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MONTOYA, M.1_{, CASTELLANO-HINOJOSA, A.}2_{, VALLEJO, A.}1_{, ÁLVAREZ, JM.1, BEDMAR, EJ.2, RECIO, J.}1_{, GUARDIA,}

G.1

1 _{ETSI Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Spain;} 2 _{Department of Soil Microbiology and}

Symbiotic Systems, Estación Experimental del Zaidín, Agencia Estatal CSIC, Spain INTRODUCTION

Fertilization with micronutrients (e.g. zinc, Zn) is essential to overcome the global nutritional problems associated with human deficiencies. An adequate nutrient balance requires the management of macronutrients (e.g. nitrogen, N) fertilization (Venterea et al., 2016) and its interactions with micronutrients (e.g. Zn), to find synergistic effects between nutrients, and improve crop quality through biofortification (Cakmak et al., 2017). However, it is

pivotal to reduce environmental pollution, e.g. the emission of nitrous oxide (N2O), without compromising food

security in a context of increasing world population. Widespread products which inhibit nitrification, such as dicyandiamide (DCD) or 3,4-dimethylpyrazole phosphate (DMPP), act as metal chelators. Therefore, chelating fertilizers, which are used to apply micronutrients with enhanced efficacy (Alvarez, 2010), could influence nitrification and other metal-dependent biochemical processes such as denitrification. The response of nitrous

oxide (N2O) emissions to N fertilization has been broadly assessed, but little is known about the effect of

micronutrient fertilizers and their interaction with nitrogen (N) on greenhouse gas (GHG) emissions and soil

microbial processes involved in N2O fluxes.

MATERIAL AND METHODS

Two field experiments were located in the National Center of Irrigation Technology, “CENTER” in the Madrid region (Spain). The soil was an alkaline Typic xerofluvent with a silt loam texture and low organic matter content

in the upper horizon (0-20 cm).The winter wheat (Triticum aestivum L. ´Ingenio`) and the maize (Zea mays L. `SY

Miami`) experiments were carried out from October 2015 to July 2016 and May 2017 to September 2017, respectively. The maize crop was irrigated through sprinklers, while no irrigation was applied to winter wheat. A

total of 12 plots (20m2 _{and 144m}2_{, respectively) were arranged in a three-replicated randomized block design.}

Each plot was a result of two N rates as urea (0, N0 and 120 kg N ha-1_{, U) with two Zn sources: control, no Zn}

application (Zn0) and Zn applied with a mixture of chelating compounds (Zn-DTPA-HEDTA-EDTA, ZnCH). GHG sampling and analyses

During the first month after fertilization, samples of gases were taken 2-3 times per week considering it is the most critical period of high gas emissions. Afterwards, the frequency of sampling was decreased progressively,

but increasing after rainfall events. The GHG (N2O, CH4 and CO2) fluxes were measured using the closed chamber

technique (Sanz-Cobena et al., 2012) and their concentrations were quantified by gas chromatography, using a gas chromatograph (GC).

DNA extraction and quantification of nitrifying and denitrifying microbial communities

Soil samples were taken from the field experiment with wheat. Total DNA was extracted from 500 mg of soil using

commercial kit PowerSoil® _{DNA Isolation and DNA concentration was measured using the Qubit}® _{ssDNA assay kit.}

The size of nitrifying communities was estimated by quantitative PCR (qPCR) of the amoA gene from ammonia- oxidizing Bacteria (AOB) and Archaea (AOA). Similarly, denitrifying communities were estimated by qPCR of the

abundance of targeted genes. Standard curves were obtained using serial dilutions (ranging from 108 _{down to 10}2

copies µl-1_{) of linearized plasmids containing the targeted genes cloned in.}

RESULTS AND DISCUSSION

In maize, cumulative N2O emissions (Fig. 1) were significantly higher in N fertilized treatments (U), independently

of Zn, than in unfertilized N treatments (N0), but small differences were found in wheat. Concerning Zn treatments, cumulative losses were significantly increased with the application of Zn chelate (ZnCH) combined

with U in maize. Contrarily, wheat crop showed a decreased in cumulative N2O emissions with U+ZnCH treatment.

Besides, we observed a decrease in the total abundance of nitrifying and denitrifying communities with U+ZnCH

treatment in wheat, except the gene involved in transforming N2O to N2 (nosZ gene) which was increased. In high

moisture conditions, Pramanik and Kim (2016) reported an increase in N2O losses after the addition of EDTA in a

submerged paddy crop which was in agreement with our results in maize. However, the understanding of how Zn chelate modulates soil nitrifying and denitrifying microbiota under irrigated conditions is still under study.

Figure 1. Total cumulative N2O-N emissions in winter wheat and maize crops amended with Zn fertilizer (Zn- DTPA-HEDTA-

EDTA, ZnCH) and without Zn (Zn0) combined with two N application rates (0, N0, and 120 kg N ha-1_{, U).}

CONCLUSION

Our results demonstrated that in the rainfed crop, the application of synthetic chelates can be a promising strategy

to decrease N2O losses. However, the opposite was observed in an irrigated crop.

REFERENCES

Alvarez JM., 2010. Influence of soil type and natural Zn chelates on flax response, tensile properties and soil Zn availability. Plant Soil, 328, 217–233.

Cakmak I., McLaughlin MJ., White P., 2017. Zinc for better crop production and human health.

Pramanik P., Kim PJ., 2017. Contrasting effects of EDTA applications on the fluxes of methane and nitrous oxide emissions from straw-treated rice paddy soils. Journal of the Science of Food and Agriculture, 97, 278-283.

Sanz-Cobena A., Sánchez-Martín L., García-Torres L., Vallejo A., 2012. Gaseous emissions of N2O and NO and NO3−

leaching from urea applied with urease and nitrification inhibitors to a maize (Zea mays) crop. Agr. Ecosyst. Environ. 149, 64-73.

Venterea R.T., Coulter J.A., Dolan M.S., 2016. Evaluation of intensive “4R” strategies for decreasing nitrous oxide emissions and nitrogen surplus in rainfed corn. J. Environ. Qual., 45, 1186-1195.