Method for enhancing plant growth

Abstract

The present invention relates to a method for enhancing plant growth, characterised by the application of a controlled release biostimulant composition consisting of at least one of hydrophobic peptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (Ile-Ile), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (Ile-Val, Val-Ile), Isoleucine-Leucine (Ile-Leu / Leu-Ile), Isoleucine-Phenylalanine (Ile-Phe / Phe-Ile), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides. The biostimulant composition can be directly applied to the crop or incorporated into an acidic solid or liquid fertiliser.

Description

[0001] METHOD FOR IMPROVING PLANT GROWTH

[0002] DESCRIPTION

[0003] The present invention relates to a method for improving plant growth in crops, that is, to increase crop yield, promote plant growth and nutrient uptake, fruit quality, plant response to abiotic stress, and plant resistance to diseases or infections.

[0004] More specifically, the invention provides a method for improving plant growth, where said method is characterized by the application of a controlled-release biostimulant composition consisting of at least one of the hydrophobic dipeptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (I-I-I), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (He-Val, Val-I-I-L), Isoleucine-Leucine (I-I-Leu / Leu-I-I), Isoleucine-Phenylalanine (I-I-Phe / Phe-I-I), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe), and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, the biostimulant composition being able to be applied directly to the crop or included in a solid or liquid fertilizer of an acidic nature.

[0005] Promoting plant growth and productivity is important in agriculture. Today, these processes are primarily managed using fertilizers, plant growth agents such as hormones, physical modifications to the soil, and so on. However, the use of these agrochemicals has long-term environmental consequences, such as resource depletion, environmental damage, and health effects.

[0006] To limit the use of environmentally hazardous chemical inputs, environmentally friendly natural products, particularly biostimulants, have been developed. Biostimulants are materials, other than fertilizers, that promote plant growth when applied in small quantities (Khan et al. 2009, Seaweed Extracts as Biostimulants of Plant Growth and Development, Journal of Plant Growth Regulation 28(4):386-399, Dol:10.1007 / s00344-009-9103-x). According to a more recent definition, plant biostimulants are substances and materials, with the exception of nutrients and pesticides, that when applied to plants, seeds, or growing media in specific formulations, have the ability to modify plant physiological processes in a way that provides potential benefits to growth, development, and / or stress response (Du Jardin P 2012, The Science of Plant Biostimulants).

[0007] Within this range of environmentally friendly products, amino acids and / or protein hydrolysates are among the most potent and widely used biostimulants. Amino acid-based biostimulants, as well as other biostimulants based on readily assimilable carbon, which are typically applied directly to the soil, have a short half-life due to the high capacity of soil microorganisms to process and utilize them. While the effect of these biostimulants applied to the soil is rapid and can last for some time even after the biostimulant itself has disappeared, due to the changes in soil microbiology they induce, repeated application is necessary in longer-cycle crops to maintain the effects throughout the growing season and achieve maximum yields.This situation is limiting in those low value added crops where there is no automated irrigation application system.

[0008] For example, document EP2618664B1 describes the use of a composition containing individual L-amino acids to influence vital processes in plants, the individual L-amino acids being selected from the group of glutamine, asparagine, histidine and combinations thereof with each other and / or with arginine and / or with glutamic acid and / or with aspartic acid and / or with lysine, and where the total amount of the selected individual L-amino acids when the composition is applied to crops is at least 0.5 g / ha and at most 250 g / ha.

[0009] Given the short half-life of these known biostimulants, the invention provides the application of a controlled-release biostimulant composition consisting of at least one of the hydrophobic dipeptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (I-I-I), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (He-Val, Val-I-I-L), Isoleucine-Leucine (I-I-Leu / Leu-I-I), Isoleucine-Phenylalanine (I-I-Phe / Phe-I-I), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, the biostimulant composition being able to be applied directly to the crop or included in a solid or acidic fertilizer.With regard to its inclusion in an acidic fertilizer, it has been found that the controlled-release biostimulant composition is more soluble in liquids with a pH lower than 3.5, which justifies its inclusion in an acidic fertilizer, so that the indicated dipeptides remain in solution, without precipitation occurring.

[0010] The biostimulant composition described here consists of dipeptides known to form self-assembling polymeric structures in aqueous solution. These structures can be nanotubes, microparticles, or similar forms. They all share the characteristic of being highly insoluble and highly stable. Due to this stability, their half-life in soil is estimated to be much longer than that of any other amino acid, dipeptide, or polypeptide not included in this list, due to their low susceptibility to microbiological degradation. This degradation is prolonged as the polymers are processed. Thus, the biostimulant effect is sustained for a much longer period compared to free amino acids or non-self-polymerizing peptides, which degrade more readily.

[0011] In this context, see articles “Self-Assembly of Unprotected Dipeptides into Hydrogels: Water-Channels Make the Difference” Ottavia Bellotto et al. ChemBioChem, November 2021 -23(2), D0l:10.1002 / cbic.202100518; “Non-zeolitic properties of the dipeptide L-leucyl-L-leucine as a result of the specific nanostructure formation”, Marat A. Ziganshin et al., Phys. Chem. Chem. Phys., 2017, 19 13788, DOI: Ol: 10.1039 / c7cp01393k; “The Phe-Phe Motif for Peptide Self-Assembly in Nanomedicine”, Silvia Marchesan et al., Molecules 2015, 20(11 ), 19775-19788; https: / / doi.Org / 10.3390 / molecules201119658).As mentioned above, the invention provides a method for improving growth characterized by the application of a controlled-release biostimulant composition consisting of at least one of the hydrophobic dipeptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (I-I-I), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (I-Val, Val-I-I-I), Isoleucine-Leucine (I-Leu / Leu-I-I), Isoleucine-Phenylalanine (I-Phe / Phe-I-I), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe), and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides, the biostimulant composition being able to be applied directly to the crop or included in a solid or liquid fertilizer acid.

[0012] In a preferred embodiment, the biostimulant composition is applied in solid form directly to the soil at a rate of 35 mg / kg of soil as a reference. The dose per kg of soil is calculated by analogy with the recommended amount per hectare. The recommended dose per hectare for these biostimulants is between 1.5 and 10 kg / ha. Thus, for example, in a horticultural hectare such as the "Cogollo de Tudela" variety, approximately 40,000 plants are sown per hectare. Therefore, the amount of biostimulant required per plant is between 35 and 250 mg. Since the biostimulation tests are carried out in 3-liter containers with 1 kg of soil sieved to 2 mm, the amount of 35 mg / kg of soil is chosen as the minimum dose at which an effect is produced.

[0013] In another embodiment, the biostimulant composition is applied using a solid fertilizer as a carrier, for example by adding the biostimulant composition during the granulation process of said fertilizer or by coating its granules with the biostimulant composition.

[0014] The invention is then explained in detail in relation to the following examples.

[0015] Example 1: Half-life of different amino acids and peptides in soil. To test the stability of the dipeptides in the biostimulant combination consisting of at least one of the hydrophobic dipeptides Val-Val, lle-lle, Leu-Leu, Phe-Phe, lle-Val / Val-llle, lle-Leu / Leu-lle, lle-Phe / Phe-lle, and Phe-Val / Val-Phe, 5.0 mg of each of these pure dipeptides per kg of soil are added to a pot containing 5 kg of soil. As a control, the amino acids Val, Leu, lle, Phe, Glu, Ala, Ser, and Asp are also added at a rate of 5.0 mg / kg of soil. Other dipeptides, different from those present in the combination described here, are also added as controls.

[0016] The soil is moistened to field capacity and maintained at a constant temperature of 25 °C. A 0.10 kg sample is taken at 3, 5, 10, and 20 days, and the dipeptides mentioned are extracted and quantified by LC-MS to study their stability. For this purpose, the 0.10 kg soil sample is placed in a 1.0 L beaker, and 0.5 L of organic extractant, preferably methanol, acetonitrile, or ethyl acetate, is added. The mixture is stirred for 2–4 hours at room temperature.

[0017] After the extraction process is complete, the mixture is filtered, and the filtrate is concentrated to dryness under reduced pressure using a rotary evaporator. The solid residue is dissolved in 25 mL of HPLC-grade acetonitrile. The solution is transferred to a 50 mL volumetric flask and diluted to the mark with HPLC-grade acetonitrile. Finally, approximately 1.0 mL of the solution, previously filtered through a syringe filter, is taken into an HPLC vial, and the sample is quantified by liquid chromatography-mass spectrometry (LC-MS) using a Waters Acquity UPLC system coupled to a Waters Acquity QDA mass spectrometer and equipped with an ACE Excel 3 Super C18 column (3 pm, 150 x 4.6 mm), under the following chromatographic conditions:

[0018] • Quantization: SI mode

[0019] • Flow rate 0.5 ml / min

[0020] • Mobile phase: A = acetonitrile with 0.1% formic acid and B = water with 0.1% formic acid • Gradient: 0 min (5% A + 95% B); 2 min (20% A + 80% B); 20 min (98% A + 2% B); 25 min (98% A + 2% B); 27 min (5% A + 95% B); 30 min (5% A + 95% B)

[0021] • Injection volume: 15 l

[0022] • Column temperature: 40 °C

[0023] • MS range: 100-800

[0024] • Ionization mode: ES+

[0025] • Cone voltage and capillary voltage: 10 V and 1.2 kV

[0026] • Solvation temperature: 600 °C

[0027] The quantification of the compounds is carried out through external calibration. For this purpose, calibration curves are prepared individually for each compound using standards between 0.1 and 15 ppm. The peak area associated with the m / zy retention time of each known compound is measured using its standard and extrapolated to the corresponding calibration curve. Applying the correct dilution factor, the concentration in mg of control amino acid and dipeptide per kg of soil is determined using this protocol.

[0028] The results are shown in Table 1 below:

[0029] Table 1

[0030] As can be seen, the dipeptides according to the invention have a prolonged half-life, being much more stable in the soil and extending their half-life beyond 20 days.

[0031] Example 2: Biostimulatory capacity

[0032] The study of biostimulant capacity is carried out in Venlo-type glass greenhouses equipped with overhead ventilation systems regulated by climate control automata.

[0033] Lettuce plants of the “Cogollo de Tudela” variety were transplanted into 3-liter containers, using a substrate of soil sieved to 2 mm. The plants were divided into 24 groups:

[0034] • a negative control without fertilizer,

[0035] • a positive control with a fertilizer consisting of a solution of monoammonium phosphate, dipotassium phosphate and urea in water until an amount of nitrogen, phosphorus and potassium equivalent to the application of 150 UFN, 80 IIFP2O5 and 120 UFK2O is achieved.

[0036] • 22 trial groups where the positive control fertilizer plus one of the dissolved dipeptides or amino acids is applied. All peptides and amino acids are dissolved in water acidified to pH 3 with hydrochloric acid, and sufficient volume is applied to each pot to achieve a final concentration of each component of 35 mg / kg of soil.

[0037] The plants were watered with mains water, its composition being as follows: 0.01 mmol / l NO 3- , 2.45 mmol / l SO4 2 ; 3.10 mmol / l HCO3; 1.61 mmol / l CT, 0.10 mmol / l K + , 1.45 mmol / l Ca 2+ , 1.46 mmol / l Mg 2+ and 1.91 mmol / l Na + .

[0038] The plants were irrigated using a drip irrigation system with a 2 L / h emitter, according to their needs. The volume of water in each irrigation and the number of irrigations were programmed to maintain a drainage rate between 20 and 30%.

[0039] The plants were distributed according to a randomized block design, with 4 replicates per treatment of 60 plants each. Subsequently, 10 plants from each replicate were taken to carry out the determinations of the parameters to be evaluated at 6 time points: at the time of transplanting, 15 days after transplanting (DAT), 30 days DAT; 45 days DAT; 60 days DAT and 75 days DAT.

[0040] For the characterization of the agronomic behavior of the plants, the fresh weight (g) of the plants was determined at each sampling time.

[0041] The results are shown in Table 2 below:

[0042] Table 2 As we can see in the table above, the application of the fertilizer generates an increase in fresh biomass 75 days after transplanting, from 496 mg per plant to 628 mg per plant. We can also observe how the incorporation of different amino acids or some dipeptides not part of the invention generates a cumulative effect of increased fresh biomass when applied together with the fertilizer solution. It is also observed that the use of the combination of the invention has a lesser impact in the first days of the trial, generating a lower amount of fresh biomass than that generated by the comparative amino acids and dipeptides at 15 and 30 days after transplanting.However, it is observed that, 45 days after transplanting, the effect of the combination of the invention is already similar to that of the other molecules, and at the end of the trial it is observed that, at 60 and 75 days post-transplanting, the fresh biomass of the plants treated with the combination of the invention is manifestly superior to the other treatments.

[0043] Example 3: Composition of the invention with a fertilizer as a vehicle

[0044] Taking as an example of a vehicle fertilizer a Urea Superphosphate fertilizer, during the production process, 450 kg of urea, 350 kg of phosphate rock, between 0.5 and 10 kg of the composition of the invention are mixed and subsequently the mixture is attacked with 200 kg of sulfuric acid of 80% purity, it is left to macerate and the fertilizer is granulated.

[0045] In another application, the combination of the invention is dissolved in monopropylene glycol to a final concentration of between 40 and 60% (w / v). This mixture is then applied as a coating over any granular fertilizer at a rate of between 3 and 20 kg / ton of fertilizer. Examples of granular fertilizers to which it can be applied include granular urea, superphosphate, triple superphosphate, superphosphate urea, or any solid granular fertilizer containing N, P, K, or other macro- and micronutrients such as calcium, magnesium, zinc, boron, copper, iron, or molybdenum, for example.

[0046] Example 4: Biostimulant capacity of a granulated solid fertilizer where the combination of the invention has been applied during the production process

[0047] The following solid fertilizers are produced:

[0048] 1 - LISP-C. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 20 kg of sulfuric acid (80% purity) are mixed. It is left to mature for 3 days and granulated. The result is a granulated fertilizer containing 20.2% nitrogen and 9.9% water-soluble P2O5;

[0049] 2- USP-Phe-Phe. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 0.5 kg of the Phe-Phe dipeptide + 20 kg of sulfuric acid (80% purity) are mixed. The mixture is left to mature for 3 days and then granulated. The result is a granulated fertilizer containing 20.7% nitrogen, 9.8% water-soluble P2O5, and 0.48% of the Phe-Phe dipeptide.

[0050] 3- USP-Leu-Leu. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 0.5 kg of the Leu-Leu dipeptide + 20 kg of sulfuric acid (80% purity) are mixed. The mixture is left to mature for 3 days and then granulated. The result is a granulated fertilizer containing 20.5% nitrogen, 10.1% water-soluble P2O5, and 0.49% of the Leu-Leu dipeptide.

[0051] 4- USP-lle-Phe / Phe-lle. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 0.5 kg of the dipeptide lle-Phe / Phe-lle + 20 kg of sulfuric acid (80% purity) are mixed. The mixture is left to mature for 3 days and then granulated. The result is a granulated fertilizer containing 20.5% nitrogen, 10.1% water-soluble P2O5, and 0.48% of the dipeptide lle-Phe / Phe-lle.

[0052] 5- USP-Glu-Glu. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 0.5 kg of Glu-Glu peptide + 20 kg of sulfuric acid (80% purity) are mixed. The mixture is left to mature for 3 days and then granulated. The result is a granulated fertilizer containing 20.9% nitrogen, 10.3% water-soluble P2O5, and 0.45% Glu-Glu dipeptide.

[0053] 6- USP-Glu. 45 kg of urea + 30 kg of Moroccan phosphate rock (P2O5 content 31%) + 0.5 kg of the amino acid Glu + 20 kg of sulfuric acid (80% purity) are mixed. The mixture is left to mature for 3 days and then granulated. The result is a granulated fertilizer containing 20.4% nitrogen, 9.8% water-soluble P2O5, and 0.47% Glu.

[0054] The field experiments were conducted in a randomized complete block design with 4 replicates. The plot area was 120 m². 2In the field experiments, the described fertilizers 1-6 were applied at a single dose of 650 kg of fertilizer / ha. This application provided 3.25 kg / ha of each peptide. To evaluate the effectiveness of the studied fertilizers, control plots without fertilization were established. The fertilizer under investigation was applied as a topdressing at tillering. The crop under investigation was Akteur quality wheat. The field experiments were established on medium-textured, light loam soils with a soil pH (KCl 1 M) in the range of 6.2 to 6.1, suitable for wheat cultivation. After harvest, wheat grain yield, thousand-seed weight, and seed N and P content on a dry matter basis were determined.

[0055] The results are shown in Table 3 below.

[0056] Table 3

[0057] Kg / Ha Weight (g) of 1,000 seeds % N sms % P sms

[0058] Control - 3,450 48.11 2.76 0.31

[0059] USP-C 5,214 49.24 3.83 0.42

[0060] USP-Phe-Phe 6,121 49.16 3.75 0.49

[0061] USP-Leu-Leu 6,314 49.29 3.91 0.47

[0062] USP-lle-Phe / Phe-lle 6,250 49.35 3.68 0.48

[0063] USP-Glu-Glu 5,821 49.84 3.69 0.41

[0064] USP-Glu 5,794 49.32 3.71 0.43

[0065] As shown in Table 3, the inclusion of fertilizer (USP-C) improves all analyzed parameters. The use of any added biostimulant increases yield per hectare compared to the control fertilizer (USP-C), although this increase is significantly greater when using the combination of the invention. No difference is observed in the 1,000-seed weight or the percentage of nitrogen (N) on a dry matter basis between the control fertilizer (USP-C) and any other biostimulant fertilizer. Finally, an increase in phosphorus (P) content is observed in the treatments with the combination of the invention compared to the control fertilizer (USP-C). This difference is not observed with biostimulants that are not part of the invention. Example 4: Biostimulant capacity of a granulated solid fertilizer where the combination of the invention is applied as a coating.

[0066] 7- USP-Coat-Phe-Phe. 800 grams of the Phe-Phe dipeptide are mixed with 1,200 grams of propylene glycol. This mixture is added as a coating onto the LISP-C fertilizer (Example 3, point 1) at a rate of 15 grams / kg.

[0067] 8- USP-Coat-Leu-Leu. 800 grams of the Leu-Leu dipeptide are mixed with 1,200 grams of propylene glycol. This mixture is added as a coating onto the USP-C fertilizer (Example 3, point 1) at a rate of 15 grams / kg.

[0068] 9- USP-Coat-lle-Phe / Phe-lle. 800 grams of the dipeptide lle-Phe / Phe-lle are mixed with 1,200 grams of propylene glycol. This mixture is added as a coating onto the USP-C fertilizer (Example 3, point 1) at a rate of 15 grams / kg.

[0069] 10-USP-Coat-Glu-Glu. 800 grams of the Glu-Glu dipeptide are mixed with 1,200 grams of propylene glycol. This mixture is added as a coating onto the USP-C fertilizer (Example 3, point 1) at a rate of 15 grams / kg.

[0070] 11 -USP-Coat-Glu. 800 grams of the amino acid Glu are mixed with 1,200 grams of propylene glycol. This mixture is added as a coating onto the USP-C fertilizer (Example 3, point 1) at a rate of 15 grams / kg.

[0071] The field experiments were conducted similarly to Example 4. Specifically, a randomized complete block design with 4 replicates was used. The plot area was 120 m². 2In the field experiments, the described fertilizers (7-11) were applied at a single dose of 650 kg fertilizer / ha. This dose provides 3.25 kg / ha of each peptide. To evaluate the effectiveness of the studied fertilizers, control plots without fertilization were established. The fertilizer under investigation was applied as a topdressing at tillering. The crop under investigation was Akteur quality wheat. The field experiments were established in medium-density, light loam soils with a soil pH (KCl 1 M) in the range of 6.2 to 6.1, suitable for wheat cultivation. After harvest, wheat grain yield, thousand-seed weight, and seed N and P content on a dry matter basis were determined. The results are shown in Table 4 below.

[0072] Kg / Ha Weight (g) of % N sms % P sms

[0073] 1,000 seeds

[0074] Control - 3450 48.11 2.76 0.31

[0075] USP-C 5214 49.24 3.83 0.42

[0076] USP-Coat-Phe-Phe 6214 49.17 3.78 0.48

[0077] USP-Coat-Leu-Leu 6189 49.35 3.95 0.49

[0078] USP-Coat-lle-Phe / Phe-lle 6278 49.58 3.65 0.51

[0079] USP-Coat-Glu-Glu 5622 49.74 3.71 0.40

[0080] USP-Coat-Glu 5645 49.62 3.82 0.41

[0081] As we can see in Table 4, the results are very similar to the previous example. Therefore, we can conclude that the way in which the biostimulants of the invention are applied to the solid fertilizer has no real impact. It should be noted, however, that there is a difference with those biostimulants that are not the subject of the invention, where the increase in yield associated with their use with the fertilizer is lower if the biostimulant is applied as a coating.

Claims

CLAIMS 1. A method for improving plant growth, wherein said method is characterized by the application of a controlled-release biostimulant composition consisting of at least one of the hydrophobic dipeptides Valine-Valine (Val-Val), Isoleucine-Isoleucine (I-I-I), Leucine-Leucine (Leu-Leu), Phenylalanine-Phenylalanine (Phe-Phe), Isoleucine-Valine (He-Val, Val-I-I), Isoleucine-Leucine (I-I-Leu / Leu-I-I), Isoleucine-Phenylalanine (I-I-Phe / Phe-I-I), Phenylalanine-Valine (Phe-Val / Val-Phe), Phenylalanine-Leucine (Phe-Leu / Leu-Phe) and Leucine-Valine (Leu-Val / Val-Leu), or any combination of said dipeptides.

2. Method according to claim 1, wherein the application of the biostimulant composition is direct to the crop or is present in a solid or liquid fertilizer of an acidic nature.

Images