Method for inhibiting the nitrification process in soil

Abstract

The invention relates to a method for inhibiting the nitrification process in soil, wherein one or more miRNAs are applied to the soil in order to inhibit the expression of one or more genes participating in the nitrification process in said soil, such as the genes encoding the AMO (ammonia monooxygenase) enzyme and / or the HAO (hydroxylamine oxidoreductase) enzyme.

Description

Title of the invention: Method for inhibiting the nitrification process in a soil DESCRIPTION Technical Field

[0001] The invention relates to a method for inhibiting the nitrification process in a soil, in which one or more miRNAs are applied to the soil in order to inhibit the expression of one or more genes that participate in the nitrification process in said soil, such as the genes encoding the enzyme AMO (Ammonia MonoOxygenase) and / or the enzyme HAO (HydroxylAmine Oxidoreductase). The invention also relates to a fertilizer composition comprising a fertilizer or an amendment, and one or more miRNAs. Prior art

[0002] In nature, due to biogeochemical processes, the oxidation state of nitrogen (N) varies considerably, ranging from -3 to +5, resulting in the formation of many nitrogen species, including N2O5, HNO3, HNO3, NO2, N2O4, HNO2, HNO2, NO, N2O, N2, NH3, NH4+, NH4Cl, and CH3NH2[1].

[0003] Nitrification is a biological process by which nitrates are produced in the environment. This occurs in several distinct stages, each under the action of different microorganisms:

[0004] Step 1) Ammonia-oxidizing bacteria (i.e., AOBs, such as Nitrosomonas spp. and Nitrosococcus spp.) initiate the nitrification process by oxidizing ammonia (NH3) to hydroxylamine (NH2OH). This step is catalyzed by the enzyme AMO (Ammonia MonoOxygenase).

[0005] Step 2) AOBs oxidize NH2OH to nitrite (NO2“) via the enzyme HAO (HydroxylAmine Oxidoreductase).

[0006] Step 3) Nitrite-oxidizing bacteria (i.e. NOB, such as Nitrobacter spp.) complete the nitrification process by producing nitrate (NO3') via the enzyme HAO (Hydroxyl Amine Oxidoreductase).

[0007] Nitrification results in the loss of about one-fifth of the nitrogen applied to the soil via leaching of NO3 and the emission of nitrous oxide (N2O) [2]. Nitrification leads to increased agricultural production costs, environmental pollution [3], human and animal diseases [4], ozone depletion, and greenhouse gas emissions. greenhouse effect that contribute to climate change [5]. About 97% of N2O emissions in crop systems are attributed to the nitrification process [6].

[0008] Therefore, controlling the nitrification process has become a major issue in agriculture in recent decades.

[0009] Currently, the control of nitrification in soil by the farmer is mainly achieved by using synthetic nitrification inhibitors. These include a wide range of compounds such as dicyandiamide (DCD), 2-chloro-6-(trichloromethyl)-pyridine (Nitrapyrin), 3,4-dimethylpyrazole phosphate (DMPP) [7], allylthiourea [8], 2-amino-4-chloro-6-methylpyrimidine [9], etc.

[0010] However, the use of these synthetic inhibitors has been limited due to the high cost which limits their large-scale use, their limited availability

[0010] , their non-targeted effect causing adverse effects on beneficial microorganisms present in the soil

[0011] and because of the environmental pollution they cause, particularly in aquatic ecosystems [2]

[0010] .

[0011] In a context where environmental protection is a major social issue, it is important to develop natural and environmentally friendly methods that can inhibit nitrification in the soil.

[0012] The inventors thus developed a new strategy for inhibiting nitrification genes by showing that certain miRNAs had the property of inhibiting the nitrification process in soil.

[0013] The invention, which finds application in the field of agriculture, relates to the discovery of microRNAs (miRNAs) having the capacity to inhibit mRNAs of different bacterial genera responsible for the regulation of the nitrification process in soils (hereinafter the invention). In particular, miRNAs have been identified having the capacity to inhibit the mRNAs coding for the AMO and / or HAO enzymes, which would make it possible to inhibit the nitrification process in soils. Summary of the invention

[0014] A first subject of the present invention relates to a method for inhibiting the nitrification process in a soil, in which one or more miRNAs of formula (I) are applied to the soil in order to inhibit the expression of one or more genes which participate in the nitrification process in said soil: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0015] A second subject of the present invention relates to the use of a miRNA of formula (I) to inhibit the expression of one or more genes which participate in the nitrification process: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0016] A third subject of the present invention relates to a composition comprising one or more isolated miRNA(s) of formula (I): [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0017] A fourth subject of the present invention relates to a culture support or a culture solution comprising one or more isolated miRNA(s) of formula (I) or a composition according to the invention. Detailed description

[0018] Method or use for inhibiting the expression of one or more genes involved in the nitrification process

[0019] The present invention relates to a method for inhibiting the nitrification process in a soil, wherein one or more miRNAs of formula (I) are applied to the soil in order to inhibit the expression of one or more genes which participate in the nitrification process in said soil: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0020] The present invention also relates to the use of a miRNA of formula (I) for inhibiting the expression of one or more genes which participate in the nitrification process: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0021] The term "soil" refers to the substrate explored by the roots of cultivated plants. The term soil includes any type of substrate on which a plant can grow, such as artificial soil, cultivated agricultural soil, uncultivated agricultural soil, peat, potting soil, rock wool, or coconut fiber. The term soil therefore also covers growing media that can be used, in particular, for so-called "soilless" cultivation, for example in pots.

[0022] The term "inhibit the nitrification process" or "inhibition of the nitrification process" means at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% reduction in the nitrification process compared to a reference condition. In the context of the invention, the inhibition of the nitrification process is calculated by measuring the nitrification process in a soil to which one or more miRNAs of formula (I) are applied, and by comparing it with a soil to which one or more miRNAs of formula (I) have not been applied. A method for measuring the inhibition is detailed in the examples.

[0023] The term "inhibiting the expression of one or more genes" or "inhibition of the expression of one or more genes" refers to the property of miRNAs to inhibit the mRNAs of said one or more genes, thereby inhibiting the synthesis of proteins encoded by said inhibited genes. Advantageously, the inhibition is at least 2%, at least 3%, at least less than 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%, of decrease in the expression of said one or more genes compared to a reference condition. In the context of the invention, the inhibition of the expression of one or more genes can be calculated by quantifying the proteins encoded by said inhibited genes and / or the corresponding mRNAs by implementing conventional techniques for those skilled in the art such as the ELISA method for quantifying proteins or RT-qPCR for quantifying mRNAs. The percentage of inhibition is calculated by measuring the inhibition of the expression of one or more genes in a soil to which one or more miRNAs of formula (I) are applied, and by comparing it with a soil to which one or more miRNAs of formula (I) have not been applied.

[0024] The term "microRNA" or "miRNA" refers to a short ribonucleic acid (RNA) molecule that acts as a post-transcriptional regulator by regulating target mRNAs. A miRNA can be 20 to 24 nucleotides (nt) in length, preferably 20 to 22 nt, for example 20, 21, or 22 nt. The preparation of a miRNA presents no particular difficulty, for example by conventional chemical synthesis.

[0025] In particular embodiments, the miRNA of formula (I) is a synthetic miRNA, i.e. it has been synthesized in vitro, for example by chemical synthesis. Examples include synthesis on a solid support by the phosphoramidite method. In vitro synthesis makes it easy to obtain the desired quantities of miRNA of formula (I).

[0026] In particular embodiments, the miRNA of formula (I) comprises from 19 to 21 nucleotides, i.e. 19 nucleotides, 20 nucleotides or 21 nucleotides.

[0027] [Sequence 3'] corresponds to the 3' part of the sequence of the miRNA of formula (I). This sequence may comprise 10, 11, 12 or 13 nucleotides. In particular embodiments, [Sequence S'] comprises at least 80% homology with a nucleotide sequence chosen from the sequences SEQ ID NO: 9-16, preferably at least 85% homology, for example at least 90% homology, at least 95% homology, at least 99% homology or even 100% homology.

[0028] When [Core Sequence] is SEQ ID NO: 1, [Sequence S'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 9. When [Core Sequence] is SEQ ID NO: 2, [Sequence S'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 10. When [Core Sequence] is SEQ ID NO: 3, [Sequence S'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 11. When [Core Sequence] is SEQ ID NO: 4, [Sequence 3'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 12. When [Core Sequence] is SEQ ID NO: 5, [Sequence S'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 13. When [Core Sequence] is SEQ ID NO: 6, [Sequence 3'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 14. When [Core Sequence] is SEQ ID NO: 7, [Sequence 3'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 15. When [Core sequence] is SEQ ID NO: 8, [Sequence S'] preferably comprises at least 80% homology with the nucleotide sequence SEQ ID NO: 16.

[0029] In particular embodiments, the miRNA of formula (I) may be selected from sbi-miR162 (SEQ ID NO: 17), sbi-miR394a (SEQ ID NO: 18), sbi-miR397-5p (SEQ ID NO: 19), sbi-miR5386 (SEQ ID NO: 20), sbi-miR5388 (SEQ ID NO: 21), sbi-miR5568d-3p (SEQ ID NO: 22), sbi-miR5568d-5p (SEQ ID NO: 23) and sbi-miR6232a-5p (SEQ ID NO: 24).

[0030] A mixture of several miRNAs of formula (I) each having a distinct [Core Sequence] can be applied to the soil. In particular embodiments, from two to eight miRNAs of formula (I) each having a distinct [Core Sequence] are applied to the soil, for example a mixture of two, three, four, five, six, seven or eight miRNAs of formula (I) each having a distinct [Core Sequence] is applied to the soil. For example, it may be a mixture comprising sbi-miR162 (SEQ ID NO: 17), sbi-miR394a (SEQ ID NO: 18), sbi-miR397-5p (SEQ ID NO: 19), sbi-miR5386 (SEQ ID NO: 20), sbi-miR5388 (SEQ ID NO: 21), sbi-miR5568d-3p (SEQ ID NO: 22), sbi-miR5568d-5p (SEQ ID NO: 23) and sbi-miR6232a-5p (SEQ ID NO: 24).

[0031] The miRNA of formula (I) inhibits the expression of a gene encoding the enzyme Ammonia MonoOxygenase (AMO) and / or inhibits the expression of a gene encoding the enzyme HydroxylAmine Oxydoreductase (HAO). In particular, the miRNA of formula (I) is capable of targeting one or more mRNAs encoding AMO and / or one or more mRNAs encoding AHO, thereby inhibiting the translation of the target mRNAs into proteins. Thus, in a preferred embodiment, said one or more genes that participate in the nitrification process in said soil encode the enzyme Ammonia MonoOxygenase (AMO) and / or the enzyme HydroxylAmine Oxydoreductase (HAO).

[0032] In the context of the present invention, said one or more genes which participate in the nitrification process in said soil is expressed by one or more bacteria present in the soil, in particular ammonia-oxidizing bacteria (AOB) and / or nitrite-oxidizing bacteria (NOB), preferably one or more bacteria present in the soil chosen from Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp., for example chosen from Nitrosomonas europaea, Nitrosomonas eutropha C-91, Nitrosomonas communis Nm2, Nitrosospira multiformis and Nitrosospira iacus APG3. The miRNA of formula (I) therefore has the property of inhibiting the nitrification activity of microorganisms involved in the nitrification process in the soil, in particular ammonia-oxidizing bacteria (AOB) and / or nitrite-oxidizing bacteria (NOB).

[0033] The miRNA of formula (I) can be applied to the soil in an amount ranging from 1 g / ha to 1000 g / ha.

[0034] In a particular embodiment, the miRNA of formula (I) may be applied to the soil with a fertilizer, for example, a solid or liquid fertilizer, an amendment, and / or a biostimulant. This may be, for example, a nitrogen fertilizer. The miRNA of formula (I) may inhibit the nitrification of a nitrogen fertilizer applied to the soil, for example, a nitrogen fertilizer that is applied to said soil before, concomitantly with, or after said miRNA. Thus, the miRNA of formula (I) and the nitrogen fertilizer may be applied to the soil simultaneously or sequentially. For example, the miRNA of formula (I) may be applied to the soil during a first treatment, and the nitrogen fertilizer may be applied to the soil during a second treatment. Several successive treatments of the soil may thus be carried out. Generally, the person skilled in the art adapts the number of treatments and the nature of the treatment, for example, depending on the nature of the soil and / or the plant grown in the soil.

[0035] The method or use according to the invention finds application in the treatment of soil in which a very wide variety of plants can be planted. Among these, we will mention in particular: - large-scale crops such as cereals (wheat, corn, barley), - protein crops (peas), - oilseeds (soybean, sunflower), - Solanaceae crops (potato), - Amaranthaceae (beet) crops, - specialized crops such as market gardening (lettuce, spinach, onion, shallot, tomato, melon), vines, arboriculture (pear, apple, nectarine), or horticulture.

[0036] In particular, the plant may belong to the order of monocotyledons, preferably to the family of Poaceae. The Poaceae, commonly called grasses, contain including most of the species commonly referred to as "grasses" and "cereals." Cereals are widely cultivated, primarily for their grains, and are used in human and animal food. Advantageously, the plant is a Poaceae, preferably selected from wheat, rice, barley, oats, rye, sugarcane, meadow or corn, preferably wheat.

[0037] In particular embodiments, the miRNA is an isolated miRNA.

[0038] Thus, in particular embodiments, the invention relates to a method for inhibiting the nitrification process in a soil, wherein one or more isolated miRNAs of formula (I) are applied to the soil in order to inhibit the expression of one or more genes which participate in the nitrification process in said soil: [Nucleotide]-[Core sequence]-[3' sequence] (I), wherein: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' Sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core Sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0039] Thus also, in particular embodiments, the present invention relates to the use of an isolated miRNA of formula (I) to inhibit the expression of one or more genes which participate in the nitrification process: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0040] An isolated miRNA corresponds to a miRNA isolated from its natural environment. For example, the miRNA may be isolated from a living organism containing said miRNA, such as a plant or an algae, for example by implementing an extraction method. Methods for extracting microRNA are described in the literature and their implementation presents no particular difficulty for the person skilled in the art. Examples include extraction with a solvent, such as water, a hydrophilic solvent or a combination thereof.

[0041] Isolated miRNA can also be chemically synthesized. This is called synthetic miRNA. Methods for chemically synthesizing nucleic acids are widely used. described in the literature and their implementation does not present any particular difficulty for the person skilled in the art.

[0042] The miRNA (or isolated miRNA) may be in the form of a composition, for example a composition comprising said miRNA and an excipient. The excipient may be chosen for its ability to stabilize the miRNA in soil, thereby reducing degradation of the miRNA in the soil. The composition may comprise from 0.01% to 99% by weight of said miRNA. When the miRNA is isolated from a plant or an algae, the composition may be in the form of a plant extract or an algae extract.

[0043] Composition and growing medium

[0044] The invention also relates to a composition comprising one or more isolated miRNA(s) of formula (I): [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide chosen from A, U, G or C, [3' sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core sequence] corresponds to a nucleotide sequence chosen from the sequences SEQ ID NO: 1-8.

[0045] In particular embodiments, the composition according to the invention further comprises a fertilizer. The fertilizer may be a fertilizer, an amendment and / or a biostimulant.

[0046] In particular embodiments, [Sequence 3'] comprises at least 80% homology with a nucleotide sequence selected from the sequences SEQ ID NO: 9-16, preferably at least 85% homology, for example at least 90% homology, at least 95% homology, at least 99% homology or even 100% homology.

[0047] In particular embodiments, the miRNA is selected from sbi-miR162 (SEQ ID NO: 17), sbi-miR394a (SEQ ID NO: 18), sbi-miR397-5p (SEQ ID NO: 19), sbi-miR5386 (SEQ ID NO: 20), sbi-miR5388 (SEQ ID NO: 21), sbi-miR5568d-3p (SEQ ID NO: 22), sbi-miR5568d-5p (SEQ ID NO: 23) and sbi-miR6232a-5p (SEQ ID NO: 24).

[0048] The composition according to the invention may be in solid form, for example in the form of granules or powder, or in liquid form, for example in the form of an aqueous solution. For example, the composition according to the invention may be in the form of granules impregnated or coated with miRNA of formula (I).

[0049] The miRNA may be a miRNA extracted from a living organism known to contain said miRNA or a synthetic miRNA, as described above.

[0050] The composition may comprise an excipient that stabilizes the miRNA in the soil, thereby reducing degradation of the miRNA in the soil. The composition may comprise from 0.01% to 99% by weight of said miRNA.

[0051] The fertilizer may be of natural or synthetic origin. In one embodiment, the fertilizer is nitrogenous. A nitrogenous fertilizer must provide at least nitrogen (N) as a nutrient.

[0052] In particular embodiments, the nitrogen fertilizer contains ammonium, ammonia, and / or urea. Examples of ammonium-containing fertilizers are calcium ammonium nitrate, calcium ammonium nitrate, ammonium sulfate nitrate, ammonium sulfate, and ammonium phosphate. An example of an ammonia-containing fertilizer is ammonium nitrate.

[0053] Nitrogen fertilizer can also provide other nutrients, for example, potassium (K) and / or phosphorus (P). It can be an inorganic fertilizer and / or an organic fertilizer. Nitrogen fertilizers providing N, as well as P or K, include so-called "NP" or "NK" fertilizers, such as monoammonium phosphate or diammonium phosphate. Nitrogen fertilizers providing N, P and K include so-called "NPK" fertilizers.

[0054] The amendment can be an organic amendment or a mineral amendment, such as calcium carbonate.

[0055] The composition according to the invention may also comprise micronutrients such as molybdenum, zinc, boron and copper. These elements are typically provided in the form of water-soluble salts.

[0056] The composition according to the invention may also comprise a solvent, a solid support, a dispersant or an emulsifier, an organic or inorganic thickener, a pesticidal agent, an antifreeze agent, an anti-foaming agent, where appropriate a colorant, a sticking agent or a binder.

[0057] In particular embodiments, the composition according to the invention comprises a mixture of two to eight miRNAs of formula (I) each having a distinct [Core Sequence], for example a mixture of two, three, four, five, six, seven or eight miRNAs of formula (I) each having a distinct [Core Sequence].

[0058] In particular embodiments, the composition according to the invention is intended for culture supports and / or culture solutions.

[0059] Thus, another subject of the present invention relates to a culture support or a culture solution comprising one or more isolated miRNA(s) of formula (I) or a composition according to the invention.

[0060] For the purposes of the present invention, the term "culture solution" means any solution which allows the cultivation of a plant such as, for example, irrigation water, liquid solution intended for fertigation by sprinkling or drip irrigation, hydroponic bath or aeroponic solution. The term "culture medium" is defined above. EXAMPLES

[0061] Example 1: miRNA to inhibit the biological nitrification process of Nitrosomonas europaea 25978

[0062] Materials and methods

[0063] The 8 miRNAs used in this example were prepared by chemical synthesis and correspond to the miRNAs presented in Table 1.

[0064] [Table 1]

[0065] A liquid culture in a 500 mL flask containing 150 mL of Skinner and Walker (SW) medium

[0012] was inoculated with Nitrosomonas europaea 25978 at a ratio of 1:100 (V / V) and grown at 28°C with shaking at 70 rpm until the OD 600nm of the culture reaches 0.3. The cell suspension was then distributed in 20 ml volumes into 50 ml Falcon tubes.

[0066] Subsequently, 1 μg of each of the 8 miRNAs in a volume of 15 μL of water was added to the bacterial cultures in three replicates and mixed gently. All cell suspensions, including control cells (treated with the same volume of deionized water containing no miRNA) and miRNA-treated cells, were cultured for 5 days with constant shaking at 28°C.

[0067] A nitrite

[0013] test was performed on all samples at different intervals: Oh (just after adding miRNAs or deionized water) and 46h. For this, the collected cells were centrifuged at 8000 rpm for 10 minutes at 4°C and the supernatants were used for nitrite assay. Nitrite concentrations were measured colorimetrically at 540 nm using a microtiter plate reader through Griess

[0013] reagent. 100 pL of 30 mM sulfanilamide was added to each sample and mixed by pipetting up and down, then 100 pL of 12 mM N-(l-naphthyl) ethylenediamine dihydrochloride was added to the mixture. In parallel, nitrite standard samples between 0 and 100 pM using 1 mM NaNO2 were prepared. The absorbance of all samples and standards was measured by a microplate reader (BMG labtech, Germany) at 540 nm.

[0068] Results:

[0069] The results are presented in Table 2.

[0070] [Table 2]

[0071] The results show that miRNAs were able to inhibit the biological nitrification of Nitrosomonas europaea 25978 by approximately 11%.

[0072] References

[0073] [1] Hanrahan, G., & Chan, G. (2005). NITROGEN. Encyclopedia of Analytical Science: Second Edition, 191-196. https: / / doi.org / 10.1016 / B0-12-369397-7 / 00401-5.

[0074] [2] Lin, B. le, Sakoda, A., Shibasaki, R., & Suzuki, M. (2001). A modeling approach to global nitrate leaching caused by anthropogenic fertilization. Water Research, 35(8). https: / / doi.org / 10.1016 / S0043-1354(00)00484-X.

[0075] [3] Anas, M., Liao, F., Verma, K. K., Sarwar, M. A., Mahmood, A., Chen, Z. L., Li, Q., Zeng, X. P., Liu, Y., & Li, Y. R. (2020). Fate of nitrogen in agriculture and environment: agronomic, eco-physiological and molecular approaches to improve nitrogen use efficiency. Biological Research 2020 53:1, 53(1), 1-20. https: / / doi.org / 10.1186 / S40659- 020-00312-4.

[0076] [4] Ma, L., Hu, L, Feng, X., & Wang, S. (2018). Nitrate and Nitrite in Health and Disease. Aging and Disease, 9(5), 938. https: / / doi.org / 10.14336 / AD.2017.1207.

[0077] [5] Portmann, R. W., Daniel, J. S., & Ravishankara, A. R. (2012). Stratospheric ozone depletion due to nitrous oxide: influences of other gases. Philosophical Transactions of the Royal Society B: Biological Sciences, 367(1593), 1256. https: / / doi.org / 10.1098 / RSTB.2011.0377.

[0078] [6] Coskun, D., Britto, D. T., Shi, W., & Kronzucker, H. J. (2017). Nitrogen transformations in modern agriculture and the role of biological nitrification inhibition. Nature Plants 2017 3:6, 3(6), 1-10. https: / / doi.org / 10.1038 / nplants.2017.74.

[0079] [7] Beeckman, F., Motte, H., & Beeckman, T. (2018). Nitrification in agricultural soils: impact, actors and mitigation. Current Opinion in Biotechnology, 50, 166-173. https: / / doi.Org / 10.1016 / J.COPBIO.2018.01.014.

[0080] [8] Tatari, K., Gülay, A., Thamdrup, B., Albrechtsen, H. J., & Smets, B. F. (2017). Challenges in using allylthiourea and chlorate as specific nitrification inhibitors. Chemosphere, 182, 301-305. https: / / doi.Org / 10.1016 / .CHEMOSPHERE.2017.05.005.

[0081] [9] Kumar, R., Parmar, B. S., Walia, S., & Saha, S. (2015). Nitrification Inhibitors: Classes and Its Use in Nitrification Management. In Nutrient Use Efficiency: From Basics to Advance (pp. 103-122). Springer, New Delhi, https: / / doi.org / 10.1007 / 978-81-322- 2169-2_8.

[0082]

[0010] Sun, L, Lu, Y., Yu, F., Kronzucker, H. J., & Shi, W. (2016a). Biological nitrification inhibition by rice root exudates and its relationship with nitrogen-use efficiency. New Phytologist, 212(3), 646-656. https: / / doi.org / 10.llll / NPH.14057.

[0083]

[0011] Opoku, A., Chaves, B., & de Neve, S. (2014). Neem seed oil: a potent nitrification inhibitor to control nitrate leaching after incorporation of crop residues. Http: / / Dx.Doi.Org / 10.1080 / 01448765.2014.885394, 30(3), 145-152. https: / / doi.org / 10.1080 / 01448765.2014.885394.

[0084]

[0012] Skinner, F. A., & Walker, N. (1961). Growth of Nitrosomonas europaea in batch and continuous culture. Archiv Für Mikrobiologie 1961 38:4, 38(4), 339-349.

[0085]

[0013] Keeney, D. R., & Nelson, D. W. (1982). Nitrogen— Inorganic Forms. In: Methods of Soil Analysis Part 2: Chemical and Microbiological Properties, 2ndedn, Page AL, Miller R. H, Keeney D.R.(Eds) Agronomy 9 / 2, American Society of Agronomy, Madison, WI, 643-698.

[0086] Listage de séquences

[0087] [Table 3]

Claims

CLAIMS

1. A method for inhibiting the nitrification process in a soil, wherein one or more miRNAs of formula (I) are applied to the soil to inhibit the expression of one or more genes that participate in the nitrification process in said soil: [Nucleotide]-[Core sequence]-[3' sequence] (I), wherein: [Nucleotide] corresponds to a nucleotide selected from A, U, G or C, [3' Sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core Sequence] corresponds to a nucleotide sequence selected from the sequences SEQ ID NO: 1-8, wherein said one or more genes which participate in the nitrification process in said soil are expressed by one or more bacteria present in said soil.

2. Use of a miRNA of formula (I) for inhibiting the expression of one or more genes that participate in the nitrification process in a soil: [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide selected from A, U, G or C, [3' Sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core Sequence] corresponds to a nucleotide sequence selected from the sequences SEQ ID NO: 1-8, wherein said one or more genes which participate in the nitrification process in said soil are expressed by one or more bacteria present in said soil.

3. A method or use according to any one of the preceding claims, wherein said one or more genes which participate in the nitrification process in said soil encode the enzyme Ammonia MonoOxygenase (AMO) and / or the enzyme HydroxylAmine Oxidoreductase (HAO).

4. A method or use according to any one of the preceding claims, wherein said one or more genes which participate in the nitrification process in said soil are expressed by one or more bacteria present in said soil selected from Nitrosomonas spp., Nitrosospira spp. and Nitrosococcus spp., for example selected from Nitrosomonas europaea, Nitrosomonas eutropha C-91, Nitrosomonas communis Nm2, Nitrosospira multiformis and Nitrosospira iacus APG3.

5. A method or use according to any preceding claim, wherein the miRNA comprises from 19 to 21 nucleotides.

6. A method or use according to any preceding claim, wherein the miRNA inhibits nitrification of a nitrogen fertilizer applied to soil, for example a nitrogen fertilizer that is applied to said soil before, concomitantly with or after said one or more miRNAs.

7. A method or use according to any preceding claim, wherein [Sequence S'] comprises at least 80% homology with a nucleotide sequence selected from the sequences SEQ ID NO: 9-16, preferably at least 85% homology, for example at least 90% homology, at least 95% homology, at least 99% homology or even 100% homology.

8. The method or use of any preceding claim, wherein the miRNA is selected from sbi-miR162 (SEQ ID NO: 17), sbi-miR394a (SEQ ID NO: 18), sbi-miR397-5p (SEQ ID NO: 19), sbi-miR5386 (SEQ ID NO: 20), sbi-miR5388 (SEQ ID NO: 21), sbi-miR5568d-3p (SEQ ID NO: 22), sbi-miR5568d-5p (SEQ ID NO: 23) and sbi-miR6232a-5p (SEQ ID NO: 24).

9. A method or use according to any preceding claim, wherein a mixture of two to eight miRNAs of formula (I) each having a distinct [Core Sequence] is applied to the soil, for example a mixture of two, three, four, five, six, seven or eight miRNAs of formula (I) each having a distinct [Core Sequence].

10. Composition comprising one or more isolated miRNA(s) of formula (I): [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide selected from A, U, G or C, [3' Sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core Sequence] corresponds to a nucleotide sequence selected from the sequences SEQ ID NO: 1-8.

11. Composition according to claim 10, wherein [Sequence 3'] comprises at least 80% homology with a nucleotide sequence selected from the sequences SEQ ID NO: 9-16, preferably at least 85% homology, for example at least 90% homology, at least 95% homology, at least 99% homology or even 100% homology.

12. The composition of claim 10 or 11, wherein the miRNA is selected from sbi-miR162 (SEQ ID NO: 17), sbi-miR394a (SEQ ID NO: 18), sbi-miR397-5p (SEQ ID NO: 19), sbi-miR5386 (SEQ ID NO: 20), sbi-miR5388 (SEQ ID NO: 21), sbi-miR5568d-3p (SEQ ID NO: 22), sbi-miR5568d-5p (SEQ ID NO: 23) and sbi-miR6232a-5p (SEQ ID NO: 24).

13. A composition according to any one of claims 10 to 12, comprising a mixture of two to eight miRNAs of formula (I) each having a distinct [Core Sequence], for example a mixture of eight miRNAs of formula (I) each having a distinct [Core Sequence].

14. A composition according to any one of claims 10 to 13, further comprising a fertilizer, for example a fertilizer, an amendment and / or a biostimulant.

15. Culture support or culture solution comprising one or more isolated miRNA(s) of formula (I) or a composition according to any one of claims 10 to 14, [Nucleotide]-[Core sequence]-[3' sequence] (I), in which: [Nucleotide] corresponds to a nucleotide selected from A, U, G or C, [3' Sequence] corresponds to a sequence of 10 to 13 nucleotides, and [Core Sequence] corresponds to a nucleotide sequence selected from the sequences SEQ ID NO: 1-8.

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