGANIC FERTILIZERS- A GREENHOUSE STUDY

PALMBORG, C.<fnref n="1" />, HAHLIN A.-H.<fnref n="1" />

## INTRODUCTION

Nutrient availability of organic fertilizers is traditionally determined by analysis of inorganic N and soluble P. However digestates can often have a large fraction of nutrients bound in bacteria. This fraction can also be easily available to plants. We have made a greenhouse study where simultaneous comparisons with increasing amounts of inorganic nitrogen and phosphorous was done to quantify the availability to barley of N and P in the organic fertilizers.

## MATERIAL AND METHODS

A 50/50 volume % mix of fine quartz sand and vermiculite was used as a substrate. Some 3 liter pots were filled to two cm from the top. The fertilizer was mixed with the upper half of the substrate and twelve barley seeds of the variety *Severi* were sown in each pot at 4 cm depth. The pots were watered to 60% of the water holding capacity three times a week. Ammonium nitrate and superphosphate was given in quantities corresponding to 10, 40 and 80 kg N ha⁻<fnref n="1" /> and 1.9, 9.3 and 18.5 kg P ha⁻<fnref n="1" />. The organic fertilizers were; (i) liquid digestate (LD) from a dairy factory, (ii) solid granulates of digestate (SD) from a municipal sewage treatment plant and (iii) deep litter sheep manure that had either been stored indoors (MI) in the stable or (iv) outdoors (MO) in a heap during summer and autumn. The target amount of the organic fertilizers was 120 kg total N ha⁻<fnref n="1" /> and the actual dose of N and P is shown in Table 1. The liquid digestate was also applied in a lower dose corresponding to 28 kg N ha⁻<fnref n="1" /> and 7 kg P ha⁻<fnref n="1" /> complemented with 80 kg of N ha⁻<fnref n="1" /> as ammonium nitrate. A solution of K, Mg, S, Ca and micro nutrients was applied at the start of the experiment to all pots (modified from Antonini et al. (2012)). Each treatment was replicated in three pots that were placed on three different tables that changed position each week to ensure equal conditions for all plants. The temperature in the greenhouse was set to 15°C at night and 20°C at day and lamps with the light intensity of 170-200 micromoles m⁻² at table height were lit during 18 h per day when the natural light was not enough (<200 micromoles m⁻²). The logged temperature varied from 13.7 to 30.0°C with a mean of 20.6°C. After 93 days the plants were harvested, measured and separated into seeds and other aboveground biomass. The fertilizer equivalency of N was obtained by linear regression estimation of the amount of mineral fertilizer required to obtain the same aboveground biomass as the organic fertilizer.

<table><caption>Table 1. Dose of P given, C/N quotient and fertilizer equivalency of the organic fertilizers treatments.</caption><thead><tr><th>Organic fertilizer</th><th></th><th>Kg total N ha<sup>-1</sup></th><th>kg P ha<sup>-1</sup></th><th>g kg<sup>-1</sup> fertilizer equivalent of N</th></tr></thead><tbody><tr><td rowspan="3">Digestate</td><td>Liquid dairy waste <b>LD80</b></td><td>80</td><td>21</td><td>0.85</td></tr><tr><td>Liquid dairy waste <b>LD28N80</b></td><td>27</td><td>7</td><td>n.d.</td></tr><tr><td>Solid municipal sewage <b>SD127</b></td><td>127</td><td>79</td><td>0.22</td></tr><tr><td rowspan="2">Sheep manure</td><td>Indoor storage <b>MI116</b></td><td>116</td><td>20</td><td>0.17</td></tr><tr><td>Outdoor storage <b>MO126</b></td><td>126</td><td>25</td><td>0.09</td></tr></tbody></table>

## RESULTS AND DISCUSSION

The total dry matter yield of the different treatments is shown in figure 1. The yield of the mineral fertilizer treatments had a linear correlation to the N fertilization dose. However the **N80P9.3** had a significantly lower yield than the **N80P18.5**. This shows that there was some P-limitation to the growth when the P fertilization dose was halved, but the relation between P dose and yield was not linear. Both liquid and solid digestates had more available N than the inorganic N, while the sheep manures had a similar plant available N as the inorganic N (figure

<footnote n="1">¹ Department of Agricultural Research for Northern Sweden, Swedish University of Agricultural Sciences, Sweden</footnote>
1). The discrepancy between the amount of inorganic N in the fertilizer and the plant available N was largest for the liquid fertilizer from the dairy factory. The feedstock to this biogas plant was largely whey, a byproduct of cheese production rich in lactose. Thus the carbon source was easily degradable and the C/N quotient of the digestate was low. The availability of the P in the liquid digestate was approximately equivalent to superphosphate since the **LD28N80** that contained 7 kg of P had somewhat lower yield than the **N80P9.3**. The fertilizer equivalent value of N was higher in the digestates than the manures (Table 1).

<figure><img src="image_84.png" /></figure>

Figure 1. Yield of aboveground barley biomass in relation to the amount of inorganic N in the fertilizer. Treatments: **N10P1.9** 10 kg N in ammonium nitrate ha⁻¹ + 1.9 kg of P in superphosphate ha⁻¹. **N40P18.5** 40 kg N in ammonium nitrate ha⁻¹ + 18.5 kg of P in superphosphate ha⁻¹. **N80P9.3** 80 kg N in ammonium nitrate ha⁻¹ + 18.5 kg of P in superphosphate ha⁻¹. **LD80** Liquid digestate 80 kg total N ha⁻¹. **SD127** Solid digestate 120 kg total N ha⁻¹. **LD28N80** Liquid digestate 40 kg total N ha⁻¹ + ammonium nitrate 80 kg N ha⁻¹. **MO126** Sheep manure stored outdoor 120 kg total N ha⁻¹. **MI116** Sheep manure stored indoor 120 kg total N ha⁻¹. The linear regression for treatments used for determining of fertilizer equivalent of N is included as a dotted line. The calculation of the fertilizer equivalent of N is illustrated with dashed lines for **LD80**.

## CONCLUSION

The plant available N and P of organic fertilizers can be assessed simultaneously in greenhouse experiments, however the number of doses and range of the reference inorganic fertilisers need to be increased in order to quantify the fertilizer equivalency of P since the relationship between P dose and biomass yield was not linear.

**Acknowledgements:** This study was financed by Botnia Atlantica, Region Västerbotten, Regional Council of Ostrobothnia and the Swedish University of Agricultural Sciences (Ekoforsk).

## REFERENCES

Antonini S., Arias M., Eichert T., Clemens J., 2012. Greenhouse evaluation and environmental impact assessment of different urine-derived struvite fertilizers as phosphorus sources for plants. Chemosphere. 89, 1202-1210.
# IMPACT OF CONVENTIONAL TILLAGE AND NO-TILLAGE COVER CROPS ON NITROUS OXIDE EMISSIONS FROM VINEYARDS IN MEDITERRANEAN PORTUGAL

MARQUES, F.J.M.<fnref n="1" />, PEDROSO, V.², TRINDADE, H.³, PEREIRA, J.L.S.<fnref n="1" />

## INTRODUCTION

Vineyards are cropped with different soil management practices such as cultivation, intermittent herbicide application, mulching and cover cropping. Nowadays, several agronomic and environmental advantages have been appointed to vineyard cover cropping such as water management, vine performance and soil conservation (Longbottom and Petrie, 2015; Garcia et al., 2018).

The aim this study was to assess the effect of conventional tillage and no-tillage cover crops on nitrous oxide (N₂O) emissions from vineyards (Vitis vinifera L.) in Mediterranean Portugal.

## MATERIAL AND METHODS

A field study was carried out at the Dão Wine Research Station (latitude: 40.51ºN, longitude: -7.85ºW) located in central Portugal (Nelas, Portugal). The experiment was established in 2010 in a mature non-irrigated vineyard, planted with the Touriga Nacional red grape variety, and with a vine spacing of 1.1 m within and 2.0 m between rows. The experiment was a randomized block factorial design with three replications and four treatments. The following four treatments were considered in this study: (i) soil tillage (100 mm depth) of the inter-row (treatment: Till); (ii) soil tillage (100 mm depth) of the inter-row and application of mineral fertiliser (50 kg N ha⁻<fnref n="1" />) (treatment: Till+N); (iii) cover crop (permanent resident vegetation) in the inter-row (treatment: NoTill); (iv) cover crop (permanent resident vegetation) in the inter-row and application of mineral fertiliser (50 kg N ha⁻<fnref n="1" />) (treatment: NoTill+N). At Mars and June of each year, the soil tillage was performed with a cultivator mounted on a tractor whereas the resident vegetation was mowed with a brush cutter.

During two consecutive growing seasons of the grapevine crop (Mars to September of 2015 and 2016), the N₂O fluxes were measured by the closed chamber technique and analysed by gas chromatography using a GC-2014 (Shimadzu, Japan). Additional information could be found in Marques et al. (2018). Data collected were analysed by analysis of variance and least-squares deviation tests were used for comparison of means by the software Statistix 7.0.

## RESULTS AND DISCUSSION

The results showed that the average direct N₂O emission factor for vineyards managed with conventional soil tillage in the inter-row was 0.57±0.12% of nitrogen input and cover cropping by permanent resident vegetation in the inter-row reduces N₂O emission in 60% (0.23±0.29% of nitrogen input) (Table 1). Thus, the vineyard cover cropping was recommended as mitigation measure in order to reduce N₂O emissions.

The defaults direct N₂O emission factor currently recommended by Intergovernmental Panel on Climate Change (IPCC) used by the Portuguese inventory was a generic fertiliser induced emission factor for agricultural crops (1% of nitrogen input). Thus, this study suggests that the N₂O emissions from vineyards are currently potentially overestimated in the Portuguese inventory and recommends the use of a specific emission factor.

<footnote n="1">¹ Polytechnic Institute of Viseu, ESAV, Quinta da Alagoa, Viseu, Portugal; ² Direcção Regional de Agricultura e Pescas do Centro, CEVDão, Quinta da Cale, Nelas, Portugal; ³ University of Trás-os-Montes and Alto Douro, ECAV, Quinta de Prados, Vila Real, Portugal</footnote>
<table><caption>Table 1. Average direct nitrous oxide emission factors from the experiment (mean ± standard deviation).</caption><thead><tr><th>Treatment</th><th>N<sub>2</sub>O emissions<br>(kg N ha<sup>-1</sup> season<sup>-1</sup>)</th><th>N<sub>2</sub>O emission factor<br>(% of N input)</th></tr></thead><tbody><tr><td>Till</td><td>0.25±0.05<sup>b</sup></td><td></td></tr><tr><td>Till+N</td><td>0.53±0.11<sup>a</sup></td><td>0.57±0.12<sup>a</sup></td></tr><tr><td>NoTill</td><td>0.24±0.09<sup>b</sup></td><td></td></tr><tr><td>NoTill+N</td><td>0.35±0.07<sup>ab</sup></td><td>0.23±0.29<sup>b</sup></td></tr></tbody></table>

Values from the treatment are presented with different superscripts within columns, are significantly different (p < 0.05) by LSD test.

## CONCLUSION

The average direct N₂O emission factor for vineyards managed with conventional soil tillage in the inter-row was 0.57±0.12% of nitrogen input and cover cropping by permanent resident vegetation in the inter-row reduces N₂O emission by 60%.

**Acknowledgements:** This study was funded by project POCI-01-0247-FEDER-003430 AMONIAVE.

## REFERENCES

* Garcia, L., Cellete, F., Gary, C., Ripoche, A., Valdés-Gómez, H., Metay, A., 2018. Management of service crops for the provision of ecosystem services in vineyards: A review. Agriculture, Ecosystems and Environment 251, 158-170.

* Longbottom, M.L., Petrie, P.R., 2015. Role of vineyard practices in generating and mitigating greenhouse gas emissions. Australian Journal of Grape Wine Research 21, 522-536.

* Marques, F.J.M., Pedroso, V., Trindade, H., Pereira, J.L.S., 2018. Impact of vineyard cover cropping on carbon dioxide and nitrous oxide emissions in Portugal. Atmospheric Pollution Research 9, 105-111.
# AMMONIA AND GREENHOUSE GAS EMISSIONS FROM A BREEDING HEN BUILDING IN PORTUGAL

PEREIRA, J.L.S.<fnref n="1" />, FERREIRA, S.², CONDE, A.³, FERREIRA, P.³, PINHEIRO, V.², TRINDADE, H.²

## INTRODUCTION

Poultry production is a significant source of ammonia (NH₃), nitrous oxide (N₂O), carbon dioxide (CO₂) and methane (CH₄) emissions and few published data are available for Mediterranean countries. The aim of this study was to assess the NH₃, N₂O, CO₂ and CH₄ emissions on commercial breeding hen's buildings under Portuguese climate conditions.

## MATERIAL AND METHODS

The study was made in the commercial breeding hen farm Quinta da Cruz located in central Portugal (Soure, Portugal). The building (length = 80 m, width = 16 m) was equipped with automatic feeding and drinking systems (Roxell), evaporative cooling pads (CELdek) and a climate control system (F37, Fancom). Ventilation was made by minimum transitional tunnel ridge system being controlled with one differential pressure (Fancom), and two sensors of temperature (SF7, Fancom) and two sensors of relative humidity (RHM.17 for inside and RHO.17 for outside, Fancom) located indoor and outdoor the building. Additional information about the production practices and management are given in Table 1 and in Pereira et al. (2017).

The NH₃, N₂O, CO₂ and CH₄ concentrations at indoor building were measured continuously in two occasions, first between 16 May and 14 June 2017 (hot season), and second from 23 November to 20 December 2017 (cold season). The concentrations of these four gases were measured with a photoacoustic field gas monitor (INNOVA 1412, Lumasense Technologies) and air samples collected, in sequence (2 minute intervals), through 6 sampling points (2 points located outdoor and 4 points located indoor), by a multipoint sampler (INNOVA 1409, Lumasense Technologies). Sampling points were made using Teflon tubes (3 mm internal diameter) equipped with PTFE-filters (1 µm pore size, Whatman) to protect from dust. The climate data were collected (1 minute intervals) from the environmental control system of the building. The average gas concentrations were defined as the average of the hourly mean concentrations measured. All data obtained from the monitored building were analysed by Excel spreadsheet using descriptive statistics and t-test was used for comparison of means by the software Statistix 7.0.

## RESULTS AND DISCUSSION

The climate data as well as the gas concentrations and emissions are shown in Table 1. To obtain fertile eggs for meat production, the breeding of hens begin when they are 20 weeks old and end when they are about 60 weeks old. As can be observed in Table 1, the NH₃, N₂O, CO₂ and CH₄ emissions were significantly higher in hot season relative to cold season, being related with age and weight of the hens, manure moisture, manure built-up litter and indoor air temperature and ventilation rate (Méda et al., 2011; Lin et al., 2017; Pereira, 2017). The annual average NH₃ (0.52 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />) and CH₄ (0.092 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />) emission rates obtained in the present study are comparable with values compiled by Méda et al. (2011) (0.26-0.92 g NH₃ day⁻<fnref n="1" /> hen⁻<fnref n="1" /> and 0.080 g CH₄ day⁻<fnref n="1" /> hen⁻<fnref n="1" />). However, the average N₂O emission rate reported in this study (0.030 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />) was higher than values reported in Méda et al. (2011) (0.0022-0.0026 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />). The differences between the studies previously referred on N₂O emission rates might be related with temperature and diet, particularly the nitrate content of the diet. The annual average CO₂ emission rate of this study (169.6 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />) was higher than emission rate reported by Lin et al. (2017) in USA (89.9 g day⁻<fnref n="1" /> hen⁻<fnref n="1" />).

<footnote n="1">¹ Polytechnic Institute of Viseu, ESAV, Quinta da Alagoa, Viseu, Portugal; ² University of Trás-os-Montes and Alto Douro, ECAV, Quinta de Prados, Vila Real, Portugal; ³ LUSIAVES, Marinha das Ondas, Figueira da Foz, Portugal</footnote>
<table><caption>Table 1. Average climatic conditions, gas concentrations and cumulative emissions (mean ± standard deviation) in a commercial breeding hen building.</caption><thead><tr><th>Parameters</th><th></th><th>Hot season</th><th>Cold season</th></tr></thead><tbody><tr><td rowspan="5">Poultry husbandry</td><td>Breeder</td><td>Cobb</td><td>Cobb</td></tr><tr><td>Average number</td><td>7053</td><td>7186</td></tr><tr><td>Age (days)</td><td>326-355</td><td>150-174</td></tr><tr><td>Liveweight (kg)</td><td>3.8-3.9</td><td>2.5-2.9</td></tr><tr><td>Litter material (2 kg m<sup>-2</sup>)</td><td>New rice hulls</td><td>New rice hulls</td></tr><tr><td rowspan="5">Climatic conditions</td><td>Outdoor temperature (°C)</td><td>20.7±1.9<sup>a</sup></td><td>8.0±2.1<sup>b</sup></td></tr><tr><td>Indoor temperature (°C)</td><td>23.2±1.2<sup>a</sup></td><td>16.2±1.1<sup>b</sup></td></tr><tr><td>Outdoor relative humidity (%)</td><td>72.7±8.2<sup>a</sup></td><td>83.3±6.6<sup>b</sup></td></tr><tr><td>Indoor relative humidity (%)</td><td>77.0±6.7<sup>a</sup></td><td>91.0±4.0<sup>b</sup></td></tr><tr><td>Ventilation rate (m<sup>3</sup> h<sup>-1</sup> hen<sup>-1</sup>)</td><td>18.1±3.9<sup>a</sup></td><td>2.5±0.4<sup>b</sup></td></tr><tr><td rowspan="3">Gas measurements</td><td>Measurement campaign</td><td>16 May to 14 June 2017</td><td>23 Nov. to 20 Dec. 2017</td></tr><tr><td>Sampling interval</td><td>Every 2 minutes</td><td>Every 2 minutes</td></tr><tr><td>Sampling equipment</td><td>INNOVA 1409+1412</td><td>INNOVA 1409+1412</td></tr><tr><td rowspan="4">Gas concentrations</td><td>NH<sub>3</sub> (mg m<sup>-3</sup>)</td><td>3±1<sup>b</sup></td><td>9±6<sup>a</sup></td></tr><tr><td>N<sub>2</sub>O (mg m<sup>-3</sup>)</td><td>1±0<sup>a</sup></td><td>1±0<sup>a</sup></td></tr><tr><td>CO<sub>2</sub> (mg m<sup>-3</sup>)</td><td>1374±94<sup>b</sup></td><td>2886±802<sup>a</sup></td></tr><tr><td>CH<sub>4</sub> (mg m<sup>-3</sup>)</td><td>0.4±0.4<sup>a</sup></td><td>0±0<sup>a</sup></td></tr><tr><td rowspan="7">Gas emissions</td><td>NH<sub>3</sub> (g day<sup>-1</sup> hen<sup>-1</sup>)</td><td>0.709±0.167<sup>a</sup></td><td>0.321±0.278<sup>b</sup></td></tr><tr><td>NH<sub>3</sub> (g day<sup>-1</sup> LU<sup>-1</sup>)</td><td>54.557±12.850<sup>a</sup></td><td>24.684±21.421<sup>b</sup></td></tr><tr><td>N<sub>2</sub>O (g day<sup>-1</sup> hen<sup>-1</sup>)</td><td>0.060±0.013<sup>a</sup></td><td>0.001±0.001<sup>b</sup></td></tr><tr><td>N<sub>2</sub>O (g day<sup>-1</sup> LU<sup>-1</sup>)</td><td>4.586±0.994<sup>a</sup></td><td>0.044±0.094<sup>b</sup></td></tr><tr><td>CO<sub>2</sub> (g day<sup>-1</sup> hen<sup>-1</sup>)</td><td>209.3±22.0<sup>a</sup></td><td>129.8±35.7<sup>b</sup></td></tr><tr><td>CO<sub>2</sub> (g day<sup>-1</sup> LU<sup>-1</sup>)</td><td>16103.6±1694.5<sup>a</sup></td><td>9985.3±2746.5<sup>b</sup></td></tr><tr><td>CH<sub>4</sub> (g day<sup>-1</sup> hen<sup>-1</sup>)</td><td>0.185±0.182<sup>a</sup></td><td>0.000±0.000<sup>b</sup></td></tr><tr><td></td><td>CH<sub>4</sub> (g day<sup>-1</sup> LU<sup>-1</sup>)</td><td>14.210±14.021<sup>a</sup></td><td>0.000±0.000<sup>b</sup></td></tr></tbody></table>

Values presented with different superscripts within rows are significantly different (p < 0.05).

One hen = 0.013 LU (livestock unit) (REAP, 2013).

## CONCLUSION

The annual average emission rates from commercial breeding hen's buildings under Portuguese climate conditions were 0.52±0.27, 0.030±0.042, 169.6±56.2 and 0.092±0.131 g day<sup>-1</sup> hen<sup>-1</sup> for NH<sub>3</sub>, N<sub>2</sub>O, CO<sub>2</sub> and CH<sub>4</sub>, respectively.

**Acknowledgements:** This study was funded by project POCI-01-0247-FEDER-003430 AMONIAVE.

## REFERENCES

Lin, X., Zhang, R., Jiang, S., El-Mashad, H., Xin, H., 2017. Emissions of ammonia, carbon dioxide and particulate matter from cage-free layer houses in California. Atmospheric Environment 152, 246-255.

Méda, B., Hassouna, M., Aubert, C., Robin, P., Dourmad, J.Y., 2011. Influence of rearing conditions and manure management practices on ammonia and greenhouse gas emissions from poultry houses. World's Poultry Science Journal 67, 441-456.

Pereira, J.L.S., 2017. Assessment of ammonia and greenhouse gas emissions from broiler houses in Portugal. Atmospheric Pollution Research 8, 949-955.

Pereira, J.L.S., Ferreira, S., Garcia, C.S.P., Conde, A., Ferreira, P., Pinheiro, V., Trindade, H., 2017. Assessment of ammonia and carbon dioxide concentrations in a breeding hen building under Portuguese winter. International Journal of Food and Biosystems Engineering 5, 1-6.

REAP, 2013. Decree number 81/2013 of 14 June regarding animal manure management (REAP). In: Diário da República, Série I, number 113 of 14 June 2013. Portugal, pp. 3304-3329.
# RESIDUAL EFFECT AND NITROGEN USE EFFICIENCY OF N FERTILIZERS IN A MAIZE/WHEAT ROTATION

VALENTIN, F.¹, CALVO, M.², GABRIEL, J.L.³, ALONSO-AYUSO, M.⁴, QUEMADA, M.⁴

¹ FUNDESCAM, Spain; ² ITAGRA, Spain, ³ INIA, Spain, ⁴ Universidad Politécnica de Madrid, Spain

## INTRODUCTION

Nitrogen fertilizers may have a residual effect in the subsequent crops that might affect N use efficiency (NUE) in the cropping system, and the use of nitrification inhibitors might enhance this residual effect [1]. The objective of this work was to study the residual effect of synthetic N fertilizers and determine how it affects NUE in a maize/wheat crop rotation. To achieve this goal we studied a traditional fertilizer and a fertilizer with a nitrification inhibitor (DMPP) under field conditions in three different locations. As a complementary objective we try to elucidate the reasons behind the residual effect by conducting laboratory experiments to determine non-readily exchangeable ammonium and N in the soil microbial biomass.

## MATERIAL AND METHODS

Field experiments were conducted from April 2016 to July 2017 at three different locations in Spain: Barrax (Albacete), Aranjuez (Madrid), Villoldo (Palencia). The crop rotation was maize/wheat and the experimental design consisted on three treatments for maize (control, fertilization with ammonium sulphate nitrate (ASN), and fertilization ASN blended with the nitrification inhibitor DMPP (3,4dimethylpyrazole phosphate (ENTEC®)), that were split in to three wheat treatments (control, ASN at recommended rate, ASN at reduced rate). Each treatment had four replications randomly distributed. The climate was Mediterranean (temperate, semi-arid and cold for each location) and the crops were irrigated. The soils had different textures (Silt loam, Clay loam, Sandy clay loam) and pH (7.9, 8.0, 6.5).

The grain yield and N grain content were assessed at harvest. The soil inorganic N content was determined in the top 40 cm of soil before sowing maize and wheat. Two components of the N use efficiency were calculated: the agronomic efficiency (ANE) that refers to the kg of grain yield increase obtained per kg of N applied, and the N use efficiency that is the ratio between the N inputs and N outputs.

Between maize harvest and wheat sowing, soil samples were taken to determine the N mineralization potential by aerobic incubation, the C and N content of microbial biomass by fumigation, and the non-exchangeable $NH_4^+$ by the potassium hypobromite dry soil combustion method.

## RESULTS AND DISCUSSION

Maize yield, grain N content and crop N uptake were lower for the control than for the fertilized treatments in the three locations, and no differences in these variables were found between ASN and ENTEC® treatments. The maximum yield of the experiments ranged from 18.4 in Barrax to 8.3 $Mg ha^{-1}$ in Villoldo, showing the difference productivity of the locations. After maize harvest, there were not significant differences in soil inorganic N content in the upper 40 cm layer between treatments in any of the locations, and Aranjuez and Villoldo had higher soil inorganic N content ($\approx 60 \text{ kg N ha}^{-1}$) than Barrax ($\approx 15 \text{ kg N ha}^{-1}$).

In Aranjuez and Villoldo grain yield in control plots (non fertilized wheat) was higher in the treatments of previous fertilized maize (ASN and ENTEC®) than in the control (Fig. 1A). In Barrax, ASN plots achieved a greater wheat yield than the control, being ENTEC® in between. In addition, the grain N content in control plots in Aranjuez and Villoldo was higher in the treatments of previous fertilized maize (ASN and ENTEC®) than in the control (Fig. 1B). In Barrax, no differences between treatments were observed. These results show a residual effect of both
fertilizers, ASN and ENTEC®, in Aranjuez and Villoldo but not a clear residual effect in Barrax. Even more, in Aranjuez the residual effect of ENTEC® was larger than in ASN as was already reported [1].

The impact on wheat ANE was observed in Aranjuez and Villoldo. In Aranjuez, ANE values in control plots where higher than in ENTEC®-low rate treatment. In Villoldo, ANE in control-low rate treatment was more efficient than in ASN-recommended rate, showing ASN residual effect. In Aranjuez, NUE>1 was observed for the wheat plots following the ASN and ENTEC® treatments, suggesting that maize residual N was uptake by the wheat.

<figure><img src="image_85.png" /><figcaption>Figure 1. Wheat yield (A) and grain N content (B) in control plots of 2017, that received either 0, ASN or ENTEC® in 2016. Different letters meaning significant differences in each location (Duncan 95%).</figcaption></figure>

In the two locations that had a clear N residual effect related to the fertilized treatments there was a tendency to increase C and N retained in the microbial biomass after application of ASN or ENTEC®, suggesting that fertilized treatments enhanced C and N retention in the organic pool. Soils with high smectites content and low available potassium had a high capacity for NH₄⁺ clay fixation. Nevertheless, the layers with a higher fixation capacity appeared only in Aranjuez and below 0.4 m in the soil profile; therefore, NH₄⁺ fixation in clay layers do not seem to be responsible for the residual effect observed.

## CONCLUSIONS

In a maize/wheat crop rotation, the residual N effect of fertilizers was clearly observed in two locations (Aranjuez and Villoldo) and suggested in the third (Barrax). The residual effect occurred as a result of N fertilizer application in the previous, either ASN or ENTEC®. The soil inorganic N presented in the fertilized plots before planting wheat was high and masked the residual N effect or possible differences between fertilized treatments.

In the two locations that had a clear N residual effect related to the fertilized treatments there was a tendency to increase C and N retained in the microbial biomass, suggesting that fertilized treatments enhanced C and N retention in the organic pool.

**Acknowledgements:** EuroChem Agro, Spanish Ministry (AGL201452310R), Comunidad Madrid (S2013/ABI2717). The funders had no role in the data collection and analysis or preparation of the manuscript.

## REFERENCES

Alonso-Ayuso M., Gabriel J.L., Quemada M., 2016. Nitrogen use efficiency and residual effect of fertilizers with nitrification inhibitors. Eur. J. Agron. 80: 1-8.
# LONG-TERM CONSEQUENCES OF UNBALANCED FERTILIZATION WITH NITROGEN AND PHOSPHORUS

RUTKOWSKA, A.

Institute of Soil Science and Plant Cultivation – State Research Institute in Puławy, Poland

## INTRODUCTION

Natural farming conditions in Poland are poor, due to prevalence of light sand-derived soils and, unfavourable climate. Soil acidity and related low content of available forms of macronutrients are the most limiting factors for crop production. However, nitrogen (N) remains most limiting factor, and application dose are currently increasing. The average fertilizer application dose is 140 kg·ha⁻¹ with a N Phosphorus (P) K (Potassium) ratio of 1: 0,3: 0,3. Considering that over 30% of Polish soils have low to very low content of available phosphorus, this practice may result in a considerable soil mining of soil P, and then, disturbance in physiological processes in plants. Long term depletion of the available P pool through crop uptake may modify the P pools in soils, by a most important fixation of soluble P after P fertilization. Thus, most of soluble P applied to the soil will not be practically utilized by crops in the first vegetation period (Sharpley, 1995, Jianbo et al., 2011). The aim of the investigation was to evaluate the long-term consequences of soil phosphorus mining while nitrogen fertilization rates are increasing.

## MATERIAL AND METHODS

Field experiments were carried out at two experimental stations of the Institute of Soil Science and Plant Cultivation in 2003-2014, at Grabów and at Baborówko. Four crops were grown each year in the rotation of winter oilseed rape - winter wheat - maize - spring barley. The first factor was P fertilization in two level doses (+ and -), and the second one were six levels of N doses. The initial content of available phosphorus in Grabów was 16 and in Baborówko 32 mg P₂O₅·100 g⁻¹ soil. Total N rates in the experiments over 12 years were: 0, 510, 1020, 1530, 2040, 2550 kg N·ha⁻¹, and phosphorus rates: 405 kg P·ha⁻¹ in P plus and 0 kg P·ha⁻¹ in P minus treatment. After harvest of barley, soil was sampled from the ploughed layer and analyzed for P₂O₅ content by Egner-Riehm DL method. The samples of grain and straw were collected at full maturity each year and analysed for the content of N and P. Statistical processing of the results was performed using the Statgraphics 5 Plus package (Statgraphics Plus, Rockville, USA). The Tukey's test has been applied to evaluate the significance of differences between the treatments. In the paper, consequences of twelve years soil phosphorus mining are presented. Yields of crops were considered as the productive effect of unsustainable N and P fertilization, and the concentration of available P forms in soil as well as balances of N and P as the environmental effects.

## RESULTS AND DISCUSSION

In the both experimental sites, twelve years without phosphorus fertilization caused only slight crop reduction of all the crops, and the differences between P plus and P minus treatments were not statistically significant. Yields were variable in the years and strongly determined by weather conditions. The average yields were as follows: oilseed rape 2,99 in P plus treatment, and 2,91 in P minus, wheat – 4,95 and 4,71, maize 7,08 and 6,90, barley – 3,80 and 3,64 t·ha⁻¹ in Grabów. In Baborówko, oilseed rape yielded on the level 2,17 in P plus treatment and 2,13 in P minus treatment, wheat – 4,21 and 4,18, maize 6,91 and 6,85, barley – 3,09 and 3,03 t·ha⁻¹. Fertilization with phosphorus increased the content of this element, mainly in straw, hence, the total uptake of P by crops. Phosphorus balance was influenced both by P and N fertilization. In the treatment P plus, the surplus of phosphorus was obtained in the whole range of nitrogen rates. In P minus treatment, the balance was the more negative, the higher nitrogen dose. In the both sites, nitrogen rates N4-N5 exclusively ensured the surplus of nitrogen through the whole 12-years period of the experiment (table 1).
<table><caption>Table 1. Nitrogen and phosphorus balances for 12 years of the experiment in Grabów and Baborówko</caption><thead><tr><th rowspan="3">N rate<br>kg · ha<sup>-1</sup></th><th colspan="4">Grabów</th><th colspan="4">Baborówko</th></tr><tr><th colspan="2">N balance</th><th colspan="2">P balance</th><th colspan="2">N balance</th><th colspan="2">P balance</th></tr><tr><th>P<br>Plus</th><th>P<br>Minus</th><th>P<br>Plus</th><th>P<br>Minus</th><th>P<br>Plus</th><th>P<br>Minus</th><th>P<br>Plus</th><th>P<br>Minus</th></tr></thead><tbody><tr><td>N0 0</td><td>-853</td><td>-830</td><td>196</td><td>-163</td><td>-530</td><td>-545</td><td>266</td><td>-103</td></tr><tr><td>N1 510</td><td>-590</td><td>-587</td><td>167</td><td>-194</td><td>-330</td><td>-353</td><td>220</td><td>-153</td></tr><tr><td>N2 1020</td><td>-496</td><td>-440</td><td>133</td><td>-225</td><td>-288</td><td>-219</td><td>151</td><td>-197</td></tr><tr><td>N3 1530</td><td>-276</td><td>-148</td><td>119</td><td>-234</td><td>-55</td><td>-32</td><td>123</td><td>-230</td></tr><tr><td>N4 2040</td><td>48</td><td>118</td><td>116</td><td>-239</td><td>288</td><td>310</td><td>104</td><td>-252</td></tr><tr><td>N5 2550</td><td>401</td><td>468</td><td>105</td><td>-247</td><td>620</td><td>654</td><td>87</td><td>-273</td></tr><tr><td>Average</td><td>-294</td><td>-236</td><td>139</td><td>-217</td><td>-49</td><td>-20</td><td>159</td><td>-201</td></tr></tbody></table>

Soil depletion from phosphorus caused the significant changes of its available forms through the 12 years. In the both sites, the decrease of available P₂O₅ was observed between the three rotations of the experiments. In Grabów, the content of P available dropped to 13,2, and in Baborówko to 27,2 mg P·100 g⁻¹ soil, but under the highest nitrogen rate to 11 and 23,2 mg P·100 g⁻¹ soil, respectively (Figure.1)

<figure><img src="image_86.png" /><figcaption>Figure 1. The average content of P2O5 in the plough layer of soil in experimental sites in the following crop rotations</figcaption></figure>

Nevertheless, the average final concentration of P₂O₅ in Grabów was classified as the medium content in Polish soils (recommended), and in Baborówko as the high content (for the environmental reason careful P fertilization recommended). According to fertilizer recommendation in Poland, the nutrient within medium and high soil fertility categories is considered as an adequate and probably not limiting crop growth.

## CONCLUSION

Twelve years without phosphorus applications to soil with medium and high P content did not affect significantly grain yields but strongly reduced concentration of P available in the plough layer. The negative balance of phosphorus in both experimental sites was enhanced by increasing nitrogen fertilization rates.
REFERENCES

Jianbo S., Lixing Y., Junling Z., Hainang L., Zhaohai B., Xinping Ch., Weifeng Z., Fusou Z., 2011. Phosphorus dynamics: from soil to plant. Plant Physiol., 156, 997-1005.

Sharpley A., N., 1995. Soil phosphorus dynamics: agronomic and environment impact. Ecol. Engin. 5, 261-279.
# NITROGEN BALANCE AND CROP NITROGEN UPTAKE IN LONG-TERM LYSIMETRIC INVESTIGATION

MIROSLAV F., SMATANOVÁ M., HYNŠT J., NĚMEC P.

Central Institute of Supervising and Testing in Agriculture, Hroznová 2, 656 06 Brno, Czech Republic

## INTRODUCTION

Many studies report the effect of different rates of N addition on crop yield and leaching of excessive mineral N. However, the effect of annual variation of N inputs and N balance in crop sequence is seldom studied. At steady rate of N fertilization, annual rates usually varies according to supposed crop demands. When annual input is applied, it does not always correspond with plant N uptake. Consequently, low N should decrease plant N uptake, mineral N accumulation in soil and losses of added N, while excessive N should increase mineral N accumulation in soil and losses of added N. Information about the short-term or long-term effect of varying annual N balance in crop sequence is limited. Short-term and long-term effects of N addition on N balance, N pools and N fluxes were studied in 20-year field investigation using lysimeters at 7 locations. Pools and fluxes of mineral N were selected as easily measurable and sensitive indicator of changes of N transformations in soil. We hypothesized that the amount of N added to soil or N lost from soil induces equivalent change of plant uptake, soil mineral N accumulation and N losses.

We also hypothesized that long-term deficit decreases and surplus increases soil Nmin, yield and losses with time.

## MATERIAL AND METHODS

Lysimetric measurements were performed at 12 locations of Central Institute of Supervising and testing in Agriculture in the Czech Republic. They were established in 1985 and to this date, 30 years of measurements were performed. For the purpose of current study, results of 20 years (1996 – 2016) of measurements at 7 locations were evaluated. At each location, lysimeters were set up at field sites with crop sequence containing cereals, legumes and row crops. Annual rate of fertilization was adjusted according to supposed demands of crops.

In each lysimeter, collectors were inserted into the wall of working hole. The structure of soil above the collector was not disturbed. Rainfall water percolates through soil profile into collector and it is drained into plastic bottle where it accumulates. Bottles are situated in the pit that is separated by wooden board from the rest of the working hole. Working hole outside of the pit was filled with soil after finishing of lysimeter construction. The amount of water in bottles was regularly determined and samples of water were collected from three depths (0-30, 30-60, 60-80 cm). Soil samples from soil layers 0 – 30 and 30 – 60 for mineral N measurements were collected in two terms during the year - in spring and after harvest. Yield of harvested products was determined and content of N was determined in plant material. Soil mineral N (Nmin) in 0 – 60 cm soil layer in spring and after harvest, leaching below 80 cm and plant N uptake were evaluated. Soil mineral N content was calculated as sum of NH4+ and NO3- content in soil. Plant N uptake was calculated from yield and N content of harvested products.

Soil mineral nitrogen was quantified colorimetrically according to Zbíral et al., (2011).

Biomass of plants was air dried and ground and dry matter yield was estimated. Nitrogen in plant biomass was estimated as Kjehldahl nitrogen (Zbíral et al., 2014).

Statistical analyses were performed using Statistica 12 software. Correlations between measured parameters were calculated using Pearson's correlation coefficient.
## RESULTS AND DISCUSSION

At the outset we expected that increasing rate of annual N inputs or N balance increases $N_{min}$ in soil, crop N uptake and leaching. We also supposed that long-term deficit decreases and surplus increases soil $N_{min}$, N uptake and losses with time. These assumptions were only partially supported by obtained data. Increasing rate of fertilization increased crop N uptake. At the same time, effect of N inputs on $N_{min}$ in soil and N leaching was not observed. Mean N balance was negative at most of studied locations. Effect of N balance on N uptake and $N_{min}$ in soil was insignificant and long-term changes of measured parameters were limited. Results therefore confirmed hypothesis, that increasing N input increased crop N uptake. However, effect of N input on $NO_3^-$ leaching and soil $N_{min}$ concentration was insignificant. Negative annual N balance was observed at most locations, but it was without effect on $N_{min}$ and leaching of $NO_3^-$.

Despite negative N balance soil sustains crop productivity and N availability at almost constant level and long-term changes were limited. Decline or accumulation of soil N were low and soil has still capacity to store and release nutrients for crop growth despite varying rate of N inputs and N losses. Level of soil $N_{min}$, losses and yield are maintained from soil reserves for relatively long time without significant changes. Possible sources of missing N may be undetected atmospheric inputs (Karimi et al., 2017), underestimated $N_2$ fixation or uptake of soil N from deeper soil layers. Stability of mineral N pools and fluxes may be also partially attributed to organic fertilization and planting of legumes at studied locations. Legumes not only provide additional N, but they also access deep leached $NO_3^-$ below 80 cm. It also confirms sustainability of cropping systems used in the experiment.

## CONCLUSION

Increasing rate of N input increased crop N uptake, but the rate of input and annual N balance had marginal effect on $NO_3^-$ leaching and soil $N_{min}$ concentration. Despite negative N balance soil sustains crop productivity and N availability at almost constant level and long-term changes were limited. Results suggest that not estimated sources of N helps to keep sustainability of studied cropping systems.

## REFERENCES

* Karimi, R., Janzen, H. H., Smith, B. H., Ellert, B. H., Kröbel, R., 2017. Nitrogen balance in century-old wheat experiment. Canadian Journal of Soil Science, in press.

* Zbíral, J., Čižmárová, E., Dočkalová, R., Fojtlová, E., Hájková, H., Holcová, H., Kabátová, N., Niedobová, E., Rychlý, M., Staňková, K., Urbánková, E., Vilamová, V., Žalmanová, A., 2014. Plant analysis. Czech Central Institute for Supervising and Testing in Agriculture, Brno, 120 pp. (in Czech).

* Zbíral, J., Malý, S., Váňa, M., Čuhel, J., Fojtlová, E., Čižmár, D., Žalmanová, A., Srnková, J., Obdržálková, E., 2011. Soil analyses, Part III. Czech Central Institute for Supervising and Testing in Agriculture, Brno, 253 pp. (in Czech)
# THE EFFECT OF NITROGEN APPLICATION METHODS ON MAIZE (ZEA MAYS L.) YIELD

SZÉLES, A.<fnref n="1" />, HORVÁTH, É.<fnref n="1" />, KITH, K.<fnref n="1" />, RAGÁN, P.<fnref n="1" />

## INTRODUCTION

The Member States of the European Union produced 62,677 thousand tons of maize in 2016. Within the EU, Hungary belongs to the most significant maize producers with its 8,730 tons of production, ranked as the 3rd highest amount (FAO, 2017). However, average yield is highly variable. Extremities caused by climate change pose a serious problem. For this reason, technological developments are needed to achieve yield increase despite the constantly changing weather conditions.

Maize yield is greatly affected by N fertilisation (Nagy, 2008; Ványiné et al., 2012). The accurate determination of nitrogen quantity and its application are of key significance from the aspect of yield, quality and the environment (Edmonds et al., 2013). The aim of this research is to examine the effect of nitrogen fertilisation management on yield.

## MATERIAL AND METHODS

Measurements were conducted at the Experiment Site of the University of Debrecen, Hungary, in an experiment established on mid-heavy calcareous chernozem soil with deep humus layers in 2011, 2012 and 2013. The experiment had a split-split-plot design and two replications. The main plots represented the different irrigation treatment (irrigated and non-irrigated), while the split plots represent fertiliser doses and hybrids. This study focuses on the examination of the non-irrigated plots of the FAO 410 hybrid.

During the growing season the mean temperature of the research area was 18.2, 18.8 and 22.8°C in 2011, 2012 and 2013, respectively. The amount of precipitation was 324, 276 and 252 mm in the three consecutive years, respectively. Based on the temperature and precipitation in the examined period and comparing them to the multiple-year average, 2011 was around the average, while the temperature in 2012 was 1.5 °C warmer and the amount of rainfall was 64 mm lower. 2013 was the driest (-88 mm) and warmest (+5.5°C) of the examined years.

In the experiment, 60 and 120 kg N ha⁻<fnref n="1" /> basic fertiliser dose was applied in addition to the non-fertilised control treatment (technology 1). This dose was applied as ammonium nitrate in all three treatments in the spring, one month before sowing. In addition to the second and third fertiliser level of all treatments (except for control plots), 30 kg N ha⁻<fnref n="1" /> was applied at the V6 growth stage (technology 2) and further 30 kg N ha⁻<fnref n="1" /> was applied at the V12 phenophase (technology 3) in the third treatment. The final fertiliser level was 0, 90 and 150 kg N ha⁻<fnref n="1" /> in the second treatment and 0, 120 and 180 kg N ha⁻<fnref n="1" /> in the third treatment.

GLM was used to demonstrate the effect of treatments on yield. To compare the mean values of the applied treatments, LSD₅% was determined. During multiple comparison, confidence intervals were corrected using Duncan's test to avoid the accumulation of alpha error. Evaluation was performed using SPSS for Windows 21.0.

## RESULTS AND DISCUSSION

Based on the statistical evaluation, there was no significant difference between yields as a result of N fertiliser treatments before sowing (A_60 and A_120) in 2011. The highest yield (14.171 t ha⁻<fnref n="1" />) resulted from applying 120 kg N ha⁻<fnref n="1" /> basic fertilisation plus 30 kg N ha⁻<fnref n="1" /> at the V6 phenophase (V6_150). The V12_180 treatment caused a significant (P<0.05) yield decrease of 1529 kg ha⁻<fnref n="1" /> compared to the V6_150 treatment. The average yield of non-

<footnote n="1">¹University of Debrecen, Faculty of Agricultural and Food Sciences and Environmental Management, Institute for Land Utilisation, Regional Development and Technology, Hungary</footnote>
fertilised plots was 12.239 t ha⁻¹ in 2012. On the contrary, the A_60 basic fertilisation resulted in a surplus of 1730 kg ha⁻¹, which was exceeded by that of treatments A_120 and V6_90, but this increase is not statistically significant. The most effective treatment was shown to be V6_150 (15.849 t ha⁻¹) (P<0.05. In 2013, the yield of the non-fertilised treatment was significantly (P<0.05) lower by 2325 kg ha⁻¹ than the yield of the lowest N dose applied as basic fertilisation (A_60). As a result of further increasing the N dose by 60 kg ha⁻¹ as basic fertilisation, the obtained yield significantly increased by 1721 kg ha⁻¹. The 30 kg N ha⁻¹ added to the A_60 N ha⁻¹ basic fertilisation at the V6 phenophase (V6_90) had a similar result to the yield of the A_60 treatment and the difference between the A_120 and V6_150 treatments did not cause any significant difference either. The highest yield was provided by the V6_150 treatment (13.090 t ha⁻¹), which showed a difference from the basic fertilisation and the V6_90 treatment at a significance level of 5%. However, the extra 30 kg N ha⁻¹ applied at the V12 phase was not successful.

The yearly technological evaluation showed that in 2011, technology 2 resulted in the highest yield (13.722 t ha⁻¹). This technology increased the yield of technology 3 by 8% (P<0.05), and it did not show significant difference from technology 1 (13.352 t ha⁻¹). There was no significant difference between each technology in 2012 and 2013.

The crop year effect evaluation showed that the use of technology 1 and 2 resulted in similar yields in 2011 and 2012, the environmental factors did not have any effect. However, this effect was significant (P<0.05) in 2013, resulting in a 20% decrease in comparison with 2012 and 11.5% compared to 2011. Technology 3 was the most successful in 2012, resulting in a 16.3% yield surplus compared to 2011 and 19.4% extra yield in comparison with 2013.

## CONCLUSION

From the aspect of the development of maize, the distribution of rainfall during the growing season was a more significant factor than the quantity of precipitation. This finding is also demonstrated by the fact that, of the three examined years, the main average yield was the highest (13.947 t ha⁻¹) in 2012, when the 276 mm rainfall during the growing season had a favourable distribution, thereby improving nutrient availability. In all three years, spring basic fertilisation improved maize development even on chernozem soil, which is properly supplied with nutrients. Although the effect of top-dressing was not shown in yield increase, it reduced the stress caused by environmental factors. The success of maize production can be favourably affected with nutrient supply adapted to the various phenophases of the crop.

**Acknowledgements:** The research was financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the 4.thematic programme of the University of Debrecen, and the projects "GINOP-2.2.1-15-2016-00001 - Developing a scale-independent complex precision consultancy system" and "EFOP-3.6.3-VEKOP-16-2017-00008".

## REFERENCES

Edmonds D.E., Tubaña B.S., Kelly J.P, Crain J.L, Edmonds M.D., Solie J.B., Taylor R.K., Raun W.R., 2013. Maize grain yield response to variable row nitrogen fertilization. J. Plant Nutr., 36.7: 1013-24.

FAO, 2017. http://www.fao.org/faostat/en/#data

Nagy J., 2008. Maize production: Food, bioenergy, forage. Akadémiai Kiadó, Budapest.

Ványiné Széles A., Megyes A., Nagy J., 2012. Irrigation and nitrogen effects on the leaf chlorophyll content and grain yield of maize in different crop years. Agr. Water Manage., 107: 133-144
AGENT BASED MODELLING OF SHEEP MOVEMENT AND URINE DEPOSITION TO SUPPORT N₂O EMISSION ESTIMATES IN UPLAND PASTURES

JOHNSON, S.C.M.<fnref n="1" />, WHELAN, M.J.<fnref n="1" />, BALZTER, H. <fnref n="1" />, LUSH, L.², KING, A.J.², TUCNY, E. ², WILSON, R.P. ², PERROTO-BALDIVIESO, H.L.³, CARDENAS, L.⁴, CHARTERIS, A.F.⁴, MARSDEN, K.A.⁵, HARRIS, I.⁵, HOLMBERG, J.⁵ CHADWICK D. ⁵

INTRODUCTION

Livestock such as sheep make important contributions to N₂O emissions via deposition of nutrients in urine [van Groenigen et al., 2005]. However, to date, most estimates of N₂O emissions from grazed pastures have been derived from lowland soils. Relatively little is known about the response of N₂O fluxes to sheep urine deposition in the uplands. N₂O emissions depend on NO₃ availability, redox conditions (principally driven by moisture content), temperature, microbial community composition (e.g. the presence and activity of nitrifiers and denitrifiers), and the availability of reduced carbon substrates. To understand the response of emissions to these factors and the influence of urine deposition, it is important to examine the spatial and temporal pattern of sheep movement, occupancy and urine deposition across heterogeneous landscapes. These patterns are expected to show diurnal patterns and be influenced by, for example, topography, vegetation, conspecific behaviour, and individuals' energy state. Here, we present an agent-based model of sheep movement and compare our model output to high resolution behavioural and position data (GPS and inertial sensors) collected from a flock of sheep in an upland pasture in North Wales. We expect extensions of this model to support the quantification of the spatial and temporal distribution of urine-associated N₂O fluxes across upland landscape.

MATERIAL AND METHODS

Agent based modelling is a form of complex systems modelling, based on individual autonomous “agents”, each with its own properties and actions. Such models have the advantage that they are conceptually simple to understand but can be used to represent the complex interactions between multiple agents in the system. A common output is the prediction of emergent whole-system behaviour, where novel and coherent structures or patterns arise from interactions between the agents. For this project, the NetLogo modelling platform was selected (Wilensky et al., 2015) to model the movement of individual sheep across a landscape. Slope data were derived from freely available airborne LiDAR datasets and a spatial layer of normalised difference vegetation index (NDVI) was derived from the RapidEye satellite system which was used as a proxy for vegetation characteristics. Sheep movement and behaviour was informed by (1) information from the literature, and (2) data collected from a flock of n=30 Welsh Mountain ewes (*Ovis aries*) over 2x30 day periods whilst grazing a semi-improved upland pasture at Henfaes Agricultural Research Station, North Wales. Sheep positional data were gathered using collar-mounted GPS (Fehlmann et al. 2017) and behavioural data derived from rear-mounted acceleration sensors (Wilson et al., 2008).

In addition to generating the statistics of occupancy and urination for a single test site, the model can be applied to predict sheep movement and associated total N₂O emissions (calibrated using experimental data on upland soil responses to sheep urine addition being collected in this project) in a wider landscape with similar characteristics.

RESULTS AND DISCUSSION

Initial comparisons of our agent-based model and empirical data show high agreement. Sheep space use was highly variable across the landscape, showing occupancy "hot spots" resulting from sheep preferences for certain vegetation and topography linked to resting and grazing. Sheep space use also altered with season, in line with changes in the type and availability of vegetation. A comparison of sheep space use as predicted from the agent-based model and our observed empirical dataset is provided in Figure 1.

<footnote n="1">¹ University of Leicester, UK; ² Swansea University, UK; ³ Texas A&M University, USA, ⁴ Rothamsted Research, UK, ⁵ Bangor University, UK</footnote>
Urination events in our empirical dataset were not simply determined by patterns of space use (above), but instead showed distinct hot spots where urination occurred more frequently.

<figure><img src="image_87.png" /><figcaption>Figure 1. Modelled spatial locations of sheep (left) vs GPS derived locations of sheep (right).</figcaption></figure>

## CONCLUSION

Given the agreement between our model and empirical data, this work presents a critical first step towards being able to accurately model the movement and urine deposition of sheep in upland environments. Application of the model over a larger upland area and implications for estimating whole landscape N₂O emission enhancement as a result of urine deposition will be presented.

**Acknowledgements:** This work was funded by the UK Natural Environment Research Council (grant NE/M013154/1). RapidEye data were supplied by Planet Team (2017). Planet Application Program Interface: In Space for Life on Earth. San Francisco, CA. LiDAR data was supplied by Natural Resources Wales.

## REFERENCES

Fehlmann, G., O'Riain, M. J., Hopkins, P. W., O'Sullivan, J., Holton, M. D., Shepard, E. L. C. King, A. J., 2017 Identification of behaviours from accelerometer data in a wild social primate. *Animal Biotelemetry* 5:6.

van Groenigen, J.W., Kuikman, P.J., De Groot, W.J.M., Velthof, G.L. 2005. Nitrous oxide emission from urine-treated soil as influenced by urine composition and soil physical conditions. Soil Biol. & Biochem. 37 463-473.

Wilensky, U., Rand, W. 2015. An introduction to agent-based modeling: Modeling natural, social and engineered complex systems with NetLogo. Cambridge, MA: MIT Press.

Wilson, R.P., Shepard, E.L.C., Liebsch, N. 2008. Prying into the intimate details of animal lives: Use of a daily diary on animals. Endangered Species Research 4, 123-137.
# COMPARISON BETWEEN DIFFERENT STABILIZED NITROGEN FERTILIZERS IN MAIZE CROP

MATEO-MARÍN, N., QUÍLEZ, D., GUILLÉN, M., ISLA, R.

Agrifood Research and Technology Centre of Aragón (CITA), Aragon Government. Av. Montañana 930, 50059 Zaragoza, Spain

## INTRODUCTION

The use of fertilizers with nitrification and urease inhibitors aims to increase the lifetime of ammonium and urea in the soil, respectively, to improve the synchronization between crop demand and available soil nitrogen (Ladha et al., 2005). One potential advantage is the reduction of the number of fertilizer applications and to enhance nitrogen use efficiency (NUE) by decreasing nitrate leaching and gas emissions. The objective of this study is to assess, in two contrasting soil types, if a single side-dress application of inhibited N fertilizer can replace the standard double side-dressing N fertilizer application of maize under semiarid irrigated conditions.

## MATERIAL AND METHODS

This study was conducted during two years (2015 and 2016) in an experimental field in the middle Ebro River Basin (Zaragoza, Spain) characterized by a semiarid Mediterranean climate (annual mean air temperature and mean precipitation of 14.5 °C and 308 mm, respectively). The study compared 4 fertilizer treatments in two soil types (deep vs. shallow) with contrasting soil water holding capacity. The N fertilizer treatments were: i) standard urea split into two applications at V6 and V13 ('Urea'); ii) a single application (V6) of urea with 3,4-dimethyl pyrazole phosphate (nitrification inhibitor) ('DMPP'); iii) a single application (V6) of urea with monocarbamide dihydrogen sulphate (urease inhibitor) ('MCDHS'); and iv) a single application (V6) of urea with N-(n-butyl) thiophosphoric triamide (urease inhibitor) ('NBPT'). The experiment was a completely randomized block design with three replicates for each soil type. All plots (5 m² each one) received a pre-planting application of 50 kg N ha⁻¹, 100 kg P₂O₅ ha⁻¹ and 150 kg K₂O ha⁻¹. The total N applied was calculated at each soil type to provide 250 kg N/ha (N fertilizer + soil nitrate at pre-planting). The total N applied each year is presented in Table 1. Maize (hybrid 'Pioneer P1758') was sprinkler irrigated and irrigation requirements were calculated using FAO methodology. The nutritional status of maize was evaluated with periodic measurements of leaf greenness (SPAD-502®, Minolta) at different vegetative and reproductive stages. Grain yield (GY), total aerial biomass (TAB) and total N uptake were measured, and the nitrogen use efficiency (NUE) was calculated as the total N uptake divided the N applied with fertilizer. Data were subjected to ANOVA and differences in treatment's means were assessed with the Tukey test (5% level of significance), using the SAS software.

## RESULTS AND DISCUSSION

Chlorophyll meter readings did not display significant differences between treatments in 2015 for the two soil types (data not shown). However, differences did show in 2016 (Fig. 1); in shallow soil, SPAD values of MCDHS treatment were 13.9 and 16.4% lower than those of NBPT treatment at VT and R3 stages, respectively. In deep soil, MCDHS and DMPP tended to present lower SPAD values than NBPT and UREA at later growing stages although only the MCDHS was significantly different at VT stage (on average 11% lower than NBPT and UREA). These differences in SPAD readings can explain some effects of the treatments on maize productive variables (Table 1). NBPT tended to have higher GY and TAB than the other treatments, especially in shallow soils in 2016 (GY 11.3% and TAB 7.8% higher); while MCDHS had the lowest GY and TAB. In spite of this, no significant differences (p<0.05) were found in GY and TAB among treatments in the two seasons and for the two soils. A significant effect of NBPT on grain yield was already described by Abalos et al. (2014) in a meta-analysis study. Differences in NUE among treatments were only detected for the shallow soils in 2016 (Table 1); MCDHS had a 19.3% lower NUE than the other treatments which was in concordance with SPAD readings after V13. NBPT and DMPP did not affect NUE, similar to Abalos et al. (2014).
<figure><img src="image_88.png" /><figcaption>Figure 1. Average (n=3) chlorophyll meter readings (SPAD) in different maize stages (V6, V10, V13, VT, and R3) during the 2016 growing season. The asterisk (*) indicates significant effects of treatments (p<0.05).</figcaption></figure>

<table><caption>Table 1. Average (n=3) of productive parameters obtained in the fertilizer treatments and soil types during two seasons.</caption><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">2015</th><th colspan="2">2016</th></tr><tr><th>Deep soil</th><th>Shallow soil</th><th>Deep soil</th><th>Shallow soil</th></tr></thead><tbody><tr><td>Total N applied, kg ha<sup>-1</sup></td><td></td><td>211</td><td>236</td><td>173</td><td>211</td></tr><tr><td rowspan="4">Grain yield (14%)<br>(Mg ha<sup>-1</sup>)</td><td>Urea</td><td>20.9</td><td>17.5</td><td>17.2</td><td>14.6</td></tr><tr><td>DMPP</td><td>20.7</td><td>18.7</td><td>16.3</td><td>14.4</td></tr><tr><td>MCDHS</td><td>20.1</td><td>17.3</td><td>16.2</td><td>12.4</td></tr><tr><td>NBPT</td><td>21.1</td><td>19.5</td><td>18.0</td><td>15.4</td></tr><tr><td></td><td><i>P-value</i></td><td>0.4191</td><td>0.0638</td><td>0.2928</td><td>0.0921</td></tr><tr><td rowspan="4">Aerial biomass yield (0%)<br>(Mg ha<sup>-1</sup>)</td><td>Urea</td><td>34.4</td><td>28.7</td><td>30.8</td><td>26.7</td></tr><tr><td>DMPP</td><td>33.9</td><td>28.6</td><td>30.0</td><td>26.6</td></tr><tr><td>MCDHS</td><td>33.3</td><td>27.7</td><td>28.9</td><td>23.8</td></tr><tr><td>NBPT</td><td>35.3</td><td>29.2</td><td>31.5</td><td>28.1</td></tr><tr><td></td><td><i>P-value</i></td><td>0.3325</td><td>0.2385</td><td>0.3731</td><td>0.0648</td></tr><tr><td rowspan="4">Nitrogen use efficiency</td><td>Urea</td><td>1.28</td><td>0.89</td><td>2.58</td><td>1.77 a</td></tr><tr><td>DMPP</td><td>1.19</td><td>0.86</td><td>2.37</td><td>1.68 a</td></tr><tr><td>MCDHS</td><td>1.15</td><td>0.84</td><td>2.40</td><td>1.42 b</td></tr><tr><td>NBPT</td><td>1.31</td><td>0.92</td><td>2.68</td><td>1.84 a</td></tr><tr><td></td><td><i>P-value</i></td><td>0.2132</td><td>0.2630</td><td>0.3255</td><td>0.0104</td></tr></tbody></table>

## CONCLUSION

The use of DMPP and NBPT allows reducing the number of side-dress N applications in maize without compromising grain yields under good irrigation practices. The novel MCDHS urease inhibitor tended to produce lower yield and NUE than the other treatments, although the differences were not always significant.

**Acknowledgements:** Spanish Ministry of Economy, Industry, and Competitiveness (AGL2013-49062-C4-3-R project) and Spanish National Institute for Agricultural Research (FPI-INIA 2015 predoctoral fellowship).

## REFERENCES

* Abalos, D., Jeffery, S., Sanz-Cobena. A., Guardia, G., Vallejo, A., 2014. Meta-analysis of the effect of urease and nitrification inhibitors on crop productivity and nitrogen use efficiency. Agric. Ecosyst. Environ. 189, 136-144.

* Ladha, J.K., Pathak, H., Krupnik, T.J., Six, J., van Kessel, C., 2005. Efficiency of fertilizer nitrogen in cereal production: Retrospect and prospects. Adv. Agron., 87, 85-156.
N₂O EMISSIONS DURING AND AFTER LEGUME CROPS CULTIVATION

¹ NICOLARDOT, B., ¹ BIZOUARD, F., ¹ COFFIN, A., ¹ GUINET, M., ² HENAULT, C., ¹ LOMBARD, F.,
¹ PAUTHENET, G., ¹ PIMET, E., ¹ VOISIN, A.S.,

¹ UMR Agroécologie, AgroSup Dijon, CNRS, INRA, Université Bourgogne Franche Comté, France, ² Unité Science du Sol INRA, Orléans, France

INTRODUCTION

Due to their ability to fix atmospheric dinitrogen thanks to symbiosis with soil bacteria, legumes provide a number of ecological services to agroecosystems. Their cultivation results in the harvest of proteins or fodder that are rich in protein, without any N fertilisation; their N rich crop residues left in the soil can be valued by the following crop. Their cultivation thus permits saving the consumption of fossil fuel energy and the emissions of greenhouse effect gas that are associated with the use of nitrogen fertilisers as reported by Rochette & Janzen (2005) and Jeuffroy et al. (2013). However, there is a lack of references on a wide range of legume crops, French research being essentially focused on pea. In the context of a French national research program (ANR Legitimes, 2018), field experiments have been set up to better characterize and quantify services related to N fluxes, for 10 legume species. Here we report first results concerning N₂O emissions during a two-year legume-cereal rotation: i) first year with different legume crops compared to fertilised cereals), and ii) second year with non-fertilised winter wheat which uptake N mineralised from crop residues coming from previous legume or cereal crops.

MATERIALS AND METHODS

Two field trials were respectively set up in 2014 and 2016 at the INRA experimental site of Bretenière (Eastern France) characterised by a semi-continental climate, the soil being classified as a Cambisol. Each field trial considered a two-year crop rotation (1st year including legume crop, followed by intercrop period and sowing of the winter wheat, and a 2nd year corresponding to the growth of unfertilised wheat crop until harvest). Legume crops were sown either on March or on May. As pre-crops, they were compared to non-fixing reference crops: Barley and Sorghum that were respectively sown in March and May and were N fertilised (135 and 70 kg N ha⁻¹ in 2014 and 2016 respectively for barley and 100 kg N ha⁻¹ for sorghum for both years). The winter wheat crop was sown on October and was not N fertilised. N₂O emissions were measured continuously using the automated chamber method (Vermue et al., 2013) during the 2-year duration of each trial. Legume crops considered during the first year were lupine, pea, faba bean, common bean, soya bean, chickpea, as well as the non-legume crops (i.e. barley and sorghum). For each treatment, emissions were measured using three automatic chambers. Soil temperature and moisture were continuously recorded for the different systems and soil bulk density and soil inorganic N were periodically measured.

RESULTS AND DISCUSSION

Most of the time for all treatments, daily N₂O emissions were less than 10 g N-N₂O d⁻¹ ha⁻¹, which is generally recorded for the pedoclimatic conditions of this experimental site (Vermue et al., 2013, 2016). Higher values (from 10 to 90 g N-N₂O d⁻¹ ha⁻¹) were measured in summer 2014 and spring 2016, respectively for field trials 1 and 2. These high emissions were related to exceptionally rainy conditions occurring during these two periods. Consequently, for field trial 1, cumulated N₂O emissions during legume crops cultivation were lower (96 to 411 g N-N₂O y⁻¹ ha⁻¹) than for intercrop + unfertilized wheat crop cultivation (92 to 487 g N-N₂O y⁻¹ ha⁻¹). For trial 2 (Figure 1), the contrary is observed with higher cumulated emissions measured during year 1 (legume crop cultivation) and lower cumulated emissions during year 2 (i.e. after incorporation of legume residues in soil and during unfertilized wheat crop). For both trials, higher emissions were measured for both fertilized crops (i.e. barley and sorghum) than for legume crops, especially during the following weeks after N fertilizer application. Consequently, for both trials, cumulated emissions for fertilized crops (barley or sorghum) were higher than respectively
corresponding cumulated emissions for legume crops sown in March or May, which is in accordance to published
data (Rochette & Janzen, 2005; Jeuffroy et al., 2013).

<figure><img src="image_89.png" /></figure>

Figure 1. Cumulated N₂O emissions during 2016-2017 field trial: a) from sowing to harvest of legume crops (first year); b) from legume harvest to following wheat harvest (second year). White bars; legume as crops (a) or -pre crops (b); Grey bars = N-fertilised non-legume crops as crops (a) or pre-crops (b), dashed-line bars = crops sown in March 2016, crossed-line bars = crops sown in May 2016.

CONCLUSION

N₂O emissions were first influenced by climatic conditions for both field trials. During the first year of both field trials, N₂O emitted during legume crops were less than for fertilised crops confirming the interest of legume crop to decrease GES emitted by cropping systems. During the second year of field trials, N₂O emissions were presumably related to soil N availability, which depends on the amounts of N present in crop residues and their mineralisation dynamics in soil. The next step of our work will consist to simulate N₂O emissions using emission's models to better precise the microbial processes responsible for emissions (i.e. nitrification and denitrification) and the role of pedoclimatic conditions.

**Acknowledgements:** M. Guinet PhD work was granted by INRA and the Ministry in charge of Agriculture. The experimental work was supported by the ANR LEGITIMES and Bourgogne-Franche-Comté Region (PSDR ProSys project).

REFERENCES

ANR Legitimes, 2018. https://www6.inra.fr/legitimes.

Jeuffroy M.H., Baranger E., Carrouée B., de Chezelles E., Gosme M., Hénault C., Schneider A., Cellier P. 2013. Nitrous oxide emissions from crop rotations including wheat, rapeseed and dry peas. Biogeosciences, 10, 1787-1797.

Rochette P., Janzen, H.H., 2005. Towards a revised coefficient for estimating N₂O emissions from legumes.
Nutrient Cycling in Agroecosystems, 73, 171-179.

Vermue A., Philippot L., Munier-Jolain N., Hénault C., Nicolardot B. 2013. Influence of integrated weed management system on N-cycling microbial communities and N₂O emissions. Plant Soil, 373, 501-514.

Vermue, A., Hénault C., Nicolardot B. 2016. High N₂O variations induced by agricultural practices in integrated weed management systems. Agronomy for Sustainable Development 36, 1-10.
# AN INCUBATION STUDY ABOUT THE POTENTIAL OF HIGH ORGANIC CARBON SOIL AMENDMENTS TO IMPROVE AGRICULTURAL NITROGEN RETENTION CAPACITY

REICHEL R<fnref n="1" />, WEI J<fnref n="1" />, ISLAM M.S.<fnref n="1" />, AMELUNG W.², SCHMID C.³ SCHRÖDER P.³, SCHLOTER M.³, BRÜGGEMANN N<fnref n="1" />

## INTRODUCTION

Nitrogen excess is a persistent problem of intensive agriculture, with consequences for climate change and ground water pollution by N₂O emission and NO₃⁻ leaching, respectively. High organic carbon soil amendments (HCA) with large C:N ratio, such as wheat straw, can stimulate microbial growth only if the microbes take up additional nitrogen from the soil in order to maintain their low C:N ratio. A static laboratory incubation study with custom-made soil columns, containing sieved agricultural sandy loam soil, was established to test the following hypotheses: (1) Can HCA such as wheat straw, spruce sawdust, and lignin help to manage temporal N excess in agricultural soil by inducing microbial growth at a relevant magnitude? (2) Can such HCA application strategies help to improve the nitrogen retention capacity of the soil by reducing an unintended N loss?

## MATERIAL AND METHODS

Soil was sampled near Kiel (Germany) from the top layer of an agricultural field (54°19'05"N, 9°58'38"E). Soil had a pH (CaCl₂) of 6.0 ± 0.1, a total organic C content of 1.3 %, and contained 0.15 % total N (Nt). Ammonium sulfate was applied, resembling an N application of 150 kg N ha⁻<fnref n="1" />. Wheat straw contained 44.4 % C and 0.28 % N. Spruce sawdust had 45.8 % C and 0.06 % total N. Both materials were ground to 1 mm. Lignin with 61.6 % organic C and 0.43 % N. All HCAs were applied at a rate of 1.5 g C kg⁻<fnref n="1" /> soil, equivalent to a field application of 4.5 t C ha⁻<fnref n="1" />. Three treatments with combined application of N fertilizer and wheat straw (SWF), or spruce sawdust (SSF), or lignin (SLF) were established. The control treatment (S) did not receive any N fertilizer or HCA, the fertilizer control (SF) received only N fertilizer. All treatments were incubated under the same experimental conditions and prepared in triplicate. Seven days prior to N fertilizer application, the first soil column section was sampled as reference, while the following samplings were conducted at 7, 21, 49, 77, and 113 days after fertilizer application (DAF).

For chemical analyses, soil and HCA of litter bags was stored at room temperature after freeze-drying. Decomposition was calculated after determining weight loss between applied and recovered HCA dry matter. Microbial biomass carbon (Cmic) was determined as described in Reichel et al. (2017). Nmic was calculated using the Cmic:Nmic ratio of 7. Residues of litter bags were washed three times with 1 M KCl at a ratio of 1:5 (w/v) to exclude adsorbed mineral N, before the freeze-dried powder was analyzed for N content (IRMS, Delta V plus, Thermo Fisher 231 Scientific, Bremen, Germany). Results were recalculated on a kilogram per hectare basis using the following input parameters of the field site: 10,000 m² × 0.2 m soil depth × bulk density of 1,500 kg m³ = 3·10⁶ kg dry soil ha⁻<fnref n="1" />.

## RESULTS AND DISCUSSION

Decomposition of OSA in N fertilized soil was reflected by microbial N retention (Fig. 1-A). Microbial biomass of the wheat straw (SWF) treatment retained 8-42 kg N ha-1, while in the sawdust treatment (SSF) 6-12 kg N ha-1 were retained in microbial biomass during the four month of incubation. As expected, pure lignin did not stimulate N immobilization.

Decomposing HCA material of wheat straw and spruce sawdust was enriched with 24 kg N ha⁻<fnref n="1" /> N, which remained in the HCA for more than four months. More about this topic will be available soon (Reichel et al., in revision).

<footnote n="1">¹ Forschungszentrum Jülich GmbH, Institute of Bio- and Geosciences, Agrosphere (IBG-3), 52425 Jülich, Germany, ² Institute of Crop Science and Resource Conservation, Soil Science & Soil Ecology, University of Bonn, 53115 Bonn, Germany, ³ Deutsches Forschungszentrum für Gesundheit und Umwelt GmbH, Research Unit Comparative Microbiome Analysis (COMI) 85764 Neuherberg, Germany</footnote>
<figure><img src="image_90.png" /></figure>

Figure 1. [A] Microbial N immobilization (kg N ha⁻¹), [B] physical and/or chemical N immobilization by the remaining HCA residues in kg N ha⁻¹ in soil of the control treatment (S, open quadrat; Fig. 1-A) without any fertilizer or HCA, the fertilizer control treatment (SF, open circle; Fig. 1-A) with mineral N fertilizer, and the HCA treatments with mineral N fertilizer plus lignin (SLF, solid triangle downwards), spruce sawdust (SSF, solid triangle upwards), or wheat straw (SWF, solid star symbol). Incubation time d = 0 divides the x-axis into a period before (-7 DBF) and after (7, 21, 49, 77, and 113 DAF) mineral N fertilization. Standard deviations of mean values (n=3) are displayed. Statistically significant differences between treatments at a certain incubation time point are depicted by different lowercase letters next to the symbols (p < 0.05).

## CONCLUSION

HCA on wheat straw basis can be effective in buffering an excess of N in a short period of time, as often reported for post-harvest sugar beet and oilseed rape fields. The N immobilization benefits might be optimized by combining different HCA with properties of wheat straw and spruce sawdust. These HCA materials retained N long enough within the microbial and HCA residue pool to serve as additional N supply for the subsequent crop. HCA can be part of cultivation strategies to improve N use efficiency of agricultural production. HCA composition, particle size, and adaption to the N fertilization regimes may improve the immobilization and re-release of N at the right time and rate in future.

**Acknowledgements:** We thank Holger Wissel, Franz Leistner, Martina Krause (Forschungszentrum Jülich GmbH, IBG-3) for their technical support, Ayhan Egmen of IBG-2-3-TAK for producing the incubation columns, Steffen Rothardt and Henning Kage at the Christian-Albrechts-Universität zu Kiel (CAU) for the provision of the soil, wheat straw, and spruce sawdust. We also thank the BMBF for funding (FKZ 031A561A).

## REFERENCES

- Reichel R., Hänsch M., Brüggemann N., 2017. Indication of rapid soil food web recovery by nematode-derived indices in restored agricultural soil after open-cast lignite mining. Soil Biol. Biochem. 115, 261-264.

- Reichel R., Wie J., Islam M.S., Schmid C., Wissel H., Schröder P., Schloter M., Brüggemann N., in revision. Potential of wheat straw, spruce sawdust, and lignin as high organic carbon soil amendments to improve agricultural nitrogen retention capacity: an incubation study. Front. Plant Sci.
# EFFECTS OF LONG-TERM APPLICATION OF MINERAL NITROGEN AND MANURE ON SELECTED SOIL PROPERTIES

PIKUŁA D.<fnref n="1" />, MARTYNIUK S.²

## INTRODUCTION

In the first half of the past century, there were hardly contradictions between the agricultural cultivation and the environment. With the increasing use of mineral fertilizers, yields have increased by more than 100% thus the quantity of roots and harvest residues on the field has increased strongly as a source of organic matter. However, in agricultural practice they are commonly removed from the field after harvest resulting in soil organic matter decrease. It results in declining of soil fertility (Mazzoncini et al. 2011).

The aim of this study was to compare selected biological and chemical characteristics soil on which for over 34 years crops were grown in two crop rotations similar with respect to N nitrogen and manure application but differing with respect to total organic matter input (mustard green manure, one year clover-grass ley).

## MATERIAL AND METHODS

The study was conducted on the basis of a three-factor long-term field experiment carried on since 1980 at the Experimental Station Grabów, on a soil classified Albic Luvisol with loamy sand texture. The experiment includes two 4-years crop rotations (I factor) with the following crops: rotation A – maize, winter wheat, spring barley and corn for silage and rotation B – maize, winter wheat, spring barley with undersown clover-grass mixture, clover-grass ley. Manure rates (II factor) was applied in the autumn preceding maize once per 4-year cycle as follows: 0, 20, 40, 60 and 80 t/ha. N fertilizer rates (III factor) (N0, N1, N2 and N3) were assigned to plots within each main plots, that is per manure rate. Total N fertilizer inputs in rotation A were: 0 (N0), 170 (N1), 340 (N2) and 510 (N3) kg N/ha per rotation and in rotation B: 0 (N0), 155 (N1), 310 (N2) and 465 (N3) kg N/ha. For the purpose of this study, in 2013, when winter wheat was grown in both rotations, soil samples were collected from selected treatments to assess the following parameters: microbial biomass (SMBC), the content of the soluble MO fraction (C extracted with hot water) and Corg, pH.

The data were subjected to the 3-way analysis of variance (ANOVA) with significance of differences assessed at P < 0.05.

## RESULTS AND DISCUSSION

There was found no significant interactions between experimental factors. The soil in rotation B with an increased input of organic matter accumulated significantly larger amounts of soil organic carbon (SOC) and soil microbial biomass (SMBC), but in all other treatments microbial biomass C decreased as N rates increased (Figure 1). In general, mineral N fertilization had also a beneficial effect on SOC accumulation. This effect was insignificant when FYM was not applied in rotation A, but in rotation B in the absence of FYM the soil treated with 100 and 150 kg N ha⁻<fnref n="1" /> contained significantly larger amounts of SOC than the unfertilized soil (N-0). When manure was used, application of 150 kg N ha⁻<fnref n="1" /> resulted in the accumulation of the maximum amounts of SOC in both rotation (Figure 1). There is also a general consensus that proper soil fertility management practices on croplands, particularly with respect to N fertilization, enhance SOC sequestration and are beneficial for soil microorganisms and their activity, mainly due to a greater return of crop residues to the soil (Liu et al., 2006, Mazzoncini et al., 2011). However, there are also reports showing that mineral N fertilizers, particularly their high rates, can reduce SMBC (Blanchet et al., 2016).

<footnote n="1">¹ Department of plant nutrition and fertilization¹, department of agriculture microbiology, ² institute of soil science and plant cultivation – state research institute in puławy, poland</footnote>
<figure><img src="image_91.png" /><figcaption>**Figure 1.** Organic C and microbial biomass C contents in soil as influenced by crop rotation (I), manure application (II) and mineral N rate (III) in a long-term field experiment. LSD(P ≤ 0.05): for C org.: I = 0.52, II = 0.65, III = 0.23, interactions – not significant; for microbial biomass C: I = 11.5, II = 10.1, III = 11.2, interactions: not significant</figcaption></figure>

## CONCLUSION

All the experimental factors had significant effects of organic C accumulation in soil. Irrespective of fertilizer N rates and manure application, the soil in rotation B with larger organic matter input (mustard green manure, clover-grass ley) contained significantly more Corg..

The high rates of mineral N fertilizers can reduce soil microbial biomass.

## REFERENCES

Blanchet, G., Gavazov, K., Bragazza, I. & Sinaj, S., 2016. Responses of soil properties and crop yields to different inorganic and organic amendments in a Swiss conventional farming system. *Agr. Ecosyst. Environ.* 230, 116–126.

Liu, X., Herbert, S. J., Hashemi, A. M., Zhang, X. & Ding, G., 2006. Effects of agricultural management on soil organic matter and carbon transformation – a review. *Plant Soil Environ.* 52, 531–543.

Mazzoncini, M., Sapkota, T. B., Barber P., Antocji, D. & Risaliti R., 2011. Long-term effect of tillage, nitrogen fertilization and cover crops on soil organic carbon and total nitrogen content. *Soil Till. Res.* 114, 165–174.
# SHORT-TERM FATE OF <sup>15</sup>N IN MAIZE ON TROPICAL SANDY SOILS AS AFFECTED BY N FORM, TILLAGE AND BIOCHAR ADDITION

MUNERA-ECHEVERRI, J.L.¹, MARTINSEN, V.¹, OBIA, A.², DÖRSCH, P.¹, MULDER, J.¹

¹ Faculty of Environmental Sciences and Natural Resource Management, Norwegian University of Life Sciences, Norway; 2 Norwegian Geotechnical Institute (NGI), Norway

## INTRODUCTION

Maize productivity in Sub-Saharan Africa (SSA) is often low due to erratic rainfall and the inherent low nutrient content of soils. Crop yields were shown to respond positively to nitrogen (N) fertilization (Palm et al., 2010). However, the high cost of fertilizers limits widespread use on smallholder farms. Therefore, strategies to reduce N losses and to optimize N uptake by crops are important for crop productivity and food security. So far, few studies in SSA have focused on the effect of tillage practice and type of N fertilizer on N use efficiency and crop yield on smallholder farms. Here, we investigated effects of conventional tillage and minimum tillage (using planting basins with and without application of biochar) on assimilation, retention and losses of N in maize on sandy soil at Kaoma, Zambia. The study involved biochar from pigeon pea and included tests with three 15-N labeled forms of N (NO3-, NH4+ and Urea). Understanding how the fate of <sup>15</sup>N is affected by soil management practices and the form of N added is important for providing farmers with recommendations on how to make a better use of N, in which losses are minimized and plant uptake optimized.

## MATERIAL AND METHODS

Three soil management practices were assessed in triplicates in 20m² plots, each with 7 rows with a spacing of 90 cm. Tillage practices included 1) Conventional: overall ploughing without residue retention, with 12 plants per row and 21 cm between plants. 2) Conservation agriculture: 4 planting basins per row (20 x 30 cm), 70 cm between basins, 3 plants per basin and residue retention between basins. 3) Conservation farming and biochar, with the same treatment as the previous one, but now with 250 g of biochar per basin (this is equivalent to 4 ton ha⁻¹). All plots received the same amount of NPK (10:20:10) fertilizer at planting (250 kg ha⁻¹). Seven weeks after planting a 15N labelling experiment with the addition of 96 g <sup>15</sup>N ha⁻¹ in the form of <sup>15</sup>NH₄NO₃, NH₄NO₃<sup>15</sup> and <sup>15</sup>N-Urea was established in the field. Water was included as a non-labelled control.

Three <sup>15</sup>N forms were applied dissolved in water and a control with the same amount of water was included. The four 15-N treatments were applied in every plot, each 15-N form in two basins per row. Thus, keeping one non-labeled row in between labelled rows, to avoid cross contamination. Two basins with three healthy plants were chosen. In the conventional plots, the <sup>15</sup>N tracers were applied to 10 cm wide strips of 60 cm length, with 3 plants in each. In all the treatments, the 15N label was applied in solution to 1200 cm² of soil. The addition of <sup>15</sup>N was small compared to the initial fertilizer dose amounting to 96 g <sup>15</sup>N ha⁻¹ as <sup>15</sup>NH₄NO₃, NH₄<sup>15</sup>NO₃ and 15N-Urea, respectively.

After 240 hours, soil samples were taken from 0 to 5 cm and 5 to 20 cm depth and extracted on site using 1 M KCl. The extracts were analysed for NO3- and NH4+. Plant samples were divided into roots, stems and new leaves and analysed for delta <sup>15</sup>N. Gas flux measurements were done 24 hours before <sup>15</sup>N addition, 0.5, 24, 48, 72, 120 and 240 hours after. Isotope analysis was done in soil, KCl extractable NO₃⁻ and plant samples.

## RESULTS AND DISCUSSION

The soil pH<sub>H2O</sub> was 6.2, the organic carbon content was 0.5%, and total N was 0.05%. The soil was sandy (89% sand and 3.5% clay). The precipitation during the 10 days of the experiment was 116mm. The residual soil recovery after 10 days was significantly different between the 3 forms of 15N added, being the highest when 15N-NH₄⁺ (71%) was added, followed by 15N-Urea (55.8%) and 15N-NO₃⁻ (21.7%). In contrast, the residual soil recovery was
not affected by the three soil management practices (CF + Biochar: 50%; CF Normal: 47%; Conventional: 50%). Very few ¹⁵N was found in the KCl extractable NO₃⁻. At the depth 5 to 20cm, it was the highest in CF + biochar (0.72%), followed by conventional (0.22%) and CF Normal (0.1%). In contrast, at 0 to 5cm depth, the recovery was neither affected by soil management practices nor the form of added N. Fluxes of N₂O were very small and the contribution of N₂O to ¹⁵N fluxes was small. The recovery in plants was significantly smaller in the Conventional treatment (3.9%) than in CF Normal (14.6%) and CF + Biochar (13.5%). Plant recovery was lower in ¹⁵N-Urea (6.4%) was applied than ¹⁵N-NO₃ (-14.3%) and ¹⁵N-NH₄⁺ (11.3%).

<figure><img src="image_92.png" /><figcaption>Figure 2. ¹⁵N recovery (%) in soil and KCl extractable NO₃⁻ from 0 to 5 cm and 5 to 20cm and maize plants after 10 days of ¹⁵N tracer addition in three soil management practices.</figcaption></figure>

In this study, the greatest ¹⁵N losses occurred in the ¹⁵N-NO₃⁻ treatments, mostly through leaching. The ¹⁵N-Urea treatments also had substantial N losses, most likely due to ammonia (NH₃) volatilization and conversion of urea into NO₃⁻. Unfortunately, we do not have results of NH₃ fluxes to compare which mechanism was more important. The low N losses for the ¹⁵N-NH₄⁺ treatments can be explained by preference of microbes for this N form (Recous & Mary, 1990) and higher adsorption to soil particles. Biochar increased the recovery of ¹⁵N in the NO₃⁻ pool and reduced plant ¹⁵N uptake and residual soil recovery. The Conventional treatment had the lowest ¹⁵N recovered in plants among the soil management practices, but it might be related to the fact that only half of the rooting environment was covered with the ¹⁵N solution in the Conventional treatment, while for the CF treatments it was completely covered

CONCLUSION

In this sandy soil N losses were greatly affected by the N form, being the highest when ¹⁵N-NO₃⁻ was applied, followed by ¹⁵N-Urea and ¹⁵N-NH₄⁺. Soil management practices marginally affected N losses. Biochar increased the amount of KCl extractable NO₃⁻. Crop showed a preference of ¹⁵N-NO₃⁻ followed by ¹⁵N-NH₄⁺ and ¹⁵N-Urea. The low ¹⁵N recovery in plants in the Conventional treatment may be due to an artifact of the application.

REFERENCES

Palm, C. A., Smukler, S. M., Sullivan, C. C., Mutuo, P. K., Nyadzi, G. I., & Walsh, M. G. (2010). Identifying potential synergies and trade-offs for meeting food security and climate change objectives in sub-Saharan Africa. *Proc Natl Acad Sci U S A*, **107**(46), 19661-19666. doi:10.1073/pnas.0912248107

Recous, S., & Mary, B. (1990). Microbial Immobilization of Ammonium and Nitrate in Cultivated Soils. *Soil Biol. Biochem.*, **22**(7), 913-922.
EFFECT OF FERTILIZER N FORMS ON ROOT GROWTH IN WINTER WHEAT (TRITICUM AESTIVUM L.)

KIRSCHKE, T.<fnref n="1" />, THIEL, E.², CHRISTEN, O.³

INTRODUCTION

Urea is the most widely used nitrogen (N) fertilizer worldwide (IFA, 2017). It undergoes rapid hydrolisation in soil, after which ammonium (NH₄⁺) is oxidized to nitrate (NO₃⁻). NO₃⁻ leaching from arable land might have significant environmental impact on ground- and surface water (Widdison and Burt, 2008). Due to the use of nitrification inhibitors, the importance of NH₄⁺ as N source for plant nutrition has increased. Different studies demonstrated that an NH₄⁺-N based fertilizer induces divergent responses in the rhizosphere, compared to a NO₃⁻-N fertilizer (Ruan et al., 2007), and e.g. promotes branching of taproots under laboratory conditions (Lima et al., 2010). Effects on root growth due to a prolonged NH₄⁺-N phase are yet not well understood under field conditions. The aim of this study was to close the existing gap in knowledge between laboratory and field scale.

MATERIAL AND METHODS

A field trial at the experimental farm in Merbitz (average annual precipitation 546 mm; average annual temperature 9.8 °C, Saxony-Anhalt, Germany) was realized in 2014–2016. The winter wheat (Triticum aestivum L.) crop was cultivated following spring barley (Hordeum vulgare L.). The soil was classified as a Chernozem from loess with a silty loam sand texture. The experimental setup showed a completely randomized design with three treatments and four replications. An unfertilized control and two different N fertilizers were tested: granular calcium ammonium nitrate (CAN) and a granular urea combined with an urease (UI) and nitrification inhibitor (NI) (urea+UI+NI). The N-application rate was 180 kg N ha⁻<fnref n="1" />. CAN fertilizer was applied in three applications of 60 kg N ha⁻<fnref n="1" /> each time at the beginning of growing season, at growth stage (GS) 31 and at late dressing (GS 51). Urea+UI+NI was applied in two split applications of 100 kg N ha⁻<fnref n="1" /> at beginning of growing season (coincident with 60 kg N ha⁻<fnref n="1" /> CAN), and 80 kg N ha⁻<fnref n="1" /> at GS 37. Wheat roots were sampled by the auger method at GS 32, 37 and 65. The soil monoliths (80 cm in deep and 9.3 cm in diameter) were subdivided into 20 cm segments. The root samples were washed free from soil on 2-mm mesh sieves. The Root length density (RLD) was measured using WinRHIZO® software (Regent Instr. Canada Inc., 2013). According to dry soil conditions in May and June, roots could be sampled at GS 65 only until 40 cm soil depth in 2014. No sampling could be realized at GS 65 in 2015. Data were subjected to analysis of variance using the F-test (p<0.05) to examine the effects of N fertilizer and N form on root growth.

RESULTS AND DISCUSSION

The high fertility of the soil was demonstrated by high soil mineral N contents in the unfertilized control in every single year. Different NH₄⁺-concentrations were produced by the used fertilizer N forms. However, the majority of the mineral N were always available as NO₃⁻. In all treatments and years, RLD decreased with increasing soil depth at each GS (Fig. 1), while constantly more than 40 % of quantified roots were measured in 0–20 cm depth. Including the soil depth 20–40 cm mostly about 75 % of the captured roots were present in the topsoil (0–40 cm). The two examined N forms caused only slightly differences in RLD. However, urea+UI+NI induced in general higher RLD at the GS 37, compared to CAN. Meanwhile the values of both treatments were almost at the same level at GS 65 (maximum root expansion). Significant differences between these treatments were measured only at GS 37 in 2014 in favor of urea+UI+NI. A treatment without N fertilizer application (control) increased rooting at GS 32, but led partially to significant lower RLD during the growing season at 0–20 cm depth (GS 65). Taking all years into consideration, the control variant was characterized by significant lower RLD (0–80 cm summed) at the GS 65, in comparison to both fertilized treatments (data not shown).

<footnote n="1">¹ Institute of Agrochemistry Piesteritz e.V. (AIP), AN-Institute of the University of Halle-Wittenberg, Germany; ² SKW Stickstoffwerke Piesteritz GmbH, Germany; ³ University of Halle-Wittenberg, Institute of Agricultural and Nutritional Sciences, Halle (Saale), Germany</footnote>
<figure><img src="image_93.png" /></figure>

Figure 1. Root length density of winter wheat at treatments with two different fertilizer N forms and one unfertilized control in different soil depths at the growth stages (GS) 32, 37 and 65 in the years 2014, 2015 and 2016. Bars represent mean values (+ SE) of four replicates per treatment, and different letters denote significant differences among means within one depth and one year at p<0.05 (F test).

Clear positive effects of an NH<sub>4</sub><sup>+</sup> based N fertilizer on root growth of *Arabidopsis* under laboratory conditions described by Lima et al. (2010), couldn't be documented in this field study. We realized less pronounced and only temporarily effects of the different fertilizer N forms on winter wheat rooting. This is primarily due to high NH<sub>4</sub><sup>+</sup>-concentrations under lab conditions (nutrient solution), which is generally not achieved at field scale.

## CONCLUSION

The results showed no statistically significant differences of RLD between the tested different N form treatments. The NH<sub>4</sub><sup>+</sup> based fertilizer tended to increase root growth during stem elongation (GS 37), while an NO<sub>3</sub><sup>-</sup> emphasised fertilizer have a more positive affect on rooting between GS 37 and flowering (GS 65). In our experiment, a two-splitted fertilizer strategy (urea+UI+NI) nevertheless obtained similar plant root growth as it achieved by a three-divided application (CAN). A scarcity on N (control) tended to stimulate root growth of young plants (GS 32), but generated declines during the growing season.

**Acknowledgement.** Participation in the 20th N Workshop was funded by the State Saxony-Anhalt and the European Union.

## REFERENCES

* IFA, 2017. International Fertilizer Industry Association, Production and International Trade https://www.fertilizer.org/En/Statistics/PIT_Excel_Files.aspx (accessed 17. Jan. 2018)

* Lima J.E., Kojima S., Takahashi H., von Wirén N., 2010. Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANS-PORTER1;3-dependent manner. The Plant cell 22 (11), 3621–3633.

* Ruan J., Gerendás J., Härdter R., Sattelmacher B., 2007. Effect of nitrogen form and root-zone pH on growth and nitrogen uptake of tea (Camellia sinensis) plants. Annals of botany 99 (2), 301–310.

* Widdison P. E., Burt T.P., 2008. Nitrogen cycle. Global Ecol, 2526-2533.
# EFFECT OF N-FERTILIZERS ON THE ABUNDANCE OF NITRIFICATION AND DENITRIFICATION GENES IN BULK AND RHIZOSPHERIC SOIL OF TOMATO AND BEANS PLANTS

CASTELLANO-HINOJOSA, A.<fnref n="1" />,², GONZÁLEZ-LÓPEZ, J.<fnref n="1" />, BEDMAR E.J.²

## INTRODUCTION

Nitrogen availability is a major limiting factor for plant growth and crop production. Root exudates and specially application of N-fertilizers from agricultural practices increase the N concentration in the soil. The effect of N-fertilizers on nitrification and denitrification gene abundances has been reported in bulk soil (Hallin et al., 2017), but comparative data on bulk and rhizospheric soil are scarce. Here we report the abundance of nitrification and denitrification genes in bulk and rhizospheric soil of tomato and common bean grown in an agricultural soil.

## MATERIALS AND METHODS

### Sampling site and experimental setup

Samples from a eutric cambisol soil were collected from an agricultural area located near Motril (Granada, Spain) and supplemented independently either with urea, ammonium sulfate or potassium nitrate and mixed with a concrete mixer. Final concentration was of 260 kg of equivalent N ha⁻<fnref n="1" />. A set of pots containing soil without fertilization was used as a control. Fertilized soils were used to fill 20-kg capacity containers (54 x 21 x 25 cm, 4 pots/treatment), then planted with either tomato (Solanum lycopersicum var. Roma) or common bean (Phaseolus vulgaris var. Kylie) and maintained under greenhouse conditions. Soils were watered every week to about 80% water filled pore space (WFPS). A set of pots containing soil without fertilization was used as a control. Four consecutive crops were recorded. The concentration of each fertilizer was determined at the end of each crop (about 4 months/crop) and when required, the soil was supplemented with the corresponding N-fertilizer to reach the initial fertilization rate. Total organic carbon (TOC), total nitrogen (TN), pH and NO₃⁻ and NH₄⁺ contents were determined along the experimental period.

### Nitrous oxide emission

To assay N₂O emissions stainless steel cylindrical corers (5 x 20 cm) were manually inserted into the ground to sample soil. Measurements were performed 24 h after watering of the pots by gas chromatography. Cumulative N₂O emissions were calculated by linear interpolation between gas sampling periods.

#### DNA extraction and quantification of nitrification and denitrification genes

Soil samples (5 g) from both bulk and rhizospheric soil were taken and total DNA extracted according to Correa-Galeote et al. (2014). The total bacterial (16SB) and archaeal (16SA) communities were quantified by qPCR using the 16S rRNA gene as molecular marker. The size of the nitrifiers was estimated by using the *amoA* gene from ammonia oxidizing bacteria (*amoA AOB*) and archaea (*amoA AOA*) and that of the denitrifiers by determination of the *napA/narG*, *nirK/nirS*, *norB* and *nosZI/nosZII* genes.

### Statistical analyses

Stepwise multiple regression analysis was performed to assess the abiotic variables (targeted genes) in both bulk and rhizospheric soil of tomato and common bean plants. For each crop, Mantel test was employed to correlate abiotic properties, plant type and targeted genes.

## RESULTS AND DISCUSSION

<footnote n="1">¹ Department of Microbiology, Faculty of Pharmacy, University of Granada, Granada, Spain; ² Estación Experimental del Zaidín, Granada, Spain.</footnote>
## Soil properties and nitrous oxide emissions

Values of pH were significantly higher in the rhizospheric soil of each tomato and common bean plants under all conditions examined. Regardless of the plant species and the N-treatment, while the ammonium detected in the bulk soil was statistically higher than that in the rhizospheric soil for the 4 consecutive crops, the content of nitrate was lower. Also, rhizospheric soil contained more TOC and TC and less TN. After N-fertilization, N₂O fluxes increased to reach a peak 20 d after fertilizers application. Afterwards, gas emission gradually decreased to a basal level. For the two plant species and each of the 4 crops, maximal N₂O fluxes were found after urea application, followed by treatment with ammonium and, finally, nitrate (Fig. 1). After 4 consecutive crops, the cumulative N₂O emissions (kg N ha⁻¹) for soils cultivated with common bean and tomato were 16.43 vs 11.97, 12.48 vs 10.56 and 3.31 vs 2.70 for urea, ammonium and nitrate, respectively.

## Abundance of nitrification and denitrification genes

For each of the 2 plants and the 4 crops, total abundance of 16SB and 16SA were higher in the rhizospheric soil regardless of the N treatment. While the *amoA* AOB gene copy number was more abundant in the rhizospheric soil, the *amoA* AOA was higher in the bulk soil. Also, all of the 7 denitrification genes showed higher abundance in the bulk soil. Moreover, all targeted genes showed higher abundance in soils cultivated with common bean.

## Multivariate relationships

A stepwise multiple regression analysis showed that the changes in the abundance of the 16S rRNA genes were explained mainly by TC and TOC in the bulk and the rhizospheric soil of each plant species. The abundance of the nitrification genes was significantly affected by NH₄⁺ and TN in the bulk soil and by pH and NH₄⁺ in the rhizospheric soil. Shifts in the biomass of the denitrification genes were mainly controlled by NO₃⁻ and pH in the bulk soil and by NO₃⁻ and TN in the rhizospheric soil. Mantel tests showed that plants influenced the abundance of targeted genes in the rhizospheric soil. N-fertilization induced an increase of the *nosZ* genes that was coupled to the reduction of N₂O emissions (Fig. 1).

<figure><img src="image_94.png" /><figcaption>Figure 1. N₂O emissions (kg N ha⁻¹ h⁻¹) by soils treated with urea (U), ammonium sulphate (A) or potassium nitrate (N) cultivated with either tomato (T, Fig. 1A) or common bean (B, Fig. 1B) during four consecutive crops (4 months each). Total abundance (log gene copy number/g soil) of *nosZI* + *nosZII* genes is also shown.</figcaption></figure>

**Acknowledgements:** This study was supported by the ERDF-cofinanced grant PEAGR2012–1968 from Consejería de Economía, Innovación y Ciencia. ACH thanks MECD for FPU grant FPU2014/01633.

## REFERENCES

* Correa-Galeote, D., Tortosa, G., Bedmar, E.J. 2014. Quantification of functional microbial nitrogen cycle genes in environmental samples. In: D.E. Marco (ed.) Metagenomics of the Microbial Nitrogen Cycle: Theory, Methods and Applications. Caister Academic Press, Norwich, UK, pp. 65-85.

* Hallin, S., Philippot, L., Löffler, F.E., Sanford, R.A., Jones, C.M. 2017. Genomics and ecology of novel N₂O reducing microorganisms. Trends in Microbiology, 26, 43-55.
# CAN N₂O ISOTOPOCULES HELP TO IMPROVE OUR UNDERSTANDING OF N₂O PROCESSES DURING GRASSLAND CONVERSION TO MAIZE CROPPING?

BUCHEN, C.<fnref n="1" />,², LEWICKA-SZCZEBAK, D.², HELFRICH, M.², FLESSA, H.², WELL, R.²

## INTRODUCTION

During the last decades, large areas of grassland were converted to cropland across Europe, mainly due to the increasing demand of cropland following the expansion of biogas plant production (e.g. maize). However, the break-up of permanent grassland is associated with the release of large amounts of carbon (C) and nitrogen (N) caused by an increased mineralisation due to decomposition of soil organic matter and the old grass sward. This additional supply of mineral N can cause enhanced N losses in form of nitrous oxide (N₂O), an important greenhouse gas. Until now, knowledge about N₂O production pathways due to grassland break-up and in particular N₂O reduction to N₂ is very limited, even though understanding of N₂O processes and identification of sources are needed in order to devise mitigation options. Therefore, natural abundance stable isotope signatures of soil-emitted N₂O (δ¹⁵N<sup>bulk</sup><sub>N₂O</sub>, δ¹⁸O<sub>N₂O</sub> and δ¹⁵N<sup>SP</sup><sub>N₂O</sub> = intramolecular distribution of ¹⁵N in the N₂O molecule) were used to identify sources of N₂O emission during the first year after grassland conversion to maize cropping.

## MATERIAL AND METHODS

### Study sites and N₂O sampling

N₂O isotopocule samples were collected periodically from manual chambers and analysed by isotope ratio mass spectrometry (IRMS) on four study sites with varying soil texture (sandy to clayey) and different annual N₂O emission in the north-western part of Germany (Table 1; Buchen et al. (2017)).

<table><caption>Table 1: Site characteristics and annual N₂O emission within the first year following grassland conversion to maize cropping</caption><thead><tr><th>Study sites</th><th>Soil type</th><th>Soil texture</th><th>Sward age [year]</th><th>N fertilisation rate [kg N ha<sup>-1</sup> year<sup>-1</sup>]</th><th>Treatment</th><th>N<sub>2</sub>O emission [kg N ha<sup>-1</sup> year<sup>-1</sup>]</th></tr></thead><tbody><tr><td rowspan="2">Oldenburg</td><td>Histic Gleysol</td><td>Peat-silty sand</td><td rowspan="2">&gt; 15</td><td rowspan="2">150</td><td rowspan="2">Chemical + Mechanical</td><td>3.3±1.2</td></tr><tr><td>Plaggic Anthrosol</td><td>Sand</td><td>1.5±0.1</td></tr><tr><td rowspan="2">Trenthorst</td><td rowspan="2">Planosol</td><td rowspan="2">Sandy loam</td><td rowspan="2">&gt; 25</td><td rowspan="2">none</td><td>Chemical</td><td>7.5±1.0</td></tr><tr><td>Mechanical</td><td>8.1±1.3</td></tr><tr><td rowspan="2">Kleve</td><td rowspan="2">Fluvic Cambisol</td><td rowspan="2">Clayey loam</td><td rowspan="2">&gt; 35</td><td rowspan="2">137</td><td>Chemical</td><td>27.9±2.4</td></tr><tr><td>Mechanical</td><td>15.9±3.0</td></tr></tbody></table>

### N₂O isotopocule mapping approach

By using a novel isotopocule mapping approach (plotting δ¹⁵N<sup>SP</sup><sub>N₂O</sub> vs. δ¹⁸O<sub>N₂O</sub>) based on the isotopic composition of N₂O produced and literature values for specific N₂O pathways, we were able to simultaneously determine the contributions of N₂O production pathways: (i) heterotrophic bacterial denitrification and/or nitrifier denitrification and (ii) nitrification and/or fungal denitrification and the contribution of N₂O reduction (Lewicka-Szczebak et al., 2017). We calculated two main scenarios with different assumptions for N₂O produced: N₂O is reduced to N₂ before residual N₂O is mixed with N₂O of various sources (Scenario a) and vice versa (Scenario b) (Figure 1).

<footnote n="1">¹ Leibniz Centre for Agricultural Landscape Research (ZALF), Germany, ² Thünen Institute of Climate-Smart Agriculture, Germany</footnote>
<figure><img src="image_95.png" /><figcaption>Figure 1: Isotopocule map, illustrating the simultaneous estimation of N₂O reduction and the contribution of different processes to soil-emitted N₂O</figcaption></figure>

## RESULTS AND DISCUSSION

The isotopic composition of soil-emitted N₂O largely resembled the known end-member values for bacterial denitrification. The isotopocule mapping approach indicated a different impact of N₂O reduction on isotopic composition of soil-emitted N₂O for the four soils under study. For the two sites in Oldenburg, differing N₂O production pathways within the season were not observed, but management events and soil conditions had a significant impact on pathway contribution and N₂O reduction. The Kleve site exhibited a close correlation between δ¹⁵N⁵Pₙ₂O and δ¹⁸Oₙ₂O suggesting that values were mainly controlled by N₂O reduction to N₂. At the Trenthorst site (organic farming) this pattern was less pronounced, possibly because processes other than bacterial denitrification (e.g. fungal denitrification and nitrification) also influence isotopocule values.

## CONCLUSION

The applied isotopocule mapping approach opens up new prospects for studying N₂O production and consumption of N₂O in soil simultaneously. Bacterial denitrification was found to be the most important process following grassland conversion to maize cropping and the extent of N₂O reduction depended on the soil type. N₂O reduction was very variable, partially very high resulting in large N₂ fluxes. However, future attempts are needed in order to properly validate the isotopocule mapping approach.

**Acknowledgements:** The studies were funded by the DFG Research Training Group 1397. We thank all our partners from the Thünen Institute of Organic Farming, as well as the Chambers of Agriculture in Oldenburg and Kleve.

## REFERENCES

Buchen, C., Well, R., Helfrich, M., Fuß, R., Kayser, M., Gensior, A., Benke, M., Flessa, H., 2017. Soil mineral N dynamics and N₂O emissions following grassland renewal. Agriculture, Ecosystems & Environment 246, 325-342.

Lewicka-Szczebak, D., Augustin, J., Giesemann, A., Well, R., 2017. Quantifying N₂O reduction to N₂ based on N₂O isotopocules – validation with independent methods (helium incubation and ¹⁵N gas flux method). Biogeosciences 14, 711-732.
# IS SOYBEAN YIELD LIMITED BY NITROGEN SUPPLY?

CAFARO LA MENZA, N.<fnref n="1" />, MONZON, J.P.², SPECHT, J. E.<fnref n="1" />, GRASSINI, P.<fnref n="1" />

## INTRODUCTION

Soybean has a large nitrogen (N) requirement: about 80 kg of N uptake are needed per Mg of seed yield produced (Salvagiotti et al., 2008). Hence, a soybean crop that produces 3 Mg ha⁻<fnref n="1" /> requires ca. 240 kg N ha⁻<fnref n="1" />, while a high-yield soybean crop that produces 6 Mg ha⁻<fnref n="1" /> requires about 480 kg N ha⁻<fnref n="1" />. Soybean rarely receives N fertilizer inputs, except for a small amount (≈20 kg ha⁻<fnref n="1" />) typically applied as 'starter' at sowing. Hence, soybean relies on two major sources of indigenous N: soil organic matter mineralization and biological fixation. To sustain yield increases, it seems critical to know the yield level at which contribution of indigenous N sources becomes insufficient to meet crop N requirements while still maintaining or increasing seed protein and oil concentration. The objective of this study was to evaluate the degree of N limitation in field grown soybean across a wide range of yield potential of the production environment.

## MATERIALS AND METHODS

It is difficult to evaluate N limitation in soybean for two major reasons. First, soybean uptakes 60% of the N after R3 stage† (Bender et al., 2015). Second, application of N fertilizer in soybean typically results in a decrease in N fixation. In order to assess soybean N limitation, we designed an experiment that included: (i) a "full-N" treatment that received an ample N supply during the entire soybean growing season, and (ii) a "zero-N" treatment that did not receive any N fertilizer, relying on indigenous N supply. Experiments were conducted at four sites in Nebraska (USA) during one season (2016) and in Balcarce (Argentina) during two crop seasons (2014/2015 & 2015/2016). Experiments in USA were replicated at four producer irrigated high-yield fields that included the two N treatments following a randomized complete design, while experiments in Argentina consisted of a combination of sowing date, variety, and N treatments following a split-split plot design. In all experiments, crops were irrigated and managed to ensure optimal water and nutrient supply (except for N in the zero-N treatment) and to avoid stress from weeds, insects and pathogens. Experiments covered a wide yield range, from 2.5 to 6.5 Mg ha⁻<fnref n="1" /> (at standard 13% seed moisture content).

We developed a novel N fertilization protocol to ensure non-N limiting conditions in field-grown soybean. Applied N fertilizer in the full N treatment was determined based on (1) a site-specific simulated yield potential of each environment, (2) a soybean N requirement (80 kg N per Mg), and (3) a N fertilizer recovery efficiency of 70-90% depending upon site. N input from fixation and soil organic matter mineralization were ignored in these calculations because of the 'trade-off' between N fertilizer application and N fixation and uncertainty in N supply from mineralization. To ensure proper synchronization between N supply and crop N demand during the entire growing season, the total amount of N fertilizer was split in five times, and proportionally adjusted to the expected accumulated crop N uptake at each stage: 10% at V2, 10% at V4, 20% R1, 30% at R3, and 30% at R5 † (Bender et al., 2015). Seed yield, protein and oil concentration, aboveground dry matter at physiological maturity (ADM), harvest index (HI), and seed number and weight were analyzed across treatments and environments using a combine analysis of variance, where each environment was defined as a combination of location x year x sowing date x variety.

## RESULTS AND DISCUSSION

On average, seed yield was 11% higher in full-N versus zero-N treatment across environments (P<0.001; Fig. 1a). However, the yield difference varied from *nil* at low-yield environments (2.5 Mg ha⁻<fnref n="1" />) to up to 900 kg ha⁻<fnref n="1" /> in high-yield environments (>6 Mg ha⁻<fnref n="1" />). Indeed, the ANOVA revealed that environment x N interaction on seed yield was significant (P=0.025). This finding suggests that N limitation in soybean increases with increasing yield level of the

<footnote n="1">¹University of Nebraska-Lincoln, USA; ²Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina</footnote>
production environment. The yield differences were attributable to an increased seed number per unit of area and a greater seed weight in the full-N versus zero-N treatment (P<0.001). The full-N treatment also produced (9%) higher ADM (P<0.001) without consistent differences in HI between N treatments (P=0.41). Full-N treatment exhibited (1.3%) higher protein (but not oil) concentration (Fig. 1b).

<figure><img src="image_96.png" /><figcaption>Figure 1. (a) Seed yield in full-N versus zero-N treatments. Each data point represents average yield for a given year x sowing date x variety (Argentina) or producer field (Nebraska, USA). Solid diagonal line indicates y = x. Inset shows the seed yield difference between the full-N and zero-N treatments relative to the full-N treatment yield. Also shown is the slope of the fitted linear regression (solid line; y = 0.2x - 0.46) and the slopes representative of the boundary functions for the 10th and 90th quantiles (dashed lines). (b) Seed protein concentration in full-N versus zero-N treatments. Seed yield and protein concentration values are reported at 13% seed moisture content basis.</figcaption></figure>

The increase in both seed yield and seed protein concentration in the full-N indicates that there is a gap between indigenous N supply (from mineralization and fixation) and crop N requirements in soybean, and that gap becomes larger in accordance with the increasing N demand needed to support higher yields. Our results also suggest that N supply will likely become (if not already) a major yield-limiting factor in high-yield soybean production systems as producer yields in those systems continue to fine-tune their agronomic practices and adopt higher yielding cultivars. This is the already the case of irrigated soybean systems in USA where producer routinely obtained yields > 4.5 Mg ha⁻¹.

## CONCLUSION

The fertilization protocol designed here allowed us to evaluate the degree of N limitation in soybean across a wide range of yield levels. Findings indicate that N limits soybean yield (as well as protein concentration) in high-yield environments where indigenous N sources seem insufficient to fully satisfy crop N requirements. Future research should be directed to increase N fixation and find agronomic practices that 'break' the trade-off between N fertilizer input and N fixation in high-yield environments.

## REFERENCES

* Bender R. R., Haegele J. W., Below F. E., 2015. Nutrient Uptake, Partitioning, and Remobilization in Modern Soybean Varieties. *Agronomy Journal* 107, 563-573.

* Cafaro La Menza N., Monzon J. P., Specht J. E., Grassini P., 2017. Is soybean yield limited by nitrogen supply? *Field Crops Research* 213, 204-212.

* Salvagiotti F., Cassman K. G., Specht J. E., Walters D. T., Weiss A., Dobermann A., 2008. Nitrogen uptake, fixation and response to fertilizer N in soybeans: A review. *Field Crops Research* 108, 1-13.
# SIMULATION OF LONG TERM C & N DYNAMICS IN A NORTHERN AGRO-ECOSYSTEM WITH DNDC

JÉGO, G.¹, CRÉPEAU, M.¹, CHANTIGNY, M.¹, D'AMOURS, E.¹, SMITH, W.², GRANT, B.², LAFOND, J.¹, ANGERS, D.¹

¹ Agriculture and Agri-Food Canada (AAFC), Quebec Research Centre, Canada; ² AAFC, Ottawa Research Centre, Canada

## INTRODUCTION

Tillage, fertilizer and cropping systems can affect the nutrient dynamics in crops and soils. For example, following long-term (21 years) liquid dairy manure application, a higher accumulation of soil organic carbon (SOC) stocks was observed in a spring barley - perennial forage (red clover - timothy) rotation than in a spring barley monoculture (Maillard et al., 2016). Yang et al. (2016) observed a higher increase of soil C and N content after 7 years of organic manure amendments compared to mineral fertilization in crop rotation of maize-soybeans. However, under northern climate conditions, reduced tillage or no-till management had insignificant impacts on SOC stocks (Angers et al. 1997). Most of these previous studies focused on C or N cycles of the cropping systems, but rarely both. Monitoring of the complete C and N cycling under long-term experiments can be expensive. Process-based models offer a cost-effective way to supplement field studies. However, the performance of these models needs to be verified with experimental data. Dutta et al., 2017 evaluated the ability of the Canadian DNDC version (DNDCv.Can) and DayCent models to simulate the long term N dynamics in mineral fertilized spring wheat systems in Saskatchewan, but the ability of process-based models, such as DNDC, to simulate the long term effect of organic fertilization on C and N fluxes and balances in crop rotations including perennials was never evaluated under the cold continental humid climate of Eastern Canada.

The objectives of this work were to: (1) evaluate the ability of the DNDCv.Can model to simulate the C and N exported in harvested crops over 21 years of a barley monoculture or a perennial grass - barley rotation fertilized with either mineral, or organic fertilization, and tilled with either a conventional moldboard plow or a chisel plow under conditions in eastern Canada and (2) evaluate the models ability to reproduce the differences in SOC and total soil nitrogen (TSN) between treatments at the end of the 21 years experiment.

## MATERIAL AND METHODS

The long-term study site was located at the Normandin Research Farm of Agriculture and Agri-Food Canada (48°50'42"N, 72°32'25"W), in Québec, Canada. The soil and site characteristics as well as crop management were described in detail in Maillard et al. (2016). In summary, the silty clay soil had a concentration of 490 g kg⁻¹ clay and 80 g kg⁻¹ sand with a pH-water of 5.6, an initial SOC content of 2.6%, a field capacity of 0.83 wfps (water filled pore space), a wilting point of 0.59 wfps, a hydro-conductivity of 0.015 cm min⁻¹ and a porosity of 48.7%. The region has a cold continental humid climate with mean annual temperature and precipitation of 1.1°C and 849mm, respectively. Field management consisted of either a spring barley monoculture (M), or a 3-year cereal-grass rotation (R) (one year of spring barley under-seeded with a forage mixture of timothy - red clover and two years of the forage mixture production). A combination of two fertilizations and two tillage management practices were studied (table 1). Experimental trials were performed for 21 years from 1990 to 2010. Yield and C and N content of harvested crops were measured each year. Soils samples at different depths (0 to 50 cm) were taken in 2010 to measure the SOC and TSN stocks.

The DeNitrification-DeComposition model (DNDC) is a process-based model that describes in detail the biochemical cycle of C and N in the agroecosystems. It can predict crop growth, N fixation, SOC, soil organic and inorganic N dynamics, N runoff and leaching as well as the flux of CO₂, CH₄, N₂O, NO and NH₃ from the crop-soil system (Giltrap et al., 2010). The Canadian version of DNDC (DNDCv.CAN) was developed to simulate the biogeochemical cycling under cold weather conditions (Dutta et al., 2016). Daily climatic data used for the simulations (maximum and minimum temperature, precipitation, wind speed, solar radiation and humidity) were
measured at a nearby weather station (Bagotville, QC [48°20' N, 71°00' O]). Simulations were run from 1990 to 2010. Model performance was evaluated by comparing measured and simulated C and N exported in harvests and measured and simulated difference of SOC (0-20 cm and 0-50 cm) and TSN (0-50 cm) in 2010 relative to the M-MP-MIN treatment (table 1).

<table><caption>Table 1. Field experiments and treatments used for calibration and validation of the model.</caption><thead><tr><th colspan="4">Grass mixture – barley rotation (R)</th><th colspan="4">Barley monoculture (M)</th></tr><tr><th colspan="2">Moldboard (MP)</th><th colspan="2">Chisel (CP)</th><th colspan="2">Moldboard (MP)</th><th colspan="2">Chisel (CP)</th></tr></thead><tbody><tr><td>MIN<sup>a</sup></td><td>LDM<sup>b</sup></td><td>MIN<sup>a</sup></td><td>LDM<sup>b</sup></td><td>MIN<sup>a</sup></td><td>LDM<sup>b</sup></td><td>MIN<sup>a</sup></td><td>LDM<sup>b</sup></td></tr><tr><td>R-MP-MIN</td><td>R-MP-LDM</td><td>R-CP-MIN</td><td>R-CP-LDM</td><td>M-MP-MIN</td><td>M-MP-LDM</td><td>M-CP-MIN</td><td>M-CP-LDM</td></tr><tr><td>Validation</td><td>Validation</td><td>Not simulated</td><td>Not simulated</td><td>Calibration</td><td>Validation</td><td>Validation</td><td>Validation</td></tr></tbody></table>

## RESULTS AND DISCUSSION

After calibration of the potential crop growth parameters, model performance in simulating C and N exported in harvest was generally good for the validation treatments. The N exports were better simulated than the C exports with normalized root mean square errors (NRMSE) of 16.7% and 22.8% for N and C, respectively. The bias was small for the two variables with normalized mean errors (NME) below 2% in both cases. On average the model tended to slightly overestimate crop yields and C and N exports of the treatments fertilized with mineral N and to slightly underestimate the yield of the LDM treatments. However, while the model was able to reproduce fairly well the difference of SOC over the 0-20 cm layer, except for the R-MP-MIN treatment (NRMSE = 40% and EF = 0.82 without this treatment), it generally failed in predicting the difference between treatments over the 0-50 cm layer (NRMSE ≥ 70% and EF < 0). The model was generally able to capture the greater SOC and TSN accumulation in the forage rotation than in the monoculture and in the LDM treatment than with MIN fertilizer, but it failed in simulating the impact of tillage, with a systematic underestimation of SOC accumulation under MP treatments. Simulation of the complete C and N balances shows that reactive N losses were small for all treatments (< 20 kg N ha⁻¹). Treatments fertilized with LDM tended to have greater simulated N losses than mineral N treatments because of greater ammonia emissions. Simulated nitrate leaching was, however, generally higher in mineral N treatments than in treatments fertilized with LDM.

These results confirm that more effort should be put in better understanding and simulating the C and N dynamics in deeper soil layers. Model improvements, such as a better description of SOC profile at model initialization, which is currently under development in DNDC, could represent a good step forward in better simulating these dynamics in deep soil layers.

## REFERENCES

* Angers D.A., Bolinder M.A., Carter M.R., Gregorich E.G., Drury C.F., Liang B.C., Voroney R.P., Simard R.R., Donald R.G., Beyaert R.P., Martel J., 1997. Impact of tillage practices on organic carbon and nitrogen storage in cool, humid soils of eastern Canada. Soil and Tillage Research 41(3): 191-201.

* Giltrap D. L., Li C., Saggar S., 2010. DNDC: A process-based model of greenhouse gas fluxes from agricultural soils. Agriculture, Ecosystems & Environment, 136(3-4), 292-300.

* Maillard É., Angers D.A., Chantigny M., Lafond J., Pageau D., Rochette P., Lévesque G., Leclerc M.-L., Parent L.-É., 2016. Greater accumulation of soil organic carbon after liquid dairy manure application under cereal-forage rotation than cereal monoculture. Agriculture, Ecosystems & Environment 233: 171-178.

* Yang R., Su Y.-Z., Wang T., Yang Q., 2016. Effect of chemical and organic fertilization on soil carbon and nitrogen accumulation in a newly cultivated farmland. Journal of Integrative Agriculture, 15(3), 658-666.

* Dutta B., Congreves K.A., Smith W.N., Grant B.B., Rochette P., Chantigny M.H., Desjardins R.L., 2016. Improving DNDC model to estimate ammonia loss from urea fertilizer application in temperate agroecosystems. Nutr. Cycl. Agroecosyst. 106(3): 275-292.

<footnote>a mineral fertilization / b liquid dairy manure fertilization</footnote>
# DIMETHYL PYRAZOL-BASED NITRIFICATION INHIBITORS EFFECT ON N CYCLE BACTERIA RESPONSIBLE OF N₂O EMISSIONS IN GRASSLAND

FUERTES-MENDIZABAL, T., HUERFANO, X., TORRALBO, F., VEGA-MAS, I., MENENDEZ, S., GONZALEZ-MURUA, C., ESTAVILLO, J.M.

University of the Basque Country UPV/EHU, Dept. Plant Biology and Ecology, Apd. 644, Bilbao, Bizkaia 48080, Spain

## INTRODUCTION

Intensive N fertilization of agricultural soils is by far the main anthropogenic source of N₂O emissions. Soil N₂O emissions are mainly the result of the biological processes of nitrification and denitrification. Although both processes occur simultaneously under most soil conditions, being the net flux to the atmosphere the result of both processes together, nitrification is the preferential source of N₂O under more aerated soils (water filled pore space WFPS < 60%) and denitrification is the dominant process in soils with WFPS of ≥ 70% (Bateman and Baggs, 2005). Among the strategies to mitigate N losses from agriculture the Intergovernmental Panel on Climate Change (IPPC) has proposed the application of ammonium-based fertilizers combined with nitrification inhibitors (NI). NIs reduce nitrification rates (blocking ammonia monooxygenase enzyme, AMO) and subsequent denitrification rates due to reduction of NO₃⁻ availability. Recent studies under laboratory conditions (Barrena et al., 2017, Torralbo et al., 2017) showed an effect of dimethyl pyrazole-based NIs also stimulating the last step of the denitrification process that consists of the reduction of N₂O to N₂, increasing nosZ gene abundance under high WFPS conditions. However, the effect of NIs decreasing N₂O emissions depends on many environmental factors such as pH, temperature and soil water content. The objective of this study was to compare the effect of 3,4-dimethylpyrazole succinic acid (DMPSA) with respect to 3,4-dimethylpyrazole phosphate (DMPP) on soil bacterial populations responsible of N₂O emissions in a grassland under Atlantic climate conditions.

## MATERIALS AND METHODS

The work was conducted in a grassland at the Basque Country sown with ryegrass (*Lolium multiflorum* Lam. var Westerwold starter) in autumn 2016. A randomized complete block design with four replicates was established with an individual plot size of 28 m². Four treatments were applied: an unfertilized control, ammonium sulphate (AS) and two treatments consisting in the combination of AS with the DMPSA and with DMPP, respectively. The N rate applied was of 80 kg N ha⁻¹ at 27th January and 60 kg N ha⁻¹ at 29th March. Soil samples were collected 15, 30 and 60 days after each fertilization event to quantify bacterial populations. Quantification of bacteria abundances (16S rRNA) and functional marker genes involved on nitrification (*amoA*) and denitrification (*nosZI*) were amplified by qPCR. Standard curves were prepared from serial dilutions of linearized plasmids with insertions of the target genes according to Torralbo et al. (2017). After test normality and homogeneity of variances, significant differences were analysed using one-way ANOVA with a Duncan post hoc test.

## RESULTS AND DISCUSSION

After the application of the first fertilizer amendment, winter rainfall allowed to maintain WFPS at values ≥60%. After the second amendment, values of soil WFPS were ≤60%. Whatever the soil WFPS, both DMPP and DMPSA avoided the ammonia oxidizer bacteria stimulation associated to AS amendment (Fig. 1), without disturbing bacteria abundance measured by means of 16S rRNA gene abundance (data not shown). Interestingly, both NIs induced a rise of N₂O-reducing bacteria at the end of the first period, 60 days after the application of 80 kg N ha⁻¹ of AS combined with NIs. This suggests that after certain period of time under conditions of WFPS favouring the denitrification process, both NIs can promote the denitrification process up to N₂.
<figure><img src="image_97.png" /></figure>

Figure 1. Abundance of amoA (top) and nosZ (bottom) genes expressed as gene copy number per gram of dry soil at 15, 30 and 60 days after fertilizations. Below each date values of WFPS are shown. Values represent the mean ± SE (n = 4). White bars = unfertilized control; black bars = AS, dark grey bars = AS+DMPP and light grey bars = AS+DMPSA. Treatments sharing the same letter within each day do not differ significantly at p≤0.05.

## CONCLUSION

Our data demonstrate under field conditions that DMPP and DMPSA have the potential to diminish N₂O emissions playing a dual role: inhibiting the nitrification process due to a decrease in ammonia oxidizer bacterial population whatever the soil WFPS values, and also increasing nosZ abundance and therefore potentially stimulating the denitrification process up to N₂ under high soil water content conditions.

**Acknowledgements:** Spanish Government (AGL2015-64582-C3-2-R MINECO/FEDER), by the Basque Government (IT-932-16) and by EuroChem Agro Iberia S.L.-UPV/EHU 2015.0248 and 2016.0339.

## REFERENCES

- Bateman, E.J., Baggs, E.M., 2005. Contributions of nitrification and denitrification to N₂O emissions from soils at different water-filled pore space. Biology and Fertility of Soils, 41, 379-388.

- Barrena, I., Menéndez, S., Correa-Galeote, D., Vega-Mas, I., Bedmar, E.J., González-Murua, C., Estavillo, J.M., 2017. Soil water content modulates the effect of the nitrification inhibitor 3,4-dimethylpyrazole phosphate (DMPP) on nitrifying and denitrifying bacteria. Geoderma 303: 1-8.

- Torralbo, F., Menéndez, S., Barrena, I., Estavillo, J.M., Marino, D., González-Murua, C., 2017. Dimethyl pyrazole-based nitrification inhibitors effect on nitrifying and denitrifying bacteria to mitigate N₂O emission. Scientific Reports, 7: 13810.
## 3,4-DIMETHYLPYRAZOLE-SUCCINIC ACID (DMPSA) NITRIFICATION INHIBITOR EFFECTIVELY REDUCES N₂O EMISSIONS IN A WINTER WHEAT CROP UNDER NO-TILLAGE MANAGEMENT

CORROCHANO-MONSALVE, M., HUÉRFANO, X., FUERTES-MENDIZABAL, T., ESTAVILLO, J.M., GONZÁLEZ-MURUA, C.

University of the Basque Country UPV/EHU, Dept. Plant Biology and Ecology, Apd. 644, Bilbao, Bizkaia 48080, Spain

### INTRODUCTION

Conventional tillage (CT) managed soils are vulnerable to nitrate leaching losses and water evaporation. No-tillage (NT) management is a way not only to retain water and nutrients in the soil surface layers but also to reduce fuel cost and fuel associated CO₂ greenhouse gas (GHG) emissions. Apart from the nitrate lost by leaching, the N applied with fertilizers can also be lost as the GHG nitrous oxide (N₂O) by means of the bacterial processes of nitrification and denitrification. Nitrification inhibitors have been developed in order to slow down the nitrification processes and consequently increase the N use efficiency. In this work, we evaluate in a winter wheat crop the effect of two different land managements (CT and NT) and the use of the NI 3,4-dimethylpyrazole-succinic acid (DMPSA) in N₂O emissions.

### MATERIALS AND METHODS

The work was conducted in a winter wheat (Triticum aestivum L., var. Cezanne) crop in Arkaute, northern Spain. To compare CT and NT managements two randomized complete blocks designs were established with four replicates and an individual plot size of 40m². Seedbed preparation consisted in mechanical tillage (disk and moldboard plow) for CT and direct sowing for NT. Wheat was sown at density of 220 kg seeds ha⁻¹ and previous crop was also winter wheat. Within each design, three treatments were applied: a control unfertilized treatment, a second treatment fertilized with ammonium sulphate 21% (AS) and a third one consisting of AS 21% combined with DMPSA (AS+DMPSA). Fertilization rate was 180 kg N ha⁻¹ applied either in a single application at beginning of tillering stage (GS21) or split in two applications of 60 kg N ha⁻¹ at beginning of tillering stage (GS21) and 120 kg N ha⁻¹ at stem elongation stage (GS30). N₂O emissions were measured using the close chamber method (Chadwick et al., 2014). Air samples were analyzed by gas chromatography (Agilent, 7890A) equipped with an electron capture detector for N₂O detection. Data were analysed using the IBM SPSS statistics 24 software (Armonk, NY, USA). Analysis of significant differences in N₂O cumulative emissions were carried out using one-way ANOVA with a Duncan post hoc test and T-student tests between AS and AS+DMPSA treatments. Significant differences were conducted at *p* < 0.05.

### RESULTS AND DISCUSSION

After first fertilization, the effect of AS application on N₂O emissions was clearer in NT than in CT probably due to the higher soil water filled pore space (WFPS) values, which were above 60% in NT (Fig. 1). These higher WFPS conditions enable not only nitrification to occur but also denitrification, thus increasing N₂O emissions (del Prado et al., 2006). After second fertilization, however, WFPS was below 60% in both managements (Fig. 1), which provided similar conditions for nitrification to occur in both CT and NT. Under these similar WFPS conditions, the control treatment showed that the tillage management induced a great increase in N₂O emissions, while higher emissions after the second fertilization than in the first were attributable to the warmer spring conditions. In CT, this higher basal emission rate induced by tillage masked the fertilizer effect on N₂O emissions, and so the effect of DMPSA reducing these emissions. On the other hand, the effect of DMPSA was clearly observed under NT conditions, being able to reduce N₂O emissions up to the unfertilized control levels (Table 1).
<figure><img src="image_98.png" /></figure>

Figure 1. N₂O emissions by management (Conventional tillage, black; No tillage, grey) and by periods (after 1st fertilization, and after 2nd fertilization). Days in brackets represent the period length. Arrows show significant differences between managements in each treatment, with percentage change above. Values in drops represent average WFPS value for each period and each management.

<table><caption>Table 1. Cumulative N₂O emissions, from day 1 to day 187 and under single (180+0) or split (60+120) fertilization. Values in brackets are the percentage of decrease of each NI with respect to AS and statistical significance. Different letters within a column indicate significant differences (P < 0.05; n = 4).</caption><thead><tr><th rowspan="2">Fertilizer dose (kg N ha<sup>-1</sup>)</th><th rowspan="2">Treatment</th><th colspan="2">g N<sub>2</sub>O-N ha<sup>-1</sup></th></tr><tr><th>Conventional tillage</th><th>No tillage</th></tr></thead><tbody><tr><td rowspan="3">180+0</td><td>Control</td><td>358,36 a</td><td>121,15 b</td></tr><tr><td>AS</td><td>368,79 a</td><td>330,45 a</td></tr><tr><td>AS + DMPSA</td><td>324,12 a (-12%) (n.s.)</td><td>164,2 b (-50%) (0.001)</td></tr><tr><td rowspan="3">60+120</td><td>Control</td><td>358,36 A</td><td>121,15 B</td></tr><tr><td>AS</td><td>408,63 A</td><td>383,31 A</td></tr><tr><td>AS + DMPSA</td><td>363,86 A (-11%) (n.s.)</td><td>166,72 B (-65%) (0.003)</td></tr></tbody></table>

## CONCLUSION

Under humid Mediterranean conditions, the tillage management induces an increase in N₂O emissions, which masks the fertilizer and DMPSA effects on them. Under no-tillage conditions, where N₂O shows a clear response to fertilization, DMPSA significantly reduce these emissions up to the unfertilized control level in both single and split fertilizations.

**Acknowledgements:** This project was funded by the Spanish Government (AGL2015-64582-C3-2-R MINECO/FEDER), by the Basque Government (IT-932-16) and by EuroChem Agro Iberia S.L.-UPV/EHU (2015.0033).

## REFERENCES

del Prado, A., Merino, P., Estavillo, J.M., Pinto, M., González-Murua, C., 2006. N range ofsoil water contents. Nutr. Cycling Agroecosyst. 74, 229–243.

Chadwick, D.R., Cardenas, L., Misselbrook, T.H., Smith, K.A., Rees, R.-M., Watson, C.J., McGeough, K.L., Williams,
J.R., Cloy, J.M., Thorman, R.E., Dhanoa, M.S., 2014. Optimizing chamber methods for measuring nitrous oxide emissions from plot-based agricultural experiments. Eur. J. Soil Sci. 65 (2), 295–307.
# EFFECT OF N FERTILIZATION RATE AND SOIL TILLAGE ON N₂O EMISSIONS FROM IRRIGATED CORN IN A MEDITERRANEAN AGROECOSYSTEM

PAREJA-SÁNCHEZ, E.<fnref n="1" />, PLAZA-BONILLA, D.², ÁLVARO-FUENTES, J.², CANTERO-MARTÍNEZ C.<fnref n="1" />

## INTRODUCTION

Most of the nitrous oxide (N₂O) emissions take place in soils and are related with agricultural activities. An adequate choice of agricultural management practices such as nitrogen (N) fertilization or tillage can help to reduce the emission of N₂O from soils, mitigating global warming, without necessarily reducing crop yields. The aim of this work was to quantify the emission of N₂O from soils and its relationship with corn yields according to different soil management practices and synthetic N fertilization rates and to analyze the impact of environmental variables on the emissions in a Mediterranean semiarid area recently transformed to irrigation.

## MATERIAL AND METHODS

The study was carried out in NE Spain (mean annual precipitation and potential evapotranspiration: 430 and 855 mm, respectively), in a long-term tillage and N rate experiment (established in 1996), which was transformed into irrigation by installing a fixed sprinkler system with corn (Zea mays L.) as cropping system in 2015. Previously, the cropping system consisted of a rainfed barley monoculture (1996-2014 period). Three types of tillage (no-tillage, NT; reduced tillage, RT; and conventional intensive tillage, CT) and three mineral N fertilization rates (0, 200, 400 kg N ha⁻<fnref n="1" />) were compared. In the NT treatment, non-selective herbicides were applied for weed control. The RT treatment consisted of one pass of a strip-till implement on the sowing line to 30 cm depth. Finally, the CT treatment consisted of one pass of a rototiller to 15 cm depth followed by a subsoiler to 35 cm depth and finished by one pass of a disc plough to 20 cm depth, according to the traditional practice for corn cultivation in the area. Nitrogen fertilizer applications were split into one pre-sowing as urea and two top-dressing applications at V5 and V10 as ammonium nitrate. The experiment was laid out in a randomized block design with three replications. During two corn growing seasons (from April to November 2015 and 2016) and the two short fallow periods between growing seasons, weekly soil N₂O emissions were measured, being more intense during fertilizer applications (i.e. 24 h. prior and 3 h. and 48h. after) using non-steady state static chambers (Hutchinson and Mosier, 1981). Gas samples were analyzed by a gas chromatography system equipped with an ECD detector. On each sampling date and close to each chamber, soil temperature and volumetric water content of the 0-5 cm depth were also quantified. The water-filled pore space (WFPS) was calculated with soil bulk density, which was quantified monthly. Corn yield was quantified at harvest to estimate yield-scaled N₂O emissions, expressed as kg of CO₂ equivalents emitted per kg of grain produced. For each year, the emission factor (EF) was also quantified.

## RESULTS AND DISCUSSION

Soil N₂O emissions were significantly affected by tillage, N fertilization rate, sampling date and their interactions. The application of 400 kg N ha⁻<fnref n="1" /> led to greater soil N₂O emissions in NT, followed by RT and CT (0.50, 0.31 and 0.23 mg N₂O-N m⁻²d⁻<fnref n="1" />, respectively as an average of the different sampling dates covered by the experiment). The use of 200 kg N ha⁻<fnref n="1" /> led to similar N₂O emissions in RT (0.16 mg N₂O-N m⁻²d⁻<fnref n="1" />) and CT (0.03 mg N₂O-N m⁻²d⁻<fnref n="1" />), and showed greater values under NT (0.28 mg N₂O-N m⁻²d⁻<fnref n="1" />). In 2015, the interaction between tillage and N fertilization rates significantly affected cumulative soil N₂O emissions, with lowest values at the rates of 200 and 400 kg N ha⁻<fnref n="1" /> under CT. The highest N rate increased soil N₂O emissions especially just after the application of fertilizer, independently of the tillage system. However, cumulative soil N₂O emissions in 2016 were only affected by N fertilization, with greatest values at rates of 200 and 400 kg N ha⁻<fnref n="1" />. The WFPS was significantly affected by tillage and N fertilization treatments, decreasing in the order NT > RT > CT for tillage and 400 > 200 > 0 kg N ha⁻<fnref n="1" /> for N fertilization, in both years. No-tillage presented greater WFPS than CT at most sampling dates, with mean values

<footnote n="1">¹ Departamento de Producción Vegetal y Ciencia Forestal, Unidad Asociada CSIC, Agrotecnio. Universidad de Lleida, 25198 Lleida, Spain; ² Departamento de Suelo y Agua. Estación Experimental de Aula Dei (EEAD-CSIC), 50056 Zaragoza, Spain.</footnote>
60.1 and 35.8 % for NT and CT, respectively. In 2015, grain yield was significantly affected by tillage and N fertilization, with greater grain yield under NT and RT (11406 and 9547 kg ha⁻¹, respectively) compared with CT (5594 kg ha⁻¹) and under the rates of 200 and 400 kg N ha⁻¹ (10394 and 8447 kg ha⁻¹) compared to the control without N (7705 kg ha⁻¹). However, grain yield was significantly affected by the interaction between tillage and N fertilization in 2016. Differences between tillage systems were only observed when applying 400 kg N ha⁻¹, with greater grain yields in NT and RT than in CT (12242, 8839 and 4260 kg ha⁻¹ respectively). The lower values of WFPS and reduction in grain yield for CT are a consequence of the lower soil structural stability under long-term CT, which would have reduced water infiltration due to soil crusting (Pareja-Sánchez et al., 2017). Therefore, soil degradation as a result of long-term use of intensive tillage caused a severe water stress to corn, with a negative influence on grain yields. Nitrogen rates significantly affected N₂O yield-scaled emissions in 2015, with values increasing with the N fertilizer rate: 0.002, 0.011 and 0.035 kg CO₂ equiv. kg grain⁻¹ for the 0, 200 and 400 kg N ha⁻¹ treatments, respectively, with no differences between treatments in 2016. In 2015, the greatest EF was observed when applying 400 kg N ha⁻¹ under NT. In 2016 the EF increased in all treatments, with highest values for NT and CT (Table 1).

<table><caption>Table 1. Soil N₂O emission factor (EF) in 2015 and 2016 as affected by N fertilization rate (200 and 400 kg N ha⁻¹) and tillage system (NT, no-tillage; RT, reduced tillage; CT, conventional intensive tillage).</caption><thead><tr><th rowspan="2">Year</th><th rowspan="2">Tillage treatment</th><th>EF (%)</th><th>EF (%)</th></tr><tr><th>200 kg N ha<sup>-1</sup></th><th>400 kg N ha<sup>-1</sup></th></tr></thead><tbody><tr><td rowspan="3">2015</td><td>NT</td><td>0.16</td><td>0.22</td></tr><tr><td>RT</td><td>0.07</td><td>0.14</td></tr><tr><td>CT</td><td>0.09</td><td>0.09</td></tr><tr><td rowspan="3">2016</td><td>NT</td><td>0.22</td><td>0.21</td></tr><tr><td>RT</td><td>0.12</td><td>0.12</td></tr><tr><td>CT</td><td>0.33</td><td>0.21</td></tr></tbody></table>

## CONCLUSION

In the Mediterranean irrigated corn system studied, NT led to higher emissions of N₂O from soils to the atmosphere than RT and CT. An increase in the fertilizer N application rate increased N₂O emissions, especially just after the application of N fertilizer. However, NT and RT increased the grain production compared to CT due to severe soil crusting causing water deficit. Nitrogen fertilizer treatments significantly affected the yield-scaled N₂O emissions in 2015, increasing with increasing fertilizer N application rate. Independently of the treatment, the EF was much lower than the 1% factor currently proposed by the IPCC. The results presented highlight that N fertilization and tillage play an important role on N₂O emissions and on corn production in irrigated Mediterranean agroecosystems prone to soil degradation.

**Acknowledgements:** This research work is financially supported by the Spanish Ministry of Education and Science (project AGL2013-49062-C4-1-R and grant BES-2014-070039).

## REFERENCES

* Hutchinson G.L., Mosier A.R., 1981. Improved soil cover method for field measure-ment of nitrous oxide fluxes. Soil Sci. Soc. Am. J., 45, 311–316.

* Pareja-Sánchez E., Plaza-Bonilla D., Ramos M.C., Lampurlanés J., Álvaro-Fuentes J., Cantero-Martínez C., 2017. Long term no-till as a means to maintain soil surface structure in an agroecosystem transformed into irrigation. Soil Tillage Res. 174, 221–230.
# EVALUATION OF THE SENSITIVITY OF PORTABLE CHLOROPHYLL METERS TO ESTIMATE LEAF CHLOROPHYLL AND N CONTENTS UNDER EXCESSIVE N CONDITIONS

PADILLA, F.M.<fnref n="1" />, PEÑA-FLEITAS, M.T.<fnref n="1" />, DE SOUZA, R.<fnref n="1" />, GIMÉNEZ, C.², THOMPSON, R.B.<fnref n="1" />, GALLARDO, M.<fnref n="1" />

## INTRODUCTION

Chlorophyll meters (CMs) are a promising approach for monitoring crop nitrogen (N) status of intensively-produced vegetable crops. To do so effectively, it is fundamental that the nature and strength of the relationships between CMs measurements and actual chlorophyll and nitrogen contents be evaluated. Particularly, little is known about these relationships when expanding the range to excessive N conditions. Given that excessive N application is a common occurrence in intensive vegetable crops, the ability of CMs to retain sensitivity under excessive crop N status is an important practical consideration. The objectives of this work were to evaluate the sensitivity of different CMs to estimate leaf chlorophyll and N contents under excessive N conditions.

## MATERIAL AND METHODS

The SPAD-502 meter (Konica Minolta, Inc., Tokyo, Japan), the atLEAF+ sensor (FT Green LLC, Wilmington, DE, USA), the MC-100 Chlorophyll Concentration Meter (Apogee Instruments, Inc., Logan UT, USA), and the Multiplex 3.6 sensor (Force-A, Orsay, France), were evaluated in a sweet pepper (*Capsicum annuum* 'Melchor') crop in Almeria, southeastern Spain. There were five N treatments of different N concentrations (very deficient, deficient, optimal, excessive and very excessive) in nutrient solutions applied by fertigation.

CMs measurements commenced on 15 September 2016 and were repeated every two weeks, on average, until 12 December 2016, for a total of six measurement dates. Measurements were always made at the same time each day (07:00 to 09:00 solar time). Individual CM measurements with each sensor were made on 6 different marked plants in each of four replicated plots of the five N treatments. One measurement per plant was made on a fully expanded and well-lit leaf, on the distal part of the adaxial (top) side of the leaf, midway between the margin and the mid-rib of the leaf. The same leaf and spot was measured with the four CMs.

Immediately after CMs measurements, each leaf was excised and a leaf disk (5.6 mm diameter) was taken in the center of the measuring spot using a metal ring, after which the leaf disk was sealed in a zip plastic bag and frozen. Chlorophyll was extracted with 80% aqueous acetone solvent; the concentration of chlorophyll a+b was calculated using the equations described by Porra et al. (1989) from absorbance at 646.5 nm and 663.5 nm. The rest of the sampled leaf was enclosed in paper bag and oven dried at 65°C until constant weight. The leaf N content (%) was determined using a Dumas-type elemental analyzer system.

Data collected on the six dates of measurement were pooled and regression analyses were conducted to evaluate the nature and the strength of the relationships between: 1) chlorophyll content and CMs measurements, 2) leaf N content and chlorophyll content, and 3) leaf N content and CMs measurements. Linear, quadratic, power, exponential and natural logarithmic regressions were considered, and the best was selected using the Akaike Information Criterion. The CurveExpert Professional® 2.6.0 software was used.

## RESULTS AND DISCUSSION

Chlorophyll content was positively and strongly related to all CMs measurements with curvilinear-like relationships (Figure 1). The relationships were quadratic for SPAD units, atLEAF units, and the Simple Fluorescence Ratio (SFR_R) index measured with the Multiplex sensor; a power relationship was found for the Chlorophyll Content Index (CCI) measured with the MC-100 Chlorophyll Concentration Meter. There was a

<footnote n="1">¹University of Almeria, Almeria, Spain; ²University of Cordoba, Cordoba, Spain</footnote>
tendency of saturation or a plateau of SPAD, atLEAF and SFR_R values at higher chlorophyll content; notably, there was no indication of saturation of CCI values over the entire range of chlorophyll measured (Figure 1).

<figure><img src="image_99.png" /><figcaption>Figure 1. Relationships between chlorophyll and different CM measurements. SFR_R is Simple Fluorescence Ratio under red excitation, and CCI is Chlorophyll Content Index.</figcaption></figure>

The CCI estimated with most accuracy, in terms of higher coefficient of determination, chlorophyll content, followed by the SPAD-502 meter and the SFR_R index (Table 1). Measurements of the atLEAF+ sensor estimated with slightly less precision chlorophyll content.

<table><caption>Table 1. Equations to estimate chlorophyll content (µg cm⁻²) from measurements with different CMs. SFR_R is Simple Fluorescence Ratio under red excitation, and CCI is Chlorophyll Content Index.</caption><thead><tr><td>Chlorophyll meter</td><td>Equation</td></tr></thead><tbody><tr><td>SPAD-502</td><td>Chl = -9.87 + 0.99 x SPAD + 0.002 x SPAD<sup>2</sup></td></tr><tr><td>atLEAF+</td><td>Chl = 0.08 x atLEAF<sup>1.63</sup></td></tr><tr><td>MC-100</td><td>Chl = 7.95 + 1.03 x CCI - 0.005 x CCI<sup>2</sup></td></tr><tr><td>Multiplex</td><td>Chl = -8.36 + 29.35 x SFR_R</td></tr></tbody></table>

## CONCLUSION

Overall, the strong relationships obtained between all four CMs and chlorophyll content confirm that these CMs can be used as non-destructive indicators of leaf chlorophyll content in sweet pepper. However, under excessive N conditions, it is suitable the use of CMs that do not exhibit a plateau response to high leaf chlorophyll and N contents. The MC-100 Chlorophyll Concentration Meter would have an advantage over the rest of CMs evaluated in this study. For non-saturating leaf chlorophyll and N contents, all four CMs evaluated are of value, but the MC-100 Chlorophyll Concentration Index and the SPAD-502 meters provided the most accurate estimation of leaf chlorophyll content. On-going analysis of leaf N content will complement the evaluation of CMs to estimate leaf N content under excessive N conditions.

**Acknowledgements:** The Spanish Ministry of Economy and Competitiveness funded this work (Project AGL2015-67076-R). FMP was supported by a Ramón y Cajal grant (RYC-2014-15815).

## REFERENCES

* Porra, R.J., Thompson, W.A. and Kriedemann, P.E., 1989. Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. BBA - Bioenergetics, 975(3): 384-394.
# LEAF NITROGEN AND δ¹⁵N FRACTIONATION DURING RESORPTION IN BEECH AND LARCH TREES IN JAPAN

LOPEZ C, M.L.<fnref n="1" />, ENTA, A.<fnref n="1" />, FUJIYOSHI, L.² YAMANAKA, T.³, OIKAWA, A.<fnref n="1" />

## INTRODUCTION

Anthropogenic N available for plants has been increasing due to human activities in recent decades (Yuan et al., 2005) and therefore it is necessary to understand its effects on N dynamics and tree internal pathways. N resorption from senescent leaves to plant storage tissues is one of the most important processes. The N stored in plant tissues becomes in addition to the soil inorganic nitrogen another source of nitrogen with a different ¹⁵N (Kolb and Evans 2002). However, until now, very few studies have used ¹⁵N to examine this process (Kolb and Evans 2002; Templer et al., 2007). Thus, the objectives of this study are to determine if fractionation occurs during leaf N resorption of senescing leaves and to evaluate the effect of protein hydrolysis in N isotope discrimination.

## MATERIAL AND METHODS

This study was conducted at the Yamagata University Research Forest (38°33′ N, 139°51′ E). The forest stands are located in the northernmost area of the Asahi Mountains and belong to the cool temperate deciduous broad-leaves zone. In this leaves N concentration and δ¹⁵N and were measured in each of the three leaf growth periods (green leaf, pre-abscission and post-abscission periods). From leaf N content values, N Resorption Efficiency (NRE) and N Resorption Proficiency (NRP) were calculated.

### Leaf protein and amino acid content

Leaves from three individuals at each of the two stands were randomly chosen and analyzed for protein and amino acids content in each of the three leaf growth periods (green leaf, pre-abscission and post-abscission).

## RESULTS AND DISCUSSION

### Nitrogen content, NRE and NRP

The decrease of N content in green to pre-abscission and from pre- to post-abscission leaves in beech trees was 19.8% and 48.8% respectively and significant differences were found in both cases, while in larch, the decrease in N content from green to pre- to post-abscission leaves was 17.5% from and 33.4% respectively. A significant difference was found for the second period but not for the first one. NRE and NRP of beech leaves were 68.7% and 14.8 mg g⁻<fnref n="1" /> while that for larch was 50.9% and 8.6 mg g⁻<fnref n="1" /> respectively (Table 1).

<table><caption>Table 1. Nitrogen concentration in green, pre-and post-abscission leaves of beech and larch tree species. Nitrogen resorption efficiency (NRE) and N resorption proficiency (NRP) were calculated from these values.</caption><thead><tr><th>Tree species</th><th>Green leaf</th><th>Pre abscission<br>Leaf</th><th>Post-<br>abscission<br>Leaf</th><th>NRE<br>(%)</th><th>NRP<br>(mg g<sup>-1</sup>)</th></tr></thead><tbody><tr><td>Beech</td><td>2.2±0.2</td><td>1.7±0.2</td><td>0.7±0.0</td><td>68.7</td><td>14.8</td></tr><tr><td>Larch</td><td>1.7±0.3</td><td>1.4±0.2</td><td>0.8±0.0</td><td>50.9</td><td>8.6</td></tr></tbody></table>

### Leaf δ¹⁵N and protein hydrolysis

Leaf ¹⁵N of green, pre-abscission and post-abscission leaves of beech and larch ranged between -1.5 ‰ to -2.7 ‰ and +0.5 ‰ to -0.9 ‰ respectively. For beech, there was a significant difference (p<0.05) in the ¹⁵N of green and pre-abscission leaves but not in the ¹⁵N of pre- to post-abscission leaves (Fig. 1). Since leaf N resorption occurs simultaneously with protein hydrolysis initially the N reabsorbed is in the form of protein N (Chapin and

<footnote n="1">¹ Faculty of Agriculture, Yamagata University, Japan; ² RIHN, Kyoto, Japan; ³ Okayama University, Okayama, Japan</footnote>
Kedrowski, 1983) and as hydrolysis progressed, indicated by the sharp increase in amino acids in pre-abscission leaves, they become the major N fraction driving leaf ♂15N. Thus, ♂15N fractionation between green and pre-abscission leaves is the withdrawal of isotopically heavier protein N than the lighter amino acids remaining in the leaves. Consequently, the sharp drop of amino acids content from pre- to post abscission leaves indicates that hydrolysis was almost negligible and amino acids was the N fraction that moved back and remained in the post-abscission leaves. The percentage of larch leaf N reabsorbed was also considerable but it appears that hydrolysis was more active as suggested from the higher amino acids content in larch leaves.

<figure><img src="image_100.png" /><figcaption>Figure 1. Mean δ¹⁵N values in August, October and November of (a) beech and (b) larch leaves.</figcaption></figure>

## CONCLUSION

In this study, N fractionation was found during leaf N resorption in beech trees, but not for larch. Protein hydrolysis in beech leaves was the lower as inferred by the lower content of amino acids found in pre-abscission leaves, suggesting that N withdrawn from leaves was in the form of proteins and thus heavier than the δ¹⁵N of the amino acids in the remaining leaves.

**Acknowledgements:** We want to thank the University Forest personnel for their support during the sampling campaigns.

## REFERENCES

* Chapin F.S., Kedrowski R.A., 1983. Seasonal changes in nitrogen and phosphorus fractions and autumn retranslocation in evergreen and deciduous taiga trees. Ecology 64, 376–391.

* Chavez-Vergara B.M., Merino A., Vázquez-Marrufo G., García-Oliva F., 2009. Organic matter dynamics and microbial activity during decomposition of forest floor under two native neotropical oak species in a temperate deciduous forest in Mexico. Geoderma 235–236, 133-145

* Kolb K.J., Evans R.D., 2002. Implications of leaf nitrogen recycling on the nitrogen isotope composition of deciduous plant tissues. New phytologist 156 (1), 57-64

* Templer P.H., Dawson T.E., 2007. Nitrogen uptake by four tree species of the Catskill Mountains, New York: Implications for forest N dynamics, Plant Soil 262, 251-261.

* Yuan A.N., Wan S., Zhou X., Subedar A.A., Wallace L.L., Luo Y., 2005. Plant nitrogen concentration, use efficiency, and contents in a tallgrass prairie ecosystem under experimental warming. Global Change Biology 11, 1733–1744.
# LINKING BIOCHAR PROPERTIES WITHS ITS ABILITY TO PROMOTE N₂O REDUCTION TO N₂ IN AGRICULTURAL SOILS

PASCUAL DE VEGA, M.B., SÁNCHEZ-MONEDERO, M.A., CAYUELA, M.L.

Centro de Edafología y Biología Aplicada del Segura CEBAS-CSIC, Murcia. Spain

## INTRODUCTION

Agricultural soils are the main anthropogenic sources of atmospheric nitrous oxide (N₂O). Biochar, a carbon-rich solid produced by pyrolysis of biomass (Atkinson et al., 2010), has been reported to reduce N₂O emissions. Its application to soils has been proposed to affect soil N₂O activity by several mechanisms, some of them biotic and others abiotic. Among them, biochar may alter N-cycling microbial community (Harter et al., 2014), structure and soil functions by reacting with various N forms (nitrate, nitrite and/or N₂O) (Lin et al., 2014) or acting as a mediator in redox reactions. In particular, recent studies have proposed biochar's involvement in N₂O reduction to N₂, the last step of the denitrification process (Cayuela et al., 2013) but, there are still very few of studies that have demonstrated this hypothesis (Quin et al., 2015). To our knowledge, no study has analysed the reduction of N₂O to N₂ in soil columns amended with several biochars with contrasting properties. Thereby, this experimental approach would allow to link biochar physical and chemical properties with its N₂O reduction rate.

## MATERIAL AND METHODS

Several column incubation experiments with soil/biochar mixtures (2% biochar) have been set up under different moisture contents (40 and 90% WFPS). N₂O was injected at the bottom of the soil column and measured at the headspace at regular time intervals. The biochars used were produced at two pyrolysis temperatures (400 and 600°C) and generated from two agricultural residues (pruning residues from olive tree and post-harvest residues from tomato plants). The evolution of N₂O concentration with time was monitored, as well as CO₂ and CH₄ concentrations in the headspace by using a gas chromatograph (VARIAN CP-4900 Micro-GC. Palo Alto, CA, USA).

Biochars were chemically and physically analysed. In all of them, pH, electrical conductivity, total N, C, H, O, metal, ash and moisture concentrations were determined. Moreover, the micro-structure, pore size distribution, porosity and total surface area of the biochars were measured by a mercury intrusion-extrusion technique. In addition, the percentage of lignin, hemicellulose, cellulose and ash on both feedstock used for biochar production were quantified.

## RESULTS AND DISCUSSION

Previous laboratory experiments have demonstrated the ability of biochar to decrease N₂O emissions; however, there is almost no information on how biochar impact specific N₂O formation and consumption pathways (Cayuela et al., 2013; Quin et al., 2015). Recently, Thomazini et al. (2015) argued that the inconsistent results found in the literature regarding biochar impact on N₂O emissions are mostly a result of the variability in biochar properties. We postulate that, in general, all biochars will increase the soil N₂O sink capacity, i.e. the reduction of N₂O to N₂. A combination of biochar factors could take part: their porosity, redox activity (capacity to accept and donate electrons), chemical structure (functional groups), physical properties, feedstock composition... Some of them are gathered in Table 1 and 2.

We contemplate that biochars with a lower molar H:C<sub>org</sub> ratio and a higher aromaticity (generally found in biochars produced at high temperatures) would facilitate the electron exchange between the biochar surface and the microorganisms involved in N₂O reduction. Biochars porosity may also play a significant role because it provides biochars the capacity to adsorb gases and modulate their diffusion to the atmosphere (Cayuela et al., 2013 and 2015). In addition, the lignocellulosic composition of the feedstock may affect the chemical groups (e.g.
hydroquinone and phenolic moieties) present in biochar and therefore its electron donor capacity (Zhao et al. 2013).

<table><caption>Table 1. Olive tree and tomato plants biochar properties at 400 and 600°C.</caption><thead><tr><th>Biochars</th><th>Feedstock</th><th>T pyrolysis (°C)</th><th>pH</th><th>CE (mS/cm)</th><th>Ash (%)</th><th>H:C</th><th>C:N</th></tr></thead><tbody><tr><td>BC-Ol400</td><td rowspan="2">Olive tree</td><td>400</td><td>9,90</td><td>0,59</td><td>4,89</td><td>0,130</td><td>107,51</td></tr><tr><td>BC-Ol600</td><td>600</td><td>11,05</td><td>0,75</td><td>4,81</td><td>0,246</td><td>105,95</td></tr><tr><td>BC-To400</td><td rowspan="2">Tomato plants</td><td>400</td><td>9,65</td><td>18,43</td><td>34,43</td><td>0,801</td><td>22,91</td></tr><tr><td>BC-To600</td><td>600</td><td>12,10</td><td>22,80</td><td>38,18</td><td>0,337</td><td>26,58</td></tr></tbody></table>

<table><caption>Table 2. Biochar feedstock content (%) in lignin, hemicellulose, cellulose and ash.</caption><thead><tr><th>Feedstock</th><th>Lignin (%)</th><th>Hemicellulose (%)</th><th>Cellulose (%)</th><th>Ash (%)</th></tr></thead><tbody><tr><td>Olive tree</td><td>25</td><td>17</td><td>56</td><td>3</td></tr><tr><td>Tomato plants</td><td>17</td><td>3</td><td>50</td><td>31</td></tr></tbody></table>

## CONCLUSION

The term biochar comprises a wide range of pyrolysed materials with contrasting physico-chemical properties. How the individual properties of biochar impact the reduction of N₂O to N₂ in soil is to date unknown. However, with the results from the incubation experiments and the detailed characterization of the biochars tested, we will be able to ascertain the properties of biochar related to its N₂O reduction capacity. This will pave the way for a future development of biochars with the environmental goal of reducing N₂O emissions in agricultural soils.

**Acknowledgements:** We thank the Ministry for the Economy and Competitiveness of Spain. Project: Ref. CTM2015-67200-R (MINECO/FEDER).

## REFERENCES

* Atkinson, C.J., Fitzgerald, J.D., Hipps, N.A. Potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review. *Plant Soil* 337, 1–18 (2010)

* Cayuela, M.L., Sánchez-Monedero, M.A., Roig, A., Hanley, K., Enders, A., Lehmann, J. Biochar and denitrification in soils: when, how much and why does biochar reduce N₂O emissions? *Sci. Rep.* 3, 1732 (2013)

* Cayuela, M. L., Jeffery, S., Van Zwieten, L. The molar H:Corg ratio of biochar is a key factor in mitigating N₂O emissions from soil. *Agr. Ecosyst. Environ.* 202, 135–138 (2015)

* Harter J., Krause, H-M., Schuettler, S., Ruser, R., Fromme, M., Scholten, T., Kappler, A., Behrens, S. Linking N₂O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community. *ISME J.* 8, 660–674 (2014)

* Lin, X., Spokas, K.A., Venterea, R.T., Zhang, R., Baker, J.M., Feyereisen, G.W. Assessing Microbial Contributions to N₂O Impacts Following Biochar Additions. *Agron.* 4, 478–496 (2014)

* Quin, P., Joseph, S., Husson, O., Donne, S., Mitchell, D., Munroe, P., Phelan, D., Cowie, A., Van Zwieten, L. Lowering N₂O emissions from soils using eucalypt biochar: the importance of redox reactions. *Sci. Rep.* 5, 16773 (2015)

* Thomazini, A, Spokas, K., Hal, K., Ippolito, J., Lentz, R., Novak, J. *Agric.*, *Ecosystems and Environ.* 207, 183-191 (2015)

* Zhao, L., Cao, X., Mašek, O., Zimmerman, A. Heterogeneity of biochar properties as a function of feedstock sources and production temperatures. *J. Hazard Mater* 256–257, 1–9 (2013)