Biological compositions for preventing nitrate leaching

Abstract

The present invention relates to microbial compositions and fertilizers containing them that reduce soil nitrate accumulation, and their use to avoid nitrogen leaching.

Description

[Technical field]

[0001] The present invention is in the field of agriculture. The present invention relates to microbial compositions and fertilizers containing them that reduce soil nitrate accumulation, and their use to avoid nitrogen leaching. [Background technology]

[0002] Leaching is the entrainment of nitrates with the flow of water or drainage through the soil profile. As a result, nitrogen is transported to deeper layers and cannot reach the crop roots. Because nitrates are soluble in water, do not interact with soil colloids, and are highly mobile, they can reach groundwater and surface waters through the leaching process, causing water pollution problems. In contrast, ammonium ions do not leach because they adsorb to soil colloids.

[0003] Nitrogen provided by fertilizers has multiple destinations when incorporated into the soil. Nitrogen use by crops rarely exceeds 60% or 70%. In addition, there is a need for more efficient nitrogen fertilizers, such as slow-release nitrogen fertilizers, which are encouraged by environmental trends regarding nitrogen losses and agricultural reasons regarding the need for nitrogen throughout the crop cycle. This objective can be achieved by using urease and nitrification inhibitors. Urease and nitrification inhibitors are the most commonly used supplements in inorganic nitrogen fertilizers aimed at increasing agricultural productivity and reducing pollutant emissions and leachates. Some of the main nitrification inhibitors identified so far are dicyanamide, 3-methylpyrazole, 1-guanyl-3-methylpyrazole nitrate, 3,4-dimethylpyrazole, 3,4-dimethylpyrazole phosphate, 1H-1,2,4-triazole, 3-amino-1H-1,2,4-triazole, 4-chloro-3-methylpyrazole, and nitrapyrin. Some of the main inhibitors of urease activity that have been identified so far are phenylphosphorodiamidate, N-(n-butyl)phosphoric diamide, N-(n-butyl)thiophosphoric triamide, N-diaminophosphoryl)-N'-(4-methoxyphenyl)-urea, N-diaminophosphoryl-urea, N-diaminophosphoryl)-N'-phenyl-urea, methyldiaminophosphorylcarbamate, benzyldiaminophosphorylcarbamate, isopropyldiaminophosphorylcarbamate and ammonium thiocyanate.

[0004] In agricultural systems, there is a series of interactions and processes that transform and transport nitrogen, all of which are part of the so-called nitrogen cycle. Nitrogen exists in various forms within this cycle. The largest source and reservoir of nitrogen is N2 gas, which makes up 78% of the Earth's atmosphere. However, higher plants extract nitrogen mainly from the soil solution, NO3 - and NH4 +The uptake of gaseous nitrogen into soil occurs through a fixation process, in which gaseous nitrogen is taken up by symbiotic and non-symbiotic microorganisms present in the soil (biological fixation), by dry and wet deposition of nitrogen compounds (during storms and by atmospheric dust), and by industrial processes for the synthesis of nitrogen fertilisers (industrial fixation). The fixation process mostly involves the reduction of N2 to NH3. The taken up nitrogen is mainly stored in soil organically, which is estimated to be more than 90% of the nitrogen present in soil. The organic forms are not directly assimilated by plants, but are assimilated after conversion to inorganic nitrogen. The main inorganic form is ammonium (NH4 + ), nitrite (NO2 - ) and nitrates (NO3 - ), which usually represents 2%-5% of the total nitrogen in soil. The conversion of organic nitrogen to inorganic nitrogen occurs in several steps: mineralization, first by the breakdown of large protein molecules (amination), then by ammonification, which converts NH4 + Ammonium is used directly by the plant, nitrified, immobilized, volatilized, or retained in soil exchange complexes.

[0005] Nitrification is the oxidation of ammonium to nitrate, which is essentially carried out in two steps by bacterial action. In the first step, bacteria convert ammonia to nitrite (NO2 - In the second step, nitrite is oxidized to nitrate. NH4 retained by the soil exchange complex + In contrast, NO2 - and NO3 - are mobile in soil solution, so they are either absorbed by plant roots with soil moisture or gradually leached out with drainage through the soil profile.

[0006] No.3 - Nitrate can be reduced by microorganisms in soil. There are various metabolic pathways for nitrate reduction, namely, denitrification, dissimilatory nitrate reduction to ammonium (DNRA) and assimilatory nitrate reduction to ammonium (ANRA).

[0007] Nitrogen is also lost from soils as inorganic forms are converted to gases that migrate into the atmosphere. This nitrogen is produced by volatilization and denitrification. Volatilization is the process by which ammonia nitrogen is lost from aqueous solution to the atmosphere as NH3 (a volatile gas). Denitrification is the conversion of nitrates and nitrites in soil to gases such as N2O or N2 by bacteria under anaerobic conditions. Nitrous oxide (NO, N2O) is an incomplete product of the conversion of nitrates to N2 and, as a greenhouse gas, contributes to climate change.

[0008] There is a need for fertilizers that prevent nitrogen leaching and are environmentally friendly. Summary of the Invention

[0009] The present invention provides a solution to the problem of nitrogen leaching in crop soils, which is also environmentally friendly. The inventors have found that aerobic rhizosphere microorganisms capable of reducing nitrate to ammonium, when added to soil alone or in combination with fertilizer, are effective in reducing nitrogen leaching from soil.

[0010] Thus, in a first aspect, the present invention relates to a composition comprising at least one aerobic rhizosphere microorganism, wherein the microorganism comprises within its genome at least one nitrate reductase gene and at least one nitrite reductase gene, and wherein when the microorganism is cultured in a minimal growth medium containing nitrate as the sole nitrogen source, the microorganism reduces nitrate to ammonium.

[0011] In a second aspect, the present invention relates to a fertilizer comprising a composition according to the first aspect.

[0012] In a third aspect, the present invention relates to the use of the composition of the first aspect or the fertiliser of the second aspect to prevent nitrogen leaching and / or to increase general crop productivity or crop yield.

[0013] In a fourth aspect, the present invention relates to the use of the composition of the first aspect or the fertilizer of the second aspect for preventing emissions of N2O. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows the evolution of total inorganic nitrogen over 60 days in soil treated with CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) strains, respectively, or untreated soil (control). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention provides a natural solution to the problem of nitrogen leaching, thus avoiding the use of chemical nitrification inhibitors in crop soils.

[0016] With respect to the microorganisms of the composition of the first aspect of the present invention, the term "aerobic" refers to microorganisms that can survive and grow in oxygen-containing environments, either facultative (alternatively, capable of surviving and growing in oxygen-containing or oxygen-free environments) or strict (capable of surviving and growing only in oxygen-containing environments). The term "rhizospheric" as used herein refers to microorganisms isolated from the rhizosphere. In a preferred embodiment, the microorganism is a plant growth-promoting rhizobacterium (PGPR).

[0017] The nitrate reductase gene is a nucleotide sequence that encodes an enzyme capable of converting nitrate to nitrite. The nitrite reductase gene is a nucleotide sequence that encodes an enzyme capable of converting nitrite to ammonium. In a preferred embodiment, the nitrate reductase gene and the nitrite reductase gene in the genome of the microorganism are expressed. The production of ammonium is tested in a minimal growth medium: 0.6 g / l KH2PO4, 0.4 g / l MgSO4 7H2O, 4 g / l glucose, 8 g / l mannitol, 8 g / l sodium pyruvate, 1 ml / l vitamin solution and 1 ml / l trace elements (Bergersen et al., 1961 Aust J Biol Sci 14: 349-360), and 1 g / l KNO3 as the sole nitrogen source. The test is performed in duplicate as follows: The microorganism is inoculated into a test tube containing 5 ml of growth medium and incubated at 28° C. for 72 hours. The presence of ammonium is tested by a colorimetric assay based on Nessler's reagent (K2HgI4), which reacts with ammonia in strongly alkaline solution to produce a yellow complex in direct proportion to the ammonia concentration, e.g., the commercial colorimetric test VACUettes KIT Ammonia (CHEMetrics).

[0018] In a preferred embodiment of the first aspect, the composition comprises 10 3 CFU~5×10 12 CFU, preferably 10 per gram of composition 5 CFU~10 11 CFU, more preferably 10 per gram of composition. 6 CFU~10 10 Contains CFU.

[0019] In a preferred embodiment of the first aspect, the microorganism does not contain the nrfA gene in its genome. This gene is considered a marker for DNRA. Therefore, it is preferred that the microorganism performs ANRA rather than DNRA.

[0020] In a preferred embodiment of the first aspect, the microorganism is not capable of producing N2 from nitrate. The production of nitrogen N2 from nitrate by the microorganism is tested as follows: The microorganism is grown in a conventional growth medium for nitrate reduction assay consisting of 5 g / l peptone, 3 g / l meat extract, and 1 g / l potassium nitrate as nitrogen source and incubated at 28°C for 48 hours. The denitrifying microorganisms convert the nitrate (NO3 - Denitrifying microorganisms can be detected by the absence of nitrates and nitrites after incubation, by reducing nitrates (0.8 g sulfanilic acid + 100 ml acetic acid 5N) + NIT 2 (0.6 g N-N-dimethyl-1-naphthylamine + 100 ml acetic acid 5N), which stains nitrites red, after incubation. If the sample does not stain, it is then treated with zinc dust, which stains nitrates red. As a result, in the case of denitrifying microorganisms, the sample does not stain in either of the two staining steps.

[0021] In a preferred embodiment of the first aspect, the microorganism comprises at least one of the genes nasB or nasC and at least one of the genes nasD and nasE. In a preferred embodiment, the genes nasB, nasC, nasD and nasE are expressed by the microorganism, such that their expression can be detected, for example, by RT qPCR.

[0022] In a preferred embodiment of the first aspect, the microorganism is Gram+.

[0023] The composition according to any one of the above claims, wherein the microorganism is a species of the genus Bacillus, preferably the microorganism is of the species Bacillus subtilis or Bacillus megaterium. In a preferred embodiment, the microorganism is strain CECT 30572 or CECT 30573, or a combination thereof. In another aspect, the invention relates to the use of the composition of the first aspect for preventing nitrogen leaching, for increasing general crop productivity or crop yield, or for preventing NO emissions, preferably wherein the microorganism is strain CECT 30572 or CECT 30573, or a combination thereof.

[0024] With respect to the fertilizer of the second aspect of the present invention, the term "fertilizer" refers to a natural or artificial substance containing chemical elements that enhance plant growth and productivity. In a preferred embodiment, the fertilizer is selected from the group consisting of nitrogen fertilizer, phosphate fertilizer, potassium fertilizer, NP compound fertilizer, PK compound fertilizer, NK compound fertilizer or NPK compound fertilizer, limestone improver, magnesium improver, sulfur improver, calcium and sulfur improver, water retention improver, silica improver, organic improver, and other soil conditioners or soil amendments. In a preferred embodiment, the fertilizer is a solid or liquid inorganic fertilizer, organic-inorganic fertilizer or organic fertilizer. In a preferred embodiment, the fertilizer is selected from the group consisting of phosphate fertilizer, potassium fertilizer, NP compound fertilizer, PK compound fertilizer, NK compound fertilizer or NPK compound fertilizer. Usually, nitrogen fertilizer is applied to the soil at 10 kg to 500 kg N / ha.

[0025] In a preferred embodiment of the fertilizer of the second aspect, the fertilizer comprises 10 2 CFU~10 10 CFU, preferably -10 per gram of fertilizer 3 CFU~10 9 CFU, preferably 10 per gram of fertilizer 4 CFU~10 8 In a preferred embodiment, the fertilizer contains 10 CFU to 10 CFU per gram of soil. 4 It is applied to the soil so that the CFU is applied.

[0026] In a preferred embodiment of the fertilizer of the second aspect, the microorganisms in the composition are protected by a microorganism-protecting compound, such as trehalose, carob gum or xanthan gum. The term "microorganism-protecting compound" as used herein refers to a compound that allows the survival of microorganisms under the physiological conditions of the fertilizer when testing the survival of the microorganism according to the most probable number method, as described in EP 3085679.

[0027] In a preferred embodiment of the third aspect of the invention, the composition or fertilizer is applied directly to the soil as such or in combination with an organic or inorganic carrier, such as peat, biochar, clay, or the composition is applied to the soil in irrigation water or compost or a soil amendment, conditioner or amendment, or the composition is applied to the seed before sowing. EXAMPLES

[0028] The following examples illustrate the invention.

[0029] Microorganisms useful in preventing nitrate leaching. Two bacterial strains (CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium)) were isolated from the rhizosphere. These strains were deposited in the Spanish Type Strain Collection (Coleccion Espanola de Cultivos Tipo (CECT)) under the accession numbers CECT 30572 and CECT 30573, respectively. Both microorganisms are Gram+ aerobic and reduce nitrate to ammonium when grown with nitrate as the sole nitrogen source. Also, neither of these microorganisms produces N2 from nitrate when tested according to the method described above.

[0030] The whole genomes of the two microorganisms were sequenced. To do so, the bacterial strains were grown on tryptic soy agar (TSA, Merck) plates at 28°C for 24 hours. Genomic DNA was obtained using a bacterial genomic DNA isolation kit (NORGEN™) following the manufacturer's protocol. Sequencing was performed on an Illumina MiSeq sequencing platform (2x250bp) by Microbes NG (UK) upon preparation of paired-end libraries.

[0031] To search for specific genes in the genomes of strains CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium), the software Geneious Prime™ 2020.2.4 (Biomatters Ltd.) was used.

[0032] None of these microorganisms contain the nrfA gene, a marker for DNRA, in their genomes, indicating that this metabolic pathway is absent in any of the microorganisms that were subjected to genome mining for the presence of the nrfA gene.

[0033] The sequences of the nrfA gene were obtained from the NCBI database of Bacillus species and searched in the corresponding genomes using the tool Blast (Megablast) by Geneious Prime. The results are shown in Table 1.

[0034] [Table 1]

[0035] Nitrate / nitrite reductase gene To explore the presence of nitrate reductase and nitrite reductase genes involved in the metabolic pathway ANRA, bacterial strains CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) were subjected to genome mining.

[0036] The whole genome sequence was obtained as described above and the search for the presence of specific genes in the strains in question was carried out using the same tools and the same procedures as above.

[0037] [Table 2] TIFF2024528044000003.tif24170

[0038] Reduction of nitrate to ammonium Both strains CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) were tested for the production of ammonium from nitrate, and both were found to produce ammonium when grown in a medium containing nitrate as the sole nitrogen source.

[0039] Bacteria with an assimilatory metabolic pathway (ANRA) reducing nitrate to ammonium can be detected by the presence of ammonium ions after incubation in minimal growth medium containing nitrate as the sole nitrogen source. Ammonium ions can also be detected as intermediate species, i.e., they are subsequently assimilated by the bacteria and converted to bacterial macromolecules (R-NH2). The composition of the minimal growth medium was 0.6 g / l KH2PO4, 0.4 g / l MgSO4 7H2O, 4 g / l glucose, 8 g / l mannitol, 8 g / l sodium pyruvate, 1 ml / l vitamin solution and 1 ml / l trace elements (Bergersen et al., 1961. Aust J Biol Sci 14: 349-360), and 1 g / l NO3K as the sole nitrogen source. The test was performed in duplicate in a test tube containing 5 ml of the above growth medium, with the bacteria incubated at 28°C for 72 hours. The presence of ammonium was detected using a commercial colorimetric test kit (VACUettes KIT Ammonia, CHEMetrics).

[0040] [Table 3]

[0041] Prevention of leaching Both strains CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) were effective in preventing leaching in column experiments measuring nitrogen leaching from agricultural soils after 60 days of continuous irrigation. The experiments were carried out by incorporating inorganic fertilizer (calcium ammonium nitrate 27% nitrogen) coated with each strain into the top soil layer of a leaching column. A control containing no microorganisms was also included. The fertilizer coated with the strains was observed to reduce nitrogen leaching by 20%-30% in both cases.

[0042] It was also observed that the addition of the strain to the soil resulted in higher inorganic nitrogen levels in the soil compared to the untreated control, as can be seen in Figure 1. In this experiment, 270 mg N / kg soil and 3 × 10 N / g soil were added. 2 CFU was applied.

[0043] We also tested ammonium nitrate fertilizer coated with both CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) strains to see if the microorganisms were able to reduce nitrate to ammonia in pots without plants subjected to the leaching process. These tests allowed us to analyze the effect of coating calcium ammonium nitrate fertilizer (27% nitrogen content) with each strain, which reduces nitrate to ammonium, on the leaching of different forms of nitrogen in real soil conditions without plants.

[0044] Treatments consisted of fertilizers coated with strains CBLUT9 (Bacillus subtilis) and RPVPMO04 (Bacillus megaterium) at a dose of 1% (volume of culture medium / weight of fertilizer), respectively. The concentration of culture medium was 6 × 10 per ml in both cases. 8 CFU was used. In all cases, the compositions containing the microorganisms also contained 1% carob gum by weight.

[0045] Nitrate leaching (mg / l in leachate) was tested after 5 and 20 days in pots containing each strain and in pots containing fertilizer only as a control, with the following results indicating that both strains reduced nitrate leaching:

[0046] [Table 4]

[0047] In this experiment, 225 mg N / kg soil and 6 × 10 3 CFU was applied.

[0048] We also tested ammonium sulphonitrate (ASN) fertilizer coated with each of the strains capable of reducing nitrate to ammonia in a column and subjected it to the leaching process. The negative control was soil and the positive control was uncoated ASN. In two tests carried out on different days, both strains significantly reduced the amount of nitrate leaching from the column:

[0049] [Table 5]

[0050] In this experiment, 148 mg N per kg of soil and 1.71 × 10 2 CFU was applied.

[0051] Biostimulation effect and improved fertilizer efficiency The microcosm test using plants was carried out under the following conditions: Agricultural soil. pH: Neutral. Fertilizer: NPK 22-10-6. Crop: Corn. Duration: 5 weeks.

[0052] The results in the table below show the NO3 - / NH4 +It demonstrates an environmental benefit due to the reduction in the ratio and a biostimulatory effect due to the biomass results. Also, a higher fertilization efficiency is observed. Two strains, CBLUT9 and RPVPMO04, were tested and a control containing only NPK fertilizer (NPK without microorganisms) was included in addition to a control where nothing was added to the plants (Control 0).

[0053] [Table 6]

[0054] In brackets is the percent difference relative to microbial-free NPK.

[0055] In this experiment, 50 mg N per kg of soil and 1.37 × 10 3 CFU was applied.

[0056] Biostimulatory Effects The first field tests were carried out under the following conditions: Maize cultivation in Valladolid, Spain, 2016. Fertilizer: NPK 18-8-10.

[0057] [Table 7]

[0058] In this experiment, 220 kg N per hectare and 5.5 × 10 1 CFU was applied.

[0059] The second field test was conducted under the following conditions: Maize cultivation in Valladolid, Spain, 2016. Fertilizer: NPK 20-5-10.

[0060] [Table 8]

[0061] The concentration of nitrates in the soil decreased and NO3 - / NH4 + The environmental benefits of lowering the ratio have been demonstrated.

[0062] In this experiment, 160 kg N per hectare and 5.5 × 10 1 CFU was applied.

[0063] Reducing N2O emissions In controlled tests, N2O emissions were measured in pots with moist soil and no plants for 14 days after fertilizer application.

[0064] Strains CBLUT9 and RPVPMO04 were able to reduce NO emissions from fertilizers when applied to soil, and we found that the addition of nitrogen fertilizers with these strains reduced NO emissions for both fertilizers used.

[0065] [Table 9]

[0066] In this experiment, 700 mg N per kg of soil and 7.77 × 10 2 CFU was applied.

[0067] The incubation support consisted of pots with a diameter of 16 cm and a height of 11.5 cm. The soil had a bulk density of 1 g / cm 32300 g of agricultural soil was added to each pot so that the N2O concentration was 60%. There were five replicates of treatments (experimental blocks) and the pots were randomly placed in each block. On day 1, water was added to each pot until a water-filled pore space (WFPS) of 60% was reached. The pots were then applied with the various selected fertilizer products. The first N2O measurements were taken on the same day, 1 and 2 hours after the fertilizer products were applied. A total of 10 N2O measurements were taken on different days over the next 14 days. Sampling consisted of inserting a PVC chamber (inserted 6 cm) into each pot, measuring 11.5 cm in diameter and 20 cm in length. Each chamber had an opening at the top, which was closed with a tight-fitting PVC lid during the sampling period. These lids had a sampling hole covered by a rubber septum, allowing a 6 ml syringe to be inserted to sample the air inside the chamber. Once the chamber was closed, the air inside was sampled at two time points: when the lid was closed (Ti) and after 20 minutes (Tf). Air collected at each time point was stored in exetainer-type evacuated vials. The concentration of N2O stored in each vial was analyzed using an Agilent 7890B gas chromatograph equipped with an electron capture detector (ECD) operating at a temperature of 280 °C and an HP-Plot Q column using He as the carrier gas. Two N2O concentration values ​​in each chamber were obtained for each sampling day, allowing the calculation of the daily emission flux from the soil in each chamber to the atmosphere. This flux value was expressed as mg / m2 of N2O-N per day. 2 Similarly, by integrating each of the daily values, it was possible to calculate the cumulative amount of N2O emitted for each chamber over the 14-day experiment. [Table 10] TIFF2024528044000012.tif60170

Claims

1. A fertilizer comprising a composition containing at least one aerobic rhizosphere microorganism, wherein the microorganism contains at least one nitrate reductase gene and at least one nitrite reductase gene in its genome, and when the microorganism is cultured in a minimal growth medium containing nitrate as the sole nitrogen source, the microorganism reduces nitrate to ammonium, and the fertilizer is selected from the group consisting of nitrogen fertilizers, phosphate fertilizers, potassium fertilizers, NP compound fertilizers, PK compound fertilizers, NK compound fertilizers or NPK compound fertilizers, limestone improvers, magnesium improvers, sulfur improvers, calcium and sulfur improvers, water retention improvers, silica improvers, and organic improvers.

2. The composition contains 10 ３ CFU to 5 × 10 １２ CFU per gram of the composition, and the fertilizer according to claim 1.

3. The fertilizer according to claim 1 or 2, wherein the microorganism does not contain the nrfA gene in its genome.

4. The microorganism is unable to generate N from nitrate ２ The fertilizer according to claim 1 or 2, which cannot be generated

5. The fertilizer according to claim 1 or 2, wherein the microorganism contains at least one of the genes nasB or nasC and at least one of the genes nasD or nasE.

6. The fertilizer according to claim 1 or 2, wherein the microorganism is Gram+.

7. The fertilizer according to claim 1 or 2, wherein the microorganism is a species of the genus Bacillus.

8. The fertilizer according to claim 1 or 2, wherein the microorganism is the CECT 30572 strain, the CECT 30573 strain, or a combination of both.

9. The fertilizer contains 10 ２ CFU to 10 １０ CFU per gram of fertilizer, and is the fertilizer according to claim 1 or 2.

10. The fertilizer according to claim 1 or 2, wherein the fertilizer is selected from the group consisting of phosphate fertilizers, potassium fertilizers, NP compound fertilizers, PK compound fertilizers, NK compound fertilizers or NPK compound fertilizers.

11. The fertilizer according to claim 1 or 2, wherein the fertilizer is a solid or liquid inorganic fertilizer, an organic-inorganic fertilizer or an organic fertilizer.

12. The fertilizer according to claim 1 or 2, wherein the microorganism is protected by a microbial protection compound such as trehalose, carob gum or xanthan gum.

13. Use of a composition containing at least one aerobic rhizosphere microorganism for preventing nitrogen leaching and / or enhancing general crop productivity or crop yield, wherein the microorganism contains at least one nitrate reductase gene and at least one nitrite reductase gene in its genome, and when the microorganism is cultured in a minimal growth medium containing nitrate as the sole nitrogen source, the microorganism reduces nitrate to ammonium, or use of the fertilizer according to claim 1 or 2.

14. The use of the composition or fertilizer according to claim 13, wherein the composition or fertilizer is applied directly to the soil as such or in combination with an organic or inorganic carrier, or the composition is applied to the soil in irrigation water, compost or a soil conditioner, or the composition is applied to seeds before sowing.

15. N ２ Use of a composition comprising at least one aerobic rhizosphere microorganism for preventing the release of NO, wherein the microorganism comprises at least one nitrate reductase gene and at least one nitrite reductase gene in its genome, and when the microorganism is cultured in a minimal growth medium containing nitrate as the sole nitrogen source, the microorganism reduces nitrate to ammonium, or use of the fertilizer according to claim 1 or 2.

16. A composition comprising at least one aerobic rhizosphere microorganism, wherein the microorganism comprises at least one nitrate reductase gene and at least one nitrite reductase gene in its genome, and when the microorganism is cultured in a minimal growth medium containing nitrate as the sole nitrogen source, the microorganism reduces nitrate to ammonium, and the microorganism is the CECT 30572 strain, the CECT 30573 strain, or a combination of both.

17. Use of the composition according to claim 16 for preventing nitrogen elution, for increasing the overall crop productivity or crop yield, or for preventing the release of N ２ O.

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