Nitrification inhibitor and use thereof in fertilisers containing ammoniacal nitrogen, urea nitrogen or both

Abstract

The present invention discloses a nitrification inhibitor for use thereof in fertilisers containing ammoniacal nitrogen, urea nitrogen or both, wherein the nitrification inhibitor is pulcherriminic acid. The pulcherriminic acid is present in a weight ratio of 0.5 to 10% relative to the amount of ammoniacal nitrogen, urea nitrogen, or both ammoniacal nitrogen and urea nitrogen.

Description

[0001] Nitrification inhibitor and its use in fertilizers containing ammoniacal nitrogen, urea nitrogen, or both

[0002] DESCRIPTION

[0003] The present invention provides a nitrification inhibitor for use in fertilizers containing ammoniacal nitrogen, ureic nitrogen or both, wherein the nitrification inhibitor is pulcherhminic acid, the pulcherhminic acid being present in a weight ratio of 0.5 to 10% relative to the amount of ammoniacal, ureic or ammoniacal plus ureic nitrogen.

[0004] Currently, chemical fertilizers based on ammonium (NH4 + ), such as urea, anhydrous NH3, (NH^SC and NH4NO3, are the most widely used worldwide, as is the case with liquid manures, whose nitrogenous fraction is mostly ammonium. This continued contribution of NH4 +to the soil stimulates the activity of nitrifying microorganisms, so most modern agricultural systems have become systems with high nitrification rates, to such an extent that nitrification is so rapid that most of the NH4 + from external supplies undergoes a complete nitrification process in a few weeks (Halvorson et al., Enhanced-Efficiency Nitrogen Fertilizers: Potential Role in Nitrous Oxide Emission Mitigation. Agronomy Journal 2014, 106, 715-722. https: / / doi.org / 10.2134 / agronj2013.0081 ; Subbarao et al., Scope and Strategies for Regulation of Nitrification in Agricultural Systems — Challenges and Opportunities. Critical Reviews in Plant Sciences 2006, 25, 303-335.

[0005] The nitrification process is divided into two stages: the first one in which the oxidation of NHs / NH4 occurs +to NO2; and a final step in which NO2 is oxidized to NOs by the action of the enzyme nitrite oxidoreductase. In turn, the first stage of nitrification is divided into two steps: the oxidation of NHs / NH4 + to hydroxylamine (NH2OH) via the enzyme ammonium monooxygenase (AMO) and the oxidation of hydroxylamine to nitrite (NO2 ) by the enzyme hydroxylamine oxidoreductase (HAO). During this process, nitrous oxide (N2O) is formed due to the chemical decomposition of hydroxylamine. The microorganisms involved in nitrification are two groups of ammonium oxidizers, ammonium-oxidizing archaea (AOA) and ammonium-oxidizing bacteria (AOB). In soil, soil AOB communities are dominated by the genera Nitrosomonas and Nitrosospira within the Betaproteobacteria. The predominance of AOA or AOB in nitrification is related to soil pH and NH4 concentration. +In acidic soils, AOA are the predominant group that carries out the nitrification process regardless of the NH4 concentration. + , while AOBs become relevant as the pH reaches neutral or alkaline values. When NH4-based fertilizers are applied + , AOA are inhibited and AOB carry out the nitrification process. As a result, alkaline and neutral soils tend to show an increase in AOB after fertilization (Castellano-Hinojosa et al., Effect of Urease and Nitrification Inhibitors on Ammonia Volatilization and Abundance of N-cycling Genes in an Agricultural Soil. J. Plant Nutr. Soil Sci. 2020, 183, 99-109. https: / / doi.org / 10.1002 / jpln.201900038).

[0006] The enzyme ammonium monooxygenase (AMO) is made up of three different subunits (AmoA, AmoB and AmoC) and contains Cu (6 Cu 2+ and 3 Cu + ), Fe (4 Fe 3+ ) and Zn (3 Zn 2+) (Gilch et al., Electron Paramagnetic Studies of the Copper and Iron Containing Soluble Ammonia Monooxygenase from Nitrosomonas Europaea. Biometals 2010, 23, 613-622. https: / / doi.org / 10.1007 / s10534-010-9308-2). However, the AMO enzyme from AOA communities saturates faster than that from AOB due to a higher affinity for NH4 + and a lower maximum oxidation rate of NH4 + . Therefore, although AOAs present higher abundances in agricultural soils (Di et al., Inhibition of Nitrification to Mitigate Nitrate Leaching and Nitrous Oxide Emissions in Grazed Grassland: A Review. J Soils Sediments 2016, 16, 1401-1420. https: / / doi.org / 10.1007 / s11368-016-1403-8), their contribution to nitrification is comparatively lower (Kits et al., Kinetic Analysis of a Complete Nitrifier Reveals an Oligotrophic Lifestyle. Nature 2017, 549, 269-272. https: / / doi.Org / 10.1038 / nature23679).

[0007] In contrast, the enzyme hydroxylamine reductase (HAO), required to complete the nitrite formation process, is an iron-dependent oxidoreductase, so it does not require Cu. 2+ nor Zn 2+ (Hendhch et al., The Active Site of Hydroxylamine Oxidoreductase from Nitrosomonas: Evidence for a New Metal Cluster in Enzymes. J.

[0008] Am. Chem. Soc. 1994, 116, 11961-11968. https: / / doi.org / 10.1021 / ja00105a041 ).

[0009] When the above processes occur rapidly, losses are increased due to the emission of N2O formed from the oxidation of hydroxylamine. In addition, nitrate (NOs') would be produced by the oxidation of NO2; in quantities that, if they exceed the needs of crops at a given time, are susceptible to leaching, with the consequent contamination of aquifers. Therefore, it is necessary to develop strategies that delay the nitrification process to avoid economic and efficiency losses (loss of nitrogen supplied to the crop) and to prevent environmental contamination (leaching of NOs' into aquifers and emission of N2O, a greenhouse gas, into the atmosphere).

[0010] One strategy for slowing the nitrification process is to add chemical compounds capable of inhibiting the nitrification process to fertilizers, generating so-called stabilized fertilizers. Nitrification inhibitors deactivate the enzyme responsible for the first step of nitrification (where the AMO enzyme intervenes), maintaining NH4 for longer periods of time. + in the soil (Gilsanz et al., Development of Emission Factors and Efficiency of Two Nitrification Inhibitors, DCD and DMPP. Agriculture, Ecosystems & Environment 2016, 216, 1-8. https: / / doi.Org / 10.1016 / j.agee.2015.09.030); Ruser et al., The Effect of Nitrification Inhibitors on the Nitrous Oxide Release from Agricultural Soils—a Review. J. Plant Nutr. Soil Sci. 2015, 178, 171-188. https: / / doi.Org / 10.1002 / j pl n.201400251 ).

[0011] Although the number of compounds described as possessing nitrification-inhibiting properties is large, only a few have been developed as products for commercial use. These include dicyandiamide (DCD), nitrapine, and 3,4-dimethylpyrazole phosphate (DMPP). These compounds have proven efficacy in reducing nitrogen oxide emissions under various climatic conditions, with DMPP being particularly effective.

[0012] Dicyandiamide 3,4-dimethylpyrazole phosphate Nitrapyrine

[0013] Although the information is not sufficient and some observations have not been conclusive, the nitrification inhibition capacity of these molecules is attributed to their chelating capacity of Cu cations. 2+ that the AMO enzyme needs to carry out the oxidation of NH4 +a NH2OH (Subbarao et al., Ruser et al. supra', Corrochano-Monsalve al., Impact of Dimethylpyrazole-Based Nitrification Inhibitors on Soil-Borne Bacteria. Science of The Total Environment 2021 , 792, 148374. https: / / doi.Org / 10.1016 / j.scitotenv.2021 .148374).

[0014] It should be remembered that in soil, chelation is not only the result of a chelator's affinity for the metal, as there are a number of competing reactions that may not favor chelation of the complex of interest. Thus, for example, a highly stable chelator with Fe 3+ , such as EDTA, may be useless in addressing iron deficiencies in calcareous soils, since the high relative abundance of Ca 2+ regarding Fe 3+It displaces it from its chelate. Furthermore, the chelation of the metal in an enzymatic system is also highly dependent on the accessibility of the chelator to the metal present in the enzyme. An example is the urease inhibitor NBPT, which binds to the Ni atoms present in the enzyme due to its high affinity and has an accessibility similar to urea. Although the mechanisms of action may be different (local modification of redox conditions, direct effects on microorganisms, etc.), it is necessary to take these premises into account when identifying the possible modes of action, and, in the context of the present invention, not only the chelation of Cu. 2+ but also of Zn 2+ and Faith 3+ .

[0015] Below, we will discuss the three most important chemical nitrification inhibitors mentioned above: diazindiamide, 3,4-dimethylpyrazole phosphate, and nitrapyrin. For example, these and other chemical nitrification inhibitors are described in US11312667B2.

[0016] Diazindiamide (PCD) Dicyandiamide, also known as cyanoguanidine or DCD, is a chemical compound capable of inhibiting the first step of nitrification, the oxidation of NHS / NH4 + to NH2OH. It is applied by mixing it with nitrogen fertilizer. Concentrations of 200-300 ppm have been described to significantly reduce the production of NCh- by Nitrosomonas europea bacteria under in vitro conditions. In soil studies, a dose of 8 mg of DCD per kg of soil was shown to be effective in inhibiting the activity of Nitrosospira in grasslands with animal urine as the NH4 substrate. +(Di et al., supra). Although in some bibliographic references its mechanism of action is attributed to the ability to chelate Cu 2+ that the AMO enzyme needs to oxidize NHs / NH4 +, the inhibition mechanism at the molecular level has not yet been confirmed. Studies have shown that DCD does not kill the bacteria because, although it temporarily inhibits the growth and activity of ammonia / ammonium oxidizing bacteria, it has been proven that once DCD is degraded in the soil, these bacteria recover their oxidation activity (Di et al., supra). It is important to note that the inhibitory capacity of DCD has been shown to be specific to this transformation, so its use in the soil does not affect other microbial communities or other enzymatic activities (Amberger, A., Research on Dicyandiamide as a Nitrification Inhibitor and Future Outlook. Communications in Soil Science and Plant Analysis 1989, 20, 1933-1955. https: / / doi.Org / 10.1080 / 00103628909368195).

[0017] However, the use of DCD can cause problems related to its absorption by plants while they remain in the soil before being degraded. Thus, Marsden et al. (Plant Acquisition and Metabolism of the Synthetic Nitrification Inhibitor Dicyandiamide and Naturally-Occurring Guanidine from Agricultural Soils. Plant So / 72015, 395, 201-214. https: / / doi.org / 10.1007 / s11104-015-2549-7) have shown that DCD can pass into grass plants and be partially metabolized in them, being a possible vehicle for its transmission to the food chain.

[0018] 3,4-Dimethylpyrazole phosphate (DMPP) 3,4-Dimethylpyrazole phosphate (DMPP) is a nitrification inhibitor developed by BASF and marketed as ENTEC® since 1999. Like DCD, it is a very effective inhibitor of the AMO enzyme in the oxidation process of NH3 or NH4 +a hydroxylamine. It has a greater inhibition capacity than DCD, so the application dose to observe satisfactory inhibition effects is lower (Pasda et al., Effect of Fertilizers with the New Nitrification Inhibitor DMPP (3,4- Dimethylpyrazole Phosphate) on Yield and Quality of Agricultural and Horticultural Crops. Biology and Fertility of Soils 2001, 34, 85-97. https: / / doi.org / 10.1007 / s003740100381 ; Zerulla et al., 3,4-Dimethylpyrazole Phosphate (DMPP) - a New Nitrification Inhibitor for Agriculture and Horticulture. Biology and Fertility of Soils 2001 , 34, 79-84. https: / / doi.org / 10.1007 / s003740100380). DMPP is broken down in the soil by microorganisms through the cleavage of the pyrazole ring, releasing CO2. Furthermore, it has been shown to remain in the soil longer than its inhibitory effect (Guardia et al. 15N-Labelled Ammonium Nitrate with or without the New Nitrification Inhibitor DMPSA in an Irrigated Maize Crop. Soil Biology and Biochemistry 2018, 116, 193-202. https: / / doi.Org / 10.1016 / j.soilbio.2017.10.013), thus being disadvantageous due to its possible absorption by the plant. On the other hand, DMPP has a lower mobility in soil than DCD. Therefore, when applied with an ammonia-N-based fertilizer, it remains together with the NH4 + released to provide the inhibitory effect.

[0019] • Nitrapyrine

[0020] Nitrapyrin is an organochlorine compound developed by Dow Chemical and sold as N-Serve® in the US. It is soluble in most organic solvents but insoluble in water, and also has a high vapor pressure. Despite these disadvantages compared to DMPP and DCD, it is a widely used nitrification inhibitor in the US due to its high volatility. Its main method of employment has been by injection into the soil (5-10 cm deep) together with anhydrous NH3, particularly in corn fields (Slangen et al., Nitrification Inhibitors in Agriculture and Horticulture: A Literature Review. Fertilizer Research 1984, 5, 1-76. https: / / doi.org / 10.1007 / BF01049492). However, due to its high volatility, it is not an effective inhibitor as a coating for broadcast solid nitrogen fertilizers.

[0021] Due to the problems that these chemical nitrification inhibitors can cause in the environment due to their persistence, studies have emerged proposing the use of biological nitrification inhibitors. These inhibitors consist of chemical compounds exuded by plants or other organisms capable of inhibiting nitrification. However, their agronomic effectiveness is not yet well quantified, and the specificity of the plant species that can generate them is a problem.

[0022] There is still not much information and knowledge about this type of inhibitors, but it is known that in Brachiaria exudates there are compounds, such as brachylactone, with the ability to inhibit AMO and HAO (Subbarao et al., Evidence for Biological Nitrification Inhibition in Brachiaria Pastures. Proc. Natl. Acad. Sci. USA 2009, 106, 17302-17307, https: / / doi.org / 10.1073 / pnas.0903694106) and in sorghum exudates compounds such as methyl 3-(4-hydroxyphenyl)propionate (MHPP) and 5,4'-dihydroxy-7-methoxyflavanone (sakuranetin) are released, which also affect AMO and HAO enzymes (Subbarao et al., Biological Nitrification Inhibition (BNI) Activity in Sorghum and Its Characterization. Plant Soil 2013, 366, 243-259. https: / / doi.org / 10.1007 / s11104- 012-1419-9).

[0023] Nitrification inhibition using biodegradable plant extracts has been proposed as a promising alternative to synthetic nitrification inhibitors. Thus, oils of Mentha spicata L. and Artemisia annua L. have been shown to inhibit nitrification (Kiran et al., Influence of Natural Essential Oils and Their By-Products as Nitrification Retarders in Regulating Nitrogen Utilization for Japanese Mint in Sandy Loam Soils of Subtropical Central India. Agriculture, Ecosystems & Environment 2003, 94, 237–245. https: / / doi.org / 10.1016 / S0167-8809(02)00027-0).

[0024] Other candidates for biological nitrification inhibitors include phenolic compounds. For example, plant extracts containing catechin have shown nitrification inhibitory capacity (Castaldi et al., Inhibition of Net Nitrification Activity in a Mediterranean Woodland: Possible Role of Chemicals Produced by Arbutus Unedo. Plant Soil 2009, 315, 273–283. https: / / doi.org / 10.1007 / s11104-008-9750-x).

[0025] Also, the inhibitory activity of alpechines (by-products rich in phenolic compounds) has been recently documented, indicating that they show a greater effect on nitrification than extracts of Mentha piperita and Artemisia annua. Di Martino et al. in “Effects of Olive Mill Wastewater and Two Natural Extracts as Nitrification Inhibitors on Activity of Nitrifying Bacteria, Soil Nitrate Leaching Loss, and Nitrogen Metabolism of Celery (Apium Graveolens L). J Plant Growth Regul 2021 , 40, 1922-1938”, state that the compounds present in alpechin are capable of reducing the activity of nitrifying bacteria, increasing the retention time in the soil of nitrogen from a (NH4)2SO4-based fertilizer and reducing NO3 leaching; with an effectiveness similar to that of DCD (synthetic nitrification inhibitor).

[0026] Caffeic acid, a hydroxycinnamic acid intermediate in lignin biosynthesis, has also been proposed to inhibit nitrification by deactivating nitric oxide through its antioxidant properties (Sauder et al., Nitric Oxide Scavengers Differentially Inhibit Ammonia Oxidation in Ammonia-Oxidizing Archaea and Bacteria. FEMS Microbiology Letters 2016, 363, 1-8. https: / / doi.org / 10.1093 / femsle / fnw052).

[0027] Considering the nature of the compounds mentioned up to this point with nitrification-inhibiting capabilities, both synthetic and of biological origin, certain compounds of microbial origin could have an effect on nitrification. For example, certain bacteria and fungi have a high capacity to produce antioxidant compounds and metabolites with the ability to complex metallic elements or surfactant compounds, which can have effects on other microorganisms and, specifically, on those involved in nitrification (Nardi et al., Biological Nitrification Inhibition in the Rhizosphere: Determining Interactions and Impact on Microbially Mediated Processes and Potential Applications. FEMS Microbiology Reviews 2020, 44, 874-908. https: / / doi.org / 10.1093 / femsre / fuaa037; Chandra et al., Antioxidant Compounds from Microbial Sources: A Review. Food Res. Int. 2020, 129, 108849. https: / / doi.Org / 10.1016 / j.foodres.2019.108849; Slama et al., Screening for Fusarium Antagonistic Bacteria from Contrasting Niches Designated the Endophyte Bacillus Halotolerans as Plant Warden Against Fusarium. Front. Microbiol. 2019, 9, 3236. https: / / doi.org / 10.3389 / fmicb.2018.03236). For example, document CN108794185 attributes the ability to inhibit nithfication to rhamnolipids of microbial origin.

[0028] Given the disadvantages of the known nithfication inhibitors, it would be desirable to have alternative nithfication inhibitors to the use of chemical inhibitors.

[0029] Thus, an object of the invention is to provide a nitric oxide inhibitor that does not entail the disadvantages of known inhibitors, said nitric oxide inhibitor being pulcherhminic acid, a compound of biological origin.

[0030] Figure 1 shows a scheme for obtaining the pulcherhminic acid used in the present invention.

[0031] Figure 2 shows the spectrum of 1 H-NMR for pulcherhminic acid obtained according to Figure 1.

[0032] Figure 3 shows the spectrum of 13 C-NMR (APT) for pulcherhminic acid obtained according to Figure 1.

[0033] Figure 4 shows the UPLC-MS chromatogram of pulcherhminic acid obtained according to Figure 1.

[0034] Figure 5 shows the mass spectrum of the peak detected at 14.06 min of pulcherhminic acid obtained according to Figure 1.

[0035] The present inventors have surprisingly found that pulcherhminic acid has demonstrated a potent nithfication inhibitory effect both in in vitro assays with Nitrosomonas bacteria and in in vivo assays performed in soil.

[0036] According to the literature, pulcherhminic acid or 2,5-dihydroxy-3,6-diisobutylenepyrazine-1,4-N,N-dioxide (1) is a precursor compound of a reddish pigment called pulcherhmin (2) (Kluyver et al., Pulcherhmin, The Pigment of Candida Pulcherhma. Proceedings of the National Academy of Sciences 1953, 39, 583-593. https: / / doi.Org / 10.1073 / pnas.39.7.583; MacDonald, JC, The Structure of Pulchemminic Acid. Can. J. Chem. 1963, 41, 165-172. https: / / doi.org / 10.1139 / v63-021).

[0037] Pulcherriminic acid is biosynthesized by some yeasts of the Metschnikowia family and some bacteria of the Bacillus family from the cyclic dipeptide derived from L-leucine (3), prepared from L-leucine. In the presence of Fe(lll), pulcherriminic acid forms a polymeric chelate known as pulcherrimin through a non-enzymatic reaction (Gore-Lloyd et al., Snf2 Controls Pulchemminic Acid Biosynthesis and Antifungal Activity of the Biocontrol Yeast Metschnikowia Pulcherrima. Mol. Microbiol. 2019, 112, 317-332. https: / / doi.org / 10.1111 / mmi.14272; Sipiczki, M., Metschnikowia Pulcherrima and Related Pulcherrimin-Producing Yeasts: Fuzzy Species Boundaries and Complex Antimicrobial Antagonism. Microorganisms 2020, 8, 1029. https: / / doi.org / 10.3390 / microorganisms8071029). Therefore, pulcherriminic acid is a compound of biological origin.

[0038] As mentioned above, the present invention provides pulcherriminic acid as a nitrification inhibitor for use in fertilizers containing ammoniacal nitrogen, urea nitrogen or both.

[0039] Preferably, pulcherriminic acid is present in fertilizers containing ammoniacal nitrogen, ureic nitrogen or both in a weight ratio of 0.5 to 10% relative to the amount of ammoniacal, ureic or ammoniacal plus ureic nitrogen.

[0040] In the present invention, fertilizers containing ammonium nitrogen, urea nitrogen, or both are not particularly limited, and may be selected from ammonium nitrate, ammonium phosphate (monoammonium phosphate, diammonium phosphate), urea, or ammonium sulfate, or combinations thereof, with ammonium sulfate being preferred. The invention will now be explained in more detail with reference to the following examples.

[0041] In the inhibition assays described here, synthetic pulcherhminic acid obtained from L-leucine is used.

[0042] Example 1: Obtaining and characterizing pulcherhminic acid.

[0043] The synthesis scheme of the pulcherhminic acid used is shown in Figure 1.

[0044] First, the synthesis of the cyclic dipeptide derived from L-leucine (3) was carried out following a thermal procedure described in the literature (Rafiemanzelat et al., Synthesis and Characterization of Hydrolysable Poly(Ether-Urethane-Urea)s Derived from l-Leucine Anhydride Cyclopeptide; a Green Synthetic Method for Monomer. Polymer Degradation and Stability 2010, 95, 901-911. https: / / doi.Org / 10.1016 / j.polymdegradstab.2010.03.030), obtaining a racemic mixture of the cyclic dipeptide derived from L-leucine. This did not pose any problem since the chirality of the α carbon of L-leucine is lost in the final product (pulcherhminic acid, 1). The second step involves the aromatization of the 2,5-diketopiperazine ring to 2,5-dichloropyrazine using POCI3. Using the procedure and conditions described in the literature (Wang et al. Large-Scale Solvent-Free Chlorination of Hydroxy-Pyrimidines, -Pyridines, -Pyrazines and -Amides Using Equimolar POCI3.Molecules 2012, 17, 4533-4544. https: / / doi.org / 10.3390 / molecules17044533), a mixture of two compounds was obtained: the desired product, 2,5-dichloro-3,6-diisobutylpyrazine, and an unwanted byproduct as the mayohtaho product, 3-chloro-2,5-diisobutylpyrazine. The byproduct could be recovered for its transformation into 2,5-dichloro-3,6-diisobutylpyrazine through a process that is divided into two stages: first, the oxidation of N in position 1 , and second, chlorination by POCI3 (Usui et al., Convergent Synthesis and Structural Confirmation of Phellodonin and Sarcodonin s. Org. Lett. 2013, 15, 2080-2083. https: / / doi.Org / 10.1021 / ol400709f).

[0045] The next step consists of the oxidation of both nitrogens of the pyrazine ring to obtain 2,5-dichloro-3,6-disobutyl-1,4-dihydroxypyrrolidine. Under the conditions described in the literature, the product mayohtaho was obtained by oxidation with hydrogen peroxide in an acidic medium (Usui, I. et al., supra). Finally, pulcherriminic acid (1) was obtained with a purity > 95% by a two-step process: aromatic nucleophilic substitution of the chlorines with potassium benzyloxide (generated in situ by the reaction of benzyl alcohol and potassium tert-butoxide) followed by deprotection of the benzyloxide groups in a strongly acidic medium (Góktürk et al., Pyrazine Chemistry. Part 13. Preparation and Reactions of Pyrazine N-Oxides Related to Mycelianamide. J. Chem. Soc. Perkin Trans. 1982, 1, 953-959. https: / / doi.org / 10.1039 / P19820000953).

[0046] The synthesized pulcherriminic acid (1) was characterized by nuclear magnetic resonance (NMR, Figures 1 and 2) and ultra-high performance liquid chromatography coupled to mass spectroscopy (UPLC-MS, Figures 3 and 4). Its purity was determined by elemental analysis (CHNS). It was also analyzed by high resolution mass spectroscopy (HRMS).

[0047] The spectra of 1 H-NMR and 13 C-NMR is consistent with that described for pulcherriminic acid (1). The characterization is shown below:

[0048] 1 H-NMR (DMSO-Ó6, or ppm, 400 MHz): 2.67 (d, 4H, J = 7.2 Hz), 2.16-2.06 (m, 2H), 0.89 (d, 12H, J = 6.6 Hz).

[0049] 13 C-NMR (DMSO-Ó6, or ppm, 100 MHz): 145.53, 129.63, 33.41, 26.22, 22.39.

[0050] Elemental analysis using CHNS has shown a purity of >95%. The results are shown below:

[0051] • Calculated: C = 54.03%, H = 7.26%, N = 10.25%

[0052] • Theoretical: C = 56.24%, H = 7.87%, N = 10.93%

[0053] Results obtained by HRMS: found (ESI+) 227.1489 [M + H + ], C12H21N2O4 (Theoretical = 227.1496).

[0054] Figures 4 and 5 show the chromatogram obtained by UPLC-MS and the mass spectrum of the detected peak (M + H + ) with an m / z = 257.

[0055] Example 2: Nitrification inhibition capacity of pulcherriminic acid in in vitro tests with Nitrosomonas europaea bacteria To determine the nitrification inhibition capacity of pulcherriminic acid in in vitro tests with Nitrosomonas europaea bacteria, the pH generated is measured 24 and 48 hours after the application of pulcherriminic acid. The generation of nitric acid by Nitrosomonas europaea bacteria through the nitrification process causes a drop in pH, so if the pH is maintained, it means that nitrification is not occurring or that the nitrification process is inhibited. Table 1 shows the pH of the medium in the in vivo nitrification tests at different concentrations of pulcherriminic acid. In the control sample (without pulcherriminic acid), it can be observed how the pH drops due to the generation of nitric acid, which indicates that nitrification is occurring.

[0056] However, when pulcherriminic acid was added at a concentration of 11.8 ppm, the pH of the medium was observed to remain unchanged after 48 hours compared to the initial pH, indicating that the nitrification process was inhibited. The same was observed when pulcherriminic acid was added at a dose of 5.9 ppm. Therefore, pulcherriminic acid inhibited the nitrifying activity of the bacteria Nitrosomonas europaea at a concentration of 5.9 ppm in in vitro inhibition assays.

[0057] Table 1. Results of the in vitro inhibition assay of pulcherriminic acid in Nitrosomonas europaea

[0058] Also, similar to this in vitro assay, the nitrification inhibition capacity of the precursor of pulcherriminic acid, i.e., the cyclic dipeptide derived from L-leucine (3), was analyzed. Table 2 shows that the cyclic dipeptide derived from L-leucine does not inhibit the nitrifying activity of Nitrosomonas europaea bacteria even at 2,000 ppm. These results indicate that the structural difference between both compounds is key to inhibiting the nitrification process.

[0059] Table 2. Results of the in vitro inhibition assay of the cyclic dipeptide derived from L-leucine in Nitrosomonas europaea

[0060] Example 3: Nitrification inhibition capacity of pulcherriminic acid in in vivo soil tests.

[0061] To analyze the nitrification inhibition activity of pulcherriminic acid in soil, an incubation test was carried out at different doses of nitrogen per hectare and with different doses of inhibitor. Three different doses of nitrogen in the form of ammonium sulfate equivalent to 100, 200, and 300 kg N / ha were applied to the same soil type. For each dose of nitrogen, different doses of pulcherriminic acid were applied as a nitrification inhibitor, as well as the following controls: control without nitrogen, control with nitrogen, and controls with nitrogen in the presence of a commercial inhibitor (diazindiamide). The percentages of inhibitor refer to the grams of inhibitor per 100 g of added nitrogen. Table 3 shows the mg of NOs per kg of dry soil at the beginning and 5 days after carrying out the incubations.

[0062] Table 3. Results of the in vivo inhibition test in soils

[0063] As can be seen in the table above, at most nitrogen doses, pulcherhminic acid reduced nitrate formation after 5 days compared to the nitrogen control. At 200 and 300 kg N / ha, the inhibitory capacity of pulcherhminic acid was equal to that of dicazindiamide, a commercial synthetic nitrification inhibitor, at the same inhibitor dose. It should be noted that when 10% pulcherhminic acid was applied, the nitrification process was completely inhibited after 5 days.

Claims

CLAIMS 1. Nitrification inhibitor for fertilizers containing ammoniacal nitrogen, urea nitrogen or both, where the nitrification inhibitor is pulcherhminic acid.

2. Nitrification inhibitor for fertilizers containing ammoniacal nitrogen, ureic nitrogen or both according to claim 1, wherein the pulcherhminic acid is present in a proportion by weight of 0.5 to 10% with respect to the amount of ammoniacal, ureic or ammoniacal plus ureic nitrogen.

3. Nitrification inhibitor for fertilizers containing ammoniacal nitrogen, urea nitrogen or both according to claim 1 wherein the fertilizers containing ammoniacal nitrogen, urea nitrogen or both are selected from ammonium nitrate, ammonium phosphate (monoammonium phosphate, diammonium phosphate), urea or ammonium sulfate, or combinations thereof.

4. Nitrification inhibitor for fertilizers containing ammoniacal nitrogen, urea nitrogen or both according to claim 3 wherein said fertilizer is ammonium sulfate.

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