Patent Description:
More specifically, in a first aspect, the invention provides a fertiliser composition that includes tyramine as an enhancer of nitrogen that can be assimilated by plants, wherein tyramine improves the levels of total nitrogen in the plant and nitrate in the sap, its application constituting an alternative to conventional nitrogen fertilisers.

In a second aspect, the invention relates to a combination of the described fertiliser composition together with an additional fertiliser and/or biostimulant.

Nitrogen is the mineral nutrient required in the greatest quantities by agricultural crops and a key factor limiting their productivity. It is estimated that, globally, agricultural systems require between <NUM> and <NUM> million tonnes of nitrogen annually, of which more than <NUM> million tonnes are obtained industrially through the Haber Bosch process and between <NUM> and <NUM> million tonnes are fixed biologically (<NPL>; <NPL>). However, the production of traditional nitrogen fertilisers is energy-intensive and their application has a low efficiency and a considerable negative impact on the environment, mainly due to losses through leaching, denitrification, fixation in organic matter and volatilisation of ammonia (<NPL>; <NPL>).

It is also difficult to deliver nutrients to the plant in a controlled way and in accordance with the physiological requirements of the crops, often causing stress due to toxicity (increased salinity) or nutrient deficiency. Finally, given that <NUM>-<NUM>% of the fertiliser applied is lost (<NPL>), the production costs for farmers increase significantly.

Consequently, the negative environmental impact associated with the excessive use of mineral nitrogen fertilisers has influenced the development of specific policies both in Spain and in the European Union, which promote the search for sustainable alternatives to traditional fertilisation and establish laws that regulate the maximum amount of fertiliser units that the farmer can apply depending on the degree of contamination existing in a specific region (this is the case in the region of Murcia, see <NPL>une; and of the EU http://ec. eu/environment/pubs/pdf/factsheets/nitrates/es.

In this sense, some solutions designed to solve the above-mentioned problems consist in the use of inhibitors of nitrification processes and urea hydrolysis (<NPL>; <NPL>) or in the application of inocula of nitrogen fixing bacteria (<NPL>; <NPL>; <CIT>).

For this reason, there is currently a clear trend in the plant nutrition sector towards the search for alternatives to conventional nitrogen fertilisation that enable the reduction of pollution derived from its use while at the same time increasing the efficiency of nitrogen use and crop productivity in a sustainable way.

Furthermore, plants exude through the roots a considerable part of the organic compounds generated in photosynthesis (between <NUM> and <NUM>%) in order to regulate the chemical composition of the rhizosphere and to promote the growth of those microorganisms that can provide benefits to the plant in a given ecosystem (<NPL>; <NPL>). Compounds present in root exudates include sugars, amino acids, organic acids, fatty acids and secondary metabolites (<NPL>). In addition to the cultivated species and its phenological stage, the composition and quantity of these exudates are mainly influenced by environmental signals, e.g. the availability of nutrients in the soil.

Indeed, root exudates can modify the composition of soil microbial communities and influence the dynamics of the nitrogen cycle, mainly through the regulation of nitrification and nitrogen fixation processes from the air (<NPL>; <NPL>).

In view of the above, the present invention builds on the above-mentioned approaches so that, on the one hand, by regulating the nitrogen transformation processes in soils, root exudates can increase the availability of nitrogen for plants and the efficiency of its use in agricultural soils and, on the other hand, it constitutes an alternative to the use of traditional nitrogen fertilisers.

Document <CIT> provides a probiotic compound organic soil preparation method of a fertilizer, including the fertilizer <NUM>-<NUM> parts of tyramine in combination with urea, amino acids, biostimulants and other organic acids, wherein the main source of nitrogen in this composition is urea, and tyramine is present in very low levels. Document <CIT> discloses a plant treatment composition in sight to act on their cell multiplication, their elongation and their vegetative, floral and gendered, which comprises, in association with an appropriate application vehicle, at least one amine capable of conjugating in situ with a phenol, in the presence of an enzyme which controls the biosynthesis of at least one of said amines in the plant, and in particular the biosynthesis of putrescine. This composition can include, among others, tyramine as amine and can be applied as a solution.

None of these prior documents discloses a fertiliser composition that includes tyramine as an enhancer of nitrogen that can be assimilated by plants, wherein tyramine improves the levels of total nitrogen in the plant and nitrate in the sap, its application constituting an alternative to conventional nitrogen fertilisers.

It would therefore be desirable to have fertilisers that mimic root exudates and have an analogous effect that can make it possible to dispense with or reduce the application of mineral fertilisers and mitigate environmental pollution and climate change, while providing an alternative to conventional nitrogen fertilisation to reduce pollution from its use and increase the efficiency of nitrogen use and crop productivity in a sustainable way.

The present invention fulfils both of the above objectives by providing a fertiliser composition that includes tyramine as an enhancer of nitrogen that can be assimilated by plants, wherein tyramine improves the levels of total nitrogen in the plant and nitrate in the sap.

Tyramine, or <NUM>-(<NUM>-aminoethyl)phenol, is an amine derived from the amino acid tyrosine that occurs naturally in plant tissues. It is involved in plant defence against biotic stress, as it can act as a neurotransmitter and neuromodulator at specific insect and herbivore receptors (<NPL>). Biochemically, tyramine is produced by the decarboxylation of tyrosine via the action of the enzyme tyrosine decarboxylase, and is obtained in the form of a white crystalline powder with a purity of more than <NUM>%.

As mentioned above, in a first aspect, the present invention provides a fertiliser composition that includes tyramine as an enhancer of nitrogen that can be assimilated by plants.

The fertiliser composition of the invention comprises between <NUM> and <NUM>% by weight of tyramine and between <NUM> and <NUM>% by weight of other components selected from the group consisting of sugars, amino acids other than tyrosine, organic acids, polyamines, glycerol, myoinositol, adenine, uracil, cytosine, guanine and combinations thereof, the fertiliser composition being in the form of a water-soluble powder.

When present in the present fertiliser composition, sugars are preferably selected from mono- and di-saccharides such as sucrose, fructose, trehalose, glucose, arabinose, maltose, as well as mixtures thereof.

When present in the present fertiliser composition, amino acids other than tyramine are preferably selected from among threonine, lysine, phenylalanine, glutamic acid, methionine, GABA, ornithine, glycine, glutamine, aspartic acid, serine, asparagine, tyrosine, tryptophan, valine, leucine, isoleucine, proline, <NUM>-hydroxyproline, arginine, histidine, alanine, cysteine, and mixtures thereof.

When present in the present fertiliser composition, the organic acids are preferably selected from lactic acid, succinic acid, oxalic acid, gluconic acid, threonic acid, fumaric acid and mixtures thereof.

Polyamines, if present in the present composition, are preferably selected from putrescine, spermidine, spermine and mixtures thereof.

The presence of these components other than tyramine in the composition of the invention is based on the fact that such components form part of the root exudates in the crops tested in the absence of nitrogen described below or are described in the literature as components of said exudates in normal conditions for plant development (Zhalnina et al. , <NUM>), and are therefore desirable for the aforementioned purpose of having a fertiliser composition that mimics the root exudates with a similar effect, that can make it possible to dispense with or reduce the application of mineral fertilisers.

The fertiliser composition of the invention is formulated in the form of a water-soluble powder, as indicated above, but can also be formulated in the form of a liquid composition by dissolving it in water or in the form of granules by adding granulating agents known to the person skilled in the art.

According to the second aspect, the invention relates to a fertiliser composition as described above in combination with another additional fertiliser selected from nitrogen fertilisers, phosphate fertilisers, potassium fertilisers, calcium fertilisers and amendments, micronutrients, boric acid and leonardite, and combinations thereof, and/or in combination with one or more biostimulants selected from the group consisting of amino acid hydrolysates, humic extracts, algae extracts, live micro-organisms or extracts of micro-organisms, e.g. Pichia guilliermondii and Azotobacter chroococcum, and combinations thereof.

In this case, the composition of the invention is present in the combination in a proportion of <NUM> to <NUM>% by weight.

In one embodiment, the additional nitrogen fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight and is selected from urea, ammonium nitrosulphate, potassium nitrate, ammonium sulphate, ammonium nitrate, calcium nitrate.

In another embodiment, the additional phosphate fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight and is selected from phosphate rock, triple superphosphate, single superphosphate, concentrated superphosphate, phosphoric acid.

In yet another embodiment, the additional potassium fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight and is selected from potassium chloride, potassium sulphate, potassium and magnesium double sulphate, potassium hydroxide.

In another embodiment, the additional calcium fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight and is selected from calcium chloride, calcium cyanamide, calcium sulphate, dolomite, limestone, calcium oxide, calcium hydroxide.

In yet another embodiment, the additional micronutrient fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight and is selected from iron sulphate, magnesium sulphate, zinc sulphate, manganese sulphate, copper sulphate, ammonium molybdate, cobalt chloride.

In a further embodiment, boric acid as an additional fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight.

In another embodiment, leonardite as an additional fertiliser is present in the combination in a proportion of <NUM> to <NUM>% by weight.

In the case of the combination of the fertiliser composition of the invention with biostimulants as described above, preferably the biostimulants are present in the combination in a proportion of <NUM> to <NUM>% by weight.

It is also the object of the invention to use the fertiliser compositions described here in the form of water-soluble powder, granulates or a liquid pre-dissolved in water for application by fertigation or foliar application.

If the composition of the invention is used in the form of a water-soluble powder by fertigation or by foliar application pre-dissolved in water, it is preferably applied in an amount of <NUM> to <NUM>/ha and <NUM> to <NUM>/ha respectively.

If the composition of the invention is used in combination with another additional fertiliser in the form of granules, preferably said combination is applied directly in an amount of <NUM> to <NUM>,<NUM>/ha.

If the composition of the invention is used in combination with biostimulants in the form of a liquid for the application thereof by fertigation or foliar application, it is preferably applied in an amount of <NUM> to <NUM>/ha and <NUM> to <NUM>/ha respectively.

In order to characterise in detail the response of crops to nitrogen deficiency and to identify the metabolites exuded by roots that have the greatest influence on nitrogen dynamics in the soil, the Applicant analysed the differential exudate profile of two crop species of agronomic interest, maize and tomato, in the absence of nitrogen. The following is a brief description of the trial to determine the root exudates emitted in the absence of nitrogen.

The method, similar to that used by other authors (<NPL>) was the same for both maize seeds (variety LG <NUM>) and tomato seeds (variety Agora Hybrid F1). The seeds were surface sterilised by washing for <NUM> minutes in <NUM>% ethanol, followed by <NUM> minutes in <NUM>% bleach. The seeds were then washed extensively and allowed to hydrate in sterile MilliQ water for <NUM> hours. For germination, the seeds were placed on a bed of filter paper moistened with sterile MilliQ water. The seeds were left to germinate in darkness for <NUM> days, after which the seedlings were placed in hydroponic culture trays with the roots immersed in standard Hoagland nutrient solution. Twelve plants were placed in each tray, with three trays (each corresponding to a biological replicate) for the control treatment and three trays for the nitrogen-free treatment. The plants were grown at a temperature and photoperiod of <NUM> and <NUM> light/<NUM> and <NUM> darkness and a light intensity of <NUM>,<NUM> lux on the surface.

The nutrient solution was replaced with fresh solution every three days and kept aerated at all times by bubbler probes. After <NUM> days of growth, the plants were subjected to nitrogen depletion treatment. For this, three trays were incubated for three days with modified Hoagland solution without nitrogen and the remaining three trays were incubated with full solution. After incubation, root exudates were obtained.

The plants were carefully removed from the culture trays and washed with plenty of water, followed by a final wash with distilled water. Plants corresponding to each tray were placed in wide-necked flasks containing <NUM> of MilliQ water, the roots being immersed in the water. The plants were incubated in the flasks for <NUM> hours. Subsequently, the plants were removed and the insoluble material was removed from the solution by filtering with <NUM> filters. The filtered material was flash-frozen in liquid nitrogen and freeze-dried. The dried material obtained was weighed and analysed by gas-mass chromatography after derivatisation with methoxyamine and N-methyl-(trimethylsilyltrifluoroacetamide).

Table <NUM> shows the metabolites exuded by the plants and their ratios under nitrogen-free versus control conditions.

Based on these results, <NUM> matching metabolites with a ratio to the control greater than <NUM> and which were present in the exudates of both cultures were selected. These metabolites were: lysine, glutamic acid, sucrose, lactic acid, urea, oxalic acid and tyramine. Each of the metabolites was applied separately at a dose of <NUM>/ha in a pot with <NUM> of soil, maize plants were planted (<NUM> pots per treatment with one plant per pot) and the effect on their dry weight was observed after <NUM> weeks. The effect of the metabolites was compared with a negative control (no treatment) and a positive control with a conventional nitrogen fertiliser (ammonium nitrosulphate at a dose of <NUM> nitrogen fertiliser units per hectare). The soil came from an agricultural soil that had been left unfertilised and uncultivated for one year, conditions under which the availability of the forms directly assimilable by plants (NH<NUM>+ and NO<NUM>-) is generally low (<NPL>). All metabolites enhanced maize growth, as shown in Table <NUM> below.

The above results indicate that tyramine is the most growth-enhancing metabolite in maize, achieving the same result as conventional nitrogen treatment with ammonium nitrosulphate.

Three fertiliser compositions in the form of a water-soluble powder were prepared according to the invention with the following composition:.

These products (A, B, C) were tested in field trials on maize and tomato plants and compared with a negative control (no treatment) and a positive control of conventional nitrogen fertilisation consisting of ammonium nitrosulphate (D). The rates and mode of application were as follows:.

The treatments significantly improved yield and nitrogen content of maize (Tables <NUM> and <NUM>) as well as tomato (Tables <NUM> and <NUM>).

In order to determine whether the increase in nitrogen and nitrate content in the sap in the treatments studied is due to an increase in the availability of assimilable nitrogen for the plants, nitrogen balances were carried out in trials with maize and tomato plants.

The analysis of the soil used for the trials determined that it contained <NUM> ppm of mineral nitrogen (NH<NUM>+ and NO<NUM>-), which corresponds to <NUM>/ha (considering <NUM> centimetres of arable soil and an average density of <NUM>,<NUM>/m3, the mass per hectare would be approximately <NUM>,<NUM> tonnes). The total nitrogen content of the soil was also determined to be <NUM>%, which corresponds to <NUM>,<NUM>/ha, of which <NUM>,<NUM>/ha is organic nitrogen, not available to the plants.

Among treatments A, B and C, the treatment that provides the highest amount of nitrogen fertiliser units would be treatment B in the following combination: <NUM>% tyramine, <NUM>% sucrose, <NUM>% glutamic acid and <NUM>% urea. The nitrogen content of this mixture would be <NUM>%, which would mean a maximum application of nitrogen fertiliser units through the treatments described here of <NUM>/ha in the case of maize and <NUM>/ha in the case of tomato.

Taking into account the dry weight of the biomass of the maize and tomato plants (see Tables <NUM> and <NUM>) and the percentage of total nitrogen in the plants (Tables <NUM> and <NUM>), the kg of nitrogen present in the biomass obtained per hectare can be calculated. As can be seen, the amount of nitrogen in the dry biomass is much higher in all cases than the sum of nitrogen available in the soil and that provided by the fertiliser treatments proposed here. Therefore, the increase in nitrogen content of the plants is necessarily due to an increase in the availability of nitrogen that can be assimilated for the plants thanks to the fertiliser composition with enhancer of nitrogen that can be assimilated.

Claim 1:
A fertiliser composition comprising an enhancer of nitrogen that can be assimilated by plants, characterised in that it comprises <NUM> to <NUM>% by weight of tyramine and <NUM> to <NUM>% by weight of other components selected from the group consisting of sugars, amino acids other than tyrosine, organic acids, polyamines, glycerol, myoinositol, adenine, uracil, cytosine, guanine and combinations thereof, the fertiliser composition being in the form of a water-soluble powder.