WO2025141276A1 - Use of an aqueous composition obtained from insects, as a biostimulant - Google Patents

Abstract

The present invention relates to the use of an aqueous composition obtained from insects, as a biostimulant.

Description

Description

TITLE: USE OF AN AQUEOUS COMPOSITION OBTAINED FROM INSECTS AS A BIOSTIMULANT

TECHNICAL FIELD OF THE INVENTION

[001] The present invention relates to a novel biostimulant.

[002] In agriculture, the use of fertilizers is widespread, particularly to maintain the fertility of agricultural soils and promote optimal yields.

[003] However, the use of fertilizers in agriculture can present many problems such as water and soil pollution, significant cost as well as a harmful impact on biodiversity.

[004] The implementation of sustainable and environmentally friendly agricultural practices such as rational fertilizer management and the use of organic fertilizers such as manure or composts can help to mitigate these problems. In addition, the use of biostimulants can improve the efficiency of nutrient use by the plant and/or the availability of nutrients in the soil. Thus, it can help to limit the use of fertilizers.

[005] Thus, there is a need to limit the use of fertilizers, particularly with the development of new biostimulants.

[006] The work of the inventors has made it possible to demonstrate that a particular aqueous composition, obtained from insects, could advantageously be used as a biostimulant on plants, in particular to improve their tolerance to abiotic stresses, such as saline stress and/or water stress.

STATEMENT OF THE INVENTION

[007] The present invention relates to the use of an aqueous composition obtained from insects, as a biostimulant.

[008] By “aqueous composition”, we mean more particularly a composition which has a humidity level of 50% to 85%, such as 50% to 70%, preferably 65% to 85%, more preferably still 70% to 80%, and even more preferably 73% to 78%.

[009] It should be noted that in the context of this application, and unless otherwise stipulated, the ranges of values indicated are understood to include the limits.

[010] Preferably, the humidity level is determined according to the method resulting from EC Regulation 152/2009, as detailed in Example 2. [011] Throughout the application, where no date is specified for a regulation, standard or directive, it means the regulation, standard or directive in force on the filing date.

[012] “Biostimulant” means a product that stimulates plant nutrition processes independently of the nutrients it contains, for the sole purpose of improving one or more of the following characteristics of plants or their rhizosphere: a) nutrient use efficiency; b) abiotic stress tolerance; c) quality characteristics; d) availability of nutrients confined in the soil or rhizosphere.

[013] A biostimulant can have an effect on plants and/or soil, in fact, it also helps to stimulate soil microbial activity and functional diversity.

[014] By "rhizosphere" we mean the region of soil surrounding the roots of a plant.

[015] Functional diversity refers to the ability of a soil microorganism to degrade a broad spectrum of organic matter. The broader the spectrum of organic matter, the greater the functional diversity.

[016] Abiotic stress means suboptimal growing conditions caused, for example, by drought (water stress), excess water, extreme temperatures, frost, wind, hail, salt stress, transplant stress, mineral deficiencies and damage following spraying with plant protection products. These conditions reduce plant growth, photosynthesis and therefore yield.

[017] Preferably, in the use according to the invention, the biostimulant is used on plants to improve their tolerance to saline stress and/or water stress.

[018] By "improving plant tolerance to salt stress and/or water stress" is meant the reduction of the effect of stress on plant growth by limiting the effect of stress through stimulation of the plant's resistance mechanisms and/or by stimulating stress-avoidance mechanisms such as stimulation of root elongation for exploration of a larger volume of soil.

[019] In agriculture, biostimulants allow for better efficiency in the use of nutrients by the plant and/or better availability of nutrients in the soil. Thus, biostimulants make it possible to limit the use of fertilizers.

[020] Advantageously, in the use according to the invention, the aqueous composition comprises between 15% and 50% by weight of dry matter, the percentage by weight being indicated on the weight of the aqueous composition. [021] Preferably the aqueous composition comprises from 15% to 35%, more preferably from 20% to 30%, and even more preferably from 22% to 27% by weight of dry matter relative to the weight of the aqueous composition.

[022] Advantageously, in the use according to the invention, the aqueous composition comprises at least 35% by weight of proteins and between 1% and 15% by weight of lipids, the percentages by weight being indicated on the dry weight of the aqueous composition.

[023] In the context of the present application, by “proteins” is meant the quantity of crude proteins. The quantification of crude proteins is well known to those skilled in the art. For example, the Dumas method or the Kjeldhal method may be mentioned. Preferably, the Kjeldhal method is used, as detailed in Example 2. By “proteins”, unless otherwise indicated, is meant not only proteins but also peptides and amino acids.

[024] Preferably, the aqueous composition comprises between 35% and 60% by weight of proteins, more preferably between 36% and 56% by weight of proteins, the percentages by weight being indicated on the dry weight of the aqueous composition.

[025] Advantageously, the aqueous composition comprises between 15% and 50% by weight of total amino acids, preferably between 18% and 45% of total amino acids, the percentages by weight being indicated on the dry weight of the aqueous composition.

[026] By total amino acids, we mean all amino acids, free or not (in the form of peptides, proteins).

[027] Preferably, the total amino acid level is determined according to ISO 13903:2005 and the method from EC Regulation 152/2009, as detailed in Example 2.

[028] Advantageously, the most abundant total amino acids are aspartic acid, glutamic acid, lysine, proline, valine, glycine and arginine.

[029] Advantageously, the aqueous composition comprises at least 1% by weight of free amino acids, the percentage by weight being indicated on the dry weight of the aqueous composition.

[030] Preferably, the aqueous composition comprises between 1% and 25% by weight, more preferably between 8% and 20% by weight, even more preferably between 9% and 18% by weight of free amino acids, the percentages by weight being indicated on the dry weight of the aqueous composition.

[031] Preferably, the level of free amino acids is determined according to ISO 13903:2005, as detailed in Example 2.

[032] By “free amino acids” is meant the following amino acids: alanine, arginine, aspartic acid, cysteine + cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine; preferably alanine, arginine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, proline, tyrosine and valine.

[033] Amino acids play a crucial role in the structure, metabolism and physiology of cells in all living things.

[034] The most abundant free amino acids are advantageously proline and arginine.

[035] Advantageously, in the use according to the invention, the aqueous composition comprises between 2% and 10% by weight of proline, the percentage by weight being indicated on the dry weight of the aqueous composition.

[036] Preferably, the aqueous composition comprises between 4% and 9% by weight of proline, more preferably between 5% and 8% by weight of proline, the percentage by weight being indicated on the dry weight of the aqueous composition.

[037] Proline is an amino acid of particular interest for improving the resistance of plants to abiotic stresses.

[038] Advantageously, the aqueous composition comprises between 4% and 9% by weight, preferably between 5% and 8% by weight of free proline, the percentage by weight being indicated on the dry weight of the aqueous composition.

[039] As indicated above, the aqueous composition comprises between 1% and 15% by weight of lipids, the percentage by weight being indicated on the dry weight of the aqueous composition.

[040] Preferably, the aqueous composition comprises between 1.5% and 12% by weight, more preferably between 1.5% and 10% by weight of lipids, the percentages by weight being indicated on the dry weight of the aqueous composition.

[041] The methods for determining the lipid content are well known to those skilled in the art. By way of example and preferably, the determination of this content will be carried out according to the method of EC Regulation 152/2009, as detailed in Example 2.

[042] Advantageously, the aqueous composition comprises between 3% and 10% by weight of nitrogen, preferably between 4% and 10% by weight of nitrogen, more preferably between 5% and 9% by weight of nitrogen, the percentages by weight being indicated on the dry weight of the aqueous composition.

[043] Methods for determining nitrogen are well known to those skilled in the art. For example, the Dumas method or the Kjeldhal method may be cited. Preferably, the Kjeldhal method is used, as detailed in Example 2.

[044] Advantageously, the aqueous composition comprises between 10% and 15% by weight of phosphorus, the percentage by weight being indicated on the dry weight of the aqueous composition. [045] Advantageously, the aqueous composition comprises between 2% and 5% by weight of potassium, the percentage by weight being indicated on the dry weight of the aqueous composition.

[046] Nitrogen, phosphorus, and potassium are essential plant nutrients. They play a crucial role in plant growth, development, and overall health.

[047] Advantageously, the aqueous composition comprises between 0.4% and 1% by weight of magnesium, the percentage by weight being indicated on the dry weight of the aqueous composition.

[048] Magnesium also performs many vital functions for plant development, growth and health.

[049] Minerals were determined by ICP/AES (inductively coupled plasma-atomic emission spectrometry), as detailed in Example 2.

[050] Advantageously, in the use according to the invention, the aqueous composition comprises between 65% and 95% by weight of organic matter, the percentage by weight being indicated on the dry weight of the aqueous composition.

[051] Preferably, the aqueous composition comprises between 70% and 90% by weight of organic matter, the percentage by weight being indicated on the dry weight of the aqueous composition.

[052] The organic matter content was determined by calculation via the determination of the ash content according to standard NF EN 13039 as detailed in example 2.

[053] Advantageously, in the use according to the invention, the aqueous composition comprises between 0.1% and 5% by weight of trehalose, the percentage by weight being indicated on the dry weight of the aqueous composition.

[054] Preferably, the aqueous composition comprises between 0.2% and 4% by weight of trehalose, the percentage by weight being indicated on the dry weight of the aqueous composition.

[055] Trehalose plays a role in plant resistance to abiotic stress, particularly in cases of drought, saline stress or cold.

[056] The trehalose content was determined by the method detailed in Example 2.

[057] Advantageously, the aqueous composition comprises at least 40% by weight of water-soluble proteins relative to the total weight of proteins.

[058] By “total protein weight” or “protein weight” without further indication of the nature of the proteins, we mean the weight of crude proteins present in the aqueous composition. This therefore includes both water-soluble and insoluble proteins. [059] Preferably, the aqueous composition comprises from 45% to 90% by weight of water-soluble proteins, the percentage by weight being expressed relative to the total weight of proteins.

[060] Therefore, the aqueous composition may comprise between 10% and 60%, preferably between 10 and 55% by weight of insoluble proteins, the percentage by weight being expressed relative to the total weight of proteins.

[061] By “water-soluble proteins” is meant, among the proteins (or crude proteins), those which are soluble in a solution consisting of 30% acetonitrile, 70% ultrapure water and 0.1% trifluoroacetic acid (“ACN/water/TFA solution”), these percentages being percentages by volume on the total volume of solution, as detailed in example 2.

[062] By “insoluble proteins” is meant proteins insoluble in the ACN/water/TFA solution, as detailed in Example 2.

[063] Advantageously, the aqueous composition comprises at least 40% by weight of water-soluble proteins having a size less than 555g/mol of the total weight of water-soluble proteins.

[064] More particularly, between 45% and 75% by weight, more preferably between 50% and 70% by weight of the water-soluble proteins of the aqueous composition have a size less than 555 g/mol, based on the total weight of water-soluble proteins.

[065] Advantageously, at least 80% by weight, preferably between 85% and 98% by weight, more preferably between 88% and 98% by weight of the water-soluble proteins of the aqueous composition have a size of less than 12,400 g/mol, based on the total weight of water-soluble proteins.

[066] Preferably, the size of the proteins is determined by HPLC-SEC, as detailed in Example 2.

[067] The aqueous composition is advantageously stabilized by the addition of additives, with at least one preservative and/or at least one pH lowerer. As a result, the aqueous composition can be stored for a period of between 6 and 12 months at a temperature of 30°C.

[068] Advantageously, the aqueous composition comprises at least one preservative chosen from potassium sorbate, sodium formate, sorbic acid, formic acid, potassium diformate, calcium formate, sodium bisulfate, acetic acid, sodium diacetate, calcium acetate and prenyl acetate.

[069] The preservative is preferably introduced into the aqueous composition at a concentration of 0.05% to 3%, preferably 0.1% to 2% by weight of the aqueous composition. [070] By “pH lowerer” is meant a product capable of lowering the pH of an aqueous solution.

[071] Preferably the pH lowerer may be chosen from an organic or inorganic acid, preferably an inorganic acid, such as for example phosphoric acid, citric acid, fumaric acid, acetic acid, sorbic acid and propionic acid.

[072] Advantageously, the pH lowerer is introduced in a quantity necessary and sufficient to lower the pH between 2 and 4, preferably between 2.5 and 3.5, more preferably between 2.8 and 3.2.

[073] In a particular embodiment, in the use according to the invention, the aqueous composition is an aqueous fraction of insects.

[074] By “aqueous fraction of insects” is meant the aqueous fraction obtained, at the end of a step of separation into three fractions (solid, oily and aqueous) of an insect pulp. Preferably, the aqueous fraction of insects is obtained by a mechanical process (without a chemical or biological step, such as, for example, hydrolysis).

[075] By “insect pulp” is meant a composition in the form of a solution or paste, comprising crushed or ground insects or parts of insects, and possibly water.

[076] According to a preferred embodiment of the particular embodiment, in the use according to the invention, the aqueous fraction of insects is a concentrated aqueous fraction of insects, capable of being obtained by the preparation process comprising the following steps: i) Separation of the cuticle from the soft part of the insects, ii) Separation of the soft part into a solid fraction, an aqueous fraction and an oily fraction, ll) Concentration of the aqueous fraction, making it possible to obtain a concentrated aqueous fraction of insects.

[077] The preparation process by which the concentrated aqueous fraction of insects is likely to be obtained or obtained may comprise a slaughtering step prior to the step of separating the cuticle from the soft part of the insects.

[078] As used herein, "insects" means insects at any stage of development, such as an adult, larval, or nymph stage.

[079] Advantageously, the insects used in the process are at a larval stage.

[080] More particularly, the insects may be chosen from the group consisting of Coleoptera, Diptera, Lepidoptera, Isoptera, Orthoptera, Hymenoptera, Blattoptera, Hemyptera, Heteroptera, Neuroptera, Ephemeroptera and Mecoptera, preferably from Coleoptera, Diptera, Orthoptera, Neuroptera and Lepidoptera.

[081] Preferably, the insects are chosen from the group consisting of Tenebrio molitor, Hermetia illucens, Galleria mellonella, Alphitobius diaperinus, Zophobas morio, Blattera fusca, Tribolium castaneum, Rhynchophorus ferrugineus, Musca domestica, Chrysomya megacephala, Locusta migratoria, Schistocerca gregaria, Acheta domesticus and Samia ricini.

[082] Preferably, in the use according to the invention, the insects are beetles.

[083] The beetles preferentially used in the process belong to the families Tenebrionidae, Melolonthidae, Dermestidae, Coccinellidae, Cerambycidae, Carabidae, Buprestidae, Cetoniidae, Dryophthoridae, or mixtures thereof, even more preferentially the insects belong to the family Tenebrionidae.

[084] More preferably, these are the following beetles: Tenebrio molitor, Alphitobius diaperinus, Zophobas morio, Tenebrio obscurus, Tribolium castaneum and Rhynchophorus ferrugineus, or their mixtures, even more preferably Tenebrio molitor and Alphitobius diaperinus.

[085] Insects are preferably farmed and not taken from the wild.

[086] For example, insects are reared on an insect farm. Rearing insects on a specific farm not only controls and eliminates risks associated with insect-borne diseases, but also limits risks associated with the toxicity of insect-derived food products due, for example, to the presence of insecticides. In addition, rearing allows the quality of the insect supply to be controlled and supply costs to be limited.

[087] The cuticle is the outer layer (or exoskeleton) secreted by the epidermis of insects. It is generally formed of three layers: the epicuticle, the exocuticle and the endocuticle.

[088] By “soft part” is meant the flesh (including in particular the muscles and viscera) and the juice (including in particular the biological fluids, water and hemolymph) of insects. In particular, the soft part does not consist of the juice of insects.

[089] Optionally, the preparation method further comprises a step of maturation of the soft part of the insects, between the step of separation of the cuticle from the soft part of the insects and the step of separation of the soft part of the insects into an oily fraction, a solid fraction and an aqueous fraction. [090] By “stage of maturation of the soft part of the insects”, we mean more particularly a stage during which the soft part of the insects is subjected to agitation.

[091] The step of concentrating the aqueous fraction is more fully described in Example 1 below.

[092] Advantageously, the preparation process comprises a step of sterilization and stabilization of the concentrated aqueous fraction. These steps are also more fully described in Example 1 below.

[093] More particularly, the concentrated aqueous insect fraction comprises between 17% and 30% by weight, preferably between 20% and 30% by weight, more preferably between 22% and 27% by weight of dry matter, the percentages by weight being indicated on the weight of the concentrated aqueous insect fraction.

[094] Advantageously, the concentrated aqueous insect fraction comprises between 35% and 55% by weight of proteins and between 1.5% and 12% by weight of lipids, the percentages by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[095] Preferably, the concentrated aqueous insect fraction comprises between 36% and 56% by weight, for example, between 36% and 54% by weight of proteins, the percentages being indicated on the dry weight of the concentrated aqueous insect fraction.

[096] Preferably, the concentrated aqueous insect fraction comprises between 1.5% and 10% by weight, more preferably between 1.5% and 9% by weight of lipids, the percentages being indicated on the dry weight of the concentrated aqueous insect fraction.

[097] Advantageously, the concentrated aqueous insect fraction comprises between 4% and 10% by weight of nitrogen, preferably between 5% and 8% by weight of nitrogen, the percentages by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[098] Advantageously, the concentrated aqueous insect fraction comprises the same phosphorus and potassium contents as the aqueous composition above.

[099] Advantageously, the concentrated aqueous insect fraction comprises between 2% and 5% by weight of potassium, the percentage by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[100] Advantageously, the concentrated aqueous insect fraction comprises between 0.4% and 1% by weight, preferably between 0.5% and 1% by weight of magnesium, the percentage by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[101] Advantageously, the concentrated aqueous insect fraction comprises between 0.1% and 4% by weight, for example, between 0.2% and 4% by weight, preferably between 0.2% and 3% by weight of trehalose, the weight percentage being indicated on the dry weight of the concentrated aqueous insect fraction.

[102] Advantageously, the concentrated aqueous insect fraction comprises between 18% and 45% by weight, preferably between 20% and 45% by weight, more preferably between 25% and 40% by weight of total amino acids, the percentages by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[103] Advantageously, the concentrated aqueous insect fraction comprises between 8% and 20% by weight, preferably between 9% and 18% by weight of free amino acids, the percentages by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[104] Advantageously, the concentrated aqueous insect fraction comprises between 4% and 9% by weight, preferably between 5% and 8% by weight of proline, the percentage by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[105] Advantageously, the concentrated aqueous insect fraction comprises between 4% and 9% by weight, preferably between 5% and 8% by weight of free proline, the percentage by weight being indicated on the dry weight of the concentrated aqueous insect fraction.

[106] Advantageously, the concentrated aqueous insect fraction comprises a mass ratio of free amino acids to total amino acids of at least 35%.

[107] Advantageously, the concentrated aqueous insect fraction comprises at least 70% by weight of water-soluble proteins relative to the total weight of proteins.

[108] Preferably, the concentrated aqueous insect fraction comprises from 70% to 90% by weight of water-soluble proteins, the percentage by weight being expressed relative to the total weight of proteins.

[109] Therefore, the concentrated aqueous insect fraction comprises between 10% and 30% by weight of insoluble proteins, the percentage by weight being expressed relative to the total weight of proteins.

[1 10] The size distribution of the water-soluble proteins of the concentrated insect aqueous fraction is as described for the aqueous composition above.

[1 11] The size distribution of water-soluble proteins helps improve resistance to abiotic stresses, particularly by providing a long-term effect.

[1 12] In the context of the present invention, by “plants” is meant any type of plant, in particular with roots, stems and leaves.

[1 13] Preferably, in the use according to the invention, the biostimulant is used on plants chosen from the group consisting of agricultural plants, ornamental plants and herbaceous plants.

[1 14] By “agricultural plants” we mean more specifically: Plants used in fruit growing, such as fruit shrubs and trees such as gooseberries, strawberries, peaches, pears, apples, melons;

Plants used in cereal crops, such as corn, rice and straw cereals such as barley, oats, wheat, wheat;

Plants used in vegetable growing, such as vegetables, fruit plants, aromatic and condiment plants.

These include: o Salads belonging to the lettuce family, such as batavia, oak leaf, arugula, watercress, lamb's lettuce, romaine; o Potatoes, aubergines, tomatoes, peppers, certain cruciferous vegetables including cabbages and cauliflowers, carrots, squash, cucumbers, green beans, beets and o Legumes such as peas, beans, lentils, field beans.

Plants used in oilseed crops, such as rapeseed (or canola), cotton, sunflower, soybean, flax and hemp.

[1 15] By “ornamental plants” we mean more specifically ornamental trees and shrubs such as conifers, deciduous trees, ornamental and/or floral plants, ornamental fruit shrubs and indoor plants, lawns.

[1 16] For example, we can cite: for conifers: pines, spruces, larches, firs; for deciduous trees: oaks, hornbeams, beeches, poplars, birches, willows, maples, lime trees, alders; ornamental and/or floral plants: cyclamens, dahlias, hydrangeas and roses.

[1 17] “Herbaceous plants” means, in particular, plants used in meadows such as clover, plants of the genus Lolium such as Lolium perenne (perennial ryegrass) or Lolium multiflorum (Italian ryegrass or annual ryegrass), lucerne, milkvetch, trefoil, sweet clover, grama, bluegrass, fescue, needlegrass, bluestem, yarrow, purple coneflower, goldenrod, black-eyed coneflower, bee balm, lupin, gaillardia, blue thistle, milkweed, ratibida, mugwort, aster, lespedeza, amorpha, sedge, rush, cattail and bulrush.

[1 18] By "used on" plants is meant an application to the plant, i.e. an application to the aerial parts of the plant, such as for example by spraying, or an application to the root parts of the plant, for example to the soil. [1 19] Preferably, in the use according to the invention, the biostimulant is applied to the soil.

[120] Preferably, the biostimulant is applied to the soil by broadcasting or spraying.

[121] By “on the ground” we mean an application directly to the ground, preferably at the foot or around the plant.

[122] Alternatively, the biostimulant may be applied to the leaves of the plant to be treated, for example, by spraying.

[123] The biostimulant can be applied before and/or after the plant has been planted.

BRIEF DESCRIPTION OF THE FIGURE

[124] Other characteristics and advantages of the invention will appear in the following examples, given for illustrative purposes, with reference to:

[Fig.1] Fresh aerial (BFA) and root (BFR) biomass and dry aerial (BSA) and root (BSR) biomass obtained according to the methods defined in example 3a.

[Fig. 2] Projected leaf area per plant of example 3a.

[Fig. 3] Root scan image of 4 plants from modalities M2 and M3 of example 3a.

[Fig. 4] Total indicators of root architecture per pot of example 3a.

[Fig. 5] Root lengths by diameter classes obtained in example 3a.

[Fig. 6] Fresh aerial biomass obtained in example 3b.

[Fig. 7] Water content at harvest of example 3b.

[Fig. 8] Projected leaf area per plant of example 3b.

[Fig. 9] Fresh and dry biomass (aerial and root) obtained according to the methods defined in example 4a.

[Fig. 10] Projected leaf area per plant of example 4a.

[Fig. 11] Total indicators of root architecture per pot of example 4a.

[Fig. 12] Root lengths by diameter classes obtained in example 4a.

[Fig. 13] Fresh aerial biomass obtained in example 4b.

[Fig. 14] Dry aerial biomass obtained in example 4b.

[Fig. 15] Water content at harvest of example 4b.

[Fig. 16] Projected leaf area of example 4b.

[Fig. 17]: Fresh aerial biomass obtained in example 5.

[Fig. 18]: Fresh root biomass obtained in example 5.

[Fig. 19]: Projected leaf area per plant of example 5.

EXAMPLES

Example 1: Preparation of the composition (concentrated aqueous fraction)

[125] First, 1 kg of Tenebrio molitor larvae are steamed and then decapitated using a twin-screw separator, thus allowing the separation of the cuticles and the pulp. [126] The pulp thus obtained is placed in a maturation tank for 1 hour at 90°C with stirring. The heated pulp is then separated via a tricanter (3-phase decanter) thus obtaining an oily fraction, an aqueous fraction and a solid protein fraction corresponding to a protein press cake.

[127] The aqueous fraction is then concentrated by evaporation in order to obtain a concentrated aqueous fraction, in order to reach a dry matter content of between 20% and 25%.

[128] The concentrated aqueous fraction is then sterilized for 30 seconds at 125°C before being stabilized by the addition of additives to reach a pH of 2.9.

[129] Additives used may include a preservative and/or a pH lowerer.

[130] The preservative may be selected from sodium formate, sorbic acid, formic acid, potassium diformate, calcium formate, sodium bisulfate, potassium sorbate, acetic acid, sodium diacetate, calcium acetate and prenyl acetate.

[131] The pH lowerer may be chosen from an organic or inorganic acid, preferably an inorganic acid, such as for example phosphoric acid, citric acid, fumaric acid, acetic acid, sorbic acid and propionic acid.

[132] The finished product obtained is a concentrated aqueous fraction having a dry matter content of between 20% and 25%.

Example 2: Characterization of the concentrated aqueous fraction

[133] The concentrated aqueous fraction prepared in Example 1 was characterized as follows:

1. Analyses

1.1 Determination of humidity level

[134] The humidity level was determined according to the method from EC Regulation 152/2009.

1.2 Determination of the organic matter content

[135] The organic matter content was determined by calculation via the determination of the ash content according to standard NF EN 13039.

1.3 Determination of the amount of protein and nitrogen

[136] The protein and nitrogen content were determined according to the Kjeldahl method, derived from EC Regulation 152/2009, with an N to protein conversion factor of 6.25.

1.4 Determination of the quantity of lipids (fats)

[137] The amount of lipids was determined according to the method under EC Regulation 152/2009.

1.5 Determination of amino acid quantities [138] The amount of total and free amino acids was determined according to the method covered by ISO 13903:2005 (for total and free amino acids except tryptophan) and EC Regulation 152/2009 (for tryptophan).

[139] The amount of free amino acids was determined according to ISO 13903:2005.

1.6 Determination of the amount of soluble and insoluble proteins and protein size

[140] The quantity of soluble proteins was determined by solubilizing said proteins in a solution consisting of 30% acetonitrile, 70% ultrapure water and 0.1% trifluoroacetic acid (“ACN/water/TFA solution”), these percentages being percentages by volume of the total volume of solution, which constitutes the mobile phase before determination by HPLC-SEC (size exclusion chromatography method known to those skilled in the art).

[141] The quantity of insoluble proteins was determined by the dry residue obtained after dissolving a sample of the concentrated aqueous fraction in the ACN/water/TFA solution, and reported to the initial dry weight of the fraction considered.

[142] Protein size was determined by HPLC-SEC (size exclusion chromatography method known to those skilled in the art).

1.7 Determination of the quantity of minerals

[143] The content of each of the minerals listed below was determined using the ICP/AES (inductively coupled plasma-atomic emission spectrometry) method.

1.8 Determination of the quantity of trehalose

[144] The amount of trehalose was determined based on a sample after lyophilization. 40 mg of dry sample was extracted using 2 mL of dimethyl sulfoxide (DMSO) for 1 hour, with stirring at 80°C. 250 pL of the extract was then mixed with 50 pL of myo-inositol, used as an internal standard (1 g/L in DMSO). After homogenization, 100 pL of this mixture was derivatized with 100 pL of BSTFA-TMCS (99:1) directly in the GC-MS vial for 30 minutes at 60°C. Before injection, 600 pL of acetonitrile was added to the GC-MS vial. The results were expressed in mg of trehalose/g dry using a trehalose standard range carried out under the same conditions.

[145] The derived extracts and the different standard range points are analyzed on a Shimadzu GC-MS-QP2010. The column used is a SH-Rxi 5 ms column (Shimadzu) with a length of 30 m, a diameter of 0.25 mm and a fineness of 0.25 μm. The GC-MS temperature program is as follows: 100°C, followed by a ramp at 10°C/min up to 300°C maintained for 2 min.

[146] The injector temperature is 280°C, the interface temperature is 250°C, the split ratio is 10, the injection volume is 1 pL. The detection is carried out in SIM (Selected Ion Monitoring) mode with specific m/z of 305 for myo-inositol and 361 for trehalose.

2. Results

[147] The concentrated aqueous fraction has the characteristics indicated in the following Table 1:

[Table 1]

‘Minimum results calculated on around ten samples of the concentrated aqueous fraction.

“Maximum results calculated on around ten samples of the concentrated aqueous fraction.

“Average results calculated on around ten samples of the concentrated aqueous fraction.

Table 1: Characteristics of the concentrated aqueous fraction.

• Proteins and amino acids

[148] The total amino acid composition of the concentrated aqueous fraction is presented in the following Table 2:

[Table 2]

*Minimum results calculated on several samples of the concentrated aqueous fraction.

**Maximum results calculated on several samples of the concentrated aqueous fraction.

***Average results calculated on several samples of the concentrated aqueous fraction.

Table 2: Total amino acid compositions of the concentrated aqueous fraction.

[149] The free amino acid composition of the concentrated aqueous fraction is presented in the following Table 3:

[Table 3]

*Minimum results calculated on several samples of the concentrated aqueous fraction.

**Maximum results calculated on several samples of the concentrated aqueous fraction.

***Average results calculated on several samples of the concentrated aqueous fraction.

Table 3: Free amino acid composition of the concentrated aqueous fraction.

[150] The size distributions of the water-soluble proteins in the concentrated aqueous fraction are shown in Table 4 below: [Table 4]

*1 Da: 1g/mol.

The results are the average results calculated on several samples of the concentrated aqueous fraction.

Table 4: Size distributions of water-soluble proteins in the concentrated aqueous fraction.

• Minerals

[151] The mineral composition of the concentrated aqueous fraction is presented in Table 5 below:

[Table 5]

* quantities are expressed as percentages by weight of the weight of the concentrated aqueous fraction.

The results are the average results calculated on several samples of the concentrated aqueous fraction.

Table 5: Mineral composition of the concentrated aqueous fraction.

Example 3: Influence of the concentrated aqueous fraction on young lettuce plants under saline stress conditions

Example 3a: Influence of the concentrated aqueous fraction on the growth and root and aerial development of young Batavia lettuce plants under saline stress conditions

[152] In the present example, the effect of the concentrated aqueous fraction was studied under semi-controlled conditions (greenhouses), on the growth and root and aerial development of young Batavia lettuce plants under saline stress conditions.

1. Materials and methods [153] Lettuce is grown on a growing medium (sand/perlite/fine vermiculite). Sowing is done in a climate chamber, in a tray, then one plant per pot is transplanted into 450 mL pots.

[154] The following Table 6 indicates the quantities of culture medium used in this example:

[Table 6]

Table 6: Quantity of materials and consumables used in example 3a.

[155] The test then takes place in a semi-controlled greenhouse (regulated temperature, natural lighting with the possibility of supplementing with a light intensity detector).

[156] The experimental device includes 3 modalities:

Modality 1 (M1): an untreated and unstressed control (TNT),

Modality 2 (M2): an untreated and stressed control with saline stress (by addition of sodium chloride (NaCl)) (TSS),

Modality 3 (M3): a stressed modality with saline stress and treated with the concentrated aqueous fraction (PSS).

[157] Modalities 1 and 2 are represented by 12 repetitions. The stressed modality treated with the concentrated aqueous fraction is represented by 11 repetitions. The total number of pots is therefore 35 in the test.

[158] The concentrated aqueous fraction is added at the beginning or end of the day, to the substrate, locally (on the mini-clod).

[159] The application dose is 24L of concentrated aqueous fraction/250L of mixture/hectare.

[160] The term “spray” refers to the concentrated aqueous fraction diluted before application. The concentrated aqueous fraction has been diluted in order to approximate real application conditions. In fact, in the field of agriculture, a product is diluted before its application. The product thus applied is called “spray” and can, for example, be applied in a quantity of 250L/hectare.

[161] The conversion between agronomic dose and experimental dose is made on the basis of a planting density of 100,000 lettuces per hectare. [162] Two treatments consisting of the supply of concentrated aqueous fraction (M3) are provided during the test:

[TTT1] Four days after transplanting at the level of the mini-root ball; and

[TTT2] one week after the first treatment.

[163] The modalities tested and the doses provided are presented in the following table 7:

calcul sur la base de 100000 laitues/ha. [Table 7]

calculation based on 100,000 lettuces/ha.

Table 7: Modalities tested and doses provided in example 3a.

[164] The trial is launched in a completely randomized design.

[165] The duration of the test is 4 weeks after transplanting the lettuces into pots.

Cultivation management a) Cultivation pots and substrate

[166] In this trial, lettuce was grown in 0.45 L pots. The substrate (sowing and growth) is light and draining to promote establishment. It is composed of a mixture of sand (20% v/v), perlite (40% v/v) and fine vermiculite (40% v/v).

[167] The characteristics of the substrate (percentage of dry matter and Maximum Water Retention Capacity (CRmax)) are determined prior to the test.

[168] The substrate is saturated with water prior to transplanting (CRmax setting without leaching). b) Sowing

[169] The trial was carried out with a commercial variety of Batavia lettuce, at a density of one plant per pot. The seeds were sown in seed trays and then transplanted (at the 2-leaf stage) into the pots. c) Fertilization

[170] Fertilization is suboptimal to ensure good establishment and growth of crops. It is composed of a complete nutrient solution, provided diluted at a rate of 50 mL of nutrient solution per pot per week.

[171] The ready-to-use nutrient solution is obtained by mixing 9 previously prepared stock solutions (concentrated).

[172] The composition of the stock solutions and the preparation of the nutrient solution are presented in the following table 8: [Table 8]

[173] With a supply of 50 ml of this nutrient solution, the equivalent in milligrams of nutrients provided per plant is shown in the following table 9:

[Table 9]

Table 9: Nutrient (mineral) intake per plant.

[174] For example, an equivalent of 1.3 kg N/ha (for a density of 100,000 plants/ha). d) Irrigation

[175] Initially (just before transplanting), the pots are brought to a humidity close to water saturation (CRmax without leaching). During sowing and a few days after transplanting, watering is optimal. The pots are then watered two to three times per week (without leaching) to maintain moisture levels at approximately 70% of maximum water holding capacity throughout the crop. e) Salt stress management

[176] The salt stress level was determined after several preliminary tests. The objective is to increase the conductivity of the substrate to a value of approximately 110 mS/m.

[177] For this purpose, in the pots subjected to saline stress (M2 and M3), the substrate is watered with 150 mL of a NaCI solution (10 g NaCI/L), i.e. a contribution of 1500 mg of NaCI per pot.

[178] After adding the saline solution, three conductivity measurements were taken and the average conductivity was 120 mS/m. Then, the pots were brought to their maximum water retention capacity (without leaching) with distilled water. The control pots were saturated with water at the same time.

Measurements and monitoring a) Observations and measurements

[179] During the test:

Health status of seedlings;

Measurement of projected leaf area (Canopeo®) once a week.

[180] At the end of the test:

The aerial biomass of lettuce is weighed fresh and dry (BFA/BSA);

The root systems are extracted from the pots and weighed fresh (BFR);

Then, the root architecture is analyzed by WinRhizo® imaging;

Then the root biomasses are weighed in dry form (BSR).

[181] b) Data analysis

[182] The data are statistically analyzed by an analysis of variance at the 95% confidence level. The conditions for applying ANOVA are verified (residual normality test and Levene's homoscedasticity test).

2. Results

[183] The objective of this example is to evaluate the effectiveness of the concentrated aqueous fraction as a biostimulant on the growth and root and aerial development of young Batavia lettuce plants grown under saline stress (NaCI).

2.1 Biomass

[184] Fresh aerial (FAB) and root (RBF) biomasses are weighed immediately at the time of harvest, they are then placed in an oven at 40°C until their mass is constant before being weighed dry (dry aerial (DAB) and root (RBF) biomasses) (Figure 1). [185] The data then undergo statistical analysis to determine significant effects.

[186] Statistical analysis shows that:

The effect of saline stress is significant: whatever the biomass considered (aerial and root; fresh and dry), we note a significant effect of saline stress between the M1 modality (the untreated and unstressed control) and the M2 modality (the untreated control but subjected to saline stress), the biomass of which is systematically negatively impacted by stress.

As for the effect of the treatment, it is noted that it is statistically significant on fresh biomass (aerial (BFA) and root (BFR)) and dry biomass (aerial (BSA) and root (BSR), giving the treated and stressed modality (M3) an aerial and root biomass statistically homogeneous with the biomass of the unstressed control modality (M1).

[187] The statistical analyses of the biomasses are presented in Figure 1, the conclusions of the tests are represented in the following Table 10:

[Table 10]

Table 10: Statistical analysis of fresh and dry biomasses obtained in example 3a.

2.2 Projected leaf area

[188] During growth, the projected leaf area of each plant was estimated once a week using a photograph taken from above. Using a 1 cm ² surface standard and Canopeo® software, the projected leaf area is calculated for each plant (see Figure 2).

[189] These data are then statistically analyzed; the analysis shows that: At the beginning of growth, the treatment modality has a significant effect. Plants in modality M1 (untreated and unstressed control) have a significantly greater projected leaf area than plants in modality M2 (salt stress control). The treated and stressed modality (M3) does not differ significantly from the other two.

One week later, the significant effect of salt stress is visible on the two stressed modalities (M2 and M3) whose surface is statistically homogeneous, but significantly lower than the surface of the non-stressed plants (M1). 5 days later, we find the same statistical result.

10 days later, the effect of salt stress is significant, but so is the effect of the treatment. Indeed, we observe that the treated modality M3 has a significantly greater leaf surface area than the control plants undergoing the same salt stress.

A fifth series of measurements 4 days later (just before harvest) gives comparable results.

[190] The results of the statistical analyses are summarized in the following Table 11:

[Table 11]

Table 11: Statistical analysis of the projected leaf areas obtained in example 3a.

2.3 Root architecture

[191] At harvest time, the lettuce roots were removed from the substrate and cleaned with water.

[192] An analysis of the root systems (root scanner and WinRhizo® analysis) is then carried out: the roots are spread on a plexiglass plate containing a water slide and then scanned in two dimensions (Figure 3). The image is digitized and then root diameter class increments are fixed for the analysis of the root architecture. The indicators measured by the software are then accessible in absolute value in total and by diameter class.

[193] Due to the root density, the root systems were cut to properly measure all the parameters of the root architecture. The accessible indicators are thus the length (L), the volume (V) and the root surface (SA). The number of ramifications (R) and the number of ends (T) are not accessible because of the dissection. a) Total indicators

[194] Statistical analysis of the data (see Figure 4) shows that the effect of salt stress is significant on the parameters of total root length, total root surface area and total root volume: the untreated and unstressed control modality (M1) presents significantly higher means than those of the untreated control modality in salt stress (M2). On the average root diameter, there is no significant difference between the unstressed control (M1) and stressed (M2) modalities.

[195] The modality treated with the concentrated aqueous fraction (M3) has, for each indicator, a mean significantly higher than the mean of the untreated control subjected to the same saline stress (M2), which validates the positive effect of the concentrated aqueous fraction on all the indicators of root architecture.

[196] Furthermore, it is noted that:

On the root surface and root volume, the treated and stressed modality (M3) is statistically homogeneous with the unstressed control modality (M1).

On the total root length, the unstressed control (M1) has a higher average than the treated modality (M3), itself higher than the stressed control modality (M2).

On the average root diameter, the average of the treated and stressed modality (M3) is significantly higher than the averages of the two control modalities (M1 and M2), which means that the treatment has a positive effect on the average root diameter, even under saline stress, compared to the control modality not subjected to saline stress.

[197] The results of the statistical analyses are shown in the following Table 12:

[Table 12]

Table 12: Statistical analysis of the total indicators obtained in example 3a. b) Root lengths by diameter classes

[198] The diameter class increments were determined visually, in order to discriminate roots by root branching level as follows:

Diameters greater than 0.4 mm correspond to the largest sections below the neck.

The roots making up the root bundle, called ^1st order, have a diameter between 0.3 and 0.4 mm.

Second-order roots (branched from the bundle) have a diameter between 0.2 mm and 0.3 mm.

Finally, the finest roots (3rd order) have a diameter < 0.2 mm. [199] The WhinRhizo® software is capable of determining the cumulative root length, by root diameter class (see Figure 5).

[200] Statistical analysis shows that all classes of root diameters are negatively impacted by saline stress (for each diameter class, the difference between unstressed control (M1) and saline stressed control (M2) are significant).

[201] As for the treatment, the effect of the treatment is positive and significant for root lengths L>0.4, 0.30<L<0.4 and 0.20<L<0.30 compared to the control subjected to the same stress. We even note that the roots of the 2 most important classes (main root bundle) are equivalent to the unstressed control plants (M1).

[202] The effect of the modalities on the lengths by classes of root diameters is represented in the following table 13:

[Table 13]

Table 13: Statistical analysis of lengths by diameter classes obtained in example 3a.

[203] The results showed that:

Salt stress had a significant impact, revealed in particular by a reduction in the aerial biomass of lettuce of 21% (9.32 g/plant in non-stressed controls and 7.36 g/plant in stressed controls without the addition of the concentrated aqueous fraction).

The concentrated aqueous fraction significantly increased the biomass of lettuce plants by: o + 26% on fresh aerial biomass (7.36 g/plant in stressed and untreated plants to 9.25 g/plant in stressed plants treated with the concentrated aqueous fraction) and + 51% on fresh root biomass compared to the controls under saline stress. o Similarly, these significant effects are found on dry biomass: + 30% in aerial and + 42% in root (compared to the stressed control).

- Three weeks after the date of transplanting, exposure to salt stress and first treatment, the concentrated aqueous fraction significantly increased the projected leaf area measured by Canopeo® by 49% compared to the stressed and untreated controls. At the root architecture level, the contribution of the concentrated aqueous fraction had a significant positive effect on all the measured indicators (compared to the stressed and untreated controls): o Total root length: + 32% (compared to the stressed control); o Total root surface area: + 41% (compared to the stressed control); o Total root volume: + 49% (compared to the stressed control); o Average root diameter: + 6% (compared to the stressed control).

Example 3b: Influence of the concentrated aqueous fraction according to two different dosages on the growth and aerial development of young lettuce plants under saline stress conditions

[204] In the present example, the efficacy of the concentrated aqueous fraction was evaluated as a biostimulant on the growth and aerial development of young lettuce plants grown under saline stress conditions. Furthermore, the present example also aims to confirm the results of example 3a by applying a treatment with the concentrated aqueous fraction consisting of two application doses (12 L/ha and 24 L/ha).

1. Materials and methods

[205] Lettuce is grown on a growing medium (sand/perlite/fine vermiculite). Sowing is done in a greenhouse, in a tray, then one plant per pot is transplanted into 450 mL pots.

[206] The following Table 14 indicates the quantities of culture medium used in this example:

[Table 14]

Table 14: Quantity of materials and consumables used in example 3b.

[207] The test then takes place in a semi-controlled greenhouse (regulated temperature, natural lighting with the possibility of supplementing with a light intensity detector).

[208] The experimental device includes 4 modalities:

Modality 1 (M1): an untreated and unstressed control (TNT), Modality 2 (M2): an untreated and stressed control with saline stress (by addition of NaCI) (TSS),

Modality 3 (M3): a stressed modality with saline stress and treated with the concentrated aqueous fraction at dose 1 (12 L/ha) (PSS D1),

Modality 4 (M4): a stressed modality with saline stress and treated with the concentrated aqueous fraction at dose 2 (24 L/ha) (PSS D2).

[209] Each modality is repeated 12 times, making a total of 48 pots in the test.

[210] The concentrated aqueous fraction is added at the beginning or end of the day, to the substrate, locally (on the mini-clod).

[211] The tested application doses are to be diluted and applied as sprays: Dose 1 = 12 L of concentrated aqueous fraction/250 L of spray/ha. Dose 2 = 24 L of concentrated aqueous fraction/250 L of spray/ha.

[212] The conversion between agronomic dose and experimental dose is made on the basis of a planting density of 100,000 lettuces per hectare.

[213] Two treatments consisting of the contribution of the concentrated aqueous fraction (M3 and M4) are provided during the test:

[TTT1] at the time of transplanting, at the level of the mini-root ball;

[TTT2] one week after the first treatment.

[214] The modalities tested and the doses provided are presented in the following table 15:

calcul sur la base de 100000 laitues/ha. [Table 15]

calculation based on 100,000 lettuces/ha.

Table 15: Modalities tested and doses provided in example 3b.

[215] The trial is launched in a completely randomized design.

[216] The duration of the test is 42 days after transplanting the potted lettuce plants.

- Cultivation a) Cultivation pots and substrate

[217] The sowing substrate is light and draining to promote implantation; it is composed of a mixture of peat (50% v/v), perlite (25% v/v) and fine vermiculite (25% v/v). [218] After 14 days, the plants were transplanted into 0.45 L pots containing a growth substrate (GS) composed of sand (20% v/v), perlite (40% v/v) and fine vermiculite (40% v/v).

[219] The characteristics of the substrate (percentage of dry matter and CRmax) are determined prior to the test.

[220] The substrate is saturated with water prior to transplanting (CRmax setting without leaching). b) Sowing

[221] The trial was carried out with a commercial variety of Batavia lettuce, at a density of one plant per pot. The seeds were sown in seed trays and then transplanted (at the 2-leaf stage) into the pots. c) Fertilization

[222] Fertilization is suboptimal to ensure good establishment and good growth of crops. It is composed of a complete nutrient solution (Hoagland type with trace elements), supplied diluted once a week.

[223] The ready-to-use nutrient solution is obtained by mixing 9 previously prepared stock solutions (concentrated).

[224] The composition of the stock solutions and the preparation of the nutrient solution are those presented in Table 8 of Example 3a.

[225] With a supply of 50 mL of this nutrient solution, the equivalent in milligrams of nutrients supplied per plant is that shown in Table 9 of Example 3a. d) Irrigation

[226] Initially (just before transplanting), the pots are brought to a humidity close to water saturation (CRmax without leaching). During sowing and a few days after transplanting, watering is optimal. The pots are then watered two to three times per week (without leaching) to maintain humidity levels at approximately 70% of the maximum water holding capacity throughout the crop. e) Salt stress management

[227] The salt stress level was determined after several preliminary tests. The objective is to increase the substrate conductivity to a value of approximately 110 mS/m.

[228] For this purpose, in the pots subjected to saline stress (M2, M3 and M4), the substrate is watered with 150 mL of a NaCI solution (10 g NaCI/L), i.e. a contribution of 1500 mg NaCI per pot.

[229] After adding the saline solution, three conductivity measurements were carried out and the average conductivity was 111.9 mS/m. Then, the pots were brought to their maximum retention capacities (without leaching) by distilled water. The control pots are saturated with water at the same time.

[230] Measurements and monitoring a) Observations and measurements

[231] During the test:

Health status of seedlings;

Measurement of projected leaf area (Canopeo®) once a week.

[232] At the end of the test:

The aerial biomass of lettuce is weighed in fresh (BFA) (marketable mass). b) Data analysis

[233] The data are statistically analyzed by an analysis of variance at the 95% confidence level. The conditions for applying ANOVA are verified (residual normality test and Levene's homoscedasticity test).

2. Results

2.1 Biomass

[234] Fresh above-ground biomass (FAB) (Figure 6) represents the marketable mass. It is weighed immediately at the time of harvest.

[235] The data then undergo statistical analysis to determine significant effects.

[236] Statistical analysis shows that:

The effect of salt stress is significant (P=0.0000) on the untreated modality (M2): plants subjected to untreated salt stress have a significantly lower biomass than unstressed plants (M1).

The effect of the treatment is significant on the fresh aerial biomass (FAB) under saline stress conditions, whatever the dose of the concentrated aqueous fraction: the biomass of the treated plants (M3 and M4) is significantly higher than the plants of the untreated modality subjected to the same saline stress (M2).

[237] These results show that the treatment including the concentrated aqueous fraction makes it possible to improve tolerance to saline stress by reducing its effects on the marketable biomass of lettuce.

[238] These results show that, under saline stress conditions, the concentrated aqueous fraction exerts an effect on fresh biomass.

2.2 Water content

[239] The water content of lettuce plants was calculated using fresh and dry biomass. For dry biomass, these are placed after harvesting at the oven at 40°C until their mass is constant before being weighed dry. The means and standard deviations by modality are shown in Figure 7.

[240] Statistical analysis of water contents shows that:

The modality factor is significant (P=0.0000),

Comparing the untreated plants (M1 and M2), we see that the water content significantly increased in the control plants in response to saline stress (M2). In the treated modalities, we note that: o the water content significantly increased compared to the stressed control (M2) for application dose 1 (M3), o significantly increased compared to the stressed control (M2) for application dose 2 (M4).

[241] Thus, the concentrated aqueous fraction, applied to plants under saline stress conditions, makes it possible to increase the water content of the plants (significantly at the application dose of 24 L/ha (M4), compared to an untreated control (M2), which validates the improvement in tolerance to saline stress.

2.3 Projected leaf area

[242] During growth, the projected leaf area of each plant was estimated once a week using a photograph taken from above. Using a 1 cm ² surface area standard and Canopeo® software, the projected leaf area is calculated for each plant (see Figure 8).

[243] These data are then statistically analyzed; the analysis shows that: From the second surface measurement (7 days after transplanting), the effect of saline stress is significant; the three modalities subjected to this stress (M2, M3 and M4) are in a homogeneous group, compared to the non-stressed control (M1).

This difference is maintained during the following weeks. We note that the growth curve of the unstressed control (M1) is rather rapid at the start and then slows down, whereas the stressed modalities (M2, M3 and M4) start more slowly but then follow a relatively linear growth, which is explained by the progressive establishment of mechanisms of tolerance to saline stress, until almost catching up with the unstressed control (M1).

At the time of harvest, we observed 3 statistically homogeneous groups: o the control under saline stress (M2) with a projected leaf area of 155 cm ² ; o the two treated and stressed modalities (M3 and M4) with a respective projected leaf area of 169 and 175 cm ² ; and o the non-stressed control (M1) with an area of 186 cm ² .

[244] The results of the statistical analyses are summarized in the following Table 16. [Table 16]

Table 16: Statistical analysis of the projected leaf areas of example 3b.

[245] The results showed that:

The level of saline stress applied in the trial is significant and results in a reduction of the aerial biomass of untreated lettuce of -23% for untreated plants (M2).

Under saline stress conditions, the concentrated aqueous fraction significantly preserved the fresh aerial biomass of lettuce plants (which constitutes the marketable mass for lettuce): o compared to untreated plants (M2), treatment with the concentrated aqueous fraction improved the biomass by +11% at dose 1 of 12 L/ha (M3) and by +15% at dose 2 of 24 L/ha (M4).

On dry aboveground biomass, the treatment effect is not significant, which means that the concentrated aqueous fraction exerts an effectiveness on the water content of the plants. Data analysis confirms that the concentrated aqueous fraction significantly improves the water content of the plants under saline stress conditions.

The harvest date was positioned in such a way as to highlight an effect of the treatment on the projected leaf surface; the effect of the concentrated aqueous fraction (compared to the stressed control (M2)) is significant on the leaf surface after 6 weeks (42 days).

Example 4: Influence of the concentrated aqueous fraction on young lettuce plants under water stress conditions

Example 4a: Influence of the concentrated aqueous fraction on the growth and root and aerial development of young lettuce plants under water stress conditions

[246] In the present example, the effect of the concentrated aqueous fraction was studied under controlled conditions (greenhouses), on the growth and root development of young lettuce plants under water stress conditions.

1. Materials and methods [247] Lettuce is grown on a growing medium (sand/perlite/fine vermiculite). Sowing is done in a climate chamber, in a tray, then one plant per pot is transplanted into 450 mL pots.

[248] The following Table 17 indicates the quantities of culture medium used in this example:

[Table 17]

Table 17: Quantity of materials and consumables used in example 4a.

[249] Sowing and growth are carried out in a semi-controlled greenhouse (regulated temperature, natural lighting with the possibility of supplementing with a light intensity detector).

[250] The experimental device includes 3 modalities:

Modality 1 (M1): an untreated and unstressed control (TNT) (optimal watering at approximately 70% of CRmax),

Modality 2 (M2): an untreated control stressed with water stress (TSH), Modality 3 (M3): a modality stressed with water stress and treated with the concentrated aqueous fraction (PSH).

[251] Each modality is repeated 12 times, making a total of 36 pots in the test.

[252] The concentrated aqueous fraction is added at the beginning or end of the day, to the substrate, locally (on the mini-clod).

[253] The application rate is 24 L of concentrated aqueous fraction/250 L of spray/ha.

[254] The conversion between agronomic dose and experimental dose is made on the basis of a planting density of 100,000 lettuces per hectare.

[255] Two treatments consisting of the supply of the concentrated aqueous fraction (M3) are provided during the test:

- [TTT1] Four days after transplanting at the level of the mini-root ball;

- [TTT2] one week after the 1st treatment.

[256] The modalities tested and the doses provided are presented in the following table 18:

[Table 18]

calcul sur la base de 100000 laitues/ha. | Modalities | Agronomic application dose

calculation based on 100,000 lettuces/ha.

Table 18: Modalities tested and doses provided in example 4a.

[257] The trial is launched in a completely randomized design.

[258] The duration of the test is 4 weeks after transplanting the lettuces into pots.

Cultivation management a) Cultivation pots and substrate

[259] In this trial, the lettuce was grown in 0.45 L pots. The substrate (sowing and growth) is light and draining to promote establishment. It is composed of a mixture of sand (20% v/v), perlite (40% v/v) and fine vermiculite (40% v/v).

[260] The characteristics of the substrate (percentage of dry matter and CRmax) are determined prior to the test.

[261] The substrate is saturated prior to transplanting (CRmax setting without leaching). b) Sowing

[262] The trial was carried out with a commercial variety of Batavia lettuce, at a density of one plant per pot. The seeds were sown in seed trays and then transplanted (at the 2-leaf stage) into the pots. c) Fertilization

[263] Fertilization is suboptimal to ensure good establishment and growth of crops. It is composed of a complete nutrient solution, provided diluted at a rate of 50 mL of nutrient solution per pot per week.

[264] The ready-to-use nutrient solution is obtained by mixing 9 previously prepared stock solutions (concentrated).

[265] The composition of the stock solutions and the preparation of the nutrient solution are those presented in Table 8 of Example 3a.

[266] With an input of 50 mL of this nutrient solution, the equivalent in milligrams of nutrients provided per plant is that represented in table 9 of example 3a.

[267] Initially (just before transplanting), the pots are brought to a humidity close to water saturation (CRmax without leaching). During sowing and a few days after transplanting, watering is optimal. The pots are then watered two to three times per week (without leaching) to maintain humidity levels at approximately 70% of the maximum water holding capacity throughout the crop. [268] Water stress sets in a few days after transplanting: watering is stopped for all stressed pots (M2 and M3), until stress symptoms appear:

Unstressed pots (M1): target 70% CRmax (setpoint between 65 and 75% CRmax), watering is carried out 2 to 3 times per week.

Water stress (M2 and M3): target 30% CRmax (setpoint between 20 and 40% CRmax).

[269] - Measurements and monitoring a) Observations and measurements

[270] During the test:

Health status of seedlings;

Measurement of projected leaf area (Canopeo®) once a week.

[271] At the end of the test:

The aerial biomass of lettuce is weighed fresh and dry (BFA/BSA);

The root systems are extracted from the pots and weighed fresh (BFR);

Root architecture is analyzed by WinRhizo® imaging;

Root biomasses are weighed in dry weight (BSR).

[272] The data are statistically analyzed by an analysis of variance at the 95% confidence level. The conditions for applying ANOVA are verified (residual normality test and Levene's homoscedasticity test).

2. Results

[273] The objective of this example is to evaluate the effectiveness of the concentrated aqueous fraction as a biostimulant on the growth and root and aerial development of young Batavia lettuce plants grown under water stress.

2.1 Characterization of water stress

[274] Watering of the plants was carried out in such a way as to maintain the substrate at approximately 70% of its maximum water retention capacity in the unstressed modality (M1) and at 30% of the maximum water retention capacity in the stressed modalities (M2 and M3). Between 06/30/2022 and harvest on 07/18/2022, the humidity level of the pots was monitored by individual weighing and watering per pot in such a way as to maintain the humidity levels within the limits defined previously.

2.2 Biomass

[275] Fresh aerial (BFA) and root (BFR) biomasses are weighed immediately at the time of harvest, they are then placed in an oven at 40°C until their mass is constant before being weighed dry (dry aerial (BSA) and root (BSR) biomasses) (Figure 9). [276] The data then undergo statistical analysis to determine significant effects.

[277] Statistical analysis shows that:

On the fresh aerial biomass (FAB): we note a significant effect of water stress (M1, the untreated and unstressed control has a fresh aerial biomass higher than the other two modalities) as well as a significant effect of the treatment (the fresh aerial biomass of M3, the modality treated with the concentrated aqueous fraction is higher than that of M2, untreated).

On dry biomasses, we note the same significant effect.

[278] The statistical analyses of fresh and dry biomasses are represented in Figure 9, the conclusions of the tests are represented in the following Table 19:

[Table 19]

Table 19: Statistical analysis of fresh and dry biomasses from example 4a.

2.3 Projected leaf area

[279] During growth, the projected leaf area of each plant was estimated once a week using a photograph taken from above. Using a 1 cm ² surface standard and Canopeo® software, the projected leaf area is calculated for each plant (Figure 10).

[280] These data are then statistically analyzed; the analysis shows that:

At the start of water stress (06/30/2022), the treatment method has no significant effect.

One week later, significant differences appear between the three modalities: the projected leaf area of the unstressed control (M1) is significantly greater than that of the stressed control (M2) while the modality treated with the concentrated aqueous fraction (M3) does not differ significantly from any of the other two modalities.

7 days later, the gap between the modalities widened and the three modalities differed significantly: M1 > M3 > M2.

[281] The results of the statistical analyses are summarized in the following Table 20:

[Table 20]

Table 20: Statistical analysis of the projected leaf areas of example 4a.

2.4 Root architecture

[282] At harvest time, the lettuce roots were removed from the substrate and cleaned with water.

[283] An analysis of the root systems (root scanner and WinRhizo® analysis) is then carried out: the roots are spread out on a plexiglass plate containing a water slide and then scanned in two dimensions. The image is digitized and then root diameter class increments are fixed for the analysis of the root architecture. The indicators measured by the software are then accessible in absolute value in total and by diameter class.

[284] Due to the root density, the root systems were cut to properly measure all the parameters of the root architecture. The accessible indicators are thus the length (L), the volume (V) and the root surface (SA). The number of ramifications (R) and the number of ends (T) are not accessible because of the dissection. a) Total indicators

[285] Statistical analysis of the data (Figure 11) shows that the effect of water stress is significant on the parameters of total root length, total root surface area and total root volume: the untreated and unstressed control modality (M1) presents significantly higher means than those of the two untreated control modalities under water stress (M2) and the modality treated with the concentrated aqueous fraction and subjected to water stress (M3).

[286] On the other hand, on the average root diameter indicator, the statistical analysis shows that the modality treated with the concentrated and stressed aqueous fraction (M3) is equivalent to the non-stressed control (M1) while the water stress control (M2) has a significantly lower average value.

[287] The results of the statistical analyses are shown in the following Table 21:

[Table 21]

Table 21: Statistical analysis of the total indicators of example 4a. b) Root lengths by diameter classes

[288] The diameter class increments were determined visually, in order to discriminate roots by root branching level as follows:

Diameters greater than 0.4 mm correspond to the largest sections below the neck.

The roots making up the root bundle, called 1st order, have a diameter between 0.3 and 0.4 mm.

2nd order roots (branched from the bundle) have a diameter between 0.2 mm and 0.3 mm.

Finally, the finest roots (3rd order) have a diameter < 0.2 mm.

[289] The WinRhizo® software is capable of determining the cumulative root length, by root diameter class.

[290] Statistical analysis (Figure 12) shows that the lengths of the thickest ^1st order roots (primary bundle) and their ramifications ( ^2nd order) do not present any significant difference between the 3 modalities. On the other hand, the thinnest roots ( ^3rd order), which are the most physiologically active roots in relation to nutrient and water uptake, are significantly impacted by water stress (M2 and M3) compared to the control (M1).

[291] The effect of the modalities on the lengths by classes of root diameters is summarized in the following table 22:

[Table 22]

Table 22: Statistical analysis of lengths by diameter classes of example 4a.

[292] The results showed that:

The water regime used generated significant water stress which resulted in a reduction in the aerial biomass of lettuce of 38% (14.77 g/plant in unstressed controls (M1) and 9.09 g/plant in stressed plants without the addition of concentrated aqueous fraction (M2).

The concentrated aqueous fraction significantly increased the fresh aerial biomass of lettuce plants from 9.09 g/plant in stressed and untreated plants (M2) to 10.17 g/plant in stressed plants treated with the concentrated aqueous fraction (M3), an increase of 11.8%. The same result was observed for the dry aerial biomass with a significant effect and an increase of 14%.

After two weeks of water stress, treatment with the concentrated aqueous fraction increased the projected leaf area measured by Canopeo® by 17% compared to stressed and untreated plants (M2).

At the root level, the contribution of the concentrated aqueous fraction significantly increased the average diameter of stressed plants treated with the concentrated aqueous fraction (M3) by 3.4% (compared to stressed and untreated plants (M2)).

Example 4b: Influence of the concentrated aqueous fraction under two different water stress conditions on the growth and aerial development of young lettuce plants

[293] In the present example, the efficacy of the concentrated aqueous fraction was evaluated as a biostimulant on the growth and aerial development of young lettuce plants grown under water stress conditions. Furthermore, the present example also aims to confirm the results of Example 4a by applying two levels of water stress (moderate and strong).

1. Materials and methods

[294] Lettuce is grown on a growing medium (sand/perlite/fine vermiculite). Sowing is done in a greenhouse, in a tray, then one plant per pot is transplanted into 450 mL pots.

[295] The following Table 23 indicates the quantities of culture medium used in this example:

Tableau 23 : Quantité de matériels et consommable utilisés en exemple 4b. [Table 23]

Table 23: Quantity of materials and consumables used in example 4b.

[296] The test then takes place in a semi-controlled greenhouse (regulated temperature, natural lighting with the possibility of supplementing with a light intensity detector).

[297] The experimental device includes 5 modalities:

Modality 1 (M1): an untreated and unstressed control (TNT) (optimal watering - 70% CRmax),

Modality 2 (M2): an untreated and “moderately” stressed control (TSH 1) (40% CRmax),

Modality 3 (M3): an untreated and “highly” stressed control (TSH 2) (20% CRmax), Modality 4 (M4): Modality treated with the concentrated aqueous fraction and “moderately” stressed (PSH 1) (40% Crmax),

Modality 5 (M5): Modality treated with the concentrated and “highly” stressed aqueous fraction (PSH 2) (20% Crmax).

[298] Each modality is repeated 12 times, making a total of 60 pots in the test.

[299] The concentrated aqueous fraction is added at the beginning or end of the day, to the substrate, locally (on the mini-clod).

[300] The application rate is 24 L of concentrated aqueous fraction/250 L of spray/ha.

[301] The conversion between agronomic dose and experimental dose is made on the basis of a planting density of 100,000 lettuces per hectare.

[302] Two treatments consisting of the supply of the concentrated aqueous fraction (M4 and M5) are provided during the test:

[TTT1] five days after transplanting at the level of the mini-root ball;

[TTT2] one week after the first treatment.

[303] The modalities tested and the doses provided are presented in the following table 24: [Table 24]

* calculation based on 100,000 lettuces/ha.

Table 24: Modalities tested and doses provided in example 4b.

[304] The trial is launched in a completely randomized design. [305] The duration of the test is 26 days after transplanting the lettuces into pots.

Cultivation management a) Cultivation pots and substrate

[306] The sowing substrate is light and draining to promote establishment; it is composed of a mixture of peat (50% v/v), perlite (25% v/v) and fine vermiculite (25% v/v).

[307] After 14 days, the plants are transplanted into 0.45 L pots containing a growth substrate (GS) composed of sand (20% v/v), perlite (40% v/v) and fine vermiculite (40% v/v).

[308] The characteristics of the substrate (percentage of dry matter and CRmax) are determined prior to the test.

[309] The substrate is saturated prior to transplanting (CRmax setting without leaching).

[310] The trial was carried out with a commercial variety of Batavia lettuce, at a density of one plant per pot. The seeds were sown in seed trays and then transplanted (at the 2-leaf stage) into the pots.

[311] Fertilization is suboptimal to ensure good establishment and growth of crops. It is composed of a complete nutrient solution (Hoagland type with trace elements), provided diluted once a week. The ready-to-use nutrient solution is obtained by mixing 9 previously prepared stock solutions (concentrated).

[312] The composition of the stock solutions and the preparation of the nutrient solution are those presented in Table 8 of Example 3a.

[313] With a supply of 50 mL of this nutrient solution, the equivalent in milligrams of nutrients supplied per plant is that shown in Table 9 of Example 3a. b) Irrigation and water stress management

[314] Initially (just before transplanting), the pots are brought to a humidity close to water saturation (CRmax without leaching). During sowing and a few days after transplanting, watering is optimal. The pots are then watered two to three times per week (without leaching) to maintain humidity levels at approximately 70% of the maximum water holding capacity throughout the crop.

[315] Water stress sets in a few days after transplanting: watering is stopped for all stressed pots, until stress symptoms appear:

consigne entre 30 et 50% CRmax. Unstressed pots (M1): target 70% CRmax setpoint between 60 and 80% CRmax. Water stress 1 (moderate) (M2 and M4): setpoint at 40%

setpoint between 30 and 50% CRmax.

consigne entre 10 et 30% CRmax. Water stress 2 (high or strong) (M3 and M5): setpoint at 20%

setpoint between 10 and 30% CRmax.

[316] - Measurements and monitoring a) Observations and measurements

[317] During the test:

Health status of seedlings;

Measurement of projected leaf area (Canopeo®) once a week.

[318] At the end of the test:

Aboveground biomass is weighed in fresh (BFA) (marketable mass) and dry (BSA). b) Data analysis

[319] The data are statistically analyzed by an analysis of variance at the 95% confidence level. The conditions for applying ANOVA are verified (residual normality test and Levene's homoscedasticity test).

2. Results

2.1 Characterization of water stress

[320] Watering of the plants was carried out in such a way as to maintain the substrate at approximately 70% of its maximum water retention capacity in the non-stressed mode (M1), at 40% of the maximum CR for the so-called “moderate” stress and around 20% for the so-called “strong” stress.

[321] Between transplanting and harvesting, the humidity level of the pots was monitored by individual weighing and watering per pot in order to maintain the humidity levels within the limits defined previously.

2.2 Biomass

[322] Fresh aerial biomass (FAB) (Figure 13) represents the marketable mass. It is weighed immediately at the time of harvest, then placed in an oven at 40°C until its mass is constant before being weighed dry (BSA) (Figure 14).

[323] The data then undergo statistical analysis to determine significant effects.

[324] Statistical analysis shows that for fresh aerial biomass:

The effect of both levels of water stress is significant on the untreated modalities: plants subjected to “strong” water stress (M3) have a significantly lower biomass than plants subjected to “moderate” stress (M2), which have- themselves a biomass significantly lower than unstressed plants (M3 < M2 < M1).

The effect of the treatment is significant on the fresh aerial biomass (FAB) at the level of moderate water stress (M4): the plants treated with the concentrated aqueous fraction have a significantly higher biomass than the untreated plants, subjected to the same stress (M2 < M4).

The effect of the treatment is significant on the fresh aerial biomass (FAB) at the level of strong water stress (M5): the plants treated with the concentrated aqueous fraction have a significantly higher biomass than the untreated plants (M3), subjected to the same stress (M3 < M5).

Statistical analysis shows that there is no significant difference between untreated control plants grown under optimal water conditions and plants treated with the concentrated aqueous fraction, at the level of moderate water stress (M4=M1). At the level of “strong” stress, plants treated with the concentrated aqueous fraction have a biomass significantly lower than the control under optimal conditions, but a biomass significantly higher than the controls, whatever their level of stress (even moderate) (M3 < M2 < M5 < M1).

[325] These results are very interesting in relation to the potential of the concentrated aqueous fraction on lettuce (fresh biomass constitutes the most relevant parameter for lettuce because it constitutes the indicator of commercial quality).

[326] In terms of dry biomass, the results are comparable: the effect of the treatments is significant (P=0.0000). We observe that:

The effect of the two levels of water stress is significant on the untreated modalities: the dry biomass of modality 3 is significantly lower than the dry biomass of modality 2, itself significantly lower than the dry biomass of the unstressed plants (M3 < M2 < M1).

The treatment effect is significant on above-ground dry biomass (ASB) at both water stress levels: M2 < M4 and M3 < M5.

There is no significant difference between untreated control plants grown under optimal water conditions and plants treated with the concentrated water fraction, regardless of the level of water stress (M1=M4=M5).

The level of water stress does not impact the dry biomass of lettuce treated with the concentrated aqueous fraction.

[327] These results show that the concentrated aqueous fraction exerts a real effect on the tolerance of lettuce to water stress (moderate and strong): the treated plants continue to grow in a manner comparable to the non-stressed control, and the treatment has an effect on the biomass of the lettuce, not only on the water content. 2.3 Water content

[328] The water content of lettuce was calculated using fresh and dry biomass. The means and standard deviations per modality are shown in Figure 15.

[329] In view of Figure 15, it appears that the water content of lettuce is extremely stable, whatever the treatment method and water regime.

[330] A statistical analysis was performed on these data. A non-parametric Kruskal-Wallis test is presented. This concludes that there is no significant difference between the medians at the 95% confidence level (P=0.1559).

2.4 Projected leaf area

[331] During growth, the projected leaf area of each plant was estimated once a week using a photograph taken from above. Using a 1 cm ² surface standard and Canopeo® software, the projected leaf area is calculated for each plant.

[332] These data are then statistically analyzed (Figure 16); the analysis shows that

At the beginning of water stress (17 and 23/11), there is no significant effect of the modalities. The following two weeks, significant differences appear between the modalities (a) control in water stress (M2 and M3) and (b) optimal control and modalities treated with the concentrated aqueous fraction (M1, M4 and M5).

Finally, in the last measurement, we observe 3 statistically homogeneous groups:

(a) the highly stressed control (M3) with a projected leaf area of 141 ^cm2 ;

(b) the moderately stressed control (M2) with a projected leaf area of 158 ^cm2 ;

(c) the unstressed control under optimal conditions (M1) (area = 183 ^cm2 ) and the two treated and stressed modalities (M4 and M5) with respective projected leaf areas of 196 and 189 ^cm2 .

[333] The results of the statistical analyses are shown in the following Table 25:

[Table 25]

* data transformed by the Log function

Table 25: Statistical analysis of the projected leaf areas of example 4b. [334] The results showed that:

The two water stress regimes (moderate and high) used generated two significant levels of water stress which resulted in a reduction in the aerial biomass of untreated lettuce of -17% and -27% (respectively at the moderate and high stress levels).

The concentrated aqueous fraction significantly increased the fresh above-ground biomass of lettuce plants (which constitutes the marketable mass for lettuce) o In moderate water stress from 12.7 g/plant in untreated plants to 15.0 g/plant in plants treated with the concentrated aqueous fraction, an increase of +18%. o In severe water stress from 11.2 g/plant in untreated plants to 14.0 g/plant in plants treated with the concentrated aqueous fraction, an increase of +25%.

On dry biomass, the effect of the treatment is always significant, which shows that the concentrated aqueous fraction exerts a real effectiveness on the biomass, which is statistically comparable to the control, whatever the level of water stress.

This is confirmed by the analysis of the water content of the plants, where it is shown that there is no significant effect of the treatment on this indicator.

From two weeks after the start of water stress, treatment with the concentrated aqueous fraction increased the projected leaf area measured by Canopeo® of lettuce plants under water stress compared to stressed and untreated plants; moreover, the leaf area remained equivalent to the unstressed control throughout the entire trial.

Example 5: Comparison of the influence of the concentrated aqueous fraction and a composition comprising proline and trehalose on young Batavia lettuce plants under water stress conditions

[335] In this example, the effectiveness of the concentrated aqueous fraction was compared with that of a comparative composition containing proline and trehalose, by evaluating their impact on the growth and development of the aerial and root parts of young Batavia lettuce plants subjected to water stress conditions.

1. Materials and methods

[336] The lettuce is grown on a seedling growing medium (peat/perlite/fine vermiculite) in trays then one plant per pot is transplanted into 1.1 L pots on a growth growing medium (sand/perlite/fine vermiculite).

[337] The following Table 26 indicates the quantities of culture medium used in this example:

[Table 26]

Table 26: Quantity of materials and consumables used in example 5.

[338] Sowing and growth take place entirely in a semi-controlled greenhouse (regulated temperature, natural lighting with the possibility of supplementing with a light intensity detector).

[339] The experimental device includes 5 modalities:

Modality 1 (M1): an untreated and stressed control (TNT SH) (30% CRmax),

Modality 2 (M2): Modality treated with the concentrated aqueous fraction at dose 1 (6 L/ha) and stressed (Y6 SH) (30% Crmax),

Modality 3 (M3): Modality treated with the concentrated aqueous fraction at dose 2 (3 L/ha) and stressed (Y3 SH) (30% Crmax),

Modality 4 (M4): Modality treated with a comparative composition including proline and trehalose at dose 1 (6 L/ha) and stressed (REF6 SH) (30% Crmax), Modality 5 (M5): Modality treated with a comparative composition including proline and trehalose at dose 2 (3 L/ha) and stressed (REF3 SH) (30% Crmax).

[340] The comparative composition (proline and trehalose) contains concentrations of proline and trehalose equivalent to those present in the concentrated aqueous fraction, with identical application rates. This corresponds to an average of 100 g/ha for proline and 14 g/ha for trehalose at a rate of 6 L/ha, and to quantities reduced by half for a rate of 3 L/ha.

[341] Each modality is repeated 12 times, making a total of 60 pots in the test.

[342] The concentrated aqueous fraction and the comparative composition are added at the start of the day, locally to the substrate (near the collar, on the mini-mound).

[343] The tested intake doses are to be diluted and applied as slurries:

Dose 1 = 6 L of concentrated aqueous fraction or comparative composition/250 L of spray mixture/ha (i.e. 24 mL of concentrated aqueous fraction or comparative composition/L). Dose 2 = 3 L of concentrated aqueous fraction or comparative composition/250 L of spray mixture/ha (i.e. 12 mL of concentrated aqueous fraction or comparative composition/L).

[344] The conversion between agronomic dose and experimental dose is based on a planting density of 100,000 lettuces per hectare (i.e. a soil application of 2.5 mL of mixture per plant).

[345] Two treatments consisting of the contribution of the concentrated aqueous fraction (M2 and M3) and the contribution of the comparative composition (M4 and M5) are provided during the test:

[TTT1] 10 days after transplanting;

[TTT2] 7 days after the first treatment.

calcul sur la base de 100000 laitues/ha. [346] The modalities tested and the doses provided are presented in the following table 27: [Table 27]

calculation based on 100,000 lettuces/ha.

Table 27: Modalities tested and doses provided in example 5.

[347] The trial is launched in a completely randomized design.

[348] The duration of the trial is 7 weeks after transplanting the lettuces into pots.

Cultivation management a) Cultivation pots and substrate

[349] The seeding substrate (SS) is composed of a mixture of peat (50% v/v), perlite (25% v/v) and fine vermiculite (25% v/v).

[350] After 27 days, the plants are transplanted into 1.1 L pots containing a growth substrate (GS) composed of sand (20% v/v), perlite (40% v/v) and fine vermiculite (40% v/v).

[351] The characteristics of the substrate (percentage of dry matter and CRmax) are determined prior to the test. [352] The substrate is saturated prior to transplanting (CRmax setting without leaching).

[353] The trial was carried out with a commercial variety of Batavia lettuce, at a density of one plant per pot. The seeds were sown in seed trays and then transplanted (at the 3-leaf stage) into the pots.

[354] Fertilization is suboptimal to ensure good establishment and growth of crops. It is composed of a complete nutrient solution (Hoagland type with trace elements), provided diluted to 50 mL per pot, once a week. The ready-to-use nutrient solution is obtained by mixing 9 previously prepared stock solutions (concentrated).

[355] The composition of the stock solutions and the preparation of the nutrient solution are those presented in Table 8 of Example 3a.

[356] With a supply of 50 mL of this nutrient solution, the equivalent in milligrams of nutrients supplied per plant is that shown in Table 9 of Example 3a. b) Irrigation and water stress management

[357] Initially (just before transplanting), the pots are brought to a humidity close to water saturation (CRmax without leaching). During sowing and a few days after transplanting, watering is optimal. The pots are then watered two to three times per week (without leaching) in order to maintain humidity levels at approximately 70% of the maximum water holding capacity throughout the crop.

[358] Water stress is established 7 days after transplanting: watering is stopped for all pots (M1 to M5), until stress symptoms appear:

Stressed pots (M1 to M5): target 30% CRmax -» setpoint between 20% and 40% CRmax.

Measurements and monitoring a) Observations and measurements

[359] During the test:

Health status of seedlings;

Measurement of projected leaf area (Canopeo®) once a week (5 measurements).

[360] At the end of the test:

The aerial biomass of lettuce is weighed in fresh (BFA) (marketable mass) and dry (BSA). b) Data analysis [361] The data are statistically analyzed by analysis of variance at the 90% confidence level. The conditions for applying ANOVA are verified (residual normality test and Levene's homoscedasticity test).

2. Results

2.1 Characterization of water stress

[362] Watering of the lettuce plants was carried out in such a way as to maintain the substrate at approximately 30% of its maximum water retention capacity for all modalities (M1 to M5).

[363] Between transplanting and harvesting, the humidity level of the pots was monitored by individual weighing and watering per pot in order to maintain the humidity levels within the limits defined previously.

2.2 Biomass

[364] Fresh aerial biomass (FAB) (Figure 17) and fresh root biomass (FRB) are weighed immediately at the time of harvest (Figure 18).

[365] The data then undergo statistical analysis to determine significant effects.

[366] Statistical analysis shows that:

On the fresh aerial biomass (FAB) (Figure 17): a significant effect of water stress is noted on the untreated and water-stressed fresh aerial biomasses (M1). The fresh aerial biomasses of the modalities treated with the concentrated aqueous fraction and stressed, at dose 1 (6L/ha) (M2) and at dose 2 (3L/ha) (M3), are higher than those of the untreated and stressed modality (M1), as well as those of the modalities treated with the comparative composition and stressed, at dose 1 (6L/ha) (M4) and at dose 2 (3L/ha) (M5). The comparative composition (proline and trehalose) even causes a decrease in the fresh aerial biomass for both doses.

On the fresh root biomass (FRB) (Figure 18): a significant effect of water stress is also noted on the untreated and water-stressed fresh root biomasses (M1). The fresh root biomasses of the modalities treated with the concentrated aqueous fraction and stressed, at dose 1 (6L/ha) (M2) and at dose 2 (3L/ha) (M3), are higher than those of the untreated and stressed modality (M1), as well as those of the modalities treated with the comparative composition and stressed, at dose 1 (6L/ha) (M4) and at dose 2 (3L/ha) (M5).

[367] These results are very interesting in terms of the effectiveness of the concentrated aqueous fraction on lettuce, in particular on fresh biomass, which constitutes the most relevant parameter for lettuce because it constitutes the indicator of commercial quality. These performances are superior to those of comparative compositions comprising proline and trehalose formulated with the same contents and administered at the same doses than in the concentrated aqueous fraction. These comparative compositions do not show any biostimulant effect at doses 1 and 2 and even lead to a reduction in the fresh aerial biomass of lettuce plants, going against the expected effect.

[368] These results demonstrate that the concentrated aqueous fraction has a biostimulant effect on lettuce plants, surpassing the effect of a comparative composition composed of proline and trehalose, by improving the tolerance of lettuce to water stress.

2.3 Projected leaf area

[369] During growth, the projected leaf area of each plant was estimated 5 times using a photograph taken from above. Using a 1 cm2 surface standard and Canopeo® software, the projected leaf area is calculated for each plant.

These data are then statistically analyzed (Figure 19); and the averages of the 12 repetitions for each modality (M1 to M5) of the projected leaf surfaces at 16 days, 23 days, 30 days and 37 days after transplanting are indicated in the following Table 28:

[Table 28]

Table 28: Average projected leaf areas at 16 days, 23 days, 30 days and 37 days after transplanting for methods 1 to 5.

[370] This analysis shows that:

At the start of water stress (9 days after transplanting (9DAR), Figure 19), there is no significant effect of the modalities.

At 16 days after transplanting (16 DAR), i.e. only 7 days after the establishment of water stress, as well as at 23 days, 30 days and 37 days after transplanting (23DAR, 30DAR and 37DAR), the stressed and treated modalities with the concentrated aqueous fraction (M2 and M3) have a significant effect on the projected leaf area compared to the untreated and stressed modality (M1) and the modalities treated with the comparative composition and stressed, at dose 1 (6L/ha) and dose 2 (3L/ha) (M4 and M5). Moreover, at 16 days, 30 days and 37 days after transplanting, the projected leaf areas obtained for the modalities treated with the comparative composition and stressed (M4 and M5) are lower than those of the untreated and stressed modality (M1).

[371] The results showed that:

The concentrated aqueous fraction significantly increased the fresh aboveground biomass of lettuce plants (which constitutes the marketable mass for lettuce) as well as the fresh root biomass.

From only 9 days after the onset of water stress, treatment with the concentrated aqueous fraction increased the projected leaf area, measured by Canopeo®, of lettuce plants under water stress compared to stressed and untreated plants and to stressed and treated modalities with the comparative composition (M4 and M5).

The comparative composition does not exert any biostimulant effect; on the contrary, it leads to a reduction in fresh aerial biomass as well as in the projected leaf surface.

On the other hand, the concentrated aqueous fraction shows a biostimulant effect at both doses (6 L/ha and 3 L/ha), on the lettuce plant, even under water stress conditions. This highlights the true effectiveness of the concentrated aqueous fraction as a biostimulant.

Claims

Claims 1. Use of an aqueous composition obtained from insects, as a biostimulant. 2. Use according to claim 1, wherein the biostimulant is used on plants to improve their tolerance to salt stress and/or water stress. 3. Use according to claim 1 or 2, in which the aqueous composition comprises between 15% and 50% by weight of dry matter, the percentage by weight being indicated on the weight of the aqueous composition. 4. Use according to any one of the preceding claims, in which the aqueous composition comprises at least 35% by weight of proteins and between 1% and 15% by weight of lipids, the percentages by weight being indicated on the dry weight of the aqueous composition. 5. Use according to any one of the preceding claims, in which the aqueous composition comprises between 2% and 10% by weight of proline, the percentage by weight being indicated on the dry weight of the aqueous composition. 6. Use according to any one of the preceding claims, in which the aqueous composition comprises between 65% and 95% by weight of organic matter, the percentage by weight being indicated on the dry weight of the aqueous composition. 7. Use according to any one of the preceding claims, in which the aqueous composition comprises between 0.1% and 5% by weight of trehalose, the percentage by weight being indicated on the dry weight of the aqueous composition. 8. Use according to any one of the preceding claims, wherein the aqueous composition is an aqueous fraction of insects. 9. Use according to claim 8, wherein the aqueous insect fraction is a concentrated aqueous insect fraction, obtainable by the preparation process comprising the following steps: i) Separation of the cuticle from the soft part of the insects, ii) Separation of the soft part into a solid fraction, an aqueous fraction and an oily fraction, iii) Concentration of the aqueous fraction, making it possible to obtain a concentrated aqueous fraction of insects. 10. Use according to any one of the preceding claims, wherein the insects are beetles. 11. Use according to any one of the preceding claims, wherein the biostimulant is used on plants selected from the group consisting of agricultural plants, ornamental plants and herbaceous plants. 12. Use according to any one of the preceding claims, wherein the biostimulant is applied to the soil. 13. Use according to any one of claims 1 to 11, in which the biostimulant is applied to the leaves of the plant.