Use of composition having trichoderma and lignin fractions as synergist for nitrogen fertilizers

Abstract

Disclosed is the use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a synergist for nitrogen fertilizers. In particular, it was observed that the composition surprisingly enhances the nitrogen absorption of plant seeds from the nitrogen fertilizer, such that the total amount of nitrogen fertilizer to be applied can be advantageously reduced to at least 50% of the recommended amount.

Description

describe Technical Field

[0001] The present invention relates to a compound comprising Trichoderma Trichoderma genus ) and a lignin fraction as a booster for nitrogen fertilizers. In particular, it was observed that the composition surprisingly enhances the nitrogen uptake by seeds from nitrogen fertilizers, so that the total amount of nitrogen fertilizer to be applied can be advantageously reduced to at least 50%. Background Art

[0002] Fertilizers, which are essentially composed of various types of nutrient components for plant growth, have been widely used around the world to improve agricultural yields. Typically, fertilizers can be in the form of liquid, suspension, or solid.

[0003] As is well known, NPK fertilizers represent the main products used to supplement the nutritional needs of flowers, trees, grasses and crops. The meaning of "NPK" reflects the three nutrients present in these fertilizers, namely nitrogen, phosphorus and potassium.

[0004] Nitrogen performs a range of different functions in plants and is therefore particularly important. Primarily, it is responsible for plant growth and regeneration. If too little nitrogen is present, plants cannot grow adequately and any yield is reduced. However, an excess has several disadvantages: it leads to delayed flowering and fruit ripening; plant tissue becomes very soft and therefore less stable; and, ultimately, diseases and pests are more likely to occur, leading to reduced yields.

[0005] Phosphorus, also a major nutrient in plants, is responsible for the transport and storage of chemical energy, as well as root formation. Furthermore, phosphorus is essential for photosynthesis. Therefore, phosphorus helps right from the start, meaning it supports seed production, such as during fertilization. This also applies later in the flowering period. A phosphorus deficiency causes plants to remain short and stunted, with spindly stems. Furthermore, leaves discolor, roots barely grow, and flowering is delayed. In contrast, an excess of phosphorus (which rarely occurs) indirectly harms plants by reducing the availability of trace elements.

[0006] Potassium, a third essential nutrient, facilitates water absorption and, therefore, maintains a proper water balance in plants. This also leads to strong plant tissues, high resilience, and resistance. Potassium also promotes resistance to disease and extreme weather conditions, such as cold weather. Potassium deficiency weakens plants and impairs root formation. Furthermore, plants evaporate more and absorb less water during dry periods. Excessive potassium leads to unfavorable salt concentrations and, consequently, reduced intake of beneficial nutrients, such as magnesium.

[0007] Therefore, it is recommended to find a good balance of nutrient concentrations in fertilizers in order to exploit their effectiveness and reduce disadvantages.

[0008] It should also be noted that the use of nitrogen fertilizers is going through a period of crisis due to high production costs, while similarly, the excessive use of phosphorus has been criticized due to its environmental impact.

[0009] Therefore, there is a felt need to reduce the overall use of fertilizers through efficient and appropriate utilization of fertilizers while providing adequate amounts of nutrients to plants while protecting human and animal health, crops, and the environment. SUMMARY OF THE INVENTION

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

[0011] In another aspect, the present invention relates to an agro-chemical kit comprising: 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 second container containing 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 a plant seed, the method comprising the steps of applying a composition to the seed soil and thereafter applying a nitrogen-containing fertilizer.

[0013] The term "seed" or "seeds" is intended to include not only plant seeds, but also tubers and bulbs.

[0014] The term "plant" means one or more plants that can be grown and harvested for profit or subsistence (thus including crops, grains, vegetables, fruits and flowers) as well as grown and harvested for horticultural or personal use.

[0015] The term "soil" refers to the earth in which the seed is sown and thus includes the ground, land and soilless substrates such as hydroculture and hydroponics. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The characteristics and advantages of the present invention will become apparent from the following detailed description, from a working example provided for illustrative purposes, and from the accompanying drawings, in which: - Figure 1-Figure 5 The present invention relates to a study on corn seedlings conducted from December 15, 2021 to January 14, 2022 according to Example 6, while comparing samples with the following different treatments: "Untreated check", that is, corn seedlings that did not receive treatment, "Conventional fertilizer", i.e. corn seedlings receiving 150 kg / ha of ammonium nitrate as fertilizer at sowing, "Comparative Invention (Comp.Inv.) 100 g / m 3 ", that is, receiving 100 g / m at sowing time 3 corn seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 100 g / m 3 , ordinary fertilizer", that is, 100 g / m 3 A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to corn seedlings. "Comparative invention 100 g / m 3 , ½ fertilizer", i.e. 100 g / m2 at sowing time 3 A mixture of Trichoderma GV41 and lignin fraction and corn seedlings with 75 kg / ha of ammonium nitrate, "Comparative invention 500 g / m 3 ", that is, receiving 500 g / m at sowing time 3 corn seedlings grown on a mixture of Trichoderma GV41 and a lignin fraction (the composition of the present invention), "Comparative invention 500 g / m 3 , ordinary fertilizer", that is, 500 g / m 3 A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to corn seedlings. "Comparative invention 500 g / m 3 , ½ fertilizer", i.e. 500 g / m2 at sowing time 3 a mixture of Trichoderma GV41 and lignin fraction and corn seedlings in the presence of 75 kg / ha of ammonium nitrate; And specifically - Figure 1 The trend of SPAD (Soil Plant Analysis Development chlorophyll meter is a rapid and non-destructive method for measuring chlorophyll content and thus determining in situ nitrogen (N) status) of corn seedlings of Example 6 is shown. - Figure 2 shows the nitrogen absorption of the corn seedlings of Example 6; - Figure 3The "PE Nitrogen" of the corn seedlings of Example 6 is shown [i.e., the physiological efficiency (PE) representing the plant's ability to convert nitrogen taken from an applied source into economic yield]; - Figure 4 The "AE Nitrogen" of corn seedlings of Example 6 is shown [i.e., the agronomic efficiency (AE) of productivity improvement obtained by using nitrogen input]; - Figure 5 "IE Nitrogen" [ie, internal use efficiency (IE) representing the plant's ability to convert nitrogen obtained from all sources into economic yield] for corn seedlings of Example 6 is shown.

[0017] - Figures 6-10 The present invention relates to a study on wheat seedlings conducted from December 15, 2021 to January 14, 2022 according to Example 7, while comparing the following differently treated samples: "Untreated control", that is, wheat seedlings that did not receive treatment, "Ordinary fertilizer", that is, wheat seedlings that received 150 kg / ha of ammonium nitrate as fertilizer at sowing, "Comparative invention 100 g / m 3 ", that is, receiving 100 g / m at sowing time 3 wheat seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 100 g / m 3 , ordinary fertilizer", that is, 100 g / m 3 mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate on wheat seedlings. "Comparative invention 100 g / m 3 , ½ fertilizer", i.e. 100 g / m2 at sowing time 3 mixture of Trichoderma GV41 and lignin fraction and 75 kg / ha of ammonium nitrate on wheat seedlings. "Comparative invention 500 g / m 3 ", that is, receiving 500 g / m at sowing time 3 wheat seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 500 g / m 3 , ordinary fertilizer", that is, 500 g / m 3 mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate on wheat seedlings. "Comparative invention 500 g / m 3 , ½ fertilizer", i.e. 500 g / m2 at sowing time 3a mixture of Trichoderma GV41 and lignin fraction and 75 kg / ha of ammonium nitrate on wheat seedlings; And specifically - Figure 6 The trend of SPAD of wheat seedlings of Example 7 is shown. - Figure 7 The figure shows the absorption of nutrients, namely nitrogen, phosphorus and potassium, by the wheat seedlings of Example 7; - Figure 8 The "PE nitrogen" of the wheat seedlings of Example 7 is shown; - Figure 9 "AE Nitrogen" of wheat seedlings of Example 7 is shown; - Figure 10 "IE Nitrogen" of wheat seedlings of Example 7 is shown; - Figure 11-13 The present invention relates to a study on soybean seedlings conducted from December 15, 2021 to January 14, 2022 according to Example 8, while comparing samples with the following different treatments: "Untreated control", i.e. soybean seedlings that did not receive treatment, "Conventional fertilizer", i.e. soybean seedlings receiving 150 kg / ha of ammonium nitrate as fertilizer at sowing, "Comparative invention 100 g / m 3 ", that is, receiving 100 g / m at sowing time 3 Soybean seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 100 g / m 3 , ordinary fertilizer", that is, 100 g / m 3 A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to soybean seedlings. "Comparative invention 100 g / m 3 , ½ fertilizer", i.e. 100 g / m2 at sowing time 3 A mixture of Trichoderma GV41 and lignin fraction and soybean seedlings with 75 kg / ha of ammonium nitrate, "Comparative invention 500 g / m 3 ", that is, receiving 500 g / m at sowing time 3 Soybean seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 500 g / m 3 , ordinary fertilizer", that is, 500 g / m 3 A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to soybean seedlings. "Comparative invention 500 g / m3 , ½ fertilizer", i.e. 500 g / m2 at sowing time 3 a mixture of Trichoderma GV41 and lignin fraction and soybean seedlings at 75 kg / ha of ammonium nitrate; And specifically - Figure 11 The trend of SPAD of soybean seedlings of Example 8 is shown. - Figure 12 shows nitrogen uptake by soybean seedlings of Example 8; and - Figure 13 “PE nitrogen” of the soybean seedlings of Example 8 is shown. Detailed Description of the Invention

[0018] The present invention therefore provides for the use of a composition comprising a fungus of the genus Trichoderma and a lignin fraction as a synergist for the uptake of nitrogen by plant seeds receiving nitrogen-containing fertilizers, wherein: - the fungus is selected from the group consisting of Trichoderma species, their protoplast fusants and mixtures thereof, - said lignin fraction comprises fragments having a weight average molecular weight as measured by size exclusion chromatography of up to 20,000 Daltons, said fragments comprising a weight average of up to 111 phenylpropane units, And the concentration of fungi is 1×10 5 to 5×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 40 wt % based on 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 Trichoderma species is selected from Trichoderma invading Trichoderma aggressivum ), Trichoderma spinulosa ( Trichoderma asperellum )、Dark green Trichoderma ( Trichoderma atroviride ), Trichoderma virens ( Trichoderma citrinoviride )、Cream Trichoderma( Trichoderma cremeum ), Trichoderma harzianum ( Trichoderma harzianum ), Trichoderma koningii ( Trichoderma koningii ), Trichoderma longifolia ( Trichoderma longibrachiatum ), Trichoderma reesei ( Trichoderma reesei )、Trichoderma viride ( Trichoderma virens )、Trichoderma viride ( Trichoderma viride ) and Trichoderma viride ( Trichoderma viridescens ).

[0021] The fungi belonging to the genus Trichoderma as defined above are capable of colonizing various ecological niches, antagonizing and controlling phytopathogenic microorganisms, and establishing direct beneficial interactions with plants, leading to enhanced growth, nutrient uptake, and systemic resistance to diseases. In particular, the improvement in plant development is generally accompanied by an increase in seed germination, root system, plant weight and leaf area, size and / or number of seeds, flowers and / or fruits, and a consequent increase in yield and, generally, the content of important nutritional factors.

[0022] Known lignin has antimicrobial activity against both fungi and bacteria. Therefore, it is expected that the processing of Trichoderma (a kind of soil-borne fungi known) will result in direct inhibition with the lignin concentration that is found to be toxic to most of the other fungi tested. Therefore, it is reasonable to expect that Trichoderma will be affected at the lignin concentration that is usually considered to be used for agricultural applications. Surprisingly and unexpectedly, the Trichoderma species listed above are not only not affected by the lignin fraction of a given concentration, but also have increased their activity in nitrogen absorption, whether naturally occurring in the soil or supplemented as nitrogenous fertilizers.

[0023] In particular, the effect on nitrogen absorption was observed to be so increased that it was possible to reduce the supplementation of nitrogen-containing fertilizers to 50%, while nevertheless achieving appreciable and comparable results.

[0024] In addition to the above, it should be understood that Trichoderma species and lignin fractions work in different ways and use different mechanisms: this makes the effect of the resulting composition more robust and appropriate under various conditions, while reducing the insurgence of resistance mechanisms (for biocontrol).

[0025] Furthermore, the composition is simple and cost-effective to produce, as the respective concentrations are advantageously very low. This also means that the composition can be prepared in a concentrated form which can be easily diluted with water to the above concentrations before use.

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

[0027] Protoplasts are cells whose cell walls are removed, and the plasma membrane is the outermost layer of this cell. Protoplasts can be obtained by removing the specific lytic enzymes of the cell wall. Protoplast fusion is a physical phenomenon, and during fusion, two or more protoplasts contact and adhere to each other spontaneously or in the presence of a fusion inducing agent. Through protoplast fusion, it is possible to transfer some useful genes from one species to another. Protoplast fusion is an important tool for bacterial strain improvement to make filamentous fungi genetic recombination and the development of hybrid strains. The improvement can include the higher yield in the production of, for example, cellulase.

[0028] The protoplast fusion used for the purpose of the present invention can be obtained according to techniques known in the art (e.g., Hassan MM (2014) Influence of protoplast fusion between two Trichoderma spp. onextracellular 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 Trichoderma harzianum ( T. Harzianum )、Dark green Trichoderma ( T. Atroviride ) and Trichoderma viride ( 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 the group consisting of Trichoderma harzianum HK2, Trichoderma amurensis HK4 and Trichoderma viride GV41 and mixtures thereof, wherein "HK2", "HK4" and "GV41" are preferred strains of each.

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

[0033] HK4 (or simply "K4") has the 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 a mixture is present in the composition, each species or strain is present 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 1×10 6 to 3×10 10 More preferably, the concentration of the fungus is 1×10 8 to 2×10 10 In a preferred embodiment, the concentration of fungi is 1×10 9 to 2×10 10 spores / g composition.

[0039] Lignin is a complex organic polymer that forms an important structural material in the supporting tissues of some algae, vascular plants (including their bark), and herbaceous plants such as wood (i.e., softwood and hardwood), straw of all cereals, bagasse, grass, flax, jute, industrial hemp, or cotton. Lignin can also have mineral origins such as peat, weathered lignite, and coal.

[0040] Chemically, lignin in its natural form is a very irregular, randomly cross-linked polymer of phenylpropane units connected by many different bonds, with a weight average molecular weight of 20,000 Daltons or more. A representative and illustrative lignin fragment (I) containing the most important bonding patterns is shown herein below: .

[0041] The polymer is the result of enzyme-mediated dehydrogenative polymerization of three phenylpropanoid monomer precursors: .

[0042] It produces the following parts respectively: .

[0043] Coniferyl alcohol is present in all species and is the predominant monomer in conifers (softwoods). Deciduous (hardwood) species contain up to 40% sinapyl alcohol units, while grasses and crops may also contain coumarin units.

[0044] Lignin can be divided into softwood lignin and hardwood lignin according to its original biomass source.For the purpose of the present invention, lignin is softwood lignin, hardwood lignin or its mixture.Preferably, lignin is hardwood lignin.

[0045] Sources of raw biomass that can serve as suitable starting materials for obtaining the relevant lignin fractions are any lignin, including substantially pure lignin as well as kraft lignin, lignin derived from biomass, lignin from an alkaline pulping process, lignin from a soda process, lignin from organosolv pulping, lignin from enzymatic processes, lignin from steam explosion processes, and any combination thereof.

[0046] The expression "substantially pure lignin" is to be understood as at least 80% pure lignin, preferably at least 90% pure lignin, more preferably at least 95% pure lignin, based on dry original biomass, the remainder being extractives and carbohydrates, such as hemicellulose, and inorganics.

[0047] The expression "kraft lignin" should be understood as lignin derived from kraft black liquor. Black liquor is an alkaline aqueous solution of lignin residue, hemicellulose and inorganic chemicals used in the kraft pulping process. The black liquor from the pulping process contains components derived from different softwood species and hardwood species in different proportions. Lignin can be separated from the black liquor by different techniques, including, for example, precipitation and filtration. Lignin usually starts to precipitate at a pH value below 11-12. Different pH values ​​can be used to precipitate lignin fractions with different properties. These lignin fractions can be distinguished by molecular weight distribution, for example, M w and M n The lignins can differ from each other in terms of their properties, polydispersity, hemicellulose and extractive content, and the content of inorganic materials. The precipitated lignin can be purified from inorganic impurities, hemicellulose, and wood extractives using an acidic washing step. Further purification can be achieved by filtration.

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

[0049] Preferably, the step of separating lignin from biomass comprises enzyme treatment.Enzyme treatment has changed the lignin to be extracted from biomass.The lignin separated from pure biomass is substantially free of sulfur (sulfur content is lower than 3%), and is therefore valuable in further processing.Preferably, the wood material is pre-treated to remove hemicellulose, and after this Mierocrystalline cellulose is hydrolyzed.The gained insoluble lignin fraction comprises the Mierocrystalline cellulose of up to 30 wt%.

[0050] The weight average molecular weight of the fragments in the lignin fraction (M w ) is measured by size exclusion chromatography (or 'SEC'). SEC uses a stagnant liquid present in the pores of the beads as the stationary phase and a flowing liquid as the mobile phase. Therefore, the mobile phase can flow between the beads and can also flow into and out of the pores of the beads. The separation mechanism is based on the size of the polymer molecules in the solution. Larger molecules will elute first. Small molecules that can enter the many pores of the beads take a long time to pass through the column and therefore leave the column slowly. In order to determine the molecular weight of the components of a polymer sample, it is necessary to calibrate with standard polymers of known weight. The values ​​from the unknown sample are then compared to the calibration chart. The retention time depends on the column material used, the eluent, and the degree of similarity of the standard used compared to the sample. Preferably, the eluent is preferably 0.1 M NaOH.

[0051] Preferably, the lignin fraction comprises fragments having a weight average molecular weight of 2,000 Da to 20,000 Da.

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

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

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

[0055] In other preferred embodiments, the lignin fraction comprises fragments having a weight average molecular weight of 9,000 Da to 11,000 Da.

[0056] Preferably, in these embodiments, the fragment comprises a weight average of 11 to 111 phenylpropane units, more preferably a weight average of 22 to 111 phenylpropane units.

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

[0058] Preferably, the lignin fraction comprises lignin having a number average molecular weight (M) of up to 2,000 Daltons. n ) fragment.

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

[0060] More preferably, the lignin fraction comprises lignins having a number average molecular weight (M n ) fragment.

[0061] In a preferred embodiment, the lignin fraction comprises fragments having a number average molecular weight of 150 Daltons to 1,300 Daltons.

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

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

[0064] Fungi of the genus Trichoderma produce cellulose-degrading enzymes such as exoglucanases (EXG), endoglucanases (EG), and beta-glucosidases (BGL). Cellulase is the most efficient enzyme system for completely hydrolyzing cellulose substrates into its monomeric glucose, a fermentable sugar. Because sugars contribute to plant cell respiration and cell growth, the presence of cellulose in the compositions of the present invention helps further improve 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 wt%, more preferably at least 70 wt%, 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 wt% to 95 wt%, 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 the group consisting of Trichoderma harzianum, Trichoderma amurensis, Trichoderma viride and mixtures thereof, - the lignin fraction comprises fragments having a weight-average molecular weight of 3,000 Daltons to 20,000 Daltons as measured by size exclusion chromatography, said fragments comprising a weight-average of 16 to 111 phenylpropane units, And the concentration of fungi is 1×10 5 to 5×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 40 wt % based on 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 the group consisting of Trichoderma harzianum, Trichoderma amurensis, Trichoderma viride and mixtures thereof, - the lignin fraction comprises fragments having a weight-average molecular weight of 3,000 Daltons to 20,000 Daltons as measured by size exclusion chromatography, said fragments comprising a weight-average of 16 to 111 phenylpropane units, And the concentration of fungi is 1×10 6 to 3×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 60 wt % based on 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 amurensis HK4, Trichoderma viride 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, said fragments comprising a weight-average of 22 to 33 phenylpropane units, And the concentration of fungi is 1×10 8 to 2×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 70 wt % based on the weight of the composition.

[0069] In other preferred embodiments, 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 amurensis HK4, Trichoderma viride 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, said fragments comprising a weight-average of 50 to 61 phenylpropane units, And the concentration of fungi is 1×10 8 to 2×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 70 wt % based on the weight of the composition.

[0070] Most preferred embodiments are those wherein the composition of the 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, said fragments comprising a weight-average of 50 to 61 phenylpropane units, And the concentration of fungi is 1×10 9 to 2×10 10 spores / g of the composition, and the concentration of the lignin fraction is at least 75 wt%-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 described above, compositions comprising Trichoderma fungi and lignin fractions have been shown to unexpectedly and significantly increase nitrogen uptake by plant seeds, such that the overall use of traditional nitrogen-containing fertilizers can be advantageously reduced, even to half the amount.

[0073] Preferably, the fertilizer may further comprise 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 comprises a nitrogen source, a phosphorus source, and a potassium source 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 further comprise macronutrients selected from the group consisting of sulfur, calcium and magnesium and / or micronutrients including boron, copper, iron, manganese, molybdenum and zinc.

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

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

[0080] In other embodiments, 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 synergist for enhancing nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers, wherein: - the fungus is selected from the group consisting of Trichoderma species, protoplast fusions thereof and mixtures thereof, - said lignin fraction comprises fragments having a weight average molecular weight as measured by size exclusion chromatography of up to 20,000 Daltons, said fragments comprising a weight average of up to 111 phenylpropane units, And the concentration of fungi is 1×10 5 to 5×10 10 spores / g of composition, and the concentration of the lignin fraction is at least 40 wt %, based on the weight of the composition. For the purposes of the present invention, the expression "essentially consisting 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, possible other components having different activities or being simple co-formulators.

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

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

[0083] In another aspect, the present invention also relates to an agricultural chemical kit, the agricultural chemical kit comprising: - a first container containing a fungus of the genus Trichoderma, - a second container containing the 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 the lignin fraction and the nitrogen-containing fertilizer, The fungus, the lignin fraction and the fertilizer are as described above.

[0084] In the embodiment comprising three containers, the ingredients are kept separate from each other, thereby better preserving them and even more easily metering each of them when they are used in combination.

[0085] In embodiments comprising two containers, the Trichoderma fungus is kept separate from the lignin fraction and the fertilizer, which are more compatible with each other while reducing overall packaging.

[0086] Therefore, according to the requirements of the present invention, the agricultural chemical kit of the present invention may be provided in a more suitable embodiment among the above alternatives.

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

[0088] Preferably, in the agrochemical kit: The first container contains solid particles a), which contain Trichoderma fungi and at least one binder, and the concentration of the Trichoderma fungi is 1×10 5 to 1×10 10 spores / g particlesa), and - said second container contains solid particles b) comprising a lignin fraction having a concentration of at least 50 wt %, based on the weight of the particles b); The particles a) and the particles b) independently of one another have a mean particle size distribution D of 0.2 mm to 4.0 mm as measured by sieve analysis according to EN 1235. 50 .

[0089] In fact, granular formulations include several advantages, such as: - No dust, - slides easily in the mechanism (without undesired packing effects), -Easy to store, - the possibility of slow-release preparations, - even distribution of nutrients, - No separation of nutrients during handling or spreading of the product, -Higher efficiency of pre-plant applications.

[0090] As mentioned above, particles a) and particles b) independently of one another have an average particle size distribution D of 0.2 mm to 4.0 mm. 50. For the purposes of the present invention, this parameter is measured by sieve analysis in accordance with EN 1235 [i.e. EN 1235: Solid fertilizers - Test sieving (modified ISO 8397:1988) (including revision A1:2003)]. Average grain size and particle size distribution are important quality characteristics of 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 related instruments and procedures are regulated by EN 1235. According to EN1235, solid fertilizers should be sieved using laboratory test sieves with a diameter of 200 mm manufactured according to the requirements of ISO 3310-1. The standard requires that a gradation test be performed using a maximum of seven test sieves, covering the complete size distribution spectrum of the sample material. The selection of sieve size should be carried out according to the R20 / 3 series of ISO 565, although the standard explicitly allows the use of additional sieves according to the R20 series. The requirements of EN 1235 are determined in a series of ring tests using woven mesh sieves with mesh widths ranging from 100 μm to 5.60 mm.

[0091] Granulated products are solid, homogeneous mixtures produced by combining various raw materials, usually in a granulation plant. Each uniformly sized granule contains all the components under analysis.

[0092] Different granulation processes are known, such as: - Drying and granulation, - wet granulation, - through a spray dryer, - fluidized bed spray, -Pan pelletizer.

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

[0094] Preferably, the concentration of Trichoderma is 1×10 5 to 1×10 10 spores / g particlesa).

[0095] Preferably, in granules a), the at least one binder is selected from kaolin, starch, modified starch, starch phosphate, pectin, modified pectin, pullulan, alginic acid, sodium alginate, guar gum, guar flour, tragacanth gum, gum arabic, xanthan gum, karaya gum, tara gum, tamarind gum, gellan gum, locust bean gum, gelatin, carob seed flour, galactomannans, glucomannans, dextran, carrageenans, mannans, arabinogalactans, pullulan, maltodextrin, cellulose, derivatized cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, soy polysaccharides, chitosan, or mixtures thereof.

[0096] More preferably, the at least one binder is selected from the group consisting of starch, modified starch, starch phosphate esters 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 the wetting agent to enter the hydrophobic soil medium and thereby facilitate penetration; their effects on water surface tension, water penetration, and water retention vary. Suitable wetting agents are selected from the group consisting of alkyl sulfates, aryl sulfonates, polyoxyalkylene alkyl ethers, alkenyl sulfonates, polyoxyethylene styrylphenyl ether, polyoxyethylene distyrylphenyl ether, polyoxyethylene tristyrylphenyl ether, polyoxyethylene styrylphenyl ether salts, polyoxyethylene distyrylphenyl ether salts, polyoxyethylene tristyrylphenyl ether salts, and N-acylamino acid salts.

[0099] Preferred wetting agents are aryl sulfonates, such as sodium isopropylnaphthalenesulfonate.

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

[0101] Dispersants are substances, usually surfactants, added to improve the separation of particles and prevent them from settling or agglomerating. Suitable dispersants are polycarboxylates, such as sodium polycarboxylate.

[0102] In some embodiments, particle b) consists essentially of the lignin fraction.

[0103] In other embodiments, particles b) consist of a lignin fraction.

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

[0105] The particles b) may also comprise at least one carrier.

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

[0107] More preferably, the at least one carrier is selected from the group consisting of carbonates of potassium or sodium or salts of ammonia or urea and mixtures thereof.

[0108] Preferably, the particles a) and the particles b) independently of each other have an average particle size distribution D of 0.5 mm to 2.0 mm. 50 .

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

[0110] In a particularly preferred embodiment, the particles a) and the particles b) have approximately the same average particle size distribution D 50 .

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

[0112] Preferably, the particles a) and the particles b) independently of one another have a bulk density (loose) of 0.3 g / ml to 0.8 g / ml, preferably 0.4 g / ml to 0.7 g / ml according to ISO 3944:1992. The following standards contain such provisions, which, by reference herein, constitute the provisions of this International Standard: - ISO 7742:1988, Solid fertilizers — Reduction of samples.

[0113] - ISO 8358:1991, Solid fertilizers — Preparation of samples for chemical and physical analysis.

[0114] For the purpose of this International Standard, the "bulk density (loose) of a fertilizer" is defined as the mass per volume of material when poured freely into a container under specified conditions. Bulk density (loose) is expressed in grams per cubic centimeter (g / cm 3 )express.

[0115] Although these standards relate to "fertilizers", the inventors also believe that these standards are suitable and adaptable references for the characterization of the present invention.

[0116] In a preferred embodiment, the particles a) and the particles b) have approximately the same bulk density (loose).

[0117] Particles a) and particles b) have similar average particle size distribution D 50 The fact that larger or heavier particles have a higher particle size and / or density is important in order to minimise the risk that larger or heavier particles will not mix uniformly, thereby leading to an unbalanced administration of the components of the composition when they are redispersed or resuspended in the liquid formulation upon use.

[0118] Preferably, the kit of the invention comprises 0.1 wt% to 20 wt% of particles a) in the first container and 80 wt% to 99.9 wt% of particles b) in the second container, based on the weight of the solid mixture of particles a) and particles b). This means that these respective "wt%" are calculated based on the sum of the weights of particles a) and particles b).

[0119] In particular, in the agrochemical kit: i) when the at least one carrier in particles b) is water-dispersible or water-soluble, the kit comprises 1 wt% to 15 wt% of particles a) and 85 wt% to 99 wt% of particles b) based on the weight of the solid mixture of particles a) and particles b), and ii) When the at least one carrier in particles b) is neither water-dispersible nor water-soluble, the kit comprises 0.1 wt% to 5 wt% of particles a) and 95 wt% to 99.9 wt% of particles b) based on the weight of the solid mixture of particles a) and particles b).

[0120] In a preferred embodiment of option i), the at least one carrier in particles b) comprises potassium carbonate, an ammonia salt, a urea salt or a mixture thereof.

[0121] In a preferred embodiment of option ii), the at least one carrier in the particles b) comprises calcium sulfate, lignin sulfite, chalk, carboxymethylcellulose or a mixture thereof.

[0122] In another preferred embodiment, the second container of the kit contains both: i) granules b), wherein the at least one carrier is water-dispersible or water-soluble, and ii) Granule b) wherein the at least one carrier is neither water-dispersible nor water-soluble.

[0123] It will be appreciated that the granules b) according to option i), ie comprising at least one water-dispersible or water-soluble carrier, may be regarded as fast-release granules; in fact, the lignin fraction is readily released upon contact with water.

[0124] Similarly, it will be appreciated that granules b) according to option ii), ie comprising at least one water-insoluble or non-dispersible carrier, may be considered to be slow-release granules.

[0125] Thus, after application, the time-dependent efficacy and activity of the resulting composition can be predetermined by adjusting the concentration of the different particles b).

[0126] An exemplary agrochemical kit according to the present invention is reported as follows: - a rapid release kit comprising, in a first container, 1 wt% of granules a) (comprising starch and 5×10 9 spores / g Trichoderma) and in a second container comprising 99 wt% of particles b) (75 wt% lignin fraction + 25 wt% potassium carbonate), - a slow-release kit comprising, in a first container, 1 wt% of granules a) (comprising starch and 5×10 9 spores / g Trichoderma) and in a second container contained 99.9 wt% of particles b) (85 wt% lignin fraction+15 wt% calcium sulfate).

[0127] Preferably, extrusion granulation is performed to prepare granules a), wherein a fine powder of Trichoderma spores and at least one binder are mixed with water (10%-20%) to produce a wet mixture. This mixture is passed through an extruder or a pan granulator to obtain wet granules, which are then dried in an oven or a fluidized bed.

[0128] In particular, the water-dispersible Trichoderma viable spore granules a) are preferably prepared by the following steps: - grinding the solid ingredients together to obtain a homogeneous premix, - add water to the premix to obtain a wet mix, - Granulate the wet mixture by any suitable technique such as extrusion, pan granulation, agglomeration, dry spray, etc.

[0129] Preferably, the premix is ​​obtained by mixing together the following solid ingredients: -Trichoderma spores 10 wt.%-90 wt.% -Wetting agent 1 wt.%-3 wt.% -Dispersant 2 wt.%-15 wt.% - Disintegrant 0-15 wt.% -Binder up to 100 wt.%.

[0130] Typically the lignin fraction used in the present invention is insoluble in water at neutral pH but soluble at alkaline pH.

[0131] It was then found that water-dispersible particles of the lignin fraction can be obtained by dissolving the lignin in water at alkaline pH by adding at least one water-dispersible or water-soluble carrier which is an alkaline compound to the lignin. b) The dissolved lignin forms a colloidal dispersion in water.

[0132] The dissolved lignin fraction is then diluted in water at neutral pH, thereby obtaining a solution / dispersion of lignin in water at plant physiological pH, ie neutral or slightly alkaline pH.

[0133] Preferably the carrier is potassium carbonate.

[0134] Therefore, the particles b) of the water-dispersible lignin fraction are preferably prepared by the following steps: - adding at least one water-dispersible or water-soluble carrier, preferably potassium carbonate, to the lignin fraction, which preferably has a dry matter content of 60% to 70%; - mixing until a homogeneous paste is obtained, thereby obtaining a water-dispersible lignin fraction paste; - Wet granulation of the water-dispersible lignin fraction paste to obtain granules b).

[0135] The term dry matter content refers to the amount of solid matter in a mixture. The dry matter content is calculated as the percentage of solid material to the total mass of the mixture, expressed in % by weight.

[0136] Pelletization of a separate lignin fraction (ie without Trichoderma spores) allows taking advantage of the good thermal and chemical stability of lignin (and avoiding exposure of Trichoderma to alkaline formulations in case of water dispersible formulations), allowing the use of very favorable process conditions in terms of cost and yield.

[0137] The water-non-dispersible particles b) of the lignin fraction are preferably prepared by the following steps: - providing a lignin fraction preferably having a dry matter content of 60% to 70%, - optionally, adding at least one water-insoluble or non-dispersible carrier, preferably lignin sulfite, chalk, carboxymethylcellulose, calcium sulfate or a mixture thereof; - pelletizing the lignin fraction, optionally mixed with said carrier, in a bladed rotor, inducing the formation of particles by mechanical action, and - Drying of the granules in a fluidized bed.

[0138] Due to the aggregation ability 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 pellets and avoids breakage of the pellets during handling, storage and use in agricultural machinery, which would otherwise produce undesirable powders.

[0139] It will be appreciated that the compositions described above for use as a synergist for enhancing nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers may similarly comprise a fungus of the genus Trichoderma and a lignin fraction in the form of granules a) and granules b), respectively, as disclosed above with respect to the agrochemical kit. It will be appreciated, therefore, that all preferred aspects of granules a) and granules b) and combinations thereof are considered to be disclosed herein, and are also considered to be similarly preferred for use of the compositions as a synergist for enhancing nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers.

[0140] In a further aspect, the present invention relates to a method for increasing nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers, the method comprising the steps of: A) applying the composition described above to the seed soil, and B) Apply nitrogen-containing fertilizers so as to provide nitrogen in an amount not greater than 50% of the recommended amount for the plants being fertilized.

[0141] Preferably, in this method, the composition and the nitrogen-containing fertilizer are provided in the form of an agrochemical kit as described above. In this respect, preferably, the agrochemical kit further comprises instructions (eg an instruction booklet) for carrying out the method reported above.

[0142] In step A), the composition described above is applied to the seed soil.

[0143] The term "seed soil" is intended to mean soil in which the seeds are to be sown or have previously been sown, such as compost, humus, peat, sand, sawdust, coconut fiber, and combinations thereof.

[0144] Preferably, the composition is mechanically mixed with the soil, wherein the seeds have already been sown or wherein the seeds are sown subsequently. This means that the seeds can be mixed together with the composition and the soil, or the seeds can be sown after the composition and the soil have been mixed.

[0145] Preferably, the composition is 0.01-1.00 kg / m 3 More preferably, the composition is applied in an amount of 0.05-0.70 kg / m 3 The amount of seed soil applied.

[0146] In a preferred embodiment, the composition is used at a rate of 0.10-0.50 kg / m 3 The amount of seed soil applied.

[0147] In step B), a nitrogen-containing fertilizer is applied to the seed soil so as to provide nitrogen in an amount not higher than 50% of the recommended amount for the plant to be fertilized.

[0148] The term "recommended amounts" should be interpreted as the amounts of nitrogen indicated as suitable for the growth of specific plants in official guidelines published by the competent agricultural authorities and fertilizer manufacturers. An example is "Guidelines on Nitrogen Management in Agricultural Systems" (IAEA-TCS-29, ISSN 1018–5518), published by the International Atomic Energy Agency, Vienna, in February 2008.

[0149] In fact, the effect of the composition of the invention on nitrogen absorption is observed to be so increased that it is possible to reduce the supplementation of nitrogen-containing fertilizers to 50%, while nevertheless achieving appreciable and comparable results.

[0150] Said effect has been confirmed by extensive tests carried out on different plants germinated and grown from seeds treated as above, in particular wheat, corn and soybeans, such as: -SPAD, or Soil Plant Analysis Development Chlorophyll Meter, is a rapid and non-destructive method for measuring chlorophyll content and thus determining nitrogen (N) status in situ. It is one of the most commonly used diagnostic tools for measuring the nitrogen status of crops, and in particular determines the relative amount of chlorophyll present by measuring the absorbance of leaves in two wavelength regions, namely the infrared region and the near-infrared region. Using these two absorbances, the instrument calculates a SPAD value, which is proportional to the amount of chlorophyll present in the leaf. The chlorophyll content, represented by the measured SPAD value, will increase proportionally to the amount of nitrogen present in the leaf. Higher SPAD values ​​indicate healthier plants.

[0151] Regarding the tests carried out on wheat, corn and soybeans, it was observed that the composition alone allowed to increase the SPAD value compared to the untreated samples; however, the combination of the composition and the fertilizer (in the amounts usually applied or in half the amount) allowed to further increase the SPAD value.

[0152] - PE nitrogen, which represents the physiological efficiency of a plant's ability to convert nitrogen taken from a given source into economic yield. In other words, PE is defined as the yield increase associated with increased uptake of nutrients from the aerial parts of the plant by the crop. This is calculated using the following formula:

[0153] Where Y = yield of the harvested portion of the crop to which nutrients were applied; Y0 = yield without applied nutrients; U = total nutrient uptake in the aboveground crop biomass to which nutrients were applied; U0 = nutrient uptake in the aboveground crop biomass without applied nutrients; units are not shown because the expressions are mass-based ratios and are therefore unitless in their standard form (values ​​of 40-60 are common).

[0154] -AE Nitrogen, which represents the agronomic efficiency (AE) of productivity improvements achieved through the use of nitrogen inputs. This is calculated as the increase in yield per unit of applied nutrient. It more closely reflects the direct production impact of applied fertilizer and is directly related to economic return. Calculating AE requires knowledge of yield without nutrient input and is therefore only known if zero nutrient input has been implemented on-farm in research plots. The following formula is used:

[0155] Where Y = yield of the harvested portion of the crop to which nutrients were applied; Y0 = yield without nutrients applied; F = amount of nutrients applied; units are not shown because the expression is a ratio based on mass and is therefore unitless in its standard form.

[0156] -IE Nitrogen, which represents the plant's internal utilization efficiency (IE) of nitrogen, a measure of its ability to convert nitrogen from all sources into economic yield. In other words, IE is defined as yield relative to total nutrient uptake. A very high IE indicates a deficiency in that nutrient. A low IE indicates poor internal nutrient conversion due to other stresses (deficiency of other nutrients, drought stress, heat stress, mineral toxicity, pests, etc.). This is calculated using the following formula:

[0157] Where Y = yield of the harvested portion of the crop to which the nutrient was applied; U = total nutrient uptake in the aboveground crop biomass to which the nutrient was applied; units are not shown because the expression is a mass-based ratio and is therefore unitless in its standard form (for N in cereals, values ​​of 30-90 are common, and values ​​of 55-65 are considered optimal).

[0158] P and K can be expressed on an elemental basis (most common in scientific literature) or on an oxide basis as P2O5 or K2O (most common in industry).

[0159] With regard to the tests carried out on wheat, corn and soybeans, it was observed that the composition alone allowed the crops to absorb more nitrogen compared to the untreated samples and to absorb similar nitrogen compared to the fertilizer-treated samples; however, the combination of the composition and fertilizer (at the amounts normally applied, but even better at half the amount) allowed further increases in total nitrogen absorption.

[0160] It will be appreciated that experimental evidence clearly supports the possibility of replacing nitrogen-containing fertilizers with a combination of "composition + half the amount of nitrogen-containing fertilizer" while achieving the same or even increased nutritional results in young plants. This allows reducing the amount of fertilizer to be used to 50% of the usual supplementary amount.

[0161] It will be understood that all preferred aspects of the compositions and agrochemical kits of the present invention are considered similarly preferred for their preparation, methods and use.

[0162] It is also to be understood that all combinations of preferred aspects of the compositions and agrochemical kits of the invention, their preparation, methods and uses as reported above are to be considered disclosed herein.

[0163] The following are working examples of the present invention provided for illustrative purposes.

[0164] Example M in these embodiments w and M n Measured by size exclusion chromatography according to the following procedure.

[0165] Unless otherwise indicated, "wt %" means weight percent based on the weight of the organic-inorganic hybrid material.

[0166] Reagents and materials -Eluent: 0.1 M NaOH, flow rate 0.5 ml / min -RI detector calibration: pullulan standard, M p :100,000-1,080 (six standards), including M p is the peak maximum molecular weight - UV detector calibration (280 nm): PSS standard, polystyrene sulfonate sodium salt, M p 65,400 – 891 (six standards). Dissolve the standards in ultrapure water to a concentration of approximately 5 mg / ml. Injection volume is 20 µl.

[0167] - Quality control samples: Use samples with known M w Distribution of lignin.

[0168] Equipment and instruments - Dionex Ultimate 3000 autosampler, column compartment, and pump - Dionex Ultimate 3000 Diode Array Detector -Reflectance index detector: Shodex RI-101 - Columns: PSS MCX columns: pre-column and two analytical columns: 1000 Å and 100,000 Å, the column material is a sulfonated divinylbenzene copolymer matrix.

[0169] -0.45 µm syringe filters and glass sample vials for STD samples. Sample Filtration: Mini Uniprep syringeless filter devices, PTFE or nylon, 0.45 µm. 5 µm syringe filters for prefiltration if required.

[0170] -Measuring bottle program - Preparation of eluent Ideally, the water used to prepare the eluent should be high-quality deionized water with low resistivity (18 MΩ•cm or better) that contains as little dissolved carbon dioxide as possible. The water must be free of biological contamination (e.g., bacteria and mold) and particulate matter.

[0171] - Rinse the needle with 10% MeOH-water -Liquid samples The strongly alkaline solution sample was diluted 1:100 and filtered into a vial using a PTFE syringe filter (0.45 µm). The solid lignin sample was diluted and dissolved in 0.1 M NaOH and filtered using a PTFE, 0.45 µm syringe filter. The prepared sample was loaded into the autosampler. The injection volume was 20 µl. Following the sample, 1 M NaOH was injected as a sample to clean the column.

[0172] Instrument parameters: -Flow rate 0.5 ml / min -Eluent 0.1 M NaOH - Column oven temperature 30°C -Isocratic operation -Running time: 48 minutes -Solid samples If necessary, dry the solid sample (lignin) in an oven at 60°C overnight. Weigh approximately 10 mg into a 10-ml measuring vial. Dissolve and dilute the sample in 0.1 M NaOH solution and fill to the mark. Filter the sample using a PTFE, 0.45 µm filter. If the sample does not dissolve properly, place it in an ultrasonic water bath, or filter it through a 5 µm syringe filter.

[0173] -Standard samples for calibration Approximately 50 mg of each standard was weighed into a 10-ml measuring vial and filled to the mark with ultrapure water. The standards were filtered through a PTFE 0.45 µm syringe filter. After running the calibration samples, the calibration results were integrated, processed, and saved in the processing method. The calibration was a linear first-order calibration.

[0174] -Quality control samples For lignin samples with known M w The distributed lignin was used as a quality control sample. Lignin was dissolved in 0.1 M NaOH and the concentration was approximately 1 mg / ml.

[0175] Example 1. Beech wood (European beech ( Fagus sylvatica )) is subjected to alkaline and enzymatic hydrolysis to obtain a lignin fraction free of hemicellulose and cellulose. The lignin fraction thus separated has the following characteristics: >95% total solids M w 9,000-11,000 Da (50-61 phenylpropane units) Essentially sulfur-free (sulfur content less than 3%) Contains 23 wt%-29 wt% cellulose.

[0176] Example 2. The following lignin fractions were extracted from Kraft black liquor, having the following characteristics: >95% total solids Single species: Southern Pine M w 4400 Da-5000 Da (24-28 phenylpropane units) M n 1200 Da-1300 Da (6-7 phenylpropane units) The structure of the OH group: Aliphatic 2.1 mmol / g 0.5 mmol / g of carboxyl group Condensed and eugenol groups 1.7 mmol / g Guaiacyl 2.0 mmol / g Catechol and p-OH-phenyl 4.0mmol / g.

[0177] Example 3. Preparation of Trichoderma granules a) Prepare a premix by grinding together the following ingredients:

[0178] Granulate the premix by extrusion granulation: First, the pre-mixed fine powder was mixed with water (15 wt%) to produce a wet mixture. The wet mixture was then passed through an extruder (i.e., a basket) to obtain wet granules having a cylindrical micropill shape. The granules were then dried in a fluidized bed dryer.

[0179] Get 1-2×10 10 Water-dispersible granules with viable spores / gram of granules and a particle size distribution of 1.5 mm 50 , sieved with a fraction between 1400 μm and 500 μm, as measured by sieve analysis according to EN 1235.

[0180] Example 4. i) Preparation of water-dispersible granules of kraft lignin fraction b) The water-dispersible granules b) of the lignin fraction of Example 2 were prepared as follows: 1) dry mixing a lignin fraction (having a dry matter content of 70%) and a powder of potassium carbonate in a weight ratio of 70:30, 2) Mix until the exothermic reaction is completely extinguished, and 3) The resulting mixture is granulated in a bladed rotor, inducing the formation of particles by mechanical action, 4) Drying the granules in a fluidized bed, the granules having a mean particle size distribution D of 1 mm sieved in the fraction between 1400 μm and 250 μm, measured by sieve analysis according to EN 1235 50 .

[0181] ii) Preparation of water-non-dispersible particles b) of the lignin fraction The water-non-dispersible particles b) of the lignin fraction are prepared as follows: 1) providing a lignin fraction having a dry matter content of 65%, 2) adding 3 wt% of lignin sulfite, 3) The mixture is granulated in a bladed rotor, inducing the formation of particles through mechanical action, 4) Drying of the granules in a fluidized bed, the granules having a mean particle size distribution D of 2 mm sieved in the fraction between 2000 μm and 250 μm, measured by sieve analysis according to EN 1235 50 .

[0182] Example 5. a) Preparation of an agrochemical kit comprising the granules a) of Example 3 and the granules b) of Example 4.i The agricultural chemical kit is prepared by combining the following: - a first bag containing 30 g of the granules a) of Example 3, - a second bag containing 970 g of the water-dispersible granules b) of Example 4.i, and - A third bag containing 150 g of ammonium nitrate.

[0183] b) Preparation of an agrochemical kit comprising the granules a) of Example 3 and the granules b) of Example 4.ii The agricultural chemical kit is prepared by combining the following: - a first bag containing 40 g of the granules a) of Example 3, A second bag containing 996 g of the water-non-dispersible granules b) from Example 4.ii and 200 g of ammonium nitrate.

[0184] Example 6. Assessment of Nitrogen Uptake in Maize Seedlings Pots containing 240 g of peat as soil for sowing corn seeds were provided.

[0185] After sowing, corn seeds were harvested and planted, and then observed from December 15, 2021 to January 14, 2022.

[0186] Prepare and test the following samples, such as Figure 1-Figure 5 Reported in: "Untreated control", that is, corn seedlings that did not receive treatment, "Conventional fertilizer", i.e. corn seedlings receiving 150 kg / ha of ammonium nitrate as fertilizer at sowing, "Comparative invention 100 g / m 3 ", that is, receiving 100 g / m at sowing time 3 corn seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 100 g / m 3 , ordinary fertilizer", that is, 100 g / m 3A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to corn seedlings. "Comparative invention 100 g / m 3 , ½ fertilizer", i.e. 100 g / m2 at sowing time 3 A mixture of Trichoderma GV41 and lignin fraction and corn seedlings with 75 kg / ha of ammonium nitrate, "Comparative invention 500 g / m 3 ", that is, receiving 500 g / m at sowing time 3 corn seedlings with a mixture of Trichoderma GV41 and lignin fractions, "Comparative invention 500 g / m 3 , ordinary fertilizer", that is, 500 g / m 3 A mixture of Trichoderma GV41 and lignin fraction and 150 kg / ha of ammonium nitrate were added to corn seedlings. "Comparative invention 500 g / m 3 , ½ fertilizer", i.e. 500 g / m2 at sowing time 3 a mixture of Trichoderma GV41 and lignin fraction and corn seedlings in the presence of 75 kg / ha of ammonium nitrate; Wherein "Comparative invention" is a composition comprising the procedure according to Example 3 and comprising 3×10 7 UFC / g of Trichoderma virens GV41 a) and water-dispersible granules b) according to the procedure of Example 4.i and comprising 100% lignin fraction.

[0187] The comparative invention was mechanically mixed with peat before sowing corn seeds.

[0188] Then, fertilizer was added to the relevant samples.

[0189] The results at the end of the observation period showed that the trend of SPAD ( Figure 1 ) is increased by using the composition of the present invention in combination with a fertilizer.

[0190] However, when the composition of the present invention is used in combination with half the amount of fertilizer, overall nitrogen uptake is better achieved, e.g. Figure 2 As shown in .

[0191] Figure 3-Figure 5 This finding is confirmed because when 100 g / m 3 The composition achieves better nitrogen uptake when combined 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 on the plants relative to the 'treated control' (ie conventional fertilizer supplementation).

[0192] Example 7. Assessment of nitrogen uptake in wheat seedlings The same procedures and tests have been repeated on wheat seeds and resulting seedlings.

[0193] The results are already in Figures 6-10 Report in.

[0194] In this case as well, all nutrient absorption indicators confirmed that the best results were achieved when the composition of the invention was used in combination with half the amount of fertilizer, which was at 500 g / m 3 This is best indicated by the results for , where the uptake of N, P and K is increased. In particular, at lower concentrations of the composition itself (i.e. 100 g / m 3 In fact, also in this case, this combination of concentrations represents the best balance between the total cost of the composition and the results achieved on the plants, relative to the 'treated control' (ie conventional fertilizer supplementation).

[0195] Example 8. Assessment of Nitrogen Uptake in Soybean Seedlings The same procedure and tests have been repeated on soybean seeds and resulting seedlings.

[0196] The results are already in Figure 11-13 Report in.

[0197] In this case as well, all nutrient absorption indicators confirmed that the composition of the invention was superior to the fertilizer when used in combination with half the amount of fertilizer, especially at the lower concentration of the composition itself (i.e. 100 g / m 3 composition) to achieve the best results.

[0198] In the case of soybeans, a legume, 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 known as "biological nitrogen fixation." This process reduces the reliance on synthetic nitrogen fertilizers. Among legumes, soybeans are the main source of protein and oil. Soybeans grow in a variety of latitudes and environments. One of the challenges in improving soybean productivity is the high demand for nitrogen compared to cereals and oilseed crops. Biological nitrogen fixation can be improved by focusing on the plants, the nitrogen-fixing bacteria, and breeding and selection to better match the plants and bacteria, however, the composition of the present invention has made a huge contribution to this.

[0199] In particular, Figure 13It was shown that "PE Nitrogen" [i.e., physiological efficiency (PE) representing the plant's ability to convert nitrogen acquired from an applied source into economic yield] was significantly negative in the treated control, i.e., conventionally fertilized samples, while, in contrast, the composition of the present invention reversed the trend of promoting nitrogen acquisition and utilization.

Claims

1. Contains Trichoderma Trichoderma genus ) as a synergist for nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers, wherein: The fungus is selected from the group consisting of Trichoderma species, protoplast fusions 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, said fragments comprising a weight average of up to 111 phenylpropane units, And the concentration of the fungus is 1×10 5 to 5×10 10 spores / g of composition, and the concentration of the lignin fraction is at least 40 wt % based on the weight of the composition.

2. The method according to claim 1, wherein the Trichoderma species is selected from the group consisting of Trichoderma spp. Trichoderma aggressivum ), Trichoderma spinulosa ( Trichoderma asperellum )、Dark green Trichoderma ( Trichoderma atroviride ), Trichoderma virens ( Trichoderma citrinoviride )、Cream Trichoderma( Trichoderma cremeum ), Trichoderma harzianum ( Trichoderma harzianum ), Trichoderma koningii ( Trichoderma koningii ), Trichoderma longifolia ( Trichoderma longibrachiatum ), Trichoderma reesei ( Trichoderma reesei )、Trichoderma viride ( Trichoderma virens )、Trichoderma viride ( Trichoderma viride ), Trichoderma viride ( Trichoderma viridescens ) and mixtures thereof.

3. The use according to claim 1 or 2, wherein the fungus is selected from Trichoderma harzianum ( T. harzianum )、Dark green Trichoderma ( T. atroviride ) and Trichoderma viride ( T. virens ) and mixtures thereof.

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

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

6. The use according to any one of claims 1 to 5, wherein the concentration of the fungus is 1×10 6 to 3×10 10 spores / g composition, preferably 1×10 8 to 2×10 10 spores / g composition, more preferably 1×10 9 to 2×10 10 spores / g composition.

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

8. The method according to claim 1, wherein the nitrogen-containing fertilizer is a fertilizer comprising urea, ammonia, ammonium nitrate, ammonium sulfate, calcium nitrate, diammonium phosphate, monoammonium phosphate, potassium nitrate, sodium nitrate or a mixture thereof as nitrogen source.

9. An agricultural chemical kit 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 the lignin fraction and the nitrogen-containing fertilizer, The fungus, the lignin fraction and the fertilizer are according to any one of claims 1-8.

10. The agricultural chemical kit according to claim 9, wherein: The first container contains solid particles a), which contain Trichoderma fungi and at least one binder. The concentration of the Trichoderma fungi is 1×10 5 to 1×10 10 spores / g particlesa), and The second container contains solid particles b) comprising a lignin fraction having a concentration of at least 50 wt %, based on the weight of the particles b). The particles a) and the particles b) independently of one another have an average particle size distribution D of 0.2 mm to 4.0 mm as measured by sieve analysis according to EN 1235. 50 .

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

12. The agrochemical kit according to claim 10 or 11, comprising 0.1 wt% to 20 wt% of particles a) and 80 wt% to 99.9 wt% of particles b) based on the weight of the solid mixture of the particles a) and the particles b).

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

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

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

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

17. A method for increasing nitrogen uptake by plant seeds receiving nitrogen-containing fertilizers, the method comprising the steps of: A) applying the composition described above to the seed soil, and B) Apply nitrogen-containing fertilizers so as to provide nitrogen in an amount not greater than 50% of the recommended amount for the plants being fertilized.

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

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