EP4048072A1

EP4048072A1 - A hydrolysate to promote plant growth, biostimulation and biocontrol, and its use in agriculture

Info

Publication number: EP4048072A1
Application number: EP20804679.7A
Authority: EP
Inventors: Roberto Verga; Silvia RAPACIOLI
Original assignee: Bict Srl
Current assignee: Bict Srl
Priority date: 2019-10-21
Filing date: 2020-10-21
Publication date: 2022-08-31
Also published as: WO2021079276A1; IT201900019412A1

Abstract

The invention describes a hydrolysate from a natural raw material which has been shown to be significantly effective in stimulating plant growth, or in protecting plants against phytopathogenic agents. The hydrolysate from a natural raw material therefore finds advantageous application in agriculture.

Description

"A HYDROLYSATE TO PROMOTE PLANT GROWTH, BIOSTIMULATION AND BIOCONTROL, AND ITS USE IN AGRICULTURE"

DESCRIPTION

LIELD OL THE INVENTION

The present invention relates to a hydrolysate from a natural raw material, which has been shown to be significantly effective in stimulating plant growth, or in protecting the plant against phytopathogenic agents. The hydrolysate from a natural raw material therefore finds advantageous application in the agricultural industry.

BACKGROUND ART

Plant growth is dictated by both internal and external factors. The internal mechanisms originate from the genetic composition of the plant and influence the extent and timing of the growth thereof. These internal mechanisms are regulated by various types of signals transmitted within the plant cells or around the plant itself. The external factors are directly related to the environment surrounding the plant. These external influences on plant growth include factors such as light, temperature, water, and nutrients. The external environment can limit the extent to which internal mechanisms enable the plant to grow and develop, with two of the most important factors being the availability of water and nutrients in the ground. Cell expansion is directly related to water supply and therefore any shortages result in a smaller plant. Mineral nutrients are necessary for the plant's biochemical processes. When nutritional substances are insufficient, growth will be less vigorous and, in extreme cases, will cease altogether. Nutrients needed for plant growth include: primary macronutrients, namely nitrogen (N), phosphorus (P), and potassium (K), secondary macronutrients, namely calcium (Ca), sulphur (S), and magnesium (Mg), and micronutrients or trace minerals, namely boron (B), chlorine (Cl), manganese (Mn), iron (Fe), zinc (Zn), copper (Cu), molybdenum (Mo), and selenium (Se).

To adequately support proper plant growth, especially in farming, the use of fertilisers is a commonly known technique. In general, fertilisers can come in the form of a neat liquid, a suspension, or a solid. They can be administered to plants by fertilising the growth medium or by applying them to the foliage of plants, by - for example - spraying, irrigation, or similar methods. Over the last few years, foliar fertilisers have gradually replaced the soil fertilisers commonly used in farming areas, as they have fewer adverse environmental impacts. Research has shown that the conventional fertilisation process - i.e. soil fertilisation - has contributed to the contamination of surface water and groundwater. This is mainly due to the leaching of soluble fertiliser nutrients, such as nitrogen, into the water table. These circumstances can lead to insufficient transport of nutrients to plant cells. Therefore, supplying nutrients to a plant directly through the foliage thereof has proved to be more desirable. Foliar fertilisers appear to overcome the drawbacks of the soil fertilisation process, however, incorrect application of said foliar fertilisers to plants, such as -for example - direct application of a high concentration of nutrients to the foliage, can result in leaf damage to the foliage consisting of necrotic areas or leaf burns, resulting in lower crop yields. It has been hypothesised that foliar fertiliser with a lower quantity of nutrients could prevent foliar damage. However, this is not very practical as fertilising plants with low-nutrient foliar fertiliser is a labour-intensive activity.

An object of the present invention is therefore to effectively promote plant growth without causing damage to foliage and the plant in general and preserve, in particular, human and animal health, as well as the crops and the environment.

SUMMARY OF THE INVENTION

Said object has been achieved by a hydrolysate from a natural raw material, as reported in Claim 1, as well as a process for the preparation thereof.

In another aspect, the present invention relates to a composition comprising said hydrolysate from a natural raw material and a microbial inoculant.

Furthermore, the present invention relates to the use of said hydrolysate from a natural raw material and likewise said composition, to promote plant growth and fruit production in agriculture, or for biocontrol.

In another aspect, the present invention relates to an agrochemical product comprising the hydrolysate from a natural raw material or the composition, and agrochemical additives.

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 the hydrolysate from a natural raw material or the composition or the agrochemical product to a plant or a growing medium for plants. The term "plant" means any plant or plants that can be grown and harvested for profit or subsistence purposes, i.e. crops, and therefore including cereals, vegetables, fruit, and flowers, as well as those grown and harvested for gardening or personal use.

The term "growing medium for plants" means the medium in which the plant is grown or in which the plant is sown or in which the plant will be sown, and therefore includes soils, earth, and soil-free media, including hydroculture and hydroponics media.

The characteristics and advantages of the present invention will become apparent from the following detailed description and from the embodiments provided for illustrative and non-limiting purposes.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a hydrolysate from a natural raw material comprising 1-25% free amino acids and up to 55% dry residue, wherein said plant raw material is selected from com steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and combinations thereof.

"%" or "percentage" means "g/100 ml".

It has surprisingly been found that the hydrolysate from a natural raw material according to the present invention not only stimulates plant growth and productivity in terms of fruit production, but also protects plants against phytopathogenic agents and pests.

Com steep liquor (or CSL) is a liquid by-product of the wet milling of corn used to make com starch and high fmctose corn symp (HFCS). CSL consists of soluble com concentrates which are extracted by a process during which the shelled and air-purified corn is soaked in water (steeped) and then separated into the main components thereof through a combination of flotation and wet-sieving. During steeping, the soluble materials dissolve, the corn softens, and its structure weakens and breaks, thereby facilitating the milling and further separations of the components thereof. The resulting concentrate is a cmde com steep liquor, which can be further combined with gluten and fibrous materials to be sold as feed or used for other purposes, with or without undergoing further processing. In addition to being used as a nutrient for ruminants, CSL has also been used in the penicillin industry as a culture medium for the production of penicillin.

Corn steep liquor (CAS n. 66071-94-1) is commercially available in an aqueous solution (about 50% water), while the rest is made up of the natural nutrients of corn, such as water-soluble proteins, amino acids (e.g. alanine, arginine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, threonine, tyrosine, valine), vitamins (e.g. B-complex), carbohydrates, organic acids (e.g. lactic acid), minerals (e.g. Mg, P, K, Ca, Ca, S), enzymes, and other nutrients.

CSL is a viscous mixture which is a light to dark brown in colour and has a pH of about

4.0.

The lupine bean is a legume originating from the Mediterranean area, East Africa, and the Americas, where it was already grown as far back as 4,000 years ago. Today, however, it is grown in Australia, Europe, and South America for food purposes, both for human and animal diets, and the plant is also used as green manure (i.e. a crop grown to enrich the soil with nutrients which are synthesised by nitrogen-fixing bacteria found on the roots of the lupine plant). In addition to its beans, lupine can also be consumed in the form of a flour obtained by milling the dry beans.

From a nutritional viewpoint, lupine features a very high content of soluble and insoluble protein and fibre and a low carbohydrate and fat content. Lupine flour contains at least 40% protein, 31% fibre, 10% sugar, 10% fat, and various micronutrients. The fats contained in lupine beans are, for the most part, "good" fats, since they amount to 67% monounsaturated fatty acids, 19% polyunsaturated fatty acids, and only 14% saturated fatty acids. Lupine beans are a good source of thiamine (vitamin Bl) and folic acid (vitamin B9), as well as certain mineral salts, in particular, manganese, copper, magnesium, phosphorus, zinc, and potassium.

The first and most commonly known bio stimulating natural raw materials include algae and, in particular, algae extracts. They have been used in agriculture for hundreds of years as soil conditioners to enhance soil fertility. Just over 50 years ago, production began on liquid extracts to enhance the bio stimulating properties of algae. Today there are numerous biostimulant products in commerce which are based on algae extracts available on the market.

The extracts are obtained from green, red, or brown algae and more specifically Ascophyllum nodosum, Ecklonia maxima, Laminaria digitata, and Fucus spp.

The algae is harvested manually or mechanically along the ocean coasts and is washed, cut, and then undergoes extraction. Extraction can be performed in various ways and using solvents of various kinds. Techniques for the production of algae extracts have also been proposed which involve microbial fermentation of the plant-derived starter. The type of algae used, the harvesting period, and the extraction process greatly influence the chemical characteristics of the extract and therefore its biostimulating properties.

Currently, the most commonly used method consists of cold extraction by using high pressure water to prevent chemical alterations of the bioactive molecules.

Algae extracts act as biostimulants by enhancing germination speed, growth, fruit set, production, product quality, and resistance to environmental stress. Furthermore, the algae extracts increase the absorption of macro and micronutrients in various crops. The biostimulating effects are mainly due to the presence of phytohormones, polysaccharides, polyphenols, and other organic molecules. The phytohormones identified in algae extracts that stimulate plant growth are auxins, cytokinins, abscisic acid, gibberellins, etc.

Of particular interest is the presence of polysaccharides in the extracts, such as laminarin, alginates, and fucoidans, which have the effect of improving environmental stress tolerance in plants. For example, the repeated application of algae extracts increases tolerance of abiotic stresses such as drought, salinity, and extreme temperatures.

There are many mechanisms underlying the increased abiotic stress tolerance induced by repeated application of algae extracts and not all of these are fully known yet. These include increase in root development and above all increase in the root/aerial ratio, improved nutritional condition of the crop, better cell turgor, reduced free radical activity, and increased enzyme defence activity against oxidative stresses.

Under the applicative point of view, the positive effects of algae extracts are more marked in crops grown in low fertile soils and with application repeated during the crop cycle. Foliar application is generally preferable due to the lower doses required and the rapidity of action.

Strictly speaking, molasses is the by-product of the production process of sucrose (commonly known as sugar), which is obtained by extraction from beet ( Beta vulgaris ) in the Mediterranean basin and sugar cane ( Saccharum ojficinarum ) in particularly in Central and South American countries. In common terms, molasses is frequently, but improperly, used to mean other by-products or residues of extraction processes for obtaining sugars other than sucrose. At commercial level, the term 'black-strap molasses' means molasses in general, regardless of its origin. More specifically though, molasses alone refers to beet molasses, while black-strap molasses refers to cane molasses. Molasses is an example of a raw material, which is widely used on an industrial scale as a source of carbon in numerous microbiological processes and in animal feed. This raw material is a brown viscous liquid, which is obtained by concentrating the mother liquors left over after sucrose extraction, within a process involving beet being cut into strips or cane being subjected to pressure. In some cases, the molasses undergoes physical-chemical pre-treatments before being used. Depending on the process technology and in particular the parameters that affect the composition of the beet/cane (plant variety, seasonal factors, time that lapses between harvesting and extraction, etc.), the composition of molasses can vary considerably. Molasses obtained from beet and that obtained from cane have a similar total sugar content, but they differ specifically in the sucrose content and the content of inverted sugar, i.e. sucrose hydrolysed into glucose and fructose. With regards to the mineral salt content and growth factors, cane molasses has a higher content of vitamins, in particular, biotin content (1-3 pg/g), than beet molasses (0.04-0.15 pg/g). For this reason, beet molasses is frequently supplemented with cane molasses, regardless of the geographical area of production and therefore of transport costs, in order to increase the biotin levels in the microbial cultures. Other by-products often referred to in general - and wrongly - as molasses include other residues such as those from the extraction of sucrose from sorghum (50% sucrose content) and corn molasses or hydrol (sugar content: 50-60%), and mother liquors from the production of glucose from com starch. High-test molasses (15 to 35% sucrose and 40 to 60% inverted sugar), on the other hand, is not a residue or by-product of sucrose production but rather the main product of the process (without the sucrose crystallisation step) as it is directly obtained through evaporation of the cane juice and partial inversion with invertase. This product can also originate from the processing of citrus fruits.

In one specific case, the product can have the following characteristics:

Coriander ( Coriandrum sativum, L. 1753) or Chinese parsley or with the Spanish name cilantro, is an annual herbaceous plant from the Apiaceae (or Umbelliferae ) family. It belongs to the same family as cumin, dill, fennel, and parsley. Coriandrum is a Latin word used by Pliny (in his Naturalis Historia ) whose roots lie in the Greek word corys or korios (meaning 'cimicid', or 'stink bug') followed by the suffix -ander (meaning 'similar'), referring to the supposed similarity of the smell released by its unripe fruits or when its leaves are rubbed with that released by said insect.

In this case, coriander can be used as seed flour but the agricultural waste originating from the preparation thereof also offers a cost-effective active starting substrate, albeit with fewer properties. Seventeen components have been identified, which make up 91.84% of the residual essential oil of Egyptian coriander.

Trans-anethole has been identified as the main compound of spent coriander oil, measuring 29.29%, followed by linalool (20.06%), butanoic acid, esters of 2-methyl- ,2- methoxy-4-(2-propyl)phenyl (14.17%), estragon (10.25%), longifolene (6.82%), and carvacrol (5.1%)

Comparison of the constituents of green herbs, dry seeds, and secondary processing materials revealed that the main constituent of the oils of green herbs and dry seeds was linalool, while trans-anethole was found to be the main constituent oil of the waste fractions, followed by linalool. The natural raw material used can have the following characteristics:

'Cocoa' means the plant and beans of Theobroma cacao Linn, an arboreal plant from the Sterculiacee family grown in tropical and subtropical regions. In addition to cocoa paste, powder, and butter, various products are obtained from the processing of cocoa beans, which differ in terms of quantity and quality and include various by-products such as the shell, the pulp, the integuments, and the germ. Each year, these by-products include vast amounts of material that could be used for new production chains and could be of help to developing countries, where the largest cocoa producers are located. The international cocoa market is split in two, i.e. the producing countries, consisting of the Southern Hemisphere, and the processing and consumer countries, consisting of the Northern Hemisphere, in particular the Netherlands and North America. The undisputed leader among the producing countries is the Ivory Coast, which accounts for about 40% of the world's production, followed by Ghana and Indonesia. Next come Ecuador, Cameroon, Nigeria, and Brazil, which account for about 37% in total. The remaining 23% is divided between the producing countries in the tropical belt. In its production cycle, cocoa needs deep, clayey soils for growth and a temperature of between 25 °C and 35 °C. It also needs abundant rainfall. The cocoa tree reaches about 8 m in height, in the most extreme cases it can even reach 18 m, with green leaves and reddish flowers arranged in panicles. It produces about 40-50 fruits over the space of a year. An individual fruit weighs about 400-500 g and is yellow or red in colour when ripe and brown when dry. Inside the fruit is a white mucilaginous acidulous pulp containing the seeds, which are enveloped by an integument consisting of a film coating around the bean.

The species farmed belongs to the Teobroma Cacao genus and the most commercially widespread cultivars are the Criollo, Forastero, and Trinitario varieties. When ripe, they are picked from the tree using a hook knife. Immediately after being picked, the fruits are broken and the cocoa beans are spread on wooden pallets or on the ground on top of a layer of banana leaves, in order to facilitate the fermentation triggered by the yeasts. At the end of fermentation, green cocoa is obtained, which is then left to dry at ambient temperature until the humidity decreases. The beans are then roasted at a temperature of between 130 °C and 140 °C. Next, special machines detach the integument from the germ. By-products of the processing consist of the shells, the pulp, the integuments, and the germ. The shells are the outermost part of the fruit and are the most important by product. Research in Japan has shown that they have an anticariogenic action, attributable to the polyphenolic compounds and unsaturated fatty acids. Furthermore, the high mineral content makes the shells suitable for the production of soap (especially black soap) and detergents, while the potassium makes them good fertilisers. Mixed with other plant by-products, they can also be used for pig and sheep feed. During fermentation the pulp is removed. The following can be obtained from the pulp: pectin for jams, alcohol for industrial and hospital use, vinegar, and soft drinks. The pulp can be frozen and then used for the production of ice cream, to give yogurt flavour, but it is only marketed locally due to the high cost. A pigment used as a food colouring can be extracted from the integuments, i.e. polyflavone glucoside. Furthermore, antioxidants such as flavonoids can be extracted using solvents. The germ is removed to enhance the finished product. Its oil is extracted for use in the food and cosmetic industry. Summing up, by-products are obtained from cocoa processing that can be used in other fields and can also be useful for economic development. The amount of by-products that originate is considerable and these can be placed on the market and be used to develop new production chains. In the case described below, the waste product used was cocoa husks, exemplificative analysis of which identified the following characteristic components:

Olive residues consist of the solid residues (stones, skin films, parts of the flesh) left after the pressing of the olive paste. It represents 30-50% of the olives processed. Its composition is:

Oil: 5-10%

Water: 25-30%

Solid fraction (30% of which is the stone): 60-75%

The acidity of the oil extracted from the olive residues ranges from 15 to 80%; high acidity should be avoided as it complicates the adjustment process, making it more expensive. Acidity increases during the time spent at the oil mill due to hydrolysis caused by lipase, which is active in the presence of thO, and to auto-oxidation caused by contact with air and the considerable surface area. Anti-fermentation agents such as NaHS0₃ (2-3%) are not recommended because they cause drawbacks upon extraction. The best preservation is achieved by drying in special rotary ovens, which are generally fed with olive residues; however, these ovens must never be used to obtain anhydrous olive residues because the cell membranes would be denatured and, as they would form an impermeable layer, the oil could not be extracted. The optimal humidity value is 7% and in industrial practice it is checked by listening to the noise produced by squeezing a handful of olive residues in one's hand. If the humidity is higher, the lipase may cause an exothermic reaction which can also lead to fires due to self-combustion of the stationary product awaiting extraction.

Whisk extraction process (no longer used).

This is a physical-mechanical process during which the re-milled olive residues are thrown into a vat of water. The light parts of the olive processing residues rise to the surface, while the stones fall to the bottom and are discarded. An agitator (whisk) is used to promote separation of the oil released from the processing residues as a result of washing in a series of cascade tanks. Just over half the oil is recovered.

Particularly preferred embodiments are those wherein said hydrolysate from a natural raw material is hydrolysate of com steep liquor.

Preferably, said hydrolysate from a natural raw material is:

- hydrolysate of corn steep liquor, comprising 6-16% of free amino acids and up to 53% dry residue,

- hydrolysate of lupine, comprising 7-20% free amino acids and up to 30% dry residue,

- hydrolysate of algae, comprising 6-8% free amino acids and up to 10% dry residue,

- hydrolysate of molasses, comprising 6-8% free amino acids and up to 55% dry residue,

- hydrolysate of coriander, comprising 1-5% free amino acids and up to 3% dry residue,

- hydrolysate of cocoa, comprising 1-2% free amino acids and up to 3% dry residue,

- hydrolysate of olive residues, comprising 6-8% free amino acids and up to 55% dry residue, or a combination thereof.

In another aspect, the present invention relates to a process for preparing said hydrolysate from a natural raw material, comprising the following steps: i) providing a natural raw material, and optionally adding water, ii) adding an enzyme complex comprising at least one peptide bond hydrolase and at least one fibre and/or carbohydrate hydrolase, and iii) obtaining a liquid mixture, i.e. the hydrolysate from a natural raw material. Preferably, in step i) water is added to the natural raw material.

Preferably, in step ii) the temperature is kept at 4-70 °C for a period of 2-20 hours, more preferably the temperature is kept at 10-50 °C for a period of 4-16 hours.

Examples of enzymes which act on the protein component: for example, enzymes in class EC 3.4, i.e. enzymes that act on the peptide bond, such as, for example, mono-, di- , or tripeptidases, or mixtures thereof, proteases, and proteinases.

Examples of enzymes which act on fibres and/or carbohydrates: for example, enzymes in class EC 3.2 - Glycosidases, i.e. enzymes that act on O- and S-glycosyl compounds or N-glycosyl compounds, such as, for example, amylases, cellulases, hemicellulases, laccases, and xylanases.

Examples of enzymes which act on lipids: for example, enzymes in class EC 3.1, i.e. enzymes that act on the ester bond, such as, for example, lipases.

Preferably, in said enzymatic complex, the hydrolase is selected from mono-, di-, or tripeptidases, proteases, proteinases, cellulases, hemicellulases, pectinases, xylanases, amylases, laccases, lipases, and mixtures thereof.

The enzymatic complex comprises a mixture of at least two hydrolases, i.e. at least one hydrolase which acts on fibres and/or carbohydrates and at least one hydrolase which acts on the protein component. Indeed, it has been observed that this mixture acts synergistically, allowing to achieve surprising results as compared to the use of a single enzyme.

Preferably in step ii), and in any case in compliance with the pH activity range of the specific enzyme, a pH adjuster is added, preferably an organic or inorganic base, such as NaOH or KOH, to adjust the pH to about 6. In some cases, an inorganic or organic acid, such as phosphoric acid, can also be used in order to reach the optimal reaction pH of the specific enzymatic mix.

According to a first particularly preferred preparation procedure, the natural raw material was treated with an anti-fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours. In the case of raw materials with a lipid part, such as olive residues, this step also included the addition of EC 3.1 enzymes, i.e. enzymes that act on the ester bond, such as lipases.

According to a second particularly preferred preparation procedure, the natural raw material was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. In the case of raw materials with a lipid part, such as olive residues, this step also included the addition of EC 3.1 enzymes, i.e. enzymes that act on the ester bond, such as lipases. Subsequently, KOH was added to bring the pH up to 5.5-6.

In another aspect, the present invention relates to a hydrolysate from a natural raw material obtainable by the process described above, wherein said plant raw material is selected from com steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and combinations thereof, in which the percentage of free amino acids in the hydrolysate is at least 0.5% higher than the percentage in the starting plant raw material, and in which the dry residue in the hydrolysate is at least 2% less than the percentage in the starting plant raw material.

"%" or "percentage" means "g / 100 ml".

Preferably, said hydrolysate from a natural raw material obtainable by the process described above is: - hydrolysate of com steep liquor, in which the percentage of free amino acids in the hydrolysate is 0.5-11% higher than the percentage in the starting corn steep liquor, and in which the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting com steep liquor,

- hydrolysate of lupine, in which the percentage of free amino acids in the hydrolysate is 2-15% higher than the percentage in the starting lupine, and in which the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting lupine,

- hydrolysate of algae, in which the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting algae, and in which the dry residue in the hydrolysate is 2-4% lower than the percentage in the starting algae,

- hydrolysate of molasses, in which the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting molasses, and in which the dry residue in the hydrolysate is 10-20% lower than the percentage in the starting molasses,

- hydrolysate of coriander, in which the percentage of free amino acids in the hydrolysate is 0.5-4.5% higher than the percentage in the starting coriander, and in which the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting coriander,

- hydrolysate of cocoa, in which the percentage of free amino acids in the hydrolysate is 0.5-1.5% higher than the percentage in the starting cocoa, and in which the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting cocoa,

- hydrolysate of olive residues, in which the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting olive residue, and in which the dry residue in the hydrolysate is 5-10% lower than the percentage in the starting olive residue, or a combination thereof.

In another aspect, the present invention also relates to the use of said hydrolysate from a natural raw material to promote plant growth and fmit production in agriculture, or for biocontrol.

In particular, this hydrolysate should preferably be used at a rate of 0.5-50 litres per hectare of soil if liquid, or in equivalent proportions for other liquid or solid formulations, such as, for example, solid granules which may be either dispersible in water or non-dispersible in water.

In a further aspect, the present invention relates to a composition comprising a microbial inoculant and hydrolysate from a natural raw material as described above, in which said microbial inoculant is selected from a bacterial inoculant, a fungal inoculant, or a combination thereof.

Microbial inoculants, also known as soil inoculants or bio-inoculants, are agricultural remedies that use beneficial rhizospheric or endophytic microbes to promote plant health. Many of the microbes involved form symbiotic relationships with the target crops which are beneficial for both parties (mutualism). While microbial inoculants are applied to improve plant nutrition, they can also be used to promote plant growth by stimulating the production of plant hormones. When added to seeds, growth media, or leaves, microbial inoculants have been shown to be useful in all crops, both in open fields and in greenhouses. Microbial inoculants can also be used to initiate systemic acquired resistance (SAR) in plant species to several common crop diseases.

As mentioned, the microbial inoculant is chosen from a bacterial inoculant, a fungal inoculant, or a combination thereof.

Preferred genera of bacterial micro-organisms are Azospirillum, Rhizobium, Bacillus, Pseudomonas, Streptomyces, Zooglia, Agrobacterium, and combinations thereof. Preferably, the bacterial inoculant belongs to one of the following species: Bacillus subtilis, Bacillus megaterium, Bacillus velezensis, Bacillus amyloliquefaciens, Azotobacter vinelandi, Azospirillum brasilense, Ensifer meliloti, Pseudomonas vancouverensis, Paenibacillus polymyxa, or combinations thereof.

For non-leguminous crops, Azospirillum has been shown to be beneficial for nitrogen fixation and plant nutrition.. Rhizobium is a genus of soil bacteria that fix nitrogen and form symbiotic associations within the nodules on the roots of pulses. This increases nitrogen nutrition and is important for the cultivation of soybeans, chickpeas, and many other pulses. Bacillus, Pseudomonas and Streptomyces provide some, if not all, of the following benefits: increased plant growth, decomposition of organic matter and pesticide residues, increased nutrient cycle and nitrogen fixation, increased resistance to biotic and abiotic stresses, greater solubility of meso- and microelements, greater production of natural hormones which are useful for plant growth, better soil structure, and better germination and vitality of seeds. Furthermore, to improve the bioavailability of phosphorus, phosphate solubilising bacteria (PSB) are also used, which act on the inorganic phosphates in the soil, transforming said phosphates into simpler forms that allow the absorption thereof by plants.

In other preferred embodiments, the composition according to the invention comprises forms of micro-organisms in a concentration of said bacterial inoculant of lxlO⁵ CFU/ml to lxlO¹⁰ CFU/ml.

Preferred genera of fungal micro-organisms are ascomycetes and basidiomycetes. Preferably, the fungal inoculant belongs to the species Trichoderma asperellum, Trichoderma longibrachiatum, Metarhizium anisopliae, Pochonia chlamidospora, and combinations thereof. These fungi provide many of the same plant health benefits as those offered by the above bacteria, including increasing the plant's resistance to environmental stresses and the production of natural hormones.

In other preferred embodiments, the composition according to the invention comprises forms of micro-organisms in a concentration of said fungal inoculant of lxlO⁵ CFU/ml to lxlO¹⁰ CFU/ml.

In preferred embodiments, said microbial inoculant comprises up to five different micro-organisms .

More preferably, said microbial inoculant comprises Bacillus subtilis, Bacillus megaterium, Bacillus velezensis, Bacillus amyloliquefaciens, Azotobacter vinelandi, Azospirillum brasilense, Ensifer meliloti, Pseudomonas vancouverensis, or a combination thereof.

In other embodiments, the composition consists essentially of a hydrolysed microbial inoculant of natural raw material, as described above. For the purposes of the present invention, the expression " consists essentially of" means that said microbial inoculant and said corn steep liquor hydrolysate are the only active ingredients that act as plant growth and fruit production promoters present in the compositions, since the other possible components show different activities which do not interfere with those of the microbial and hydrolysed inoculant, or are simply co-formulants.

In still further embodiments, the composition consists of a hydrolysed microbial inoculant of natural raw material, as described above.

Preferably, the composition according to the invention further comprises corn steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and combinations thereof.

Optionally, the composition according to the invention further comprises additional ingredients such as glycerol, humates (from humic acid), fulvates (from fulvic acid), acetic acid, propionic acid, citric acid, lactic acid, or combinations thereof.

Optionally, the composition according to the invention further comprises additional ingredients such as botanical extracts, fermented plant extracts, or combinations thereof. Optionally, the composition according to the invention further comprises additional ingredients such as amino acids. Amino acids can act as an energy source to increase plant metabolism and improve plant nutrient absorption. Said additional amino acids may be tryptophan, asparagine, glutamine, glycine, selenocysteine, serine, ornithine, taurine, or combinations thereof.

Optionally, the composition according to the invention further includes additional ingredients such as vitamins and minerals. Vitamins act as catalysts for beneficial enzymes and improve plant metabolism. Folic acid and biotin (two components of the vitamin B complex) also work to improve microbial and plant growth. These vitamins and minerals may be boron, copper, iron, manganese, zinc, molybdenum, chlorine, phosphorus, potassium, calcium, magnesium, sulphur, or combinations thereof. Optionally, the composition according to the invention further comprises additional ingredients such as carbohydrates. Said carbohydrates may be glucose, galactose, galactose, fructose, arabinose, xylose, sucrose, lactose, maltose, amylose, amylopectin, glycogen, glyceraldehyde, ribose, or combinations thereof.

Optionally, the composition according to the invention further comprises additional ingredients such as enzymes. Said enzymes may be phospholipases, lipases, proteases, amylases, cellulases, catalases, laccases, or combinations thereof.

Preferably, the composition may be applied at a rate of 0.5-50 litres/hectare, more preferably with periodic treatments, depending on the crop and the season.

In another aspect, the present invention relates to an agrochemical product comprising the composition and agrochemical additives.

Suitable additives include pH regulators, acidity regulators, water hardness regulators, mineral oils, plant oils, fertilisers, natural leaf fertilisers, and combinations thereof.

It should be understood that all the possible combinations of the preferred aspects of the components of the composition are described herein and therefore are also preferred, as stated above.

It should also be understood that all the aspects identified as preferred and advantageous for the composition and its components are to be considered similarly preferred and advantageous also for the preparation and uses of said composition. Below are working examples of the present invention provided for illustrative purposes. EXAMPLES

Example 1.

Preparation of the hydrolysates according to the invention

The natural raw material was analysed to establish the content of organic nitrogen and starches and fibres, in order to assess what kind of enzymatic digestion of the components performs best. On the basis of the results, different hydrolases which act on the components were tested, deciding on the amounts required according to the composition.

Examples of enzymes which act on fibres and carbohydrates: for example, enzymes in class EC 3.2 - Glycosidases, i.e. enzymes that act on O- and S-glycosyl compounds or N-glycosyl compounds, such as, for example, amylase, cellulase and xylanase. Each hydrolase was used within the optimum pH and temperature ranges, by developing different processes which could all lead to the hydrolysis of the different components present, regardless of the pathway.

1A) Hydrolysis of CSL

For the hydrolysis of CSL, one of the enzyme mixtures that was used effectively comprised: The formulation is as follows:

According to a first preparation procedure, the CSL was treated with an anti fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours.

According, meanwhile, to a second preparation procedure, the CSL was treated with an anti-fermentation agent and a mixture of enzymes EC 3.2 and 3.4 for 6 hours at about 52 °C and at pH about 4. Subsequently, KOH was added to bring the pH up to 5.5-6. Amino acid analysis showed a general increase in free amino acids of between 0.5 and 11% and, more specifically, that these are the amino acids which become more bioavailable.

Said results were produced when the class 3.4 enzymes (which act on the peptide bond) were used both alone and in a mixture with enzymes from the other classes. The following is a specific example of total CSL analysis before and after hydrolysis, in which, more specifically, the free amino acids using Process 1 reach 7%. Following treatment with enzymes from class 3.2 (which act on fibres and complex carbohydrates), the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased:

IB) Hydrolysis of lupine

For the hydrolysis of lupine, one of the enzyme mixtures that was used effectively comprised: The formulation is as follows:

According to a first preparation procedure, the lupine was treated with an anti fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours.

According, however, to a second preparation procedure, the lupine was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6.

Following treatment with enzymes from class 3.2 (which act on fibres and complex carbohydrates), the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes.

Amino acid analysis showed a general increase in free amino acids of 2 to 15%.

Said results were produced when the class 3.4 enzymes (which act on the peptide bond) were used both alone and in a mixture with enzymes from the other classes.

1C) Hydrolysis of Ascophyllum algae

For the hydrolysis of algae, one of the enzyme mixtures that was used effectively comprised: The formulation is as follows:

According to a first preparation procedure, the algae is treated with an anti-fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours.

According, however, to a second preparation procedure, the algae was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6. Following treatment with enzymes from class 3.2 (which act on fibres and complex carbohydrates), the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes.

Amino acid analysis showed a general increase in free amino acids of 4 to 6%.

Said results were produced when the class 3.4 enzymes (which act on the peptide bond) were used both alone and in a mixture with enzymes from the other classes. ID) Hydrolysis of molasses

For the hydrolysis of molasses, one of the enzyme mixtures that was used effectively comprised:

The formulation is as follows: According to a first preparation procedure, the molasses was treated with an anti fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours. According, however, to a second preparation procedure, the CSL was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6. Following treatment with enzymes from class 3.2 (which act on fibres and complex carbohydrates), the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes.

Amino acid analysis showed a general increase in free amino acids of 4 to 6%. Said results were produced when the class 3.4 enzymes (which act on the peptide bond) were used both alone and in a mixture with enzymes from the other classes.

IE) Hydrolysis of coriander

For the hydrolysis of coriander, one of the enzyme mixtures that was used effectively comprised: The formulation is as follows:

According to a first preparation procedure, the coriander was treated with an anti fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours.

According, however, to a second preparation procedure, the coriander was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6.

Following treatment with enzymes from class 3.2, which act on fibres and complex carbohydrates, the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes Amino acid analysis showed a general increase in free amino acids of 0.5 to 4.5%.

IF) Hydrolysis of cocoa

For the hydrolysis of cocoa, one of the enzyme mixtures that was used effectively comprised:

The formulation is as follows:

According to a first preparation procedure, the cocoa was treated with an anti- fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC 3.2 and 3.4 enzymes for a further 6 hours.

According, however, to a second preparation procedure, the cocoa was treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6.

Following treatment with enzymes from class 3.2, which act on fibres and complex carbohydrates, the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes.

Amino acid analysis showed a general increase in free amino acids of 0.5 to 1.5%.

1G1 Hydrolysis of olive residues For the hydrolysis of olive residues, one of the enzyme mixtures that was used effectively comprised:

According to a first preparation procedure, the olive residues were treated with an anti fermentation agent and a mixture of EC 3.2 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 6, followed by a mixture of EC enzymes 3.2 and 3.4 and 3.1 for a further 6 hours.

According, however, to a second preparation procedure, the olive residues were treated with an anti-fermentation agent and a mixture of EC 3.2 and E 3.4 enzymes for 6 hours at about 52 °C and at about pH 4. Subsequently, KOH was added to bring the pH up to 5.5-6.

Following treatment with enzymes from class 3.2, which act on fibres and complex carbohydrates, the dry residue - which indicates the presence of insoluble material - decreased and the amount of polysaccharides or simple sugars present in the mixture increased. Viscosity decreased and, probably due to this, the protein part became more available for the action of proteolytic enzymes

Example 2.

Hydrolysates of CSL and efficacy test A greenhouse test was carried out on cyclamens; 18 plants were tested for each test mixture. Each text mixture was treated in fertigation, with about 25 ml per plant, corresponding to the doses/hectare specified below. The treatment was carried out three times over three weeks, one treatment per week. The following fertigation protocol was applied 45PPM N - NPK (1:0.7: 1.5) - 400ML/PLANT x DAY (3 IRR.) - PH 7 1. CSL - 2.5 1/ha 2. CSL hydrolysed solely with enzymes that act on carbohydrates - 2.5 1/ha

3. CSL hydrolysed solely with enzymes that act on the protein fraction - 2.5 1/ha

4. CSL hydrolysed with both enzymes - 2.51/ha

The hydrolysate preparation methods used were the same as for Example 1.

Example 3.

Hydrolysates of lupine and efficacy test

A greenhouse test was carried out on cyclamens; 18 plants were tested for each test mixture. Each text mixture was treated in fertigation, with about 25 ml per plant, corresponding to the doses/hectare specified below. The treatment was carried out three times over three weeks, one treatment per week. The following fertigation protocol was applied 45PPM N - NPK (1:0.7: 1.5) - 400ML/PLANT x DAY (3 IRR.) - PH 7

1. lupine - 2.5 1/ha

2. lupine hydrolysed solely with enzymes that act on carbohydrates - 2.5 1/ha 3. lupine hydrolysed solely with enzymes that act on the protein fraction - 2.51/ha

4. lupine hydrolysed with both enzymes - 2.51/ha

The hydrolysate preparation methods used were the same as for Example 1. Lupin that ac

Lupin that ac

Lupin enzym

Example 4.

Hydrolysates of Ascophyllume algae and efficacy test

A greenhouse test was carried out on cyclamens; 18 plants were tested for each test mixture. Each text mixture was treated in fertigation, with about 25 ml per plant, corresponding to the doses/hectare specified below. The treatment was carried out three times over three weeks, one treatment per week. The following fertigation protocol was applied 45PPM N - NPK (1:0.7: 1.5) - 400ML/PLANT x DAY (3 IRR.) - PH 7 1. algae extract - 2.51/ha 2. algae extract hydrolysed solely with enzymes that act on carbohydrates - 2.5 1/ha

3. algae extract hydrolysed solely with enzymes that act on the protein fraction - 2.51/ha

4. algae extract hydrolysed with both enzymes - 2.51/ha f Average wet j Average dry j Increase _% f Increase _% weight /plant weight /plant j wet WEIGHT dry WEIGHT

Example 5. Synergistic mixture of hydrolysate of CSL, molasses, and algae

A greenhouse test was carried out on cyclamens; 18 plants were tested for each test mixture. Each text mixture was treated in fertigation, with about 25 ml per plant, corresponding to the doses/hectare specified below. The treatment was carried out three times over three weeks, one treatment per week. The following fertigation protocol was applied. 45PPM N - NPK (1:0.7:1,5) - 400ML/PLANT x DAY (3 IRR.) - PH 7 1. CSL hydrolysed with both enzymes - 2.51/ha

2. Algae extract hydrolysed with both categories of enzyme - 2.5 1/ha

3. molasses - 2.51/ha

4. algae extract - 2.51/ha

5. CSL hydrolysed with both enzymes in dose reduced to 33% 6. Algae extract hydrolysed with both categories of enzyme in dose reduced to 33%

7. Molasses in dose reduced to 33%

8. 33% hydrolysed CSL + 33% hydrolysed algae + 33% molasses

Example 6. Synergistic mixture of hydrolysate of lupine, molasses, and algae A greenhouse test was carried out on cyclamens; 18 plants were tested for each test mixture. Each text mixture was treated in fertigation, with about 25 ml per plant, corresponding to the doses/hectare specified below. The treatment was carried out three times over three weeks, one treatment per week. The following fertigation protocol was applied: 45PPM N - NPK (1 :0.7: 1.5) - 400ML/PLANT xD A Y (3 IRR.) - PH 7

1. Lupine hydrolysed with both enzymes - 2.5 1/ha

2. Algae extract hydrolysed with both categories of enzyme - 2.5 1/ha

3. molasses - 2.5 1/ha

4. algae extract - 2.5 1/ha 5. Lupine hydrolysed with both enzymes in dose reduced to 33%

6. Algae extract hydrolysed with both categories of enzyme in dose reduced to 33%

7. Molasses in dose reduced to 33%

8. 33% hydrolysed lupine + 33% hydrolysed algae + 33% molasses

Average wet Average dry j Increase % Increase % weight /plant weight /plant j wet WEIGHT dry WEIGHT Example 7.

Synergistic mixture of hydrolysed lupine and coriander

The test was performed on greenhouse tomatoes subjected to SALINE STRESS CROP: "CUORBENGA" OXHEART TOMATO PLACE: GREENHOUSE (AUTUMN)

TREATMENTS, DOSE: The compounds and the hydrolysates were diluted within a range of 1:100 to 1:2000. 16 PLANTS/TEST MIXTURES were used and the saline stress was provided by means of a WATER + NACL 300 mm solution IN A SAUCER, to which the biostimulant mixtures were added during the treatment. TREATMENTS, FREQUENCY: IRRIGATIONS WITH 2.5 1/SAUCER

TREATMENTS, NUMBER: 2 (ONE PER WEEK)

TREATMENTS, APPLICATION: ROOTS (SAUCER)

PLANTS, purchased from a NURSERY, were REPOTTED IN PEAT + PERLITE POTS IRRIGATION was performed WITH 2.5L NACL 300 mm (TAP WATER) +

NPK (N 50ppm, 1:0.5:1.7) - The growth temperature was the following: 15-25 °C measured in the GREENHOUSE

At the end of the test, the wet weight and dry weight of each plant and the total weight for each test mixture were assessed. From the results listed below it is clear that the plants in which the two compounds were mixed were more vigorous than those where a single compound was used, which indicates a greater ability to overcome the strong saline stress caused.

Example 8.

Efficacy of hydrolysates of algae, coriander, lupine, and molasses on tomato

The test was carried out on stressed tomatoes in a climatic growth chamber, on 5-week- old seedlings transplanted into a soil composed of 40% sand.

CROP: "CUORBENGA" OXHEART TOMATO

PLACE: Climatic test chamber

TREATMENTS, DOSE: 10 litres/hectare

TREATMENTS , FREQUENCY: once a week TREATMENTS, NUMBER: 4 weeks

TREATMENTS, APPLICATION: ROOTS (SAUCER)

At the end of the test, the seedlings were phenotyped according to the height of the aerial part and root development.

The results are summarised in the following table: The results show how the hydrolysates according to formulations such as Ascophyllum algae, hydrolysed coriander, hydrolysed lupine, hydrolysed molasses and hydrolysed CSL, give significant results in phenotyping, confirming the quality of the process developed and the effectiveness of the products produced in accordance with the claims.

Example 9.

Efficacy test of hydrolysates of Ascophyllum algae, cocoa, hydrolysed cocoa, hydrolysed molasses, and hydrolysed olive residues

The test was carried out on stressed tomatoes in a climatic growth chamber, on 2- month-old seedlings transplanted into a growth medium made up of 40% sand.

CROP: "CUORBENGA" OXHEART TOMATO

PLACE: Climatic test chamber

TREATMENTS, DOSE: 15 litres/hectare

TREATMENTS, FREQUENCY : once upon repotting, once one week later TREATMENTS, NUMBER: 2 weeks

TREATMENTS, APPLICATION: ROOTS (SAUCER)

At the end of the test, the seedlings were phenotyped according to the height of the aerial part and root development. The aerial part showed no significant variations, while the root part, showed the following significant results in sandy growth medium. The positive results of root phenotyping are summarised in the table below: The results show how the hydrolysates according to formulations such as Ascophyllum algae, cocoa, and hydrolysed cocoa, hydrolysed molasses and hydrolysed olive residues, give significant results in phenotyping, confirming the quality of the process developed and the effectiveness of the products produced according to the claims.

Example 10.

Association of hydrolysed CSL and micro-organisms

The results of a study on leaf physiology are reported here in relation to plots of com, the seeds of which were treated using different experimental coating processes.

To evaluate the effect of the composition according to the invention, the corn field was divided as follows:

- Area A, cultivated with seeds coated with hydrolysed CSL (Ex. 1A)

Zone al : dose: 4 g/kg seed Zone a2: dose: 16 g/kg seed Zone a2: dose: 20 g/kg seed

- Area B, cultivated with seeds coated with Trichoderma asperellum

Zone bl : dose: 4 g/kg seed Zone b2: dose: 16 g/kg seed Zone b3: dose: 20 g/kg seed

- Area C, cultivated with seeds coated with Trichoderma asperellum + hydrolysed CSL (Ex.lA)

Zone cl: dose: 4 g/kg CSL + 16 g/kg Trichoderma asperellum Zone c2: dose: 16 g/kg Trichoderma asperellum + 4 g/kg CSL -Area D, Azospirillum brasilense

Zone dl : dose: 4 g/kg seed Zone d2: dose: 16 g/kg seed Zone d3 : dose: 20 g/kg seed

- Area E, cultivated with seeds coated with Azospirillum brasilense + hydrolysed CSL (Ex. 1A)

Zone el: dose: 4 g/kg CSL + 16 g/kg Azo spirillum brasilense Zone e2: dose: 16 g/kg Azo spirillum brasilense + 4 g/kg CSL

- Area L, cultivated with seeds coated with Bacillus megaterium, Zone/ : dose: 4 g/kg seed Zone/2: dose: 16 g/kg seed Zona f3: dose 20 g/kg seme

- Area G, cultivated with seeds coated with Bacillus megaterium + hydrolysed CSL (Ex. 1A)

Zone gl : dose: 4 g/kg CSL + 16 g/kg Bacillus megaterium Zone g2: dose: 16 g/kg Bacillus megaterium, + 4 g/kg CSL

- Area H, cultivated with seeds coated with Glomus intraradices

Zone hi : dose: 4 g/kg seed Zone h2 : dose: 16 g/kg seed Zone /z5: dose: 20 g/kg seed

-Area I, cultivated with seeds coated with Glomus intraradices + hydrolysed CSL (Ex.lA)

Zone il: dose: 4 g/kg CSL + 16 g/kg Glomus intraradices Zone z2: dose: 16 g/kg Glomus intraradices + 4 g/kg CSL

- Area L, cultivated with seeds coated with a combination of Trichoderma asperellum, Azospirillum brasilense, Bacillus megaterium, Glomus intraradices + hydrolysed CSL (Ex. 1A)

Zone 11: dose: 4 g/kg Trichoderma asperellum seed, + 4 g/kg Azospirillum brasilense seed, + 4 g/kg Bacillus megaterium seed + 4 g/kg Glomus intraradices seed

Zone 12: dose: 4 g/kg Trichoderma asperellum seed, + 4 g/kg Azospirillum brasilense seed, + 4 g/kg Bacillus megaterium seed + 4 g/kg Glomus intraradices seed + 4 g/kg CSL

- NT area, cultivated with untreated seeds.

The microbial cultures used were brought to a concentration of 1c10^L8 CLU/ml, apart from the Glomus intraradices, which were employed in a concentration of 500 spores/g. 150 kg/ha of N, in the form of urea, was distributed in coverage over the study areas. No irrigation was necessary during the growth cycle.

The data was collected at three seasonal measurement times, namely at stem elongation, at flowering, and at the ripening of the cobs (hard dough stage).

Leaf physiology Plant physiology was measured directly in the field by choosing 10 treated plants per area at random. The measurements were taken using a leaf which had been exposed to the sun and was located on the stalk above the highest cob.

Two portable instruments were used: 1- Fluorimeter (model OS5p), i.e. an instrument featuring an LED lamp which emits light radiation at different frequencies and with different intensities in order to verify two parameters:

- Fv/Fm (known as maximum photosynthetic efficiency), whose values oscillate within a range of between 0 to 1; it is measured on dark-adapted leaves and shows how promptly a leaf responds to light radiation (Baker N. et ah, 2004).

- ETR (Electron Transport Rate) is measured on leaves exposed to light and shows the efficiency of Photosystem II (Genty B. et ah, 1989).

2- Portable gas exchange analyser (ADC pro-SD model), i.e. an instrument that measures changes in the concentration of carbon dioxide caused by photosynthetic activity through the "Assimilation rate" parameter (A).

As regards leaf physiology, starting from the parameters measured in the field, the photosynthetic efficiency (given by the average of several leaves) was calculated using the ratio between ETR (Electron Transport Rate) and A (Assimilation rate). This ratio expresses the number of pmoles of electrons required by PII (Photosystem II) to organise one pmole of CO2.

Results

The leaf ecophysiology measured in the field shows appreciable differences in values in plants from different areas.

The stomatal conductance (gs), which was correlated with the photosynthesis activity, confirmed that the plants were not in water stress conditions. Example 11.

Biocontrol of Fusarium oxysporum cubense Test method

The tests were performed on PCA (0.5% peptone and 0.25% yeast extract) for bacteria, bacteria with hydrolysed citrus molasses, and hydrolysed citrus molasses alone, and on PDA for fungi, fungi with hydrolysed citrus molasses, and hydrolysed citrus molasses alone. As regards the fungal cultures, the fungal cultures supplemented with hydrolysed citrus molasses, and the hydrolysed citrus molasses alone, the dual tests were performed placing the F. oxysporum strain on one side of the petri dish on the surface of the growth medium while the strain or product for which the biocontrol activity was to be assessed was placed on the other side. The surface of the dish occupied by F. oxysporum was measured at 10 days of growth at 27 °C. The percentage of inhibition was calculated using the ratio between the surface area occupied by F. oxysporum in the presence of the test strains and the surface area occupied by F. oxysporum grown in the absence of a biocontrol agent. Tests were performed with the bacterial cultures alone, with the bacterial cultures supplemented with hydrolysed citrus molasses, and with hydrolysed citrus molasses alone by placing the phytopathogen in the centre of the petri dish on the surface of the growth medium, while the strain or product for which the biocontrol activity was to be assessed was streaked onto the two opposite sides of the dish. The surface area of the dish occupied by F. oxysporum was measured at 10 days of growth at 27 °C and the percentage of inhibition calculated as described earlier. Concentration of use of bacterial cultures: 1c10^L9 CFU/ml Concentration of use of fungal cultures: 5c10^L7 CFU/ml Hydrolysed citrus molasses/microbial culture mixture ratio: 50%:50% Product dose per dish: 0.1 g/dish Results

The table below shows the results for the strains that have a biocontrol activity against Fusarium oxysporum cubense (FOC)

Compound/strain Inhibition

(%)

Hydrolysed citrus molasses 15

50

Bacillus subtilis + hydrolysed citrus molasses 81

Bacillus velezensis 67 Bacillus velezensis + hydrolysed citrus molasses 93

Pseudomonas vancouverensis 48

Pseudomonas vancouverensis + hydrolysed citrus 88 molasses

Trichoderma asperellum 68

Trichoderma asperellum + hydrolysed citrus molasses 91

Example 12.

Botrytis cinerea biocontrol Test method Tests were performed on PCA (0.5% peptone and 0.25% yeast extract) for bacteria, bacteria with hydrolysed algae extract, and hydrolysed algae extract alone and on PDA for fungi, fungi with hydrolysed algae extract, and hydrolysed algae extract alone. As regards the fungal cultures, the fungal cultures supplemented with hydrolysed algae extract, and the hydrolysed algae extract alone, the dual tests were performed placing the Botrytis cinerea strain on one side of the petri dish on the surface of the growth medium while the strain or product for which the biocontrol activity was to be assessed was placed on the other side. The surface of the dish occupied by Botrytis cinerea was measured at 10 days of growth at 27 °C. The percentage of inhibition was calculated using the ratio between the surface area occupied by Botrytis cinerea in the presence of the test strains and the surface area occupied by Botrytis cinerea grown in the absence of a biocontrol agent. Tests were performed with the bacterial cultures alone, with the bacterial cultures supplemented with hydrolysed algae extracts, and with hydrolysed algae extracts alone by placing the phytopathogen in the centre of the petri dish on the surface of the growth medium, while the strain or product for which the biocontrol activity was to be assessed was streaked onto the two opposite sides of the dish. Furthermore, commercially available products Serenade® and Amylo-x® products were also tested for comparison purposes. The surface area of the dish occupied by Botrytis cinerea was measured at 10 days of growth at 27 °C and the percentage of inhibition calculated as described earlier. Concentration of use of bacterial cultures: 1c10^L9 CFU/ml Concentration of use of fungal cultures: 5c10^L7 CFU/ml Hydrolysed algae extract/microbial culture mixture ratio: 50%:50%

Product dose per dish: 0.1 g/dish Results

The table below shows the results for the strains that have a biocontrol activity against

Botrytis cinerea.

Compound/strain Inhibition

Hydrolysed algae extract 12

Serenade 77

Bacillus subtilis 68

Bacillus subtilis + hydrolysed algae extract 85

Amylo-x 71

Streptomices griseus 70

Streptomices griseus + hydrolysed algae 88 extract

Example 13. Biocontrol of Fusarium fujikuroi

The tests were performed on PC A (0.5% peptone and 0.25% yeast extract) for bacteria, bacteria with polyphenols, and polyphenols alone, and on PDA for fungi, fungi with polyphenols, and polyphenols alone. As regards the fungal cultures, the fungal cultures supplemented with polyphenols, and the polyphenols alone, the dual tests were performed placing the F. fujikuroi strain on one side of the petri dish on the surface of the growth medium while the strain or product for which the biocontrol activity was to be assessed was placed on the other side. The surface of the dish occupied by F. fujikuroi was measured at 10 days of growth at 27 °C. The percentage of inhibition was calculated using the ratio between the surface area occupied by F. fujikuroi in the presence of the test strains and the surface area occupied by F. fujikuroi grown in the absence of a biocontrol agent. Tests were performed with the bacterial cultures alone, with the bacterial cultures supplemented with polyphenols, and with polyphenols alone by placing the phytopathogen in the centre of the petri dish on the surface of the growth medium, while the strain or product for which the biocontrol activity was to be assessed was streaked onto the two opposite sides of the dish. The polyphenols used derive from various plant sources, such as hydrolysate of olive residues, extracts of Vitis vinifera, or derivatives of lignocellulose. The surface area of the dish occupied by F. fujikuroi was measured at 10 days of growth at 27 °C and the percentage of inhibition calculated as described earlier.

Concentration of use of bacterial cultures: 1c10^L9 CFU/ml Concentration of use of fungal cultures: 5c10^L7 CFU/ml Polyphenol/microbial culture mixture ratio: 50%:50%

Product dose per dish: 0.1 g/dish

Results

The table below shows the results for the strains that have a biocontrol activity against Fusarium oxysporum cubense (FOC).

Compound/strain Inhibition (%)

Polyphenols 20

Paenibacillus polymyxa 54

Paenibacillus polymyxa + 81 polyphenols

Bacillus velezensis 47

Bacillus velezensis + polyphenols 82

Trichoderma asperellum 70

Trichoderma asperellum + 94 polyphenols

Claims

1. Hydrolysate from a natural raw material comprising 1-25% of free amino acids and up to 55% of dry residue, wherein said natural raw material is selected from corn steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and their combinations.

2. The hydrolysate of claim 1, wherein said hydrolysate from natural raw material is:

- hydrolysate of corn steep liquor, comprising 6-16% of free amino acids and up to 53% of dry residue,

- hydrolysate of lupine, comprising 7-20% of free amino acids and up to 30% of dry residue,

- hydrolysate of algae, comprising 6-8% of free amino acids and up to 10% of dry residue,

- hydrolysate of molasses, comprising 6-8% of free amino acids and up to 55% of dry residue,

- hydrolysate of coriander, comprising 1-5% of free amino acids and up to 3% of dry residue,

- hydrolysate of cocoa, comprising 1-2% of free amino acids and up to 3% of dry residue,

- hydrolysate of olive residues, comprising 6-8% of free amino acids and up to 55% of dry residue, or a combination thereof.

3. Process for preparing the hydrolysate of claim 1 or 2, comprising the steps of: i) providing a natural raw material, and optionally adding water, ii) adding an enzyme complex comprising at least one peptide bond hydrolase and at least one fiber and carbohydrate hydrolase, and iii) obtaining a liquid mixture, i.e. the hydrolysate from a natural raw material.

4. The process of claim 3, wherein said at least one peptide bond hydrolase is selected from monopepdidase, dipepdidase, tripepdidase, protease, proteinase, and their combinations, and said at least one fiber and carbohydrate hydrolase is selected from amylase, cellulase, hemicellulases, laccases, xylanases, and their combinations.

5. The process of claim 3 or 4, wherein the enzyme complex further comprises at least one ester bond hydrolase, preferably selected from lipases.

6. The process of any one of claims 3-5, wherein in step ii) the temperature is 4-70°C for a time period of 2-20 hours, preferably the temperature is 10-50°C for a time period of 4-16 hours.

7. Hydrolysate from natural raw material obtainable from the process of any one of claims 3-6, wherein said natural raw material is selected from corn steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and their combinations, wherein the percentage of free amino acids in the hydrolysate is at least 0.5% higher than the percentage in the starting natural raw material, and wherein the dry residue in the hydrolysate is at least 2% lower than the percentage in the starting natural raw material.

8. The hydrolysate from natural raw material of claim 7, said hydrolysate being:

- hydrolysate of com steep liquor, wherein the percentage of free amino acids in the hydrolysate is 0.5-11% higher than the percentage in the starting com steep liquor, and wherein the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting corn steep liquor,

- hydrolysate of lupine, wherein the percentage of free amino acids in the hydrolysate is 2-15% higher than the percentage in the starting lupine, and wherein the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting lupine,

- hydrolysate of algae, wherein the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting algae, and wherein the dry residue in the hydrolysate is 2-4% lower than the percentage in the starting algae,

- hydrolysate of molasses, wherein the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting molasses, and wherein the dry residue in the hydrolysate is 10-20% lower than the percentage in the starting molasses,

- coriander hydrolysate, wherein the percentage of free amino acids in the hydrolysate is 0.5-4.5% higher than the percentage in the starting coriander, and wherein the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting coriander,

- cocoa hydrolysate, wherein the percentage of free amino acids in the hydrolysate is 0.5-1.5% higher than the percentage in the starting cocoa, and wherein the dry residue in the hydrolysate is 10-15% lower than the percentage in the starting cocoa, - hydrolysate of olive residues, wherein the percentage of free amino acids in the hydrolysate is 4-6% higher than the percentage in the starting olive residues, and wherein the dry residue in the hydrolysate is 5-10% lower than the percentage in the starting olive residues, or a combination thereof.

9. Agro-chemical product comprising at least one hydrolysate of claim 1, 2, 7, or 8, and agro-chemical additives, and optionally also a natural raw material selected from com steep liquor, lupine, algae, molasses, coriander, cocoa, olive residues, and their combinations.

10. Use of the hydrolysate of claim 1, 2, 7, or 8, or the agro-chemical product of claim 9, as a promoter of vegetable growth and production of fruits in agriculture.