Use of a composition of Trichoderma and lignin fractions as a booster of nitrogen fertilizers

Abstract

The use of a composition comprising a Trichoderma fungus and a lignin fraction as a booster for nitrogen fertilizer is disclosed. Specifically, the composition has surprisingly been shown to enhance nitrogen uptake by plant seeds from nitrogen fertilizer. As a result, the total amount of the latter used can be advantageously reduced by at least 50% of the recommended amount.

Description

[Technical Field]

[0001] The present invention relates to the use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a booster of nitrogen fertilizer. Specifically, it has been surprisingly observed that said composition enhances seed uptake of nitrogen from nitrogen fertilizer. As a result, the total amount of the latter used can be advantageously reduced by at least 50%. [Background technology]

[0002] Fertilizers, which essentially consist of various types of nutritional components for plant growth, are widely used around the world to improve agricultural yields. Generally, fertilizers can be in the form of liquids, suspensions, or solids.

[0003] It is well known that NPK fertilizers represent the primary product used to supplement the nutritional requirements of flowers, trees, pastures, and crops. The "NPK" designation reflects the three nutrient elements found in these fertilizers: nitrogen, phosphorus, and potassium.

[0004] Nitrogen is particularly important because it has a variety of different functions in plants. It is primarily responsible for plant growth and reproduction. If too little nitrogen is present, the plant will not grow well and yields will be reduced. However, too much nitrogen has several drawbacks. Excess nitrogen can delay flowering and fruit ripening, and the plant tissue will become very weak and unstable. This increases the likelihood of disease and pest infestation, leading to reduced yields.

[0005] Phosphorus, another major nutrient in plants, is involved in the transfer and storage of chemical energy and in root formation. It is also essential for photosynthesis. This means that phosphorus is available early on, supporting seed fertilization and later flowering. A phosphorus deficiency results in small, stunted plants with thin stems. Furthermore, leaves become discolored, roots grow poorly, and flowering is delayed. On the other hand, an excess of phosphorus (which rarely occurs) indirectly damages the plant by reducing the amount of available trace elements.

[0006] The third essential nutrient, potassium, supports water absorption and maintains water balance in plants. Potassium strengthens plant tissues, making them more resilient and resistant, while also promoting resistance to disease, cold, and other extreme weather conditions. A potassium deficiency can cause plants to wilt and inhibit root formation. Additionally, plants transpire more during dry periods and take up less water. Excess potassium can lead to undesirable salt concentrations, thus reducing the uptake of desirable nutrients such as magnesium.

[0007] Therefore, it is desirable to find a good balance of nutrient concentrations in fertilizers to take advantage of their effectiveness and reduce their drawbacks.

[0008] It should also be noted that the use of nitrogen fertilizers has come under attack due to high production costs, and similarly, the overuse of phosphorus has been criticized due to its environmental impacts.

[0009] Therefore, there is a felt need to reduce the overall use of fertilizers by utilizing them effectively and appropriately while providing sufficient amounts of nutrients to plants while protecting human and animal health, crops, and the environment. Summary of the Invention

[0010] The above object has been achieved by using a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a booster of nitrogen uptake by plant seeds from nitrogen-containing fertilizers, as claimed in claim 1.

[0011] In another aspect, the present invention relates to an agrochemical kit comprising: a first container containing a fungus of the genus Trichoderma; a second container comprising a lignin fraction; and a third container comprising a nitrogen-containing fertilizer, or a second container comprising a lignin fraction and a nitrogen-containing fertilizer.

[0012] In a further aspect, the present invention relates to a method for increasing nitrogen uptake by plant seeds, said method comprising the steps of applying a composition to the seed soil followed by the application of a nitrogen-containing fertilizer.

[0013] The term "seed" is meant to encompass not only plant seeds but also tubers and bulbs.

[0014] The term "plants" refers to plants that can be grown and harvested for profit or subsistence, including plants grown and harvested for horticultural or personal use, and includes crops, grains, vegetables, fruits, and flowers.

[0015] The term "soil" means the soil in which the seeds are sown and thus encompasses land, plots, and soilless media, for example, hydroponics and hydroponic culture media.

[0016] The features and advantages of the present invention will become apparent from the following detailed description, the examples provided for illustrative purposes, and the accompanying drawings. [Brief explanation of the drawings]

[0017] [Figure 1]Figures 1 to 5 relate to a study of corn seedlings according to Example 6 carried out from December 15, 2021 to January 14, 2022, comparing the following differently treated samples: "Untreated control", i.e., corn seedlings that received no treatment; "Normal fertilizer", i.e., corn seedlings that received 150 kg / ha of ammonium nitrate as fertilizer at the time of sowing; "100 g / m³ of the inventive composition", i.e., corn seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction at the time of sowing; and "100 g / m³ of the inventive composition, normal fertilizer", i.e., corn seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction and 150 kg / ha of ammonium nitrate at the time of sowing. "100g / m³ of the composition of the present invention, 1 / 2 fertilizer", i.e., corn seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 100g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the composition of the present invention", i.e., corn seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ (the composition of the present invention) at the time of sowing. "500g / m³ of the composition of the present invention, normal fertilizer", i.e., corn seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 150kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the composition of the present invention, 1 / 2 fertilizer", i.e., corn seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. Specifically, Figure 1 shows the SPAD (i.e., Soil, Crop, and Produce Analysis System Commercialization Project Chlorophyll Meter, a rapid and non-destructive approach to measure chlorophyll content and, therefore, determine in situ nitrogen (N) status) trends for the corn seedlings in Example 6. [Figure 2] FIG. 2 shows nitrogen uptake by corn seedlings of Example 6. [Figure 3] FIG. 3 shows the "PE Nitrogen" [i.e., physiological efficiency (PE) which refers to the plant's ability to convert nitrogen obtained from an applied source into economic yield] of the corn seedlings of Example 6. [Figure 4]FIG. 4 shows the "AE Nitrogen" [ie, agronomic efficiency (AE) meaning productivity improvements obtained through the use of nitrogen inputs] for the corn seedlings of Example 6. [Figure 5] FIG. 5 shows the "IE Nitrogen" [ie, internal use efficiency (IE) which refers to the plant's ability to convert nitrogen obtained from all sources into economic yield] of the corn seedlings of Example 6. [Figure 6] Figures 6 to 10 relate to a study of wheat seedlings according to Example 7 carried out from December 15, 2021 to January 14, 2022, comparing the following differently treated samples: "Untreated control", i.e., wheat seedlings that received no treatment; "Normal fertilizer", i.e., wheat seedlings that received 150 kg / ha of ammonium nitrate as fertilizer at the time of sowing; "100 g / m³ of the composition of the invention", i.e., wheat seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction at the time of sowing; and "100 g / m³ of the composition of the invention, normal fertilizer", i.e., wheat seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction and 150 kg / ha of ammonium nitrate at the time of sowing. "100g / m³ of the present composition, ½ fertilizer," i.e., wheat seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 100g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the present composition," i.e., wheat seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ at the time of sowing. "500g / m³ of the present composition, normal fertilizer," i.e., wheat seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 150kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the present composition, ½ fertilizer," i.e., wheat seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. Specifically, Figure 6 shows the SPAD trends of the wheat seedlings of Example 7. [Figure 7] FIG. 7 shows the uptake of nutrients, namely nitrogen, phosphorus, and potassium, by wheat seedlings of Example 7. [Figure 8] FIG. 8 shows the "PE Nitrogen" of wheat seedlings in Example 7. [Figure 9]FIG. 9 shows the "AE Nitrogen" of wheat seedlings in Example 7. [Figure 10] FIG. 10 shows the "IE Nitrogen" of wheat seedlings in Example 7. [Figure 11] Figures 11 to 13 relate to a study of soybean seedlings according to Example 8 carried out from December 15, 2021 to January 14, 2022, comparing the following differently treated samples: "Untreated control", i.e., soybean seedlings that received no treatment; "Normal fertilizer", i.e., soybean seedlings that received 150 kg / ha of ammonium nitrate as fertilizer at the time of sowing; "100 g / m³ of inventive composition", i.e., soybean seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction at the time of sowing; and "100 g / m³ of inventive composition, normal fertilizer", i.e., soybean seedlings that received a mixture of Trichoderma GV41 and 100 g / m³ of the lignin fraction at the time of sowing, and 150 kg / ha of ammonium nitrate at the time of sowing. "100g / m³ of the composition of the present invention, 1 / 2 fertilizer", i.e., soybean seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 100g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the composition of the present invention", i.e., soybean seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ at the time of sowing. "500g / m³ of the composition of the present invention, normal fertilizer", i.e., soybean seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 150kg / ha of ammonium nitrate at the time of sowing. "500g / m³ of the composition of the present invention, 1 / 2 fertilizer", i.e., soybean seedlings receiving a mixture of Trichoderma GV41 and the lignin fraction at 500g / m³ and 75kg / ha of ammonium nitrate at the time of sowing. Specifically, FIG. 11 shows the SPAD trends of soybean seedlings in Example 8. [Figure 12] FIG. 12 shows nitrogen uptake by soybean seedlings of Example 8. [Figure 13] FIG. 13 shows the "PE Nitrogen" of soybean seedlings in Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0018] A subject of the present invention is therefore the use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers, wherein: the fungus is selected from the group consisting of Trichoderma, protoplast fusion products thereof, and mixtures thereof; the lignin fraction comprises fragments having a weight average molecular weight of up to 20,000 Daltons as measured by size exclusion chromatography, the fragments comprising a weight average of up to 111 phenylpropane units; Here, the concentration of fungi is 1 × 10 5 ~5×10 10 spores / g composition, the concentration of the lignin fraction is at least 40% by weight relative to the weight of the composition.

[0019] Preferably, the composition is in the form of an aqueous solution, dispersion or suspension, or alternatively in the form of a solid mixture.

[0020] Preferably, the fungus of the genus Trichoderma is selected from the group consisting of Trichoderma aggressiveum, Trichoderma asperellum, Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma cremeum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, and Trichoderma viride. viride, and Trichoderma viridescens.

[0021] The above-mentioned fungi belonging to the genus Trichoderma can colonize various niches, antagonize and control plant pathogenic microorganisms, and establish direct beneficial interactions with plants, resulting in enhanced growth, nutrient uptake, and systemic disease resistance. Specifically, improved plant development is generally associated with increased seed germination, root system, plant weight, and leaf area, as well as the size and / or number of seeds, flowers, and / or fruits, resulting in increased yield and, in many cases, the content of important nutritional factors.

[0022] Lignin is known to have antimicrobial activity against both fungi and bacteria. Therefore, it was expected that Trichoderma, a well-known soil-dwelling fungus, would be directly inhibited when treated with lignin concentrations previously found to be toxic to the majority of other fungi tested. Thus, it was reasonably expected that Trichoderma would be affected at lignin concentrations typically considered for agricultural use. Surprisingly and unexpectedly, however, the above-listed Trichoderma fungi were not only unaffected by the lignin fractions at the given concentrations, but also increased their activity in terms of nitrogen uptake, either naturally occurring in the soil or supplemented as a nitrogen-containing fertilizer.

[0023] Specifically, it was found that the effect on nitrogen uptake was increased, such that it was possible to reduce nitrogen-containing fertilizer supplementation by up to 50% and still achieve appreciable and comparable results.

[0024] In addition to the above, it should be understood that Trichoderma and the lignin fraction work in different ways and utilize different mechanisms, and that doing so can make the resulting composition more robust and suitable in a variety of conditions while reducing the over-exploitation of resistance mechanisms (for biocontrol).

[0025] Moreover, advantageously, the compositions are simple and cost-effective to manufacture, as the concentrations of each are very low, which also means that the compositions may be prepared in concentrated form that can be easily diluted with water to the above concentrations before use.

[0026] The term "protoplast fusion" is meant to encompass hybrid strains of Trichoderma spp. obtained by protoplast fusion.

[0027] Protoplasts are cells from which the cell wall has been removed, with the cytoplasmic membrane being the outermost layer of the cell. Protoplasts can be obtained by specific lytic enzymes to remove the cell wall. Protoplast fusion is a physical phenomenon during which two or more protoplasts come into contact and adhere to each other either spontaneously or in the presence of a fusogenic agent. Protoplast fusion can transfer several useful genes from one species to another. Protoplast fusion is an important tool in strain improvement to cause genetic recombination in filamentous fungi and generate hybrid strains. The improvement may include, for example, higher yields of cellulase production.

[0028] Protoplast fusions for the purposes of the present invention can be obtained according to techniques known in the art (e.g., Hassan MM, Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity, Biotechnology & Biotechnological Equipment, 28:6, 1014-1023).

[0029] In a preferred embodiment of the composition of the present invention, the fungus of the genus Trichoderma is selected from T. harzianum, T. atroviride, and T. virens, and mixtures thereof.

[0030] In some embodiments, the composition comprises a mixture of Trichoderma species.

[0031] In a more preferred embodiment, the fungus is selected from T. harzianum HK2, T. atroviride HK4, and T. virens GV41, and mixtures thereof, where "HK2," "HK4," and "GV41" are each preferred strains.

[0032] HK2 (or simply "K2") has ATCC number PTA-9708 and is disclosed in US Pat. No. 8,716,001 (strain RR17Bc).

[0033] HK4 (or simply "K4") has ATCC number PTA-9707 and is disclosed in US Pat. No. 8,877,480 (strain WW10TC4).

[0034] GV41 (or simply "G41") is commercially available from BioWorks Inc. NY 14564, USA.

[0035] In some embodiments, the composition comprises a mixture of Trichoderma strains.

[0036] When the mixture is present in a composition, each genus or strain is at the same or about the same concentration.

[0037] In a preferred embodiment, the composition comprises two Trichoderma species or two Trichoderma strains in a concentration ratio of 2:1 to 1:2, preferably 1:1.

[0038] Preferably, the concentration of fungi is 1 x 10 6 ~3×10 10 More preferably, the concentration of the fungus is 1 x 10 spores / g composition. 8 ~2×10 10 In a preferred embodiment, the concentration of fungi is 1 x 10 spores / g composition.9 ~2×10 10 Spores / g composition.

[0039] Lignin is a class of complex organic polymers that form the important structural material in the support tissue of some algae, vascular plants and their bark, and herbaceous plants, such as trees (i.e., softwoods and hardwoods), the culms of all cereals, sugarcane residue (bagasse), grasses, linen, jute, hemp, or cotton. Lignin may also have mineral sources, such as peat, leonardite, and coal.

[0040] Chemically, in its native form, lignin is a highly irregular, randomly cross-linked polymer of phenylpropane units joined by many different linkages, with a weight average molecular weight of 20,000 daltons or greater. A representative and exemplary lignin fragment (I) containing the most significant bond patterns is shown herein below. [ka]

[0041] The polymer is made from three phenylpropanoid monomer precursors [ka] is the result of an enzyme-mediated dehydrogenative polymerization, yielding the following moieties:

[0042] [ka]

[0043] Coniferyl alcohol is present in all species and is the major monomer in coniferous (softwood) trees. Deciduous (hardwood) species contain up to 40% sinapyl alcohol units, and grasses and crops may also contain coumaryl alcohol units.

[0044] Lignins can be classified according to their raw biomass source as softwood lignins and hardwood lignins. For purposes of the present invention, the lignin is softwood lignin, hardwood lignin, or a mixture thereof. Preferably, the lignin is hardwood lignin.

[0045] Raw biomass sources that may be suitable starting materials for obtaining the lignin fractions of interest are essentially pure lignin and any lignin including kraft lignin, biomass-derived lignin, lignin from an alkaline pulping process, lignin from a soda process, lignin from organosolv pulping, lignin from an enzymatic process, lignin from a steam explosion process, and any combination thereof.

[0046] The expression "essentially pure lignin" should be understood as at least 80% pure lignin, preferably at least 90% pure lignin, more preferably at least 95% pure lignin, based on dry raw biomass, the remainder being extractives and carbohydrates, such as hemicellulose, and inorganic materials.

[0047] The expression "kraft lignin" should be understood as lignin originating from kraft black liquor. Black liquor is an alkaline aqueous solution of lignin residues, hemicellulose, and inorganic chemicals used in the kraft pulping process. Black liquor from the pulping process contains components originating from different softwood and hardwood species in varying proportions. Lignin can be separated from black liquor by different techniques, including, for example, precipitation and filtration. Lignin usually begins to precipitate at pH values ​​below 11-12. Different pH values ​​can be used to precipitate lignin fractions with different properties. These lignin fractions are characterized by their molecular weight distribution, e.g., M w and M nThe precipitated lignin may differ in polydispersity, hemicellulose and extractive content, and inorganic material content. The precipitated lignin may be purified from inorganic impurities, hemicellulose, and wood extractives using an acid wash step. Further purification may be achieved by filtration.

[0048] Alternatively, lignin can be separated from pure biomass. The separation process can begin by liquefying the biomass with strong alkali, followed by a neutralization process. After alkali treatment, lignin can be precipitated in a similar manner as described above.

[0049] Preferably, the separation of lignin from biomass includes an enzymatic treatment step. The enzymatic treatment modifies the lignin extracted from the biomass. The lignin separated from the pure biomass is essentially sulfur-free (less than 3% sulfur content) and is therefore valuable for further processing. Preferably, the wood material is pretreated to remove hemicellulose, followed by cellulose hydrolysis. The resulting insoluble lignin fraction contains up to 30% cellulose by weight.

[0050] The weight average molecular weight (M w ) is measured by size exclusion chromatography (or "SEC"). SEC uses stagnant liquid present in the pores of the beads as the stationary phase and flowing liquid as the mobile phase. Therefore, the mobile phase can flow between the beads and also into and out of the pores of the beads. The separation mechanism is based on the size of the polymer molecules in solution. Larger molecules elute first. Smaller molecules, which can enter the many pores of the beads, take longer to pass through the column and therefore exit the column slowly. To determine the molecular weights of the components of a polymer sample, a calibration must be performed with standard polymers of known weight. Values ​​from unknown samples are then compared to the calibration graph. Retention times depend on the column material used, the eluent, and how similar the standards used are compared to the sample. Preferably, the eluent is 0.1 M NaOH.

[0051] Preferably, the lignin fraction contains fragments having a weight-average molecular weight of 2,000 to 20,000 Da (Daltons).

[0052] More preferably, the lignin fraction contains fragments having a weight-average molecular weight of 3,000 to 20,000 Da.

[0053] Even more preferably, the lignin fraction comprises fragments having a weight average molecular weight of 4,000 to 15,000 Da.

[0054] In some preferred embodiments, the lignin fraction contains fragments with a weight-average molecular weight of 4,000 to 8,000 Da.

[0055] In another preferred embodiment, the lignin fraction contains fragments having a weight-average molecular weight of 9,000 to 11,000 Da.

[0056] Preferably, in these embodiments, the fragments contain, on average by weight, 11 to 111 phenylpropane units, more preferably, on average by weight, 22 to 111 phenylpropane units.

[0057] The molecular weights of the three phenylpropanoid monomer precursors vary between 150 Da for coumaryl alcohol, 180 Da for coniferyl alcohol, and 210 Da for sinapyl alcohol. Therefore, the average weight was 180 Da, and this value was used as the "phenylpropane unit." M w The value was divided by 180 Da to obtain the weight average number of phenylpropane units.

[0058] Preferably, the lignin fraction has a number average molecular weight (M n ) up to 2,000 daltons.

[0059] For the purposes of the present invention, the number average molecular weight (M n) is measured by size exclusion chromatography.

[0060] More preferably, the lignin fraction has a number average molecular weight (M n ) up to 1,500 daltons.

[0061] In a preferred embodiment, the lignin fraction has a number average molecular weight (M n ) contains fragments with a length between 150 daltons and 1,300 daltons.

[0062] In a further embodiment, the lignin fraction has a polydispersity index (PDI) of 1.25-12.

[0063] Polydispersity index (PDI), or heterogeneity index, or simply dispersity, is a measure of the distribution of molecular masses in a given polymer sample. PDI is the number average molecular weight (M n ) divided by the weight average molecular weight (M w ) which describes the distribution of individual molecular masses in a batch of polymer.

[0064] Fungi of the genus Trichoderma produce cellulolytic enzymes, such as exoglucanase (EXG), endoglucanase (EG), and β-glucosidase (BGL). Cellulase is the most efficient enzyme system for completely hydrolyzing cellulosic substrates into the fermentable sugar monomer glucose. Because carbohydrates aid in plant cellular respiration and cell growth, the presence of cellulose in the compositions of the present invention is advantageous for further improving the overall efficiency of promoting plant growth.

[0065] Preferably, the composition of the present invention comprises a lignin fraction at a concentration of at least 60% by weight, more preferably at least 70% by weight, based on the weight of the composition. In a preferred embodiment, the composition of the present invention comprises a lignin fraction at a concentration of 75-95% by weight, based on the weight of the composition.

[0066] In a preferred embodiment, the composition of the present invention comprises a fungus of the genus Trichoderma and a lignin fraction, wherein: the fungus is selected from Trichoderma harzianum, Trichoderma atroviride, Trichoderma virens, and mixtures thereof; the lignin fraction comprises fragments having a weight-average molecular weight of 3,000 to 20,000 daltons as measured by size exclusion chromatography, the fragments comprising, on average, 16 to 111 phenylpropane units; Here, the concentration of fungi is 1 × 10 5 ~5×10 10 spores / g composition, the concentration of the lignin fraction is at least 40% by weight relative to the weight of the composition.

[0067] More preferably, the composition of the present invention comprises a fungus of the genus Trichoderma and a lignin fraction, wherein: - the fungus is selected from Trichoderma harzianum, Trichoderma atroviride, Trichoderma virens, and mixtures thereof; the lignin fraction comprises fragments having a weight-average molecular weight of 3,000 to 20,000 daltons as measured by size exclusion chromatography, the fragments comprising, on average, 16 to 111 phenylpropane units; Here, the concentration of fungi is 1 × 10 6 ~3×10 10 spores / g composition, the concentration of the lignin fraction is at least 60% by weight relative to the weight of the composition.

[0068] In some preferred embodiments, the compositions of the present invention comprise a fungus of the genus Trichoderma and a lignin fraction, wherein: - the fungus is selected from Trichoderma harzianum HK2, Trichoderma atroviride HK4, Trichoderma virens GV41, and mixtures thereof; the lignin fraction comprises fragments having a weight average molecular weight of 4,000 to 6,000 daltons as measured by size exclusion chromatography, the fragments comprising, on average, 22 to 33 phenylpropane units; Here, the concentration of fungi is 1 × 10 8 ~2×10 10 The concentration of spores / g composition, the lignin fraction is at a concentration of at least 70% by weight relative to the weight of the composition.

[0069] In another preferred embodiment, the composition of the present invention comprises a fungus of the genus Trichoderma and a lignin fraction, wherein: - the fungus is selected from Trichoderma harzianum HK2, Trichoderma atroviride HK4, Trichoderma virens GV41, and mixtures thereof; the lignin fraction comprises fragments having a weight average molecular weight of 9,000 to 11,000 daltons as measured by size exclusion chromatography, the fragments comprising a weight average of 50 to 61 phenylpropane units; Here, the concentration of fungi is 1 × 10 8 ~2×10 10 spores / g composition, the concentration of the lignin fraction is at least 70% by weight relative to the weight of the composition.

[0070] In a most preferred embodiment, the composition of the present invention comprises a fungus of the genus Trichoderma and a lignin fraction, wherein: - the fungus is Trichoderma virens GV41, the lignin fraction comprises fragments having a weight average molecular weight of 9,000 to 11,000 daltons as measured by size exclusion chromatography, the fragments comprising a weight average of 50 to 61 phenylpropane units; Here, the concentration of fungi is 1 × 10 9 ~2×10 10 Spores / g composition, the concentration of the lignin fraction is 75-95 wt% based on the weight of the composition.

[0071] The nitrogen-containing fertilizer is a fertilizer containing urea, ammonia, ammonium nitrate, ammonium sulfate, calcium nitrate, diammonium phosphate, monoammonium phosphate, potassium nitrate, sodium nitrate, or a mixture thereof as a nitrogen source.

[0072] As discussed above, compositions comprising Trichoderma fungi and lignin fractions have been shown to unexpectedly and significantly increase nitrogen uptake by plant seeds, so that the overall use of conventional nitrogen-containing fertilizers can be advantageously reduced by as much as half.

[0073] Preferably, the fertilizer may also include a phosphorus source, a potassium source, or a mixture thereof.

[0074] Suitable phosphorus sources include diammonium phosphate, monoammonium phosphate, monopotassium phosphate, dipotassium phosphate, tetrapotassium pyrophosphate, potassium metaphosphate, and mixtures thereof.

[0075] Suitable potassium sources include potassium chloride, potassium nitrate, potassium sulfate, monopotassium phosphate, dipotassium phosphate, tetrapotassium pyrophosphate, potassium metaphosphate, and mixtures thereof.

[0076] In a preferred embodiment, the fertilizer contains nitrogen, phosphorus, and potassium sources in an N:P:K ratio selected from the group consisting of 29-3-4, 16-4-8, 10-10-10, 15-5-10, 15-0-15, 22-3-14, 20-28-5, and 12-6-6.

[0077] Optionally, the fertilizer may also include macronutrients selected from the group consisting of sulfur, calcium, and magnesium, and / or micronutrients including boron, copper, iron, manganese, molybdenum, and zinc.

[0078] The nitrogen-containing fertilizer may be in the form of a liquid, a suspension, or a solid, such as a powder or granules.

[0079] The composition may be used in an amount of 1 to 1,000 kg per hectare (ha) of soil, preferably 1 to 100 kg / ha, more preferably 1 to 10 kg / ha.

[0080] In another embodiment, the present invention relates to the use of a composition consisting essentially of a fungus of the genus Trichoderma and a lignin fraction as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizer, wherein: the fungus is selected from the group consisting of Trichoderma, protoplast fusion products thereof, and mixtures thereof; the lignin fraction comprises fragments having a weight average molecular weight of up to 20,000 Daltons as measured by size exclusion chromatography, the fragments comprising a weight average of up to 111 phenylpropane units; Here, the concentration of fungi is 1 × 10 5 ~5×10 10 spores / g composition, the concentration of the lignin fraction is at least 40% by weight relative to the weight of the composition. For the purposes of the present invention, the expression "consisting essentially of" means that the fungus and the lignin fraction are the only active ingredients present in the composition that act as plant growth and fruit production promoters, and that any other components that may be present have different activities or are simply adjuvants.

[0081] In a further embodiment, the present invention relates to the use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction, as described above, as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers.

[0082] It should be noted that all preferred aspects of the use of the compositions of the present invention are considered equally preferred for embodiments defined by the terms "consisting essentially of" and "consisting of".

[0083] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a first container containing a fungus of the genus Trichoderma; a second container containing a lignin fraction, and - a third container containing nitrogen-containing fertilizer, or a first container containing a fungus of the genus Trichoderma, and a second container containing a lignin fraction and a nitrogen-containing fertilizer, wherein the fungus, the lignin fraction, and the fertilizer are as described above.

[0084] In the embodiment comprising three containers, the components are separated from one another, thus better storing them and making it easier to weigh each of the components when combined.

[0085] In an embodiment comprising two containers, the Trichoderma fungus is separated from the lignin fraction and the fertilizer, the latter being more compatible with each other while reducing the overall package.

[0086] Therefore, depending on the needs at the time, the agrochemical kit of the present invention may be provided in a more suitable embodiment of the above alternatives.

[0087] The container may be a pouch, bag, envelope, box, drum, bottle, or can.

[0088] Preferably, the agricultural chemical kit comprises: the first container contains 1 x 10 5 ~1×10 10 The method comprises the steps of: (a) solid granules a) comprising a fungus of the genus Trichoderma in a concentration of spores / g granules a) and at least one binder; - the second container contains solid granules b) comprising a lignin fraction in a concentration of at least 50% by weight relative to the weight of the granules b), Average particle size distribution D of granules a) and granules b) determined by sieve analysis according to EN 1235 50 are independently between 0.2 and 4.0 mm.

[0089] Indeed, granular formulations offer several advantages, e.g. -No dust generation - easy sliding in mechanical devices (no unwanted compaction effect), -Ease of storage, -Possibility of slow release formulations, -Uniform nutrient distribution, - No nutrient segregation during product handling or application; - higher pre-plant application efficiency, Includes.

[0090] As described above, the average particle size distribution D of granules a) and granules b) 50are independently between 0.2 and 4.0 mm. For purposes of this invention, this parameter is measured by sieve analysis based on EN 1235 [i.e., EN 1235: Solid Fertilizers - Test Sieving (ISO 8397:1988, modified) (including Amendment A1:2003)]. Mean particle size and particle size distribution are important quality attributes for solid fertilizers and related products. Sieve analysis has been declared a mandatory process for determining the particle size distribution of solid fertilizer products sold in the European Union, and all relevant equipment and procedures are specified by EN 1235. Based on EN 1235, solid fertilizers are subjected to sieve analysis using a 200-millimeter diameter laboratory test sieve manufactured in accordance with the requirements of ISO 3310-1. The standard requires a maximum of seven test sieves to be used in a gradient test covering the complete size distribution spectrum of the sample material. The selection of the mesh size should be made from the R20 / 3 series of ISO 565, although the standard explicitly allows the use of additional sieves from the R20 series. The requirements of EN 1235 were determined in a series of ring tests using woven wire sieves with mesh widths ranging from 100 μm to 5.60 mm.

[0091] Granular products are homogeneous solid mixtures typically produced in granulation plants by combining various raw materials. Each uniformly sized particle contains all the components of the analysis.

[0092] Various granulation processes are known, for example: -dry granulation, -Wet granulation, -spray dryer, -Fluid bed spray, -Bread granulator.

[0093] In a preferred embodiment, the Trichoderma fungus and the lignin fraction are granulated separately in different and distinct granulation processes to obtain granules a) and b), respectively, which can be mixed to obtain a solid mixture that can be stored for long periods of time and then redispersed or resuspended in a liquid formulation at the time of use while retaining their activity and effectiveness.

[0094] Preferably, the concentration of Trichoderma fungus is 1 x 10 5 ~1×10 10 spores / g granules a).

[0095] Preferably, in granules a), the at least one binder is selected from kaolin, starch, modified starch, starch phosphate, pectin, modified pectin, amylopectin, alginic acid, sodium alginate, guar gum, guar powder, tragacanth, gum arabic, xanthan gum, karaya gum, tara gum, tamarind gum, gellan gum, locust bean gum, gelatin, carob seed powder, galactomannan, glucomannan, dextran, carrageenan, mannan, arabinogalactan, pullulan, maltodextrin, cellulose, derivatized cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, soy polysaccharide, chitosan, or mixtures thereof.

[0096] More preferably, the at least one binder is selected from starch, modified starch, starch phosphate, and mixtures thereof.

[0097] In a preferred embodiment, the granules a) further comprise a wetting agent, a disintegrant, a dispersant, or a mixture thereof.

[0098] Wetting agents can reduce the surface tension of water, allowing them to penetrate hydrophobic soil media and thereby facilitate penetration. They differ in their effects on water surface tension, water penetration, and water retention. Suitable wetting agents are selected from the group consisting of alkyl sulfates, allyl sulfonates, polyoxyalkylene alkyl ethers, alkenyl sulfonates, polyoxyethylene styrylphenyl ethers, polyoxyethylene distyrylphenyl ethers, polyoxyethylene tristyrylphenyl ethers, polyoxyethylene styrylphenyl ether salts, polyoxyethylene distyrylphenyl ether salts, polyoxyethylene tristyrylphenyl ether salts, and N-acylamino acid salts.

[0099] A preferred wetting agent is an allyl sulfonate, such as sodium isopropyl naphthalene sulfonate.

[0100] Disintegrants are additives incorporated into granules to facilitate their disintegration when they come into contact with a liquid or fluid substance. Suitable disintegrants include water-soluble polymers and polysaccharides.

[0101] Dispersants are substances, typically surfactants, added to improve the separation of particles and to prevent their settling or agglomeration. Suitable dispersants are polycarboxylates, such as sodium polycarboxylate.

[0102] In some embodiments, the granules b) consist essentially of the lignin fraction.

[0103] In another embodiment, the granules b) consist of a lignin fraction.

[0104] Preferably, the granules b) contain the lignin fraction in a concentration of 65 to 95% by weight, more preferably 70 to 90% by weight, based on the weight of the granules b).

[0105] The granules b) may further comprise at least one carrier.

[0106] Preferably, in granules b), the at least one carrier is selected from lignosulfite, chalk, carboxymethylcellulose, carbonates, bicarbonates, sulfates, phosphates, oxides or hydroxides of potassium, sodium, lithium, calcium, magnesium, zinc or ammonium, or urea salts, or mixtures thereof.

[0107] More preferably, said at least one carrier is selected from potassium or sodium carbonate salts, or ammonia or urea salts, and mixtures thereof.

[0108] Preferably, the average particle size distribution D of the granules a) and the granules b) 50 are independently between 0.5 and 2.0 mm.

[0109] In a preferred embodiment, the average particle size distribution D of the granules a) 50 and the average particle size distribution D of the granules b) 50 The ratio is 3:1 to 1:3, more preferably 2:1 to 1:2.

[0110] In a particularly preferred embodiment, the average particle size distribution D 50 are approximately the same.

[0111] In the most preferred embodiment, no particles less than 0.2 mm in size are present in the solid mixture.

[0112] Preferably, the bulk density (coarse) of said granules a) and said granules b) according to ISO 3944:1992, independently of one another, is between 0.3 and 0.8 g / ml, preferably between 0.4 and 0.7 g / ml. The following specifications include provisions which, by reference in this document, constitute provisions of this International Standard. -ISO 7742:1988, Solid fertilizers - Reduction of samples. -ISO 8358:1991, Solid fertilizers -- Preparation of samples for chemical and physical analysis.

[0113] For the purposes of this International Standard, "crude bulk density of fertilizer" is defined by the mass per volume of material after it has been poured freely into a container under specified conditions. Crude bulk density is expressed in grams per cubic centimetre (g / cm 3 )

[0114] These standards refer to "fertilizers", which the inventors considered to be suitable and applicable references also for the present characterization.

[0115] In a preferred embodiment, the bulk density (coarse) of said granules a) and said granules b) is approximately the same.

[0116] Granules a) and b) have similar mean particle size distributions D 50 and / or density is important to minimize the risk that larger or heavier granules will not be homogeneously mixed and thus will lead to unbalanced dosing of the composition components when redispersed or resuspended in a liquid formulation at the time of use.

[0117] Preferably, the kit of the present invention contains 0.1 to 20 wt % of granules a) in a first container and 80 to 99.9 wt % of granules b) in a second container, based on the weight of the solid mixture. The "wt %" of each of granules a) and granules b) means that it is calculated based on the total weight of granules a) and granules b).

[0118] Specifically, in agricultural chemistry kits, i) when the at least one carrier of granules b) is water-dispersible or water-soluble, the kit comprises 1 to 15 wt. % of granules a) and 85 to 99 wt. % of granules b), based on the weight of the solid mixture thereof; ii) When the at least one carrier of granules b) is neither water-dispersible nor water-soluble, the kit comprises 0.1 to 5 wt. % of granules a) and 95 to 99.9 wt. % of granules b), based on the weight of the solid mixture.

[0119] In a preferred embodiment of option i), said at least one carrier of granules b) comprises potassium carbonate, an ammonia salt, a urea salt, or a mixture thereof.

[0120] In a preferred embodiment of option ii), said at least one carrier of granules b) comprises calcium sulfate, lignosulfite, chalk, carboxymethylcellulose, or a mixture thereof.

[0121] In a further preferred embodiment, the second container of the kit comprises: i) granules b), wherein the at least one carrier is water-dispersible or water-soluble; and ii) granules b), wherein said at least one carrier is neither water-dispersible nor water-soluble; Includes both.

[0122] It should be understood that option i), i.e., granules b), including at least one water-dispersible or water-soluble carrier, can be considered fast-acting granules. In fact, the lignin fraction is readily released upon contact with water.

[0123] Similarly, it should be understood that option ii), ie, granules b) including at least one non-water-dispersible or non-water-soluble carrier, may be considered slow-release granules.

[0124] Thus, by adjusting the concentration of the different granules b), the efficacy and activity over time of the resulting composition once used may be preset.

[0125] The agricultural chemical kit according to the present invention is as follows: - by weight of the solid mixture, starch in the first container and 5 x 10 9 A fast-acting kit comprising 1% by weight of granules a) containing spores / g Trichoderma and 99% by weight of granules b) (75% by weight lignin fraction + 25% by weight potassium carbonate) in a second container. - by weight of the solid mixture, starch in the first container and 5 x 10 9 A slow release kit comprising 0.1 wt. % granules a) containing spores / g Trichoderma and 99.9 wt. % granules b) (85 wt. % lignin fraction + 15 wt. % calcium sulfate) in a second container.

[0126] Preferably, granules a) are prepared by extrusion granulation, in which a fine powder of Trichoderma spores and at least one binder are mixed with water (10-20%) to obtain a wet mixture. This mixture is passed through an extruder or pan granulator to obtain wet granules, which are then dried in an oven or fluidized bed.

[0127] Specifically, the water-dispersible Trichoderma live spore granules a) are preferably - milling the solid ingredients together to obtain a homogeneous premix; - adding water to the premix to obtain a wet mixture; - granulating the wet mixture by any suitable technique, such as extrusion, pan granulation, agglomeration, spray drying, etc., It is prepared by

[0128] Preferably, the premix is ​​obtained by mixing together the following solid ingredients: - Trichoderma spores 10-90% by weight - Wetting agent 1 to 3% by weight Dispersant 2 to 15% by weight Disintegrant 0 to 15% by weight - Binder: 100% by weight.

[0129] The lignin fractions typically used in the present invention are not soluble in water at neutral pH, but are soluble at basic pH.

[0130] It has then been found that water-dispersible granules of the lignin fraction b) can be obtained by adding at least one water-dispersible or water-soluble carrier, for example an alkaline compound, to the lignin, thus solubilizing the lignin in water at a basic pH. The solubilized lignin forms a colloidal dispersion in water.

[0131] The solubilized lignin fraction is then diluted with water at neutral pH, thus obtaining a solution / dispersion of lignin in water at a pH physiological for plants, ie neutral or slightly basic pH.

[0132] Preferably, the carrier is potassium carbonate.

[0133] Thus, the granules b) of the water-dispersible lignin fraction are preferably - adding at least one water-dispersible or water-soluble carrier, preferably potassium carbonate, to the lignin fraction, the lignin fraction preferably having a dry matter content of 60-70%, - mixing until a uniform paste is formed to obtain a water-dispersible lignin fraction paste; - wet granulating the water-dispersible lignin fraction paste to obtain granules b), It is prepared by

[0134] The term dry matter content means the content of solid matter in a mixture. It is calculated as the percentage, by weight, of solid material in the total mass of the mixture.

[0135] Granulation with the lignin fraction alone (i.e., without Trichoderma spores) allows for the utilization of the good thermal and chemical stability of lignin (and, in the case of water-dispersible formulations, avoiding exposure of Trichoderma to alkaline formulations), allowing for process conditions that are highly advantageous in terms of cost and yield.

[0136] The non-water-dispersible granules b) of the lignin fraction are preferably - providing a lignin fraction preferably having a dry matter content of 60-70%, - optionally adding at least one non-water-dispersible or non-water-soluble carrier, preferably lignosulfite, chalk, carboxymethylcellulose, calcium sulfate, or a mixture thereof, - granulating the lignin fraction, optionally mixed with the carrier, in a blade rotor, inducing the formation of granules by mechanical action; and - drying the granules in a fluidized bed, It is prepared by

[0137] Due to the agglomerating properties of lignin, the use of a non-water-dispersible or non-water-soluble carrier is not strictly necessary, however, the addition of a carrier improves the texture of the granules and prevents break-up of the granules during handling, storage, and use with agricultural machinery that would otherwise result in undesirable dust.

[0138] It should be understood that the composition used as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizer can comprise the Trichoderma fungus and the lignin fraction in the form of granules a) and b), respectively, as described above in connection with the agricultural chemical kit. Accordingly, it should be understood that all preferred embodiments of granules a) and b), and combinations thereof, are also disclosed herein and should be considered equally preferred for use of the composition as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizer.

[0139] In a further aspect, the present invention relates to a method for increasing nitrogen uptake by a plant seed receiving a nitrogen-containing fertilizer, said method comprising: A) applying the composition to the seed soil; and B) applying a nitrogen-containing fertilizer to provide nitrogen at an amount not exceeding 50% of the recommended amount for the plant being fertilized; Includes:

[0140] Preferably, in this method, the composition and the nitrogen-containing fertilizer are provided in the form of the above-described agricultural chemical kit. In this regard, preferably, the agricultural chemical kit further includes instructions (e.g., an instruction leaflet) for implementing the above-described method.

[0141] In step A), the composition is applied to the seed soil.

[0142] The term "seed soil" is intended to mean the soil in which seeds will be or have previously been sown, such as compost, humus, peat, sand, sawdust, coconut fiber, and combinations thereof.

[0143] Preferably, the composition is mechanically mixed with the soil, where the seeds have already been sown or the seeds will be sown later, meaning that the seeds can be mixed together with the composition and soil, or the seeds can be sown after the composition and soil have been mixed.

[0144] Preferably, the composition has a density of 0.01 to 1.00 kg / m 3 More preferably, the composition is used in an amount of 0.05 to 0.70 kg / m of seed soil. 3 Seed soil is used in the amount.

[0145] In a preferred embodiment, the composition has a density of 0.10 to 0.50 kg / m 3 Seed soil is used in the amount.

[0146] In step B), a nitrogen-containing fertilizer is applied to the seed soil to provide nitrogen in an amount not exceeding 50% of the recommended amount for the plant used.

[0147] The term "recommended amount" should be interpreted as the amount of nitrogen that official guidelines issued by appropriate agricultural authorities and fertilizer manufacturers indicate is appropriate for the particular plant being cultivated. An example is "Guidelines on Nitrogen Management in Agricultural Systems" published by the International Atomic Energy Agency, Vienna, February 2008 (IAEA-TCS-29, ISSN 1018-5518).

[0148] In fact, it has been observed that the effect on nitrogen uptake is increased by the compositions of the present invention, so that it is possible to reduce the supplementation of nitrogen-containing fertilizers by up to 50% and still achieve appreciable and comparable results.

[0149] Said effect has been demonstrated by a number of tests carried out on different plants, in particular wheat, corn and soybean, germinated and grown from seeds treated as described above, for example:

[0150] -SPAD, or Soil, Plant, Soil, Crop, and Produce Analysis System Commercialization Project Chlorophyll Meter. It is a rapid and non-destructive approach to measure chlorophyll content and therefore determine in-situ nitrogen (N) status. It is one of the most commonly used diagnostic tools for measuring crop nitrogen status; specifically, it determines the relative amount of chlorophyll present by measuring leaf absorbance in two wavelength regions: red and near-infrared. Using these two absorbances, the meter calculates a SPAD number, which is proportional to the amount of chlorophyll present in the leaf. The chlorophyll content, represented by the measured SPAD value, increases proportionally to the amount of nitrogen present in the leaf. A higher SPAD value indicates a healthier plant.

[0151] Tests carried out on wheat, corn and soybeans showed that the composition alone was able to increase the SPAD value compared to untreated samples, however, the combination of the composition and fertilizer (either at the amount typically applied or at half the amount) further increased the SPAD value.

[0152] - PE nitrogen, i.e. physiological efficiency, which means the plant's ability to convert nitrogen obtained from applied sources into economic yield. In other words, PE is defined as the yield increase per increase in crop uptake of nutrients in the above-ground parts of the plant. It is calculated according to the following formula: PE=(Y-Y0) / (U-U0) where Y = yield of the harvested portion of the nutrient-fed crop, Y0 = yield without nutrient treatment, U = total nutrient uptake of the aboveground crop biomass with nutrient treatment, and U0 = nutrient uptake of the aboveground crop biomass without nutrient treatment. Units are not given (values ​​of 40-60 are common) because the expressions are ratios on a mass basis and are therefore unitless in their standard form.

[0153] -AE Nitrogen, i.e. agronomic efficiency, which refers to the productivity improvement obtained through the use of nitrogen inputs. It is calculated in units of yield increase per unit of nutrient applied. AE Nitrogen more closely reflects the direct production effect of the fertilizer used and is directly related to economic return. Calculating AE requires knowledge of the yield without nutrient application and is therefore only known when study plots without nutrient application are conducted in the field. The following formula is used: AE=(Y-Y0) / F where Y = yield of the crop portion that received the nutrient, Y = yield without the nutrient, and F = amount of nutrient applied. No units are shown because the expression is a ratio by mass and is therefore unitless in its standard form.

[0154] - IE nitrogen, i.e., the internal use efficiency, which means the plant's ability to convert nitrogen obtained from all sources into economic yield. In other words, IE is defined as the yield relative to the total nutrient uptake. A very high IE indicates a deficiency of that nutrient. A low IE indicates a low internal nutrient conversion due to other stresses (deficiency of other nutrients, drought stress, heat stress, mineral toxicity, pests, etc.). It is calculated according to the following formula: IE=Y / U where Y = yield of the harvested portion of the nutrient-fed crop and U = total nutrient uptake of the aboveground crop biomass that was nutrient-fed. Units are not given because the expression is a mass-based ratio and is therefore unitless in its standard form (values ​​of 30-90 are common for N in cereals, with 55-65 considered optimal).

[0155] P and K can be expressed either on an elemental basis (most common in the scientific literature) or on an oxide basis as P2O5 or K2O (most common in industry).

[0156] Tests conducted on wheat, corn, and soybeans showed that the composition alone allowed the crops to take up more nitrogen than untreated samples and similar nitrogen compared to fertilized samples. However, the combination of the composition and fertilizer (at typically used amounts, or even better, half the amount) further increased total nitrogen uptake.

[0157] It should be appreciated that experimental evidence clearly supports that the same or even increased nutritional results can be achieved in plant seedlings while replacing nitrogen-containing fertilizer with the "composition + half the amount of nitrogen-containing fertilizer" combination, thereby allowing the amount of fertilizer used to be reduced by up to 50% of the typical supplemental amount.

[0158] It is to be understood that all preferred aspects of the compositions and agricultural chemical kits of the present invention are considered equally preferred for the processes for their preparation, methods, and uses.

[0159] It should also be understood that all combinations of the preferred embodiments of the compositions of the present invention reported above, as well as of the agrochemical kits, preparation processes, methods, and uses thereof, are to be considered as disclosed herein.

[0160] The following are examples of the present invention provided for illustrative purposes. [Example]

[0161] In these examples, M w and M n was determined by size exclusion chromatography according to the following procedure.

[0162] "Wt %" means weight percentage based on the weight of the organic-inorganic hybrid material, unless otherwise specified.

[0163] Reagents and materials -Eluent: 0.1M NaOH, flow rate 0.5ml / min -RI detector calibration: pullulan standard, M p : 100,000~1,080 (6 standards), where M p is the peak maximum molecular weight - Calibration of UV detector (280 nm): PSS standard, polystyrene sulfonate sodium salt, M p 65,400 to 891 (6 standards). The standards should be dissolved in ultrapure water and the concentration should be approximately 5 mg / ml. The injection volume is 20 μl. -Quality control sample: known M w Lignin having a distribution is used.

[0164] Equipment and instruments -Dionex Ultimate 3000 autosampler, column compartment, and pump -Dionex Ultimate 3000 Diode Array Detector -Differential refractive index detector: Shodex RI-101 - Columns: PSS MCX column: pre-column and two analytical columns: 1000 Å and 100000 Å, the column material is a sulfonated divinylbenzene copolymer matrix. - Syringe filter 0.45 μm and glass sample bottle for STD samples. Sample filtration: Mini-Uniprep syringeless filter device PTFE or nylon, 0.45 μm. Optionally, 5 μm syringe filter for pre-filtration. -Measuring bottle

[0165] procedure - Preparation of eluent Ideally, the water used to prepare the eluent should be high-quality deionized water with low resistivity (greater than 18 MΩ·cm) and contain as little dissolved carbon dioxide as possible. The water should be free of biological contaminants (e.g., bacteria and fungi) and particulate matter. - Needle cleaning with 10% MeOH-water

[0166] -Liquid samples The strong alkaline solution sample is diluted 1:100 and filtered into a vial through a PTFE syringe filter (0.45 μm). The solid lignin sample is diluted, dissolved in 0.1 M NaOH, and filtered through a PTFE 0.45 μm syringe filter. The resulting sample is loaded into the autosampler. The injection volume is 20 μl. After the sample, 1 M NaOH is injected as a sample to clean the column.

[0167] Equipment parameters: -Flow rate 0.5ml / min -Eluent 0.1M NaOH -Column oven temperature 30℃ -Isocratic orchids -Runtime: 48 minutes

[0168] -Solid samples If necessary, dry the solid sample (lignin) in a 60°C oven overnight. Weigh out approximately 10 mg into a 10 ml measuring bottle. Dissolve and dilute the sample in 0.1 M NaOH solution and make up to volume. Filter the sample through a PTFE 0.45 μm filter. If the sample does not dissolve properly, it can be placed in an ultrasonic water bath or the sample can be filtered through a 5 μm syringe filter.

[0169] -Standard samples for calibration Approximately 50 mg of each standard is weighed into a 10 ml measuring bottle and made up to volume with ultrapure water. The standard is filtered through a PTFE 0.45 μm syringe filter. After the calibration sample is run, the calibration results are waveform analyzed in the analysis method and saved. The calibration is a linear first-order calibration.

[0170] -Quality control samples For lignin samples, the known M w Lignin with the distribution is used as a quality control sample. The lignin is dissolved in 0.1 M NaOH at a concentration of approximately 1 mg / ml.

[0171] Example 1 Beech wood (Fagus sylvatica) was subjected to alkaline and enzymatic hydrolysis, which resulted in the production of a hemicellulose- and cellulose-free lignin fraction. The lignin fraction thus isolated has the following characteristics: >95% total solids M w 9,000-11,000 Da (50-61 phenylpropane units) Essentially sulfur-free (less than 3% sulfur content) It contains 23 to 29% by weight of cellulose.

[0172] Example 2. The following lignin fractions were extracted from kraft black liquor, said lignin fractions having the following characteristics: >95% total solids Single species: Southern pine M w 4400-5000 Da (24-28 phenylpropane units) M n 1200-1300 Da (6-7 phenylpropane units) OH group structure: Aliphatic 2.1mmol / g Carboxyl 0.5mmol / g Condensed and syringyl 1.7mmol / g Guaiacyl 2.0mmol / g Catechol and p-OH-phenyl 4.0mmol / g

[0173] Example 3. Preparation of Trichoderma fungal granules a) A premix was prepared by milling the following ingredients together:

[0174] [Table 1]

[0175] The premix was granulated by extrusion granulation: First, the fine powder premix was mixed with water (15 wt%) to produce a wet mixture. The wet mixture was then passed through an extruder (i.e., basket) to obtain wet granules having the shape of cylindrical micropellets. The granules were then dried in a fluidized bed dryer.

[0176] 1-2·10 per gram of granules 10 Water-dispersible granules containing viable spores were obtained. The particle size distribution D of 1.5 mm was determined by sieve analysis according to EN 1235. 50 and was sieved to a fraction of 1400 microns to 500 microns.

[0177] Example 4. i) Preparation of water-dispersible granules b) of kraft lignin fraction Water-dispersible granules b) of the lignin fraction of Example 2 were prepared as follows. 1) dry mixing the lignin fraction (having a dry matter content of 70%) and potassium carbonate powder in a weight ratio of 70:30; 2) Mix until the exothermic reaction has completely disappeared. 3) Granulating the mixture in a blade rotor, inducing the formation of granules by mechanical action; 4) Dry the granules in a fluidized bed. The average particle size distribution D of 1 mm is determined by sieve analysis according to EN 1235. 50 and is sieved to a fraction of 1400 microns to 250 microns.

[0178] ii) Preparation of non-water-dispersible granules b) of the lignin fraction Non-water-dispersible granules of the lignin fraction b) were prepared as follows. 1) providing a lignin fraction having a dry matter content of 65%, 2) adding 3% by weight of lignosulfite; 3) Granulating the mixture in a blade rotor, inducing the formation of granules by mechanical action; 4) Dry the granules in a fluidized bed. The granules have an average particle size distribution D of 2 mm, as determined by sieve analysis according to EN 1235. 50 and is sieved to a fraction of 2000 microns to 250 microns.

[0179] Example 5. a) Preparation of an agricultural chemical kit containing granules a) of Example 3 and granules b) of Example 4.i Agricultural chemistry kit, a first bag containing 30 g of granules a) of Example 3, a second bag containing 970 g of water-dispersible granules b) of Example 4.i, and - a third bag containing 150 g of ammonium nitrate, was prepared by combining

[0180] b) Preparation of an agricultural chemical kit containing granules a) of Example 3 and granules b) of Example 4.ii Agricultural chemistry kit, a first bag containing 40 g of granules a) of Example 3, a second bag containing 996 g of non-water-dispersible granules b) of Example 4.ii and 200 g of ammonium nitrate, was prepared by combining

[0181] Example 6 Assessing nitrogen uptake in maize seedlings Pots containing 240 g of peat were prepared as soil for sowing corn seeds.

[0182] After sowing, the corn seeds were cultivated and then observed from December 15, 2021 to January 14, 2022.

[0183] The following samples were prepared and tested as shown in Figures 1-5. "Untreated control", i.e., corn seedlings that received no treatment; "Normal fertilizer", i.e., maize seedlings that received 150 kg / ha of ammonium nitrate as fertilizer at the time of sowing; "100g / m of the composition of the present invention 3 ", i.e., 100 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 Corn seedlings fed a mixture of "100g / m of the composition of the present invention 3 , normal fertilizer”, i.e., 100 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 and corn seedlings given 150 kg / ha of ammonium nitrate. "100g / m of the composition of the present invention 3 , 1 / 2 fertilizer”, i.e., 100 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 and corn seedlings given 75 kg / ha of ammonium nitrate. "500g / m of the composition of the present invention 3 ", i.e., 500 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 Corn seedlings fed a mixture of "500g / m of the composition of the present invention 3 , normal fertilizer”, i.e., 500 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 and corn seedlings given 150 kg / ha of ammonium nitrate. "500g / m of the composition of the present invention 3 , 1 / 2 fertilizer”, i.e., 500 g / m of Trichoderma GV41 and lignin fraction at the time of sowing. 3 and corn seedlings given 75 kg / ha of ammonium nitrate. Here, the "composition of the present invention" refers to a method for producing a composition of matter containing 3×10 Trichoderma virens GV41 according to the procedure of Example 3. 7 A composition comprising granules a) containing UFC / g and water-dispersible granules b) according to the procedure of Example 4.i and containing 100% of the lignin fraction.

[0184] The composition of the invention was mechanically mixed with the peat before sowing the corn seeds.

[0185] Fertilizer was then added to the target samples.

[0186] The final results of the observation period showed that the SPAD trend (Figure 1) was increased by the combined use of the composition of the present invention and fertilizer.

[0187] However, as shown in Figure 2, the total uptake of nitrogen was better when the composition of the present invention was used in combination with half the amount of fertilizer.

[0188] Figures 3-5 support this finding, and 100 g / m 3 This shows that better nitrogen uptake is achieved when the composition is used in combination with half the amount of fertilizer. In fact, this concentration combination represents the best balance between the total cost of the composition and the results achieved for the plants compared to the "treatment control" (i.e., conventional fertilizer supplementation).

[0189] Example 7 Assessing nitrogen uptake in wheat seedlings The same procedure and test was repeated with wheat seeds and seedlings derived therefrom.

[0190] The results are shown in Figures 6 to 10.

[0191] Again, all nutrient uptake indicators showed that the best results were achieved when the composition of the invention was used in combination with half the amount of fertilizer, which was 500 g / m 3This is most evident in the results of 100 mg / ml, which resulted in increased uptake of N, P, and K. The increased N uptake was particularly evident at lower concentrations of the composition itself (i.e., 100 g / m 3 Indeed, again, this concentration combination represents the best balance between the total cost of the composition and the results achieved for the plants relative to the "treatment control" (i.e., conventional fertilizer supplementation).

[0192] Example 8 Assessing nitrogen uptake in soybean seedlings The same procedure and test was repeated with soybean seeds and seedlings derived therefrom.

[0193] The results are shown in Figures 11 to 13.

[0194] Again, all nutrient uptake indices were significantly higher when the composition of the present invention was used in combination with half the amount of fertilizer, especially at the lower concentration of the composition itself (i.e., 100 g / m 3 It has been shown that the best results are achieved with the composition

[0195] In the case of soybeans as legumes, the results are even more surprising. In fact, legumes such as soybeans capture atmospheric nitrogen through a symbiotic relationship with soil bacteria in a process called "biological nitrogen fixation." This process reduces dependence on synthetic nitrogen fertilizers. Among legumes, soybeans are a major source of protein and oil. Soybeans are grown in a wide range of latitudes and environments. One of the challenges in improving soybean productivity is its high nitrogen requirement compared to grain and oilseed crops. Biological nitrogen fixation can be improved through breeding and selection, focusing on plants, nitrogen-fixing bacteria, and more compatible plants and bacteria. However, the composition of the present invention has made a significant contribution to this goal.

[0196] Specifically, Figure 13 shows that "PE Nitrogen" (i.e., physiological efficiency (PE), which refers to the plant's ability to convert nitrogen obtained from applied sources into economic yield) is significantly negative in the treated controls, i.e., conventionally fertilized samples, but conversely, the composition of the present invention reverses the trend and promotes nitrogen absorption and utilization.

Claims

1. 1. Use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a booster of nitrogen uptake by plant seeds receiving nitrogen-containing fertilizer, wherein: - the fungus is selected from the group consisting of Trichoderma, protoplast fusion products thereof, and mixtures thereof; the lignin fraction comprises fragments having a weight average molecular weight of up to 20,000 Daltons as measured by size exclusion chromatography, the fragments comprising a weight average of up to 111 phenylpropane units; Here, the concentration of the fungus is 1 x 10 ５ ~5 x 10 １０ spores / g composition, the concentration of the lignin fraction being at least 40% by weight relative to the weight of the composition; use.

2. The fungus of the genus Trichoderma is selected from the group consisting of Trichoderma aggressiveum, Trichoderma asperellum, Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma cremeu m, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma truncatulatum ...

2. The use according to claim 1, wherein the active ingredient is selected from the group consisting of Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, Trichoderma viridescens, and mixtures thereof.

3. 3. The use according to claim 1 or 2, wherein the fungus is selected from T. harzianum, T. atroviride, and T. virens, and mixtures thereof.

4. 4. Use according to any one of claims 1 to 3, wherein the lignin fraction comprises fragments with a weight average molecular weight of 2,000 to 20,000 Da, preferably 3,000 to 20,000 Da, more preferably 4,000 to 15,000 Da.

5. The use according to claim 4, wherein the lignin fraction comprises fragments having a weight average molecular weight of 4,000 to 8,000 Da.

6. The concentration of the fungus is 1 x 10 ６ ~3 x 10 １０ Spores / g composition, preferably 1 x 10 ８ ~2 x 10 １０ Spores / g composition, more preferably 1 x 10 ９ ~2 x 10 １０ The use according to any one of claims 1 to 5, which is a spore / g composition.

7. 7. Use according to any one of claims 1 to 6, wherein the concentration of the lignin fraction is at least 60% by weight, preferably at least 70% by weight, more preferably between 75 and 95% by weight, relative to the weight of the composition.

8. The use according to any one of claims 1 to 7, wherein the nitrogen-containing fertilizer is a fertilizer containing urea, ammonia, ammonium nitrate, ammonium sulfate, calcium nitrate, diammonium phosphate, monoammonium phosphate, potassium nitrate, sodium nitrate, or a mixture thereof as a nitrogen source.

9. - a first container containing a fungus of the genus Trichoderma, a second container containing a lignin fraction, and - a third container containing a nitrogen-containing fertilizer, or a first container containing a fungus of the genus Trichoderma, and a second vessel containing the lignin fraction and a nitrogen-containing fertilizer, Including, The fungus, the lignin fraction, and the fertilizer are as described in any one of claims 1 to 8. Agricultural chemistry kit.

10. - the first container is 1 x 10 ５ ~1 x 10 １０ solid granules a) comprising the fungus of the genus Trichoderma in a concentration of spores / g granule a) and at least one binder, - said second container contains solid granules b) comprising said lignin fraction in a concentration of at least 50% by weight relative to the weight of the granules b), The average particle size distribution D of the granules a) and granules b) measured by sieve analysis according to EN 1235 ５０ are, independently of each other, 0.2 to 4.0 mm; The agricultural chemical kit according to claim 9.

11. granules a), wherein the at least one binder is selected from kaolin, starch, modified starch, starch phosphate, pectin, modified pectin, amylopectin, alginic acid, sodium alginate, guar gum, guar powder, tragacanth, gum arabic, xanthan gum, karaya gum, tara gum, tamarind gum, gellan gum, locust bean gum, gelatin, carob seed powder, galactomannan, glucomannan, dextran, carrageenan, mannan, arabinogalactan, pullulan, maltodextrin, cellulose, derivatized cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, soy polysaccharide, chitosan, or mixtures thereof; the granules b) further comprise at least one carrier, said at least one carrier being selected from chalk, carboxymethylcellulose, carbonates, bicarbonates, sulfates, phosphates, oxides or hydroxides of potassium, sodium, lithium, calcium, magnesium, zinc or ammonium, or mixtures thereof; The agricultural chemical kit according to claim 10.

12. 12. The agrochemical kit according to claim 10, comprising 0.1 to 20% by weight of granules a) and 80 to 99.9% by weight of granules b), based on the weight of the solid mixture.

13. i) when the at least one carrier of granules b) is water-dispersible or water-soluble, the kit comprises 1 to 15% by weight of granules a) and 85 to 99% by weight of granules b), based on the weight of the solid mixture; ii) if the at least one carrier of granules b) is neither water-dispersible nor water-soluble, the kit comprises 0.1 to 5 wt. % of granules a) and 95 to 99.9 wt. % of granules b), based on the weight of the solid mixture; The agricultural chemical kit according to claim 12.

14. 14. The agricultural chemical kit according to any one of claims 11 to 13, wherein the at least one carrier of granules b) is water-dispersible or water-soluble and comprises potassium carbonate.

15. The agricultural chemical kit according to any one of claims 11 to 13, wherein the at least one carrier of granules b) is neither water-dispersible nor water-soluble and comprises lignosulfite, chalk, carboxymethylcellulose (CMC), calcium sulfate, or a mixture thereof.

16. The second container comprises: i) granules b) in which the at least one carrier is water-dispersible or water-soluble; and ii) granules b), wherein said at least one carrier is neither water-dispersible nor water-soluble; The agricultural chemical kit according to any one of claims 11 to 15, comprising both of the above.

17. 1. A method for increasing nitrogen uptake by a plant seed receiving a nitrogen-containing fertilizer, said method comprising: A) applying the composition to the seed soil; and B) using a nitrogen-containing fertilizer to provide nitrogen in an amount not exceeding 50% of the amount recommended for the plant being fertilized; A method comprising:

18. The method according to claim 17, wherein the composition and the nitrogen-containing fertilizer are provided in the form of an agricultural chemical kit according to any one of claims 9 to 15.

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