Granular biostimulant as a plant growth promoter, its preparation and use

Abstract

The present invention discloses a biostimulant comprising a solid mixture consisting of a) granules containing a fungus of the genus Trichoderma and at least one binder, and b) granules containing a lignin fraction and optionally at least one carrier, as well as a method for producing the same and its use as a plant growth and fruit production promoter in agriculture. In further embodiments, a pesticide product comprising the biostimulant and pesticide additives is also disclosed.

Description

[Technical field]

[0001] The present invention relates to a granular biostimulant comprising a fungus of the genus Trichoderma and a lignin fraction, as well as its method of manufacture and use as a plant growth and fruit production promoter in agriculture. [Background technology]

[0002] Fertilizers, which essentially consist of various nutrients necessary for plant growth, are widely used throughout the world to improve agricultural yields. In general, fertilizers can be in the form of pure liquids, suspensions or solids. Fertilizers can be provided to plants by soil application or by application to the foliage of plants by spraying, irrigation, etc.

[0003] It is known that many fertilizers are sold in powder form and dissolved in water when used, however, powders are difficult to handle, have the risk of spreading in the environment and are dangerous for workers, so that said powders are unattractive and undervalued, despite their theoretical convenience from the standpoint of storage and transportation.

[0004] In recent years, foliar-applied fertilizers have gradually replaced the common use of soil-applied fertilizers in agricultural fields. This is because foliar-applied fertilizers have less adverse effects on the environment. Research has shown that traditional fertilization methods by soil application lead to the contamination of surface and groundwater. This is mainly due to the seepage of soluble nutrients from fertilizers, such as nitrogen, into the aquifer. This situation can hinder the transport of nutrients to plant cells. It is therefore more desirable to provide nutrients directly to plants through the foliar surface of the plant. Although foliar-applied fertilizers seem to overcome the shortcomings of soil-applied methods, inappropriate application to plants, such as spraying high concentrations of nutrients directly onto the foliar surface, can cause damage to the foliar surface as the formation of necrotic areas and leaf scorch, which can result in reduced crop yields. It is believed that foliar damage can be prevented by reducing the amount of nutrients in foliar-applied fertilizers. However, fertilizing plants with foliar-applied fertilizers with low nutrient content is not practical because it requires labor-intensive operations. For example, US Patent No. 6,475,258 discloses a foliar fertilizer composition for promoting plant growth by foliar application. The composition is an aqueous solution of at least one coenzyme, preferably vitamin B, more preferably folic acid and / or pyridoxine, at least one carbohydrate source, a complexing agent and a preservative. The subject fertilizer can substantially improve the absorption of nutrients by plants by increasing the metabolic activity of the plants, but the availability of nutrients taken up by plant cells from soil or foliar fertilizer remains to be resolved.

[0005] Another disadvantage is that although aqueous solutions and suspensions are preferred and particularly suitable for application as fertilizers to crops, at the same time the overall storage and transportation costs as well as long-term instability can adversely affect commercial use.

[0006] A class of plant products called plant biostimulants are known to have complementary properties in crop nutrition and crop protection. Plant biostimulants are substances or microorganisms that are applied to plants with the aim of increasing nutrient efficiency, abiotic stress tolerance and / or crop quality attributes, regardless of nutrient content.

[0007] According to the European Biostimulants Industry Council (EBIC), biostimulants are distinguished from conventional crop applications in two main ways. - Biostimulants work by a different mechanism than fertilizers, regardless of whether nutrients are present in the product. - Biostimulants only affect plant vitality.

[0008] As mentioned above, the nature of a biostimulant is not limited and can be a substance or a microorganism. Rather, the agricultural function is central to the definition. Biostimulants are defined by their intended agricultural output. "Nutrient efficiency" may include nutrient mobilization and uptake from the soil, root development, nutrient transport, storage and assimilation (i.e., conversion from inorganic to organic forms) within the plant. "Abiotic stress" refers to physical or chemical stressors of non-biological origin, such as drought, salinity, or low temperature. "Quality attributes" are diverse and may range from nutritional value to shelf life or flower color expression. These effects should be distinguished from those resulting from the nutrient content of the biostimulant. Biostimulants are not fertilizers in the sense that they do not contain nutrients intended to be delivered to the plant. However, biostimulants may enhance nutrient acquisition by exploiting new pathways for nutrient acquisition, such as elemental mobilization in the rhizosphere or fixation of atmospheric nitrogen (N) by supplementing bacterial endophytic symbionts.

[0009] Technical challenges in the development of biostimulants include their formulation and blending with other fertilizer materials and / or plant protection products. Many biostimulants aim to improve nutrient use efficiency, which requires optimizing the fertilizer-biostimulant combination. Biofertilizer formulation design is particularly complex and requires exploring positive interactions between the microbiological components of the biostimulant mixture on the one hand and between the biostimulant inoculant and the resident rhizobial / endophytic microbiota on the other hand.

[0010] Therefore, a need is felt for a product that effectively promotes plant growth without foliar damage, while at the same time exhibiting long-term storage stability, compatibility with fertilizers and pesticides, and a more sustainable overall cost. Summary of the Invention

[0011] As claimed in claim 1, the object is achieved by providing a solid mixture of: a) granules containing a fungus of the genus Trichoderma, and b) Granules containing a lignin fraction This was achieved through the use of biostimulants including

[0012] For the purposes of the present invention, the terms "granule", "particulate", "granular" or "particulate" refer to particles having regular or irregular shapes, such as spheres, pellets, flakes or tablets, and having a mean particle size distribution D 50 This refers to small solid particles with a particle size of 0.2 to 4.0 mm.

[0013] In another aspect, the present invention relates to the use of said biostimulants as plant growth and fruit production promoters in agriculture.

[0014] In a further aspect, the present invention relates to an agrochemical product comprising said biostimulant and an agrochemical additive.

[0015] In a further aspect, the present invention relates to a method for promoting plant growth and fruit production comprising the step of applying a biostimulant or agrochemical product to the plant or plant soil.

[0016] The term "plant" refers to a plant or group of plants that can be grown and harvested for profit or subsistence, and includes crops, grains, vegetables, fruits, flowers, as well as seed, tuber and bulb plants, and plants grown and harvested for gardening or personal use.

[0017] The expression "on plants" means that the biostimulant or pesticide product can be applied to any part of the plant, including the roots, trunk, branches, twigs, leaves, flowers and fruits.

[0018] The term "plant soil" refers to the soil in which a plant is growing, in which a plant is sown, or in which a plant is to be sown, including ground, soil, and soilless media such as in hydroponics and hydroponics.

[0019] The features and advantages of the present invention will become apparent from the following detailed description and examples provided for purposes of illustration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The subject of the present invention is therefore a solid mixture consisting of: a) 1×10 per 1g of granules 5 ~1×10 10 A granule comprising a fungus of the genus Trichoderma in a spore concentration and at least one binder; and b) Granules containing at least 50% by weight of a lignin fraction relative to the weight of the granules b). A biostimulant comprising: - the fungus is selected from 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 (Da) as measured by size exclusion chromatography, the fragments comprising a weight average of up to 111 phenylpropane units; The granules a) and the granules b) each independently have an average particle size distribution D 50 A biostimulant with a pH of 0.2-4.0 mm.

[0021] The solid mixture of the present invention has surprisingly been able to achieve many technical results and advantages while at the same time overcoming some of the disadvantages of known granular products.

[0022] Granules are certainly the most widely used physical form for all agricultural products that constitute fertilizers and other products that fall under the broad category of fertilizers but have a specific action, such as micronutrients, soil conditioners, fungal and mycorrhizal inoculants, pH correctors, etc. In fact, granular formulations have several advantages, such as: - No dust is generated. - Easy sliding in machinery (no undesirable packing effects), - Ease of storage, - Possibility of sustained release formulations; - uniform distribution of nutrients, - no separation of nutrients during handling or application of the product; - High efficiency of pre-sowing application, Examples include:

[0023] As described above, the granular material a) and the granular material b) each independently have an average particle size distribution D 50 is between 0.2 and 4.0 mm. For the purposes of the present invention, this parameter is measured by sieve analysis according to EN 1235 [i.e. EN 1235: Solid fertilizers - Test sieves (ISO 8397:1988, revised) (including Addendum A1:2003)]. The average particle 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 marketed in the European Union, and all relevant equipment and procedures are specified in EN 1235. According to EN 1235, solid fertilizers are subjected to sieve analysis using laboratory test sieves with a diameter of 200 millimeters manufactured according to the requirements of ISO 3310-1. This standard requires that up to seven test sieves are used in the grading test in order to cover the complete particle size distribution range of the sample material. The aperture dimensions must be selected from the R20 / 3 series of ISO 565, although the standard explicitly allows for the use of additional sieves from the R20 series. The requirements of EN 1235 were determined in a series of ring trials using woven wire sieves with aperture widths ranging from 100 µm to 5.60 mm.

[0024] Granular products are homogenous solid mixtures typically produced in granulation plants by combining various raw materials. Each uniformly sized particle contains all the components in the analysis.

[0025] Various granulation processes are known, e.g. - Dry granulation, - Wet granulation, - spray drying, - Fluidized bed atomization, - Rolling granulation, etc.

[0026] In all the various processes the parameters involved are those which generate more or less intense (thermal, mechanical, rheological) stresses on the components forming the granules.

[0027] In addition to the process parameters (mixing time and intensity, amount of water / solvent, temperature and drying time and method, etc.), it is possible or necessary to introduce granulating or binding agents to give the granules an appropriate hardness to maintain their structure during handling, storage and application, and to give them an appropriate (and rapid) solubility in the ground or dispersed water. Furthermore, it should be noted that during the granulation process, strong and violent interactions occur between the different components, which must be evaluated in advance in order to avoid undesired chemical reactions, i.e. denaturation, of live microbiological components (even in a dormant state).

[0028] Thus, even if in principle a granular formulation would be advantageous, the operating conditions of granulation may in any case adversely affect the ingredients present therein and reduce the overall efficacy and stability over time of the resulting granular product, particularly when microorganisms such as fungi are involved as ingredients.

[0029] In the case of the present invention, the challenge was not only to incorporate fungi into the granular biostimulant, but also to combine it with the lignin fraction.

[0030] Lignin is known to have antimicrobial activity against both fungi and bacteria, and the lignin fractions detailed below exhibit even greater antimicrobial activity due to the more active and reactive fragments generated by the depolymerization process.

[0031] Nevertheless, surprisingly and unexpectedly, Trichoderma sp. in the liquid formulation was not only not affected at all by the lignin fraction, but also showed increased activity both in terms of plant productivity and in terms of plant growth.

[0032] In addition to the above, it should be appreciated that Trichoderma species and lignin fractions act in different ways and use different mechanisms, making the efficacy of the resulting composition more robust and suitable under a variety of conditions while at the same time reducing the rebellion of resistance mechanisms (for biological control).

[0033] However, when the same formulation was granulated, the above mentioned stressful conditions caused problems with fungal viability and activity, especially after long term storage.

[0034] Thus, the solid mixture of the present invention surprisingly makes it possible to utilize the advantages of the combination of Trichoderma with a lignin fraction, as well as the advantages of a granular formulation, without falling into the known disadvantages of the granulation process.

[0035] In fact, the Trichoderma species and the lignin fraction can be granulated as granules a) and b) in different separate granulation processes, respectively, mixed together to form a solid mixture, stored for a long period of time, and then redispersed or resuspended in a liquid formulation at the time of use, while still maintaining their activity and efficacy.

[0036] The Trichoderma species include Trichoderma aggressivum, Trichoderma asperellum, Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma cremeum, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, and Trichoderma It is preferred that the host cell is selected from Trichoderma viridescens.

[0037] Fungi belonging to the genus Trichoderma as defined above are able to colonize various niches, to combat and control phytopathogenic microorganisms and to establish direct beneficial interactions with the plant, resulting in enhanced growth, nutrient uptake and systemic disease resistance. In particular, improved plant development is generally associated with an increase in seed germination, root system, plant weight and leaf area, size and / or number of seeds, flowers and / or fruits, and thus an increase in yield, as well as an increase in the content of important nutritional factors.

[0038] The term "protoplast fusion" is intended to include hybrid strains of Trichoderma species obtained by protoplast fusion.

[0039] Protoplasts are cells from which the cell wall has been removed and the cytoplasmic membrane is 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 attach to each other spontaneously or in the presence of a fusogenic agent. Protoplast fusion allows the transfer of useful genes from one species to another. Protoplast fusion results in genetic recombination in filamentous fungi and is an important tool in strain improvement to develop hybrid strains. The improvement may include, for example, improved yield of cellulase production.

[0040] Protoplast fusions for the purposes of the present invention can be obtained according to techniques known in the art (see, for example, Hassan MM (2014) Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity, Biotechnology & Biotechnological Equipment, 28:6, 1014-1023).

[0041] In a preferred embodiment of the biostimulant of the present invention, the fungus of the genus Trichoderma is selected from T. harzianum, T. atroviride and T. virens, and mixtures thereof.

[0042] In some embodiments, the biostimulant comprises a mixture of Trichoderma species. 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.

[0043] HK2 has ATCC number PTA-9708 and is disclosed in US Pat. No. 8,716,001. HK4 has ATCC number PTA-9707 and is disclosed in US Pat. No. 8,877,480. GV41 is commercially available from BioWorks Inc. NY 14564, USA.

[0044] In some embodiments, the biostimulant comprises a mixture of strains of the genus Trichoderma.

[0045] When a mixture is present in the biostimulant, each species or strain is at the same or approximately the same concentration.

[0046] In a preferred embodiment, the biostimulant comprises two different Trichoderma species or two strains of the genus Trichoderma in a concentration ratio of 2:1 to 1:2, preferably 1:1. The fungi are present in the granules a) at a concentration of 1×10 5 ~1×10 10 It is preferably present in a concentration of spores / g.

[0047] Lignin is a family of complex organic polymers that form the important structural material in the supporting tissues of algae, vascular plants including bark, and herbaceous plants such as wood (i.e. conifers and hardwoods), all cereal straw, sugarcane bagasse, grasses, flax, jute, hemp or cotton. Lignin can also be derived from mineral sources such as peat, leonardite and coal.

[0048] Chemically, in its natural form, lignin is a highly irregular, randomly cross-linked polymer of phenylpropane units linked by many different linkage modes, with a weight average molecular weight of over 20,000 Daltons. Representative and exemplary lignin fragments (I) containing the most important linkage modes are shown below:

[0049] [ka]

[0050] The polymer is made from three phenylpropanoid monomer precursors:

[0051] [ka]

[0052] which results in the enzyme-mediated dehydrogenative polymerization of

[0053] [ka]

[0054] Coniferyl alcohol is present in all species and is the major monomer in conifers (softwoods). Hardwood (hardwood) species contain up to 40% sinapyl alcohol units, while grasses and crops may also contain coumaryl alcohol units.

[0055] Lignin can be classified according to the source of the raw biomass as softwood lignin and hardwood lignin.

[0056] Suitable starting raw biomass sources for obtaining the relevant lignin fractions include essentially pure lignin, as well as 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.

[0057] The expression "essentially pure lignin" should be understood to mean at least 80% pure lignin, preferably at least 90% pure lignin, more preferably at least 95% pure lignin, based on the dry feedstock biomass, the remainder being carbohydrates such as extractives and hemicellulose, and inorganic matter.

[0058] The expression "kraft lignin" is understood to mean lignin derived 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 derived from different softwood and hardwood species in various proportions. Lignin can be separated from the black liquor by various techniques such as precipitation and filtration. Lignin usually starts to precipitate at pH values ​​below pH 11-12. Various pH values ​​can be used to precipitate lignin fractions with different properties. These lignin fractions can differ from each other in terms of molecular weight distribution, e.g. Mw and Mn, polydispersity, hemicellulose and extractives content, inorganic content, etc. The precipitated lignin can be purified from inorganic impurities, hemicellulose and wood extractives using an acidic washing process. Further purification can be performed by filtration.

[0059] Alternatively, lignin can be separated from pure biomass. The separation process can begin with liquefaction of the biomass with strong alkali followed by a neutralization process. After alkali treatment, lignin can be precipitated in a similar manner as described above.

[0060] The separation of lignin from biomass preferably 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 (sulfur content less than 3%) and is therefore useful for further processing. It is preferred to pretreat the wood to remove hemicellulose and then hydrolyze the cellulose. The resulting insoluble lignin fraction contains up to 30% cellulose by weight.

[0061] The separated lignin is preferably also subjected to a depolymerization process to further reduce the weight average molecular weight of the fragments.

[0062] In some embodiments, the separated lignin is also subjected to a depolymerization process to further reduce the weight average and number average molecular weight of the fragments.

[0063] Suitable depolymerization processes include base-catalyzed depolymerization, acid-catalyzed depolymerization, metal-catalyzed depolymerization, ionic liquid-assisted depolymerization, and supercritical fluid-assisted lignin depolymerization.

[0064] In a preferred embodiment, said lignin fraction is obtained by base-catalyzed depolymerization.

[0065] Preferably, said lignin fraction is obtained by subjecting the separated lignin to a base-catalyzed depolymerization at a temperature below 300° C. and a pressure below 30 MPa.

[0066] The pH is set to 11-14 by adding a base such as NaOH, KOH, Ca(OH)2, LiOH, K2CO3, or mixtures thereof.

[0067] The weight average molecular weight (Mw) of the fragments in the lignin fraction is measured by size exclusion chromatography (or "SEC"). In SEC, a stationary liquid present in the pores of the beads is used as the stationary phase and a flowing liquid as the mobile phase. The mobile phase can therefore flow between the beads and into and out of the pores in the beads. The separation mechanism is based on the size of the polymer molecules in the solution. Large molecules elute first. Small molecules, which can fit into the many pores of the beads, will take a long time to pass through the column and therefore will exit the column slowly. 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 ​​of the unknown sample are compared to a calibration curve. The retention time depends on the column material used, the eluent, and how similar the standards used are to the sample. The eluent is preferably 0.1 M NaOH.

[0068] The lignin fraction preferably contains fragments having a weight average molecular weight of 2,000 to 20,000 daltons. More preferably, the lignin fraction contains fragments having a weight average molecular weight of 3,000 to 20,000 daltons. More preferably, the lignin fraction contains fragments having a weight average molecular weight of 4,000 to 15,000 daltons. In some preferred embodiments, the lignin fraction comprises fragments having a weight average molecular weight of 4,000 to 8,000 daltons. In another preferred embodiment, the lignin fraction comprises fragments having a weight average molecular weight of 9,000 to 11,000 daltons.

[0069] In these embodiments, preferably the fragments contain, on average, from 11 to 111 phenylpropane units, more preferably, on average, from 22 to 111 phenylpropane units.

[0070] The molecular weights of the three phenylpropanoid monomer precursors vary between 150 Daltons for coumaryl alcohol, 180 Daltons for coniferyl alcohol, and 210 Daltons for sinapyl alcohol. Therefore, the average weight is 180 Daltons, and this value is used as the "phenylpropane unit." Dividing the Mw value by 180 Daltons gives the weight average number of phenylpropane units. Preferably, the lignin fraction comprises fragments with a number average molecular weight (Mn) of up to 2,000 Daltons.

[0071] For the purposes of the present invention, the number average molecular weight (Mn) of the fragments in the lignin fraction is measured by size exclusion chromatography. More preferably, the lignin fraction comprises fragments with a number average molecular weight (Mn) of up to 1,500 Daltons.

[0072] In a preferred embodiment, the lignin fraction comprises fragments having a number average molecular weight of 150 to 1,300 daltons.

[0073] Without wishing to be bound by theory, it is believed that a lower number average molecular weight means a more active molecule. This is presented with the idea that lower molecular weight means smaller fragments, which means fewer cross-linked / shortened fragments, which means more free functional groups on the fragments, and therefore more reactive fragments.

[0074] Furthermore, it is believed that small molecules can easily diffuse through the pathogen cell membrane and into the interior, thereby significantly increasing the overall effectiveness of the lignin fraction.

[0075] In these embodiments, preferably, the fragments contain a number average of up to 11 phenylpropane units, more preferably a number average of up to 8 phenylpropane units.

[0076] The molecular weights of the three phenylpropanoid monomer precursors vary between 150 Daltons for coumaryl alcohol, 180 Daltons for coniferyl alcohol, and 210 Daltons for sinapyl alcohol. Therefore, the average weight is 180 Daltons, and this value is used as the "phenylpropane unit." Dividing the Mn value by 180 Daltons gives the number average number of phenylpropane units.

[0077] In a preferred embodiment, the lignin fraction comprises fragments with a weight average molecular weight (Mw) of 150 to 2,500 Daltons and fragments with a number average molecular weight (Mn) of up to 2,000 Daltons.

[0078] More preferably, the lignin fraction comprises fragments having a weight average molecular weight (Mw) of 150 to 2,500 Daltons and a weight average of 2 to 13 phenylpropane units, and fragments having a number average molecular weight (Mn) of up to 2,000 Daltons and a number average of up to 11 phenylpropane units.

[0079] In a further embodiment, the lignin fraction has a polydispersity index (PDI) of 1.25-12.

[0080] Polydispersity Index (PDI) or heterogeneity index, or simply dispersity, is a measure of the distribution of molecular weights in a given polymer sample. PDI is the weight average molecular weight (Mw) divided by the number average molecular weight (Mn). It indicates the distribution of individual molecular weights in a batch of polymer.

[0081] The lignin fraction preferably further comprises up to 30% by weight, more preferably 10-30% by weight, of cellulose based on the weight of the lignin fraction. 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 the complete hydrolysis of cellulosic substrates to glucose, a monomeric fermentable sugar. The presence of cellulose in the biostimulant of the present invention is advantageous in further increasing the overall efficiency of promoting plant growth, since sugars aid in plant cellular respiration and cell growth.

[0082] In the granules a), it is preferred that the at least one binder is selected from kaolin, starch, modified starch, phosphorylated starch, pectin, modified pectin, amylopectin, alginic acid, sodium alginate, guar gum, guar flour, tragacanth, gum arabic, xanthan gum, karaya gum, tara gum, tamarind gum, gellan gum, locust bean gum, gelatine, carob seed flour, galactomannan, glucomannan, dextran, carrageenan, mannan, arabinogalactan, pullulan, maltodextrin, cellulose, derivatized cellulose, carboxymethylcellulose, sodium carboxymethylcellulose, soy polysaccharides, chitosan, or mixtures thereof.

[0083] More preferably, the at least one binder is selected from starch, modified starch, phosphorylated starch, and mixtures thereof.

[0084] In a preferred embodiment, the granulate a) further comprises a wetting agent, a disintegrant, a dispersant, or a mixture thereof.

[0085] Wetting agents reduce the surface tension of water, allowing them to penetrate into the hydrophobic soil medium and promote penetration. Wetting agents vary in their effect on water surface tension, water penetration and water retention. Suitable wetting agents are selected from the group consisting of alkyl sulfates, aryl sulfonates, polyoxyalkylene alkyl ethers, alkenyl sulfonates, polyoxyethylene styryl phenyl ethers, polyoxyethylene distyryl phenyl ethers, polyoxyethylene tristyryl phenyl ethers, polyoxyethylene styryl phenyl ether salts, polyoxyethylene distyryl phenyl ether salts, polyoxyethylene tristyryl phenyl ether salts, and N-acyl amino acid salts.

[0086] A preferred wetting agent is an aryl sulfonate such as sodium isopropyl naphthalene sulfonate.

[0087] Disintegrants are excipients incorporated into granules to facilitate their disintegration when contacted with a liquid or fluid substance. Suitable disintegrants include water-soluble polymers and polysaccharides.

[0088] Dispersants are substances, typically surfactants, that are added to improve particle separation and prevent particle settling or agglomeration. Suitable dispersants are polycarboxylates, such as sodium polycarboxylate. In some embodiments, the particulate material b) consists essentially of a lignin fraction.

[0089] In another embodiment, the particulate matter b) consists of a lignin fraction.

[0090] The granular material b) preferably contains a lignin fraction at a concentration of 65 to 95% by weight, more preferably 70 to 90% by weight, based on the weight of the granular material b).

[0091] The particulate material b) may further comprise at least one carrier.

[0092] In granules b), the at least one carrier is preferably selected from lignosulfites, chalk, carboxymethylcellulose, potassium, sodium, lithium, calcium, magnesium, zinc or ammonium carbonates, bicarbonates, sulfates, phosphates, oxides or hydroxides, or urea salts, or mixtures thereof.

[0093] More preferably, the at least one carrier is selected from potassium or sodium carbonate, or ammonium or urea salts, and mixtures thereof.

[0094] The granules a) and b) each independently have an average particle size distribution D 50 It is preferable that the thickness is 0.5 to 2.0 mm.

[0095] In a preferred embodiment, the average particle size distribution D 50 and the average particle size distribution D of the granular material b) 50 The ratio of is 3:1 to 1:3, more preferably 2:1 to 1:2. In a particularly preferred embodiment, the granules a) and the granules b) have approximately the same average particle size distribution D 50 has. In a most preferred embodiment, there are no particles in the solid mixture less than 0.2 mm in size.

[0096] Preferably, said granules a) and said granules b) each independently have a bulk density (loose) of 0.3-0.8 g / ml, preferably 0.4-0.7 g / ml, according to ISO 3944:1992. This International Standard specifies a method for the measurement of bulk density (loose) of solid biostimulants, excluding powdered biostimulants. This method is only applicable to dry biostimulants. If the biostimulant absorbs moisture during transportation or storage, it must be dried in a constant low humidity environmental chamber before measurement. This method is not suitable for materials containing a large amount of particles larger than 5 mm in diameter. The following standards contain provisions which, by reference in the text, constitute provisions of this International Standard: - ISO 7742:1988, Solid fertilizers - Sample reduction - ISO 8358:1991, Solid fertilizers - Preparation of samples for chemical and physical analysis

[0097] For the purposes of this International Standard, "bulk density of a fertilizer (loose)" is defined as the mass per unit volume of material after free application to a container under defined conditions. Bulk density (loose) is expressed in grams per cubic centimetre (g / cm 3 )

[0098] Although these standards refer to "fertilizers", the inventors have considered them as well as suitable and adoptable references for the characterization of the present biostimulants.

[0099] In a preferred embodiment, said granules a) and said granules b) have approximately the same bulk density (loose).

[0100] Granules a) and b) have similar mean particle size distribution D 50 and / or density is important to minimize the risk of large or heavy particulates separating out during transportation and storage, resulting in uneven application of the biostimulant components when redispersed or resuspended in a liquid formulation at the time of use.

[0101] The biostimulant of the present invention preferably contains 0.1 to 20% by weight of granular material a) and 80 to 99.9% by weight of granular material b), based on the weight of the solid mixture.

[0102] In particular, in biostimulants, i) when the at least one carrier in the granules b) is water-dispersible or water-soluble, the solid mixture comprises 1-15% by weight of the granules a) and 85-99% by weight of the granules b), ii) When the at least one carrier in granules b) is neither water-dispersible nor water-soluble, the solid mixture comprises 0.1-5% by weight of granules a) and 95-99.9% by weight of granules b). These weight percentages are based on the weight of the solid mixture.

[0103] In a preferred embodiment of option i), said at least one carrier in granule b) comprises potassium carbonate, an ammonia salt, a urea salt, or a mixture thereof.

[0104] In a preferred embodiment of option ii), said at least one carrier in the granules b) comprises calcium sulfate, lignosulfite, chalk, carboxymethylcellulose, or a mixture thereof.

[0105] In a more preferred embodiment, the solid mixture comprises both of the following: i) a granule b), in which the at least one carrier is water-dispersible or water-soluble; and ii) a particulate material in which the at least one carrier is neither water-dispersible nor water-soluble;

[0106] Naturally, the granules b) according to option i), i.e. the granules b) comprising at least one water-dispersible or water-soluble carrier, can be considered as fast-release granules. In fact, the lignin fraction is readily released on contact with water. Thus, the biostimulant comprising the granules b) according to option i) is particularly suitable for application to plants or plant soil, either upon addition of water or directly to the plant soil as such.

[0107] Likewise, naturally, granules b) according to option ii), i.e. granules b) comprising at least one water-non-dispersible or water-insoluble carrier, can be considered as sustained release granules. Thus, biostimulants comprising granules b) according to option ii) are particularly suitable for application directly to plant soil.

[0108] Thus, the effectiveness and duration of activity of the biostimulant after application can be preset by adjusting the concentrations of the different granules b) in the solid mixture.

[0109] Exemplary biostimulants according to the present invention are shown below. - Starch and 5x10 9 A fast release biostimulant comprising a solid mixture of a) 1% by weight of granules containing Trichoderma spores / g and b) 99% by weight of granules (75% by weight lignin fraction + 25% by weight potassium carbonate), said weight percentages being based on the weight of the solid mixture. - Starch and 5x10 9 A sustained release biostimulant comprising a solid mixture of a) 0.1% by weight of granules containing Trichoderma spores / g and b) 99.9% by weight of granules (85% by weight lignin fraction + 15% by weight calcium sulfate), said weight percentages being based on the weight of the solid mixture.

[0110] In a preferred embodiment, the biostimulant 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.

[0111] In some preferred embodiments, the biostimulant 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 4,000 to 8,000 daltons as measured by size exclusion chromatography.

[0112] In another preferred embodiment, the biostimulant 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 contains fragments having a weight average molecular weight of 9,000 to 11,000 daltons as measured by size exclusion chromatography.

[0113] In a most preferred embodiment, the biostimulant 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.

[0114] In other embodiments, the biostimulant consists essentially of a solid mixture of: a) 1 x 10 Trichoderma fungi 5 ~1×10 10 granules containing spores / g (based on granule a) and containing at least one binder; and b) a granule comprising a lignin fraction in a concentration of up to 90% by weight, based on the weight of the granule b), and optionally comprising at least one carrier; Where: - the fungus is selected from 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, the fragments comprising a weight average of up to 111 phenylpropane units; The granules a) and the granules b) each independently have an average particle size distribution D 50 is 0.2 to 4.0 mm.

[0115] For purposes of the present invention, the term "consisting essentially of" means that the fungus and the lignin fraction are the only active ingredients present in the biostimulant that act as plant growth and fruit production promoters, with possible other ingredients having different activities or simply being co-formulants.

[0116] In a further embodiment, the biostimulant consists of the solid mixture described above.

[0117] All combinations of the preferred aspects, methods of manufacture, and uses of the biostimulants of the present invention disclosed above are to be understood as being described herein, and likewise preferred are those embodiments having the expressions "consisting essentially of" and "consisting of".

[0118] In another aspect, the present invention relates to a method for producing a biostimulant, said method comprising the steps of: 1) providing a granule a) obtained by mixing and granulating Trichoderma spores and at least one binder; 2) providing a granulate b) obtained by wet granulation of the lignin fraction and, optionally, at least one carrier; 3) Mixing granules a) and b) to obtain a solid mixture.

[0119] Preferably, step 1) involves extrusion granulation, in which a fine powder is mixed with water (10-20%) to produce a wet mixture, which is passed through an extruder or tumbling granulator to obtain a wet granulate, which is then dried in an oven or in a fluidized bed.

[0120] In particular, the water-dispersible granules a) of viable Trichoderma spores are preferably prepared by the following steps: - grinding the solid ingredients together to obtain a homogeneous premix; - adding water to the premix to obtain a wet mix; - granulating the wet mixture by any suitable technique such as extrusion, tumbling granulation, agglomeration, dry spraying, etc.

[0121] The premix is ​​preferably obtained by mixing together the following solid ingredients: - Trichoderma spores 10-90% by weight - Wetting agent 1-3% by weight - Dispersant 2-15% by weight - Disintegrant 0-15% by weight - Binder remainder.

[0122] The lignin fractions typically used in the present invention are insoluble in water at neutral pH, but are soluble at basic pH.

[0123] It has now been found that by adding at least one water-dispersible or water-soluble carrier (alkaline compound) to the lignin, the lignin can be solubilized in water at a basic pH, thereby obtaining water-dispersible granules of the lignin fraction b). The solubilized lignin forms a colloidal dispersion in water.

[0124] The solubilized lignin fraction is then diluted with water at neutral pH, thereby obtaining an aqueous solution / dispersion of lignin at a pH physiological for plants, ie neutral or slightly basic pH. Preferably, the carrier is potassium carbonate.

[0125] Thus, the water-dispersible lignin fraction granules b) are preferably prepared by the following process: - 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 60-70%; - mixing until a uniform paste is formed, thereby obtaining a water-dispersible lignin fraction paste; - Wet granulating the water-dispersible lignin fraction paste, thereby obtaining granules b).

[0126] The term dry matter content refers to the solids content in a mixture, calculated as the percentage of solids relative to the total mass of the mixture, in % by weight.

[0127] Granulation of only the lignin fraction (i.e., without Trichoderma spores) makes it possible to take advantage of the superior thermal and chemical stability of lignin (and avoid exposing Trichoderma to alkaline formulations in the case of water-dispersible formulations), thereby allowing the use of very advantageous process conditions in terms of cost and yield.

[0128] The non-water-dispersible lignin fraction granules b) are preferably prepared by the following process. - providing a lignin fraction preferably having a dry matter content of 60-70%; - optionally adding at least one water-non-dispersible or water-insoluble carrier, preferably lignosulfite, chalk, carboxymethylcellulose, calcium sulfate, or mixtures thereof; - granulating the lignin fraction, optionally mixed with said carrier, in a blade rotor, while inducing the formation of granules by mechanical action; and - Drying granular material in a fluidized bed.

[0129] Due to the flocculating ability of lignin, the use of a water-non-dispersible or water-insoluble carrier is not strictly necessary, however, the addition of a carrier can improve the texture of the granules and prevent breakage of the granules during handling, storage and use with agricultural machinery that would otherwise result in undesirable dust.

[0130] In another aspect, the present invention relates to the use of said biostimulants as plant growth and fruit production promoters in agriculture.

[0131] Preferably, the biostimulant is applied in an amount of 0.1-10% by weight in water, at a rate of 100-300 ml per plant every 5-15 days. Preferably, the biostimulant is present in an amount of 0.5-5% by weight in water.

[0132] The biostimulant in granular form may be applied in an amount of from 1 to 1,000 kg per hectare (ha), preferably from 1 to 100 kg per hectare, more preferably from 1 to 10 kg per hectare.

[0133] In a further aspect, the present invention relates to an agrochemical product comprising said biostimulant and an agrochemical additive.

[0134] Suitable additives are pH adjusters, acidity adjusters, water hardness adjusters, mineral oils, vegetable oils, fertilizers, foliar fertilizers, and combinations thereof.

[0135] Exemplary additives include 2-ethylhexanol EO-PO, alkoxylated alcohols, alkoxylated fatty amines, alkoxylated triglycerides, alkyl polyglycosides, sodium alkyl ether sulfates, alkylphenol ethylene oxide condensates, alkylphenyl hydroxypolyoxyethylenes, allyl polyethylene glycol methyl ethers, amphoteric dipropionic acid surfactants, di-lp-menthene, dimethylpolysiloxanes, esterified vegetable oils, ethylene oxide condensates, fatty acid esters, fatty alcohol ethylene oxide condensates, fatty alcohol polyalkoxy Ingredients that may be used include but are not limited to: glyceryl ether, lecithin (soybean), methylated rapeseed oil, n-dodecylpyrrolidone, n-methylpyrrolidone, n-octylpyrrolidone, nonionic surfactants, nonylphenol ethylene oxide condensates, paraffin oil, poly(vinylpyrrolidone / 1-hexadecene), polyacrylamide, polyalkylene glycols, polyalkylene oxides, polyether modified trisiloxanes, polyethylene polypropylene glycols, polyoxyethylene monolaurate, propionic acid, styrene-butadiene copolymers, synthetic latex, tallow amine ethoxylates, vegetable oils, and mixtures thereof.

[0136] In view of the fact that the biostimulant is effective even at very low concentrations of the fungal and lignin fractions, the agrochemical product advantageously contains said biostimulant preferably at a concentration of 1 to 500 grams per kg of agrochemical product. The agrochemical product may be in solid or liquid form.

[0137] When the agrochemical product is in a solid form, said solid form may be tablets, mini-tablets, micro-tablets, granules, micro-granules, pellets, multi-particulates, fine particles, or powder. If the agrochemical product is a liquid, the liquid form may be a solution, suspension, emulsion, dispersion, droplets or a sprayable fluid, and may be in the form of an aqueous or oil-based liquid. The liquid form may include a solvent. Suitable solvents are water, glycols, alcohols, polyalcohols, organic acids, and combinations thereof.

[0138] Preferred solvents are water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, allyl alcohol, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-ethylene glycol, polyethylene glycol (PEG), glycerol, lactic acid, polylactic acid, and mixtures thereof. More preferred solvents are water, 1,2-propylene glycol, 1,3-propylene glycol, 1,2-ethylene glycol, polyethylene glycol (PEG), and mixtures thereof.

[0139] When the agricultural chemical product is a liquid, the liquid form preferably has a pH of 5-9, more preferably 6-8.

[0140] If the agrochemical product is a liquid, the liquid form contains 1-50% by weight of the biostimulant, which means that the agrochemical product is a concentrate that can be diluted appropriately or mixed directly with other chemicals before use, if necessary.

[0141] The agrochemical products include granular bait, aerosol can, liquid (without dilution), bulk bait, matrix, concentrated bait, oil miscible fluid concentrate, encapsulated granules, capsule suspension, dispersible concentrate, powder, powder for dry tanning seeds, emulsified concentrate, electrostatic liquid, water in oil emulsion, seed tanning emulsion, oil in water emulsion, smoke bottle, microgranule, smoke candle, smoke cartridge, smoke bar, tanning concentrate suspension, smoke tablet, smoke agent (fumigant), smoke granule (or pellet), gas (under pressure), granular bait, gasifiable product, microgranule, slippery powder, granule, oil paste, hot smoke concentrate, solid / liquid combination package, liquid / liquid combination package, low The composition may be a hot smoke concentrate, a solid / solid combination package, a lacquer, a solution for seed tanning, a microemulsion, a microgranule, a dispersible oil, an oil miscible concentrated suspension, an oil miscible liquid, an oil suspension, a paste, a flat bait, a concentrated paste or gel, a pour-on liquid, a plant stick, a treated or coated seed, a ready to use bait, a topical liquid, a piece bait, a concentrated suspension, a suspension-emulsion, a water soluble granule, a water soluble concentrate, a film forming oil, a water soluble powder, a soluble powder for seed tanning, a suspension, a tablet, a technical material, a technical concentrate, a powder for tracing, an ultra low volume liquid, a water dispersible microgranule, a water dispersible granule, a wettable powder, a wettable powder for seed tanning, a self-adhesive patch, and combinations thereof.

[0142] Advantageously, the agrochemical product may further comprise a fertilizer comprising a nitrogen, phosphorus, potassium compound, or mixtures thereof.

[0143] In a further aspect, the present invention relates to a method for promoting plant growth and fruit production, said method comprising the step of applying to the plant or to the plant soil the biostimulant or agrochemical product.

[0144] As mentioned above, the expression "on the plant" means that the biostimulant or pesticide product can be applied to any part of the plant, including the roots, trunk, branches, twigs, leaves, flowers and fruits. When using a biostimulant, it is preferable to add water before applying it to the plant.

[0145] The biostimulant or pesticide product may be applied by one or more of the following methods: - A method for mixing biostimulants or agrochemical products with seeds in the hopper of a sowing machine; - Spreading biostimulant or pesticide products next to the seed furrow; - Applying biostimulant or pesticide products to the entire field before or after the final soil tillage.

[0146] If the pesticide product is a liquid, it may also be applied by one or more of the following methods. - Methods of spraying pesticide products on tubers, bulbs and seeds; - Methods of spraying agrochemical products on the above-ground parts, leaves and stems of plants; - Immersing the roots of plants in an aqueous solution containing the pesticide product.

[0147] The agrochemical product may be applied in an amount to achieve 1,000g to 10,000kg of biostimulant per hectare (ha), preferably 1,000g to 1,000kg per hectare, more preferably 1,000g to 10,000g per hectare.

[0148] Moreover, all combinations of the preferred embodiments of the biostimulant of the present invention described above, and the methods for producing and using same, should be understood as being disclosed in the present specification.

[0149] All combinations of the preferred embodiments, methods of manufacture and uses of the biostimulants of the invention disclosed above are to be understood as being described herein.

[0150] The following examples of the present invention are given for illustrative purposes. EXAMPLES

[0151] Mw and Mn in these examples were determined by size exclusion chromatography according to the following procedure. "Weight %" means weight percentage based on the weight of the organic-inorganic hybrid material, unless otherwise specified.

[0152] Reagents and Materials - Eluent: 0.1M NaOH, flow rate 0.5ml / min - For calibration of RI detector: Pullulan standard, Mp: 100,000-1,080 (6 types of standards). Mp is the peak maximum molecular weight. - For calibration of the UV detector (280 nm): PSS standards, sodium polystyrene sulfonate, Mp 65,400-891 (6 types of standards). The standards are dissolved in ultrapure water to a concentration of approximately 5 mg / ml. The injection volume is 20 μl. - Quality control samples: Use lignin with known Mw distribution.

[0153] Equipment and Instruments - Dionex Ultimate 3000 autosampler, column compartment, and pump - Dionex Ultimate 3000 Diode Array Detector - Refractive index detector: Shodex RI-101 - Column: PSS MCX column: precolumn and two analytical columns: 1000Å and 100000Å, column material is sulfonated divinylbenzene copolymer matrix - Syringe filter 0.45μm and glass sample bottles for STD samples. Sample filtration: Mini-Uniprep syringeless filter device, PTFE or nylon, 0.45μm. Optionally, 5μm syringe filter for prefiltration - Volumetric flask

[0154] procedure - Preparation of eluent Ideally, the water used to prepare the eluent should be high-quality deionized water with a resistivity of 18 MΩ·cm or greater and as little dissolved carbon dioxide as possible. The water should be free of biological contaminants (e.g., bacteria and mold) and particulates. - Cleaning the needle with 10% MeOH-water - Liquid samples The strong alkaline liquid sample is diluted 1:100 and filtered through a PTFE syringe filter (0.45 μm) into a vial. The solid lignin sample is diluted and dissolved in 0.1 M NaOH and filtered through a PTFE, 0.45 μm syringe filter. The prepared sample is placed in the autosampler. The injection volume is 20 μl. After the sample, 1 M NaOH is injected as a sample to wash the column.

[0155] Equipment parameters: - Flow rate 0.5ml / min - Eluent 0.1M NaOH - Column oven temperature 30°C - Isocratic Analysis - Analysis time 48 minutes

[0156] - Solid samples If necessary, dry the solid sample (lignin) in a 60 °C oven overnight. Weigh approximately 10 mg into a 10 ml volumetric flask. 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 filtered through a 5 μm syringe filter.

[0157] - Calibration standards Approximately 50 mg of each standard is weighed into a 10 ml volumetric flask and made up to volume with ultrapure water. The standards are filtered through a PTFE, 0.45 μm syringe filter. After the calibration samples are analyzed, the calibration results are integrated and processed using the processing method and stored. The calibration is a linear first order calibration.

[0158] - Quality control samples For lignin samples, lignin with known Mw distribution is used as a quality control sample. Lignin is dissolved in 0.1 M NaOH to a concentration of approximately 1 mg / ml.

[0159] [Example 1] Beech wood (Fagus sylvatica) was subjected to alkaline and enzymatic hydrolysis to obtain a hemicellulose- and cellulose-free lignin fraction. The lignin fraction thus isolated has the following characteristics: > >95% total solids Mw 9,000-11,000Da (phenylpropane units 50-61) Essentially sulfur free (less than 3% sulfur content) Contains 23 to 29% by weight of cellulose.

[0160] [Example 2] The following lignin fractions were extracted from kraft black liquor, said lignin fractions having the following characteristics: > >95% total solids Single species: Tropical pine Mw 4400~5000Da (phenylpropane units 24~28) Mn 1200-1300Da (phenylpropane units 6-7) Structure of OH group: Aliphatic 2.1mmol / g Carboxyl group 0.5mmol / g Condensed type and syringyl 1.7mmol / g Guaiacyl 2.0mmol / g Catecholic and p-OH phenyl groups: 4.0mmol / g

[0161] [Example 3] Production of Trichoderma granules a) A premix was prepared by grinding the following ingredients together:

[0162] [Table 1]

[0163] The premix was granulated by extrusion granulation. First, a pre-blend of fine powders 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 in the form of cylindrical micropellets. The granules were dried in a fluidized bed dryer. 1-2×10 per gram of granules 10 Water-dispersible granules were obtained containing viable spores of 0.01 g / ml, which had a mean particle size distribution D 50 was 1.5 mm and sieved into a fraction of 1400 μm to 500 μm.

[0164] [Example 4] i) Production of water-dispersible granules of kraft lignin fraction b) Water-dispersible granules b) of the lignin fraction of Example 2 were prepared as follows. 1) Dry mix the powder of the lignin fraction (dry matter content 70%) with potassium carbonate in a weight ratio of 70:30. 2) Mix until the exothermic reaction is complete. 3) The resulting mixture is granulated in a blade rotor, inducing the formation of granules by mechanical action. 4) Dry the granulate in a fluidized bed. The average particle size distribution D 50 was 1 mm and distributed in the sieved fraction of 1400 μm to 250 μm.

[0165] ii) Production of water-indispersible granules of lignin fraction b) A water-non-dispersible granule of the lignin fraction b) was prepared as follows. 1) It provides a lignin fraction with a dry matter content of 65%. 2) Add 3% by weight of lignosulfite. 3) Granulating the mixture in a blade rotor and inducing the formation of granules by mechanical action. 4) Dry the granulate in a fluidized bed. The average particle size distribution D 50 was 2 mm and distributed in the sieved fraction of 2000 μm to 250 μm.

[0166] [Example 5] a) Preparation of a biostimulant comprising granules a) of Example 3 and granules b) of Example 4.i A granular product was prepared by mixing the following: 30 g of the granules of Example 3 a) and 970 g of the water-dispersible granules of Example 4.i containing the lignin fraction and potassium carbonate b) b) Production of a biostimulant comprising granules a) of Example 3 and granules b) of Example 4.ii Lignin fraction and 3 × 10 6 A granular product containing viable spores of Trichoderma at a concentration of spores / g was prepared by mixing. In particular, the product includes: 4 g of the granules a) of Example 3 and 996 g of the water-non-dispersible granules b) of Example 4.ii

[0167] [Example 6] Evaluation of the biostimulant activity of the biostimulant prepared in Example 5 on endive escarole

[0168] The biostimulant activity of the biostimulant product prepared in Example 5a was evaluated in Endive Escarole plants.

[0169] Transplanting was carried out on September 10th using seedlings grown in 104-hole cell trays filled with nursery soil.

[0170] The experimental design was a randomized block of three replicates as follows:

[0171] [Table 2]

[0172] [Table 3]

[0173] The pesticide product of the present invention was applied prior to transplanting along the rows where the seedlings were to be transplanted, and then 9 days later by hand irrigation.

[0174] Treatment with the comparative product was performed by immersing the cell tray containing the seedlings in an aqueous solution of the product and then hand watering 9 days after transplanting.

[0175] During the growing period, the applied biostimulant products were checked for possible phytotoxicity (no phytotoxicity was observed). At the commercial "optimum harvest time", the vigor of the plants was assessed on a scale of 0 to 100. Harvesting was done manually on October 28th, approximately 7 weeks after transplanting. A total of 20 plants were harvested per plot. To accelerate bleaching, 5 of the remaining plants were tied with rubber bands per plot. The tied plants were harvested on November 11th, 2 weeks after tying.

[0176] Harvested plants were weighed both intact (fresh weight) and after removing the leaves. The data obtained were statistically analyzed by analysis of variance using the MSTATC program. Analysis of variance revealed statistically significant differences only for the trait “vigor,” with the treatment area passing the test (Table 1).

[0177] The pesticide product of the present invention was shown to be superior in all properties examined.

[0178] [Table 4]

[0179] Considering the yields (t / ha) of bunches before and after defoliation, it can be seen that the treatment with the product of the invention showed the highest yields, 29.28t / ha raw and 22.855t / ha net, followed by the comparative commercial biostimulant (28.4 and 20.58t / ha) and the control (22.84 and 17.22t / ha).

[0180] [Example 7] Evaluation of the biostimulant activity of the biostimulant prepared in Example 5a on tomato The test was carried out in mulched soil using the processing tomato variety PIETRAROSSA.

[0181] Transplanting was performed on June 18 using seedlings grown in 104-well cell trays filled with nursery soil. The experimental design was a randomized block with three replicates as follows:

[0182] [Table 5]

[0183] [Table 6]

[0184] The pesticide product of the present invention was applied to mulch holes prior to transplanting and then by hand irrigation on days 9 and 13 thereafter. For the commercial product, the cell tray containing the seedlings was immersed in the product solution and applied by hand irrigation 9 days after transplanting.

[0185] Crop growth was delayed due to late transplanting and the use of old seedlings. Little growth was observed in the transplanted seedlings for the first 15 days after transplanting. Thereafter, growth was good.

[0186] During the cultivation period, the applied biostimulant products were checked for phytotoxicity, but no phytotoxicity was found. The start of flowering was confirmed when at least 5-10 flowers were seen on all plants in the test plots, which was an average of 33 days after transplanting. At full maturity, the vigor of the plants was judged on a scale of 0 to 100.

[0187] Harvesting was done manually on 20 September, 13 weeks after transplanting, and the fruits were separated into marketable (red), unripe (green) and discarded. The first two categories were weighed, while the third was counted only. Average fruit weight was calculated from a sample of 30 marketable fruits. Three fruits per plot were used for Brix measurements.

[0188] The data obtained were statistically analyzed by analysis of variance using the MSTATC program. Analysis of variance showed a statistically significant difference only in plant vigor (Table 2), which supports the fact that the total yield and marketable yield obtained with the product of the present invention are significantly higher than those of the control (Table 3).

[0189] [Table 7]

[0190] [Table 8]

[0191] The vitality of the plants, which was judged on a scale of 0 to 100 at the fully ripened stage, is shown in Table 2. The most vigorous plants were observed in the comparative commercial biostimulant group (93.3%) and the product group of the present invention (90%), while the control group was significantly lower (70%). Table 2 also shows the sugar content (Brix) of the fruit, plant height at full maturity, and average marketable fruit weight, which were similar for all treatments.

[0192] [Example 8] Evaluation of the biostimulant activity of the biostimulant prepared in Example 5b against birch Add 50g or 500g of Biostimulant product to 1m of seedling soil (peat-based). 3 and mixed.

[0193] The seedling soil was dispensed into flowerpots, and birch seeds were sown in each pot (cultivation period: 3.5 months from March to June).

[0194] 81-well seedling trays were subjected to the analysis. The length and thickness of the stem (Figure 1A) and the amount of nutrients absorbed from the leaves (Figure 1B) were analyzed, and the results are summarized in Table 4 below.

[0195] [Table 9]

[0196] From these results, it is clear that the pesticide product of the present invention promoted the growth of birch seedlings, increasing stem thickness and leaf number.

Claims

1. A solid mixture consisting of: a) 1 x 10 per 1 g of granules a) ５ ~1 x 10 １０ granules comprising a fungus of the genus Trichoderma in a spore concentration and at least one binder; and b) a granule containing at least 50% by weight of a lignin fraction based on the weight of the granule b), - the fungus is selected from Trichoderma species, protoplast fusion products thereof, and mixtures thereof; - the lignin fraction comprises fragments having a weight average molecular weight of up to 20,000 Daltons (Da) as measured by size exclusion chromatography, the fragments comprising a weight average of up to 111 phenylpropane units; - the granules a) and the granules b) each independently have a mean particle size distribution D measured by sieve analysis in accordance with EN 1235 ５０ A biostimulant having a particle size of 0.2 to 4.0 mm.

2. The Trichoderma species is selected from the group consisting of Trichoderma aggressivum, Trichoderma asperellum, Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma cremeu m, Trichoderma harzianum, Trichoderma koningii, Trichoderma 2. The biostimulant of claim 1, selected from Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma virens, Trichoderma viride, Trichoderma viridescens, and mixtures thereof.

3. 2. The biostimulant of claim 1, wherein the fungus is selected from T. harzianum, T. atroviride, and T. virens, and mixtures thereof.

4. 4. The biostimulant of claim 3, wherein the fungus is selected from T. harzianum HK2, T. atroviride HK4, and T. virens GV41, and mixtures thereof.

5. The fungi are present in the granules a) at a concentration of 1 x 10 ５ ~1 x 10 １０ 10. The biostimulant of claim 1, present at a concentration of spores / g.

6. 2. Biostimulant according to claim 1, wherein the lignin fraction is present in a concentration of 65 to 95% by weight, preferably 70 to 90% by weight, based on the weight of the particulate material b).

7. 2. The biostimulant of claim 1, wherein the lignin fraction comprises fragments having a weight average molecular weight of 2,000 to 20,000 daltons, preferably 3,000 to 20,000 daltons, more preferably 4,000 to 15,000 daltons.

8. 2. The biostimulant of claim 1, wherein the lignin fraction comprises fragments having a weight average molecular weight of 4,000 to 8,000 daltons.

9. 2. The biostimulant of claim 1, wherein the at least one binder in the granules a) is selected from kaolin, starch, modified starch, phosphorylated starch, pectin, modified pectin, amylopectin, alginic acid, sodium alginate, guar gum, guar flour, tragacanth, 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, carboxymethylcellulose, sodium carboxymethylcellulose, soy polysaccharides, chitosan, or mixtures thereof.

10. 2. The biostimulant of claim 1, wherein the granules b) further comprise at least one carrier, and the at least one carrier is selected from chalk, carboxymethylcellulose, carbonates, bicarbonates, sulfates, phosphates, oxides or hydroxides of potassium, sodium, lithium, calcium, magnesium, zinc or ammonium, or mixtures thereof.

11. The granules a) and the granules b) each independently have an average particle size distribution D ５０ The biostimulant of claim 1, wherein the particle size is 0.5 to 2.0 mm.

12. 2. The biostimulant of claim 1, 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 in granules b) is water-dispersible or water-soluble, the solid mixture comprises 1 to 15 wt. % of granules a) and 85 to 99 wt. % of granules b), based on the weight of the solid mixture; ii) when the at least one carrier in granules b) is neither water-dispersible nor water-soluble, the solid mixture 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 biostimulant of claim 10.

14. 14. The biostimulant of claim 13, wherein the at least one carrier in granules b) is water-dispersible or water-soluble and comprises potassium carbonate.

15. 14. The biostimulant of claim 13, wherein the at least one carrier in granule b) is neither water-dispersible nor water-soluble and comprises lignosulfite, chalk, carboxymethylcellulose (CMC), calcium sulfate, or a mixture thereof.

16. 14. The biostimulant of claim 13, wherein the solid mixture comprises both of the following: i) a particulate material b) in which the at least one carrier is water-dispersible or water-soluble, and ii) Particulate material b) in which the at least one carrier is neither water-dispersible nor water-soluble.

17. Use of the biostimulant according to any one of claims 1 to 16 as a plant growth and fruit production promoter in agriculture.

18. 17. A pesticide product comprising the biostimulant of any one of claims 1 to 16 and a pesticide additive.

19. 17. A method for promoting plant growth and fruit production, said method comprising applying to a plant or plant soil a biostimulant according to any one of claims 1 to 16.