Biocontrol product using a co-product of leek

Abstract

The present invention proposes a biocontrol product comprising or consisting of a leek powder having a particle size of less than 700 µm. The leek used in the biocontrol product is derived from the co-product of the harvesting of leek and exhibits a very high antifungal activity when it is brought into contact with an agricultural product to be protected, such as a seed, a crop or a fruit that has been harvested. The invention also relates to aqueous extract obtained from the biocontrol product of the invention and to an optimised method for obtaining said biocontrol product. Finally, the invention also proposes the combined use of a biocontrol agent, such as copper salts, lipopeptides and detergents, with the biocontrol product of the invention and in order to obtain an antifungal synergistic effect.

Description

[0001] BIOCONTROL PRODUCT FROM LEEK COPRODUCT

[0002] Field of invention

[0003] The invention relates to the field of biocontrol molecules and the recovery of agricultural waste. The invention relates in particular to the recovery of leek co-product as a biocontrol product, and the method for obtaining it.

[0004] State of the art

[0005] The cultivation of cereals, fruits and vegetables is subject to attacks by pests (insects, weeds, microorganisms, etc.) that affect crop yields and thus reduce producers' profits. To overcome these disadvantages, farmers regularly, if not massively, use pesticides to protect crops and thus increase their yields. However, the use of pesticides is not without risks to the health or sustainability of cultivated soils. In addition, their selectivity is also lacking; pests are certainly destroyed effectively, but other species of insects or microorganisms beneficial to plants can be affected. It should be noted that since their use, certain pesticides are now banned from sale.

[0006] To address the shortcomings of pesticides, farmers have sought alternatives and turned to so-called biocontrol products, which are more selective and less toxic to the environment. Most often, these are natural products present in plants or microorganisms; application WO2022047396 demonstrates that microbial compositions have beneficial effects on agriculture. Although effective, the production of natural products by microorganisms remains low, and the quantities needed to protect crops may not be sufficient.

[0007] In application WO2021236799 reference is made to a biological control composition based on an intact minicell containing a nucleic acid that targets a transcription product encoding a polypeptide and a method for controlling pests and pathogens with novel systems and compositions for administering RNA molecules within crops. Here again, the use of an intact minicell, although effective, may encounter concerns about stability of use within soils.

[0008] Thus, there is a clear need for alternatives to pesticides, and biocontrol products are one of these alternatives that solve the disadvantages of said pesticides. However, the quantity of biocontrol agents obtained and their stability remains a problem that must be resolved in order to make them a daily use that positively impacts crop yields while preserving them and the environment.

[0009] In particular, there is a need for effective biocontrol agents to address fungal diseases that are involved in many plant diseases and cause significant production losses. For example, diseases such as gray mold, alternaria, and fusarium wilts affect various plant productions, from seed to post-harvest product.

[0010] Plant extracts can at least partially meet these needs. For example, the allium family, and garlic in particular, has been known for many years for its antibacterial effects, as well as its antifungal and insecticidal activity. Its effectiveness has been widely demonstrated, and garlic extract is now recognized in France in the list of biocontrol plant protection products, under Article L. 253-5 and L. 253-7 of the Rural and Maritime Fisheries Code. Garlic extract is marketed in concentrations of 45% w / w as a biocontrol product.

[0011] However, it remains desirable to have other biocontrol products that can effectively combat fungal diseases and molds at different stages of agricultural production. It is also desirable to have new biocontrol agents available in large volumes, at low cost, and without monopolizing agricultural land for their production.

[0012] Summary of the invention

[0013] One aim of the invention is therefore to propose a new biocontrol product effective against fungal diseases, which can be produced in large volumes and at a moderate price.

[0014] To this end, the invention provides a biocontrol product comprising or consisting of leek powder (Allium porrum) whose average particle size is equal to or less than 700 μm, and preferably between 10 μm and 700 μm. The average particle size of the powder may also be from 90 to 500 μm, from 90 μm to 300 μm, or preferably from 90 μm to 180 μm. In one embodiment, the particle size is between 90 and 700 μm.

[0015] Indeed, it is the inventors' merit to have demonstrated that dried leek in powder form with a particle size < 700 pm has antifungal properties against a significant spectrum of pathogenic molds and their spores. Surprisingly, the inventors found that at the same dose of leek powder tested, a particle size greater than 700 pm strongly limits the antifungal activity and does not prevent mold growth.

[0016] The antifungal effect can be obtained by using leek powder placed in direct contact with an agricultural product to be protected, such as a seed, a crop or a harvested fruit. The leek powder according to the invention therefore constitutes a biocontrol product in itself, or it can be incorporated into a formulation comprising excipients, preservatives and / or other active ingredients, or be used in the form of an extract.

[0017] The biocontrol product according to the invention is derived from the upper part and / or the lower part of the leek. The upper part corresponds to the leaves of the green part of the leek and the lower part corresponds to the leaves of the white part of the leek typically used as food.

[0018] Advantageously, the biocontrol product of the invention is derived from the co-product of the leek harvest, and is composed essentially, mainly or exclusively of the green part. It will thus be possible to recover a plant waste whose source is sustainable and abundant. Indeed, the annual production of leeks in France is approximately 165,000 tonnes and it is estimated that 15% or 20% of the production is lost as a co-product resulting from post-harvest peeling. The present invention therefore makes it possible to recover as a biocontrol product this part of the leek harvest which is currently unexploited.

[0019] In order to facilitate the application of the biocontrol product, the invention proposes to produce an aqueous extract from the leek powder described above. Advantageously, a simple aqueous extract exhibits high antifungal activity when brought into contact with the product to be treated. Moreover, unlike garlic extracts, the aqueous extract of the invention does not exhibit antifungal activity when it is at a distance from the product to be treated.

[0020] In one embodiment, the invention provides an aqueous extract having a concentration of leek powder to solvent volume of between 5 g / L and 50 g / L, preferably 10 g / L to 25 g / L. In particular, a concentration of between 10 g and 50 g of leek powder per 1 L of water allows approximately 60 to 90% of the antifungal activity of the powder to be extracted. The extract may be used with the leek powder suspended in water or as a filtrate to facilitate the use of the extract.

[0021] The pH of the simple aqueous extract is between 6 and 7 without pH adjustment, especially between 6.5 and 6.8 and exhibits antifungal activity. Preferably, the pH of the extract is between 3.5 and 5.5, and preferably between 3.7 and 4.7. The antifungal activity can thus be significantly increased. Advantageously, the extract comprises an organic acid, such as acetic acid, to acidify the pH and further improve the antifungal activity.

[0022] The invention also relates to a biocontrol, insecticidal or antifungal composition comprising a biocontrol product according to the invention or an aqueous extract of said biocontrol product.

[0023] Advantageously, the inventors have demonstrated the synergistic effect of leek powder or its extract according to the invention with other biocontrol agents, such as copper salts, cationic and anionic detergents, lipopeptides and acetic acid. The invention therefore provides a composition comprising a biocontrol product according to the invention or an aqueous extract of said biocontrol product and also comprising one of the following compounds or any combination of these compounds:

[0024] - a copper salt chosen from copper sulfate, cuprous oxide, copper hydroxide, copper oxychloride, tribasic copper sulfate, copper acetate, copper tallate and expanded copper carbonate or mixtures thereof, and making it possible to envisage a reduction of at least two in the quantities conventionally used in the treatment of crops with copper;

[0025] - an antiseptic or disinfectant surfactant, preferably an anionic surfactant compound, for example sodium lauryl sulfate, and / or a cationic surfactant, for example cetrimonium bromide, and making it possible to envisage a reduction of at least 5 in the use of leek co-product biomass and 2 in the quantity of detergent required;

[0026] - a lipopeptide-type compound exhibiting antimicrobial properties, preferably antifungal activity, for example caspofungin, or surfactins, iturins, and fengycins produced by different strains of Bacillus sp, and making it possible to envisage a reduction of at least 5 times the use of leek co-product biomass while reducing by at least 2 times the required concentrations of lipopeptides to achieve an antifungal effect.

[0027] - an organic acid chosen from acetic acid, propionic acid or their mixtures and making it possible to envisage a reduction of at least 5 times the use of leek co-product biomass while reducing by at least 2 times the concentrations required to achieve an antifungal effect.

[0028] Of course, the invention also relates to the combined use of the biocontrol product of the invention or the aqueous extract of the invention with one of the compounds mentioned above and their mixtures, in particular when they are applied as an antifungal treatment on crops, seeds and / or plants.

[0029] Due to the synergistic effect of the biocontrol product of the invention with other biocontrol products, the respective concentration of the copper salt, the antiseptic surfactant and / or the lipopeptide compound present in the composition is lower than the active dose required for the use of said compounds alone as biocontrol products. Effective antifungal concentrations can therefore be envisaged from 0.5% to 5% w / v (5 to 50g / L if V= 1 L), preferably 1% to 2.5% (10g / L to 25g / L if V= 1 L) for the leek green powder according to the invention or the aqueous extract resulting from said concentration. The leek powder can also be combined with surfactant compounds such as sodium lauryl sulfate in an amount of between 0.01% and 0.1% of the mass of leek powder included in the biocontrol composition.Leek powder can also be combined with fungicidal compounds such as copper salts which may be present in an amount of between 5% and 20% of the mass of leek powder included in the biocontrol composition. Leek powder can also be combined with a mixture of fungicides and surfactants at the aforementioned concentrations.

[0030] Empirically, the combined use of the extract of the invention with a copper salt on a crop makes it possible to apply a dose of copper of less than 4 kg / ha / year (dose authorized per year as recommended by the European Commission), and preferably less than 3 kg / ha / year or 2 kg / ha / year. The dose per application of copper salt can of course also be reduced by at least a factor of 2.

[0031] In preferred embodiments, the composition or use comprises one of the following combinations:

[0032] - an aqueous extract according to the invention, copper sulfate and a surfactant chosen from sodium lauryl sulfate and cetrimonium bromide;

[0033] - an aqueous extract according to the invention, sodium lauryl sulfate, and a lipopeptide chosen from iturin, surfactin, fengycin or their mixtures.

[0034] The composition of the invention is in liquid form, perhaps presented in the form of a powder to be incorporated into an aqueous solvent before use, or in the form of a kit comprising the different biocontrol agents to be mixed and dissolved in water in a predefined order for use.

[0035] The invention provides the use of a biocontrol product according to the invention, an extract from a biocontrol product according to the invention and / or the composition according to the invention for biocontrol, insecticide or antifungal purposes. In particular, the invention provides said use on seeds, crops or plants such as post-harvest fruits or vegetables.

[0036] The invention also relates to a seed coated by film-coating or spraying with a biocontrol product, an extract or a composition according to the invention.

[0037] The invention also relates to a method for obtaining a biocontrol product according to the invention, said method comprising the following steps:

[0038] 1) Harvesting the green or green and white parts of the leek;

[0039] 2) Cutting of parts according to 1);

[0040] 3) Drying of the said parts of the leek;

[0041] 4) Grinding of the parts after drying to obtain an average particle size

[0042] < 700 pm, and preferably between 10 pm and 700 pm, preferably 90 pm and 500 pm, even more preferably 90 pm and 180 pm.

[0043] 5) Optionally Sieving of the extract obtained in step 4.

[0044] Advantageously, the invention proposes to optimize the drying step of obtaining said extract by one or all of the following actions: - cutting into sections or strips the green or green and white parts of the leek before step 2) drying. Preferably the sections are approximately 1 to 5 cm, and the strips 0.5 cm by 2 cm. On the contrary, a loss of activity is observed when the leek parts are directly dried.

[0045] - drying step 2) is carried out at a temperature below 70°C, preferably below 60°C and more preferably at a temperature between 30 and 50°C or between 30 and 40°C. On the contrary, a loss of activity is observed with a drying temperature above 70°C.

[0046] - step 2) of drying is carried out for a drying time varying from approximately 24 hours to 72 hours, using a ventilated dehydrator. Advantageously, the drying step can thus be carried out with a means available on farms, and so as to maintain optimal activity of the biocontrol product of the invention.

[0047] - step 2) of drying is carried out until a humidity content of the order of 5% (± 1% or 2%) is obtained and so as to stabilize the components exhibiting antimicrobial activity in the biocontrol product of the invention.

[0048] The process of the invention comprises an aqueous extraction step in order to obtain the aqueous extract of the leek powder of the invention. Preferably, the duration of the extraction step is less than 60 minutes, and preferably is between 2 and 30 minutes.

[0049] Optionally, the method also comprises a step of adjusting the pH between 3.5 and 5.5, preferably 3.7 to 4.7 by adding an organic acid such as acetic acid.

[0050] Optionally, the process includes a filtration step to collect a filtrate of said extraction and thus facilitate the use of the extract. The unfiltered extract can also be used with the suspended powder.

[0051] Definitions

[0052] In the present invention, powder is understood to mean a form of matter consisting of more or less fine dry solid particles, in a free or agglomerated state.

[0053] The white or lower part of the leek, or white part of the leek, refers to the white leaves of Allium porrum, also known as Allium ampeloprasum var. porrum.

[0054] The green or upper part of the leek, or the green of the leek, refers to the green leaves of Allium porrum, also known as Allium ampeloprasum var. porrum.

[0055] In the figures, treatment means the use of leek powder or aqueous extract according to the invention.

[0056] By aqueous extract is meant the liquid from a maceration of leek powder in water, possibly filtered, and its concentration is expressed as the quantity of leek powder per volume of solvent used in said maceration, and before filtration. In the experimental part, the aqueous extract is obtained from leek green powder without this being limiting for the invention.

[0057] Figures

[0058] Fig. 1 Antifungal activity of the leek green powder according to the invention on common phytopathogens. Fig. 2 Impact of particle size on antifungal activity. Particle sizes greater than 700 pm (A), between 180 and 700 pm (B), 90 and 180 pm (C) and less than 90 pm (D).

[0059] Fig. 3 (A) Sporicidal and fungicidal effect of leek extract on Alternaria. (B) Antifungal efficacy as a function of growth stage.

[0060] Fig. 4 Post-harvest treatment of infected fruits. (A) Treatment of clementines with leek green powder, (B) treatment of clementines with aqueous extract, and (C) treatment of tomatoes with aqueous extract.

[0061] Fig. 5 Treatment of broccoli seeds with an aqueous extract of leek green. (A) Broccoli seeds were infected with spores of Alternaria alternate or Botrytis cinera and then treated with leek green extract before germination and growth for 7 days. (B) Measurement of seedlings at 7 days

[0062] Fig. 6 Antifungal activity of the aqueous extract in combination with a detergent. (A) Extract + anionic detergent, sodium lauryl sulfate, (B) Extract 50g / L diluted by 5 or 10 + cationic detergent CTAB. The wells were inoculated with a strain of A. Alternata for incubation at room temperature for 5 days.

[0063] Fig. 7 Antifungal activity of the aqueous extract in combination with a biocontrol product. (A) Extract + copper sulfate, (B) 50g / L extract diluted by 5 or 10 + a lipopeptide, surfactin (Bacillus Subtilis). The wells were inoculated with a strain of A. Alternata for incubation at room temperature for 4 to 6 days.

[0064] Fig. 8 Impact of pH on the antifungal activity of the aqueous extract. (A) Effect of the pH of the culture medium. (B) Antifungal activity of the aqueous extract prepared in water with 2 mM acetic acid (0.012%).

[0065] Fig. 9 Combined antifungal effects of aqueous extract with detergent (0.01% lauryl sulfate) and copper sulfate. Initial concentration of extract 50g / L. Wells were inoculated with A. Alternata strain for incubation at room temperature for 3 days.

[0066] Fig. 10 Combined antifungal effects of the aqueous extract with a detergent (lauryl sulfate 0.01%) and a lipopeptide (fengycin 50mg / mL). Initial concentration of the extract 50g / L. The wells were inoculated with an A. Alternata strain for incubation at room temperature. (A) Microscopic observation after 20h of treatment, (B) Macroscopic observations after 6 days of treatment.

[0067] Fig. 1 1 Combined antifungal effects of aqueous extract with detergent (lauryl sulfate 0.01%) and lipopeptide (Iturin A 25pg / ml). Initial concentration of extract 50g / L. Wells were inoculated with A. Alternata strain for incubation at room temperature for 24 hours, followed by microscopy observation.

[0068] Fig. 12 Impact of the processing method on antifungal activity. (A) Impact of the cutting method on antifungal activity. Leek leaves were cut using the indicated approaches and then subjected to the following process steps (drying at 40°C, grinding, and extraction). Alternaria growth was assessed by crystal violet quantification after incubation for 4.5 to 5 hours in the presence of extract (3.125 to 50%) in the culture medium and then compared to the negative control. (B) Impact of drying temperature on antifungal activity. Leek leaves were cut into sections and then dried at different temperatures before being subjected to the following process steps. Alternaria growth was assessed by crystal violet quantification. Fig. 13 Impact of powder concentration (10, 20, and 50 g / L) during maceration on the extraction efficiency of antifungal activity.Antifungal activity is quantified by measuring the radial growth of a Trichoderma strain after incorporation of leek powder into a nutrient agar medium. The residual activity after extraction is compared to the initial activity to deduce the percentage of activity extracted.

[0069] Fig. 14 A and 14B Impact of maceration duration on the extraction level of antifungal activity. (A) Quantification by growth measurement after crystal violet staining. (B) Germination of Alternaria in the presence of 12.5% ​​extract obtained after maceration of 2 (panel B), 30 (panel C), 60 (panel D) and 120 (panel E) min.

[0070] Detailed description of the invention

[0071] The present invention aims to enhance the value of market garden by-products, in particular leeks as a biocontrol product. In particular, the invention aims to propose a form of leek ensuring that biocontrol effects are obtained, as well as to propose forms of preparation of leeks making it possible to obtain said effect and facilitating its application to plant products such as crops, seeds and harvests.

[0072] For these purposes, the invention provides a biocontrol product comprising or consisting of leek powder having a particle size of less than 700 μm. Indeed, the inventors have found that the use of leek powder with such a particle size allows antifungal effects to be obtained against a broad spectrum of phytopathogenic molds. The leek powder of the invention can therefore be used directly as a biocontrol product by sprinkling or spraying a product to be protected. By biocontrol product is meant a product of biological origin used as a treatment in the protection of plants against diseases of microbial origin and insects.

[0073] In particular, the invention proposes to enhance the value of the green part of the leek because it is a co-product of the leek harvest. The white part of the leek is also active and can be used for the purposes of the invention; it will have the advantage of exhibiting greater activity than the green part. Indeed, leek white powder exhibits an antifungal activity approximately 5 to 10 times greater than leek green powder (results not shown). A mixture of white and green parts of leek can therefore be considered, and the ratio will depend on the percentage of each part in the co-product to be enhanced. The experimental results presented below are based exclusively on leek green powder.

[0074] In order to facilitate the use of leek, the invention proposes to produce an aqueous extract of the leek powder according to the invention. Surprisingly, a simple aqueous maceration makes it possible to obtain an extract with high antifungal efficacy, and does not require the use of particular co-solvents or a pH adjustment step. The extract of the invention can therefore be obtained very easily, and with basic equipment available on farms. The leek green extract is active from concentrations of 5 g / L and will preferably be used at a concentration of 10 g / L to 25 g / L. A concentrated leek green extract of approximately 50 g / L can also be considered to be diluted before its use as a treatment on plant products. In order to optimize the activity of the extract, it is suggested to adjust its pH between 3.5 and 5.5, preferably using an organic acid such as acetic acid.

[0075] The inventors have also demonstrated the synergistic effect of the combination of the leek powder according to the invention and other control agents whose doses used are to be reduced for environmental reasons. Synergy has been found in particular with copper salts, detergents and lipopeptides exhibiting antimicrobial activity. The invention thus makes it possible to significantly reduce the doses used of said agents when combined with the antifungal effects of leek powder or its extract.

[0076] The invention also relates to the optimization of the process for obtaining the leek powder and the aqueous extract of the invention. As already mentioned, the particle size <700pm is essential to obtain an effective antifungal effect with a low dose of leek powder. On the other hand, the use of a powder, i.e. a form of the particles in the dry state, necessarily implies the drying of the fresh form of the leek before or after its grinding.

[0077] Drying the leek is in fact a step in the process for obtaining the biocontrol product according to the invention. The inventors have highlighted the importance of this drying step in stabilizing the antifungal activity. Consequently, the invention proposes the use of leek in powder form as a biocontrol product and an extract obtained from this powder.

[0078] The invention proposes in particular a method for obtaining the biocontrol product comprising or consisting of leek powder with a particle size <700 pm. This method proposes to optimize the drying step to maintain the highest possible activity. The drying step can in particular be optimized by first cutting the fresh parts of the leek into sections or strips, and drying said pre-cut parts to approximately 5% moisture content, using temperatures below 70°C, and for a duration of 24 to 72 hours. Grinding is then carried out so as to obtain the desired particle size. To prepare the extract, it will be sufficient to carry out an aqueous maceration at room temperature for a duration of 2 to 30 minutes of the leek powder thus obtained. The extract can be used with the powder in suspension or be filtered to facilitate its use.

[0079] The following experimental section demonstrates the many advantages of valorizing the leek co-product when it is put in the form of powder and a specific particle size, or an extract obtained from this powder. The potential as a biocontrol product has been highlighted by means of the antifungal activity, but use as an insecticide is also envisaged.

[0080] EXPERIMENTAL PART

[0081] I. Processing of leeks

[0082] 1. 1 Obtaining leek powder

[0083] The green leek leaves are separated and washed before being dried at a low or medium temperature. Drying will be carried out in such a way as to preserve the activity of the molecules sensitive to its degradation and the conditions will vary depending on the drying method chosen. According to a non-limiting embodiment, the drying step is carried out using a ventilated dehydrator allowing gradual drying of the leek leaves in less than 72 hours, at a temperature below 70°C and until a preferred humidity level of around 5% is obtained.

[0084] The leek leaves are then roughly crushed and then ground indifferently using a hammer mill or a blade mill until reduced to powder form. Preferably, this grinding is carried out in cycles (generally 3 times 15 sec) with a short rest period to limit heating of the powder and down to a particle size of less than 700 pm. The objective here is to reduce the size of the biomass in order to increase the contact surface with the medium and to achieve an optimal level of extraction of the active ingredients, i.e. diffusion of active ingredients from the powder to the product to be treated, while retaining the activity of the active ingredients. In order to optimize the drying step and the preservation of the active molecules, the invention proposes cutting the leek parts into sections of approximately 1 to 5 cm or into strips of approximately 0.5 to 2 cm wide to guarantee optimal antifungal activity.This cutting step allows for faster drying at low temperature and leads to stabilization of the active forms. The strips or sections are then left to dry for 24 to 72 hours in a ventilated dehydrator at a temperature below 70°C and ideally between 30 and 50°C. In the context of the present invention, it is not recommended to grind the fresh parts, also called "fresh grinding", of the leek parts because in this case the antifungal activity decreases more than significantly, or even risks disappearing.

[0085] I.2 Aqueous extract

[0086] Antifungal activity can be extracted by maceration in water at leek green powder concentrations ranging from 10 to 50 g / L to obtain a fraction containing sufficiently concentrated active ingredients to avoid adding an additional step. Concentrations of 20 to 25 g / L are generally used to consider subsequent dilutions while maintaining a high extraction yield (around 90%) (Figure 4). The optimal maceration time to preserve antifungal activity is less than 1 h, and preferably from 2 minutes to 30 minutes.

[0087] Maceration is usually followed by a vacuum or atmospheric pressure filtration step to remove insoluble residues and facilitate the use of the extract. Generally, it is advisable to use the extract quickly after preparation.

[0088] II. Materials and methods

[0089] 2.1 Determination of the antifungal activity of the dry form

[0090] The strains used Penicillum digitatum, Fusarium oxysporum, Penicillum expansum, Alternaria alternata, Botrytis cinera and / or Trichoderma are cultured on PDA medium for 4 to 5 days at 25 or 30°C before harvesting in a 0.05% Tween solution, then adjusted to a concentration of 10 5 spores / ml.

[0091] The leek powder is weighed and then mixed with Potato Dextrose Agar (PDA) medium brought to a temperature between 40 and 50°C in order to obtain powder concentrations varying from 5 to 50 g / L. After homogenization, the mixture is placed in a Petri dish. 10 pL of spores at a concentration of 10 5 spores / ml are placed in the center of the Petri dish before incubation for 1 to 5 days at room temperature. Antifungal efficacy is assessed by measuring the growth diameter using the following formula:

[0092] % growth inhibition = (Negative control diameter - Radial diameter on PDA containing leek powder) *100 / Negative control diameter.

[0093] The negative control corresponds to a culture on PDA without treatment.

[0094] 2.2 Determination of the antifungal activity of the extract a) Quantification with crystal violet

[0095] The protocol implemented is suitable for phytopathogenic fungi with the ability to adhere to polystyrene surfaces, for example the genera Alternaria or Botrytis. Crystal violet interacts with protein structures and DNA and allows the correlation of the coloration intensity with the biomass produced. The leek extract is prepared according to the described method from a powder concentration of 25 g / L.

[0096] 100 μl of spores suspended in Potato Dextrose Broth (PDB) medium at a concentration of 10 5spores / mL are distributed in a 96-well plate and then placed for 50 minutes at 25°C. The planktonic cells are then removed by washing with 100 pL of PBS, then 100 pL of PDB2X are added and mixed with 100 pL of crude or diluted extract to obtain extract percentages varying in the medium from 3.125% to 50%. The cultures are placed for 4.5 hours at 25°C before a first observation under the microscope and quantification with crystal violet. The latter can also be set up after a 20-hour incubation.

[0097] For crystal violet quantification, a wash with 100 μL of PBS is carried out and then the culture is fixed with 100 μL of methanol before a 20-minute staining step with 0.5% crystal violet. The cultures are then washed extensively to remove excess crystal violet. 150 μL of a 33% acetic acid solution is added to resolubilize the crystal violet before an absorbance measurement at 590 nm. The growth is compared to that of an untreated culture and then quantified:

[0098] % growth = (Absorbance experimental point - Absorbance positive control) x 100 / Absorbance negative control

[0099] The negative control corresponds to a culture without treatment

[0100] The positive control corresponds to a culture containing 50% leek extract, sporicidal condition.

[0101] 2.3 Post-harvest fruit treatment

[0102] • Treatment with the extract

[0103] Organically grown mandarins underwent surface cleaning with clean water and then a 1 cm incision was made with a scalpel in the fruit skin before inoculating it with 3 μL of a suspension comprising 1000 Penicillium Digitatum spores in a 0.9% NaCl physiological solution. Hydrated leek green powder (50 g / L) in a vehicle solution (0.9% NaCl physiological solution) or the vehicle solution was introduced into the wound and the fruit was incubated at room temperature in a transparent airtight enclosure.

[0104] Halved tomato pulp was inoculated with a mixture comprising 1000 Rhizopus Stolonifer spores. The spores were pre-incubated for 24 h in a mixture of PDB medium and leek spawn extract or vehicle solution (0.9% NaCl) before being inoculated. The tomatoes were incubated in a sealed, transparent plastic container.

[0105] • Treatment with powder

[0106] Mandarin slices 0.5 cm thick were inoculated with a suspension of Penicillium Digitatum spores in physiological solution (1000 spores / slice). Leek green powder was then spread on the surface of the sliced ​​fruit and incubated at room temperature in a sealed, transparent plastic container.

[0107] 2.4 Seed treatment

[0108] Broccoli seeds were chosen for their sensitivity to the two pathogens Alternaria, the causative agent of early blight, and Botrytis, the causative agent of gray mold. The broccoli seeds were soaked for 3 minutes in a 2% bleach solution and then rinsed 3 times in water. 500 μL of a 10% Alternaria or Botrytis spore solution were added. 4spores / mL diluted in sterile water are added to approximately 70 broccoli seeds which are infected for 1 h - 1 h 30 at room temperature with gentle stirring. After removing the solution, 1 mL of water or extract prepared from an aqueous maceration of powder at 25 g / L is added to the broccoli seeds and left for approximately 4 hours at room temperature. The water or extract is removed and the seeds are placed on the surface of a Petri dish containing 1.5% agar. They are then germinated in a dark room for 2 days and then in room light for a further 5 days. After 7 days of culture, the seedlings are isolated and then measured (stems and roots).

[0109] 2.5 Evaluation of the antifungal effects of active substances in solid media

[0110] Lipopeptides were previously solubilized in solvents recommended by the suppliers. Briefly, surfactin and Iturin A were solubilized in absolute ethanol at 10 g / L. Fengycine was dissolved at 1 g / L in a phosphate buffer containing 0.1% DMSO. Caspofungin was solubilized at 10 g / L in distilled water. Growth control conditions were carried out in the presence of adequate amounts of solvents when necessary to exclude possible synergistic effects attributable to them. Copper sulfate was dissolved in distilled water at 200 g / L.

[0111] To evaluate the antifungal activities of substances of interest used in combination, the different agents were premixed in the following order: water, extract, SDS (if necessary), solvent (if necessary) and lipopeptide. The mixture was then mixed with nutrient agar (PDA) and then placed in 24-well cell culture plates. After solidification, the media were surface-inoculated with 1000 Alternaria alternata spores (dissolved in a mixture of water and 0.05% Tween 80). The plates were incubated at room temperature for the various times indicated and then photographed. When possible, the growth diameter of the fungus was measured. For the evaluation of combined effects in solid media, the concentrations of leek extract, lipopeptide and lauryl sulfate selected were subtoxic when these substances were applied individually.

[0112] III. Results

[0113] 3.1 Antifungal activity of dry powder

[0114] Figure 1 shows the effectiveness of the powder dispersed in an agar culture medium against Penicillium digitatum and expansum, Fusarium oxysporum and Alternaria alternata. The radial growth of molds is slowed down in the presence of 1 g / L of leek green powder (treatment) showing a different sensitivity from one phytopathogenic agent to another, it is completely inhibited from 5 g / L of powder in the agar medium.

[0115] 3.2 Effect of particle size

[0116] In Figure 2, no germination of Alternaria spores is observed by optical microscopy (X20) after treatment with an extract obtained from fractions between 90 pm and 700 pm (images B, C, and D) while germination of spores is comparable to the negative control (untreated) after treatment with an extract obtained from a dry fraction with a particle size greater than 700 pm (image A). The black arrows indicate germination.

[0117] 3.3 Sporicidal and fungicidal effect of the aqueous extract

[0118] Figure 3 shows the sporicidal effect of leek extract on Alternaria alternata observed after 4 hours of treatment of a culture medium containing spores, as well as a fungicidal effect obtained after initiation of hyphal elongation (Figure 3, panel A). It should be noted that the extract exhibits superior antifungal activity if added to the spores and that this efficacy decreases when the extract is added after germination and initiation of mold growth. Figure 3, panel B thus shows a complete inhibition of Alternaria spore germination when 12.5% ​​of extract is added to the culture medium, whereas at this concentration, growth is only inhibited by approximately 50% at more advanced stages of development (germ tubes, early hyphae, and late hyphae). Similarly, a concentration of 25% of extract no longer allows total inhibition of growth on late hyphae.These results therefore suggest that early application of the extract is desirable to optimize its protective effect.

[0119] 3.4 Post-harvest fruit treatment

[0120] Figure 4 shows the protective effects of leek powder sprinkled on clementine slices infected with Penicillium digitatum and then left at room temperature for 4 to 5 days (see panel A). Similar observations are made if the aqueous leek extract is applied to infected clementines by introducing the spores through an incision (Figure 4, panel B). Protection of tomatoes infected with Rhizopus stoloniferes is also observed when the extract is co-applied with the spores on tomatoes (Figure 4, panel C).

[0121] 3.5 Seed treatment

[0122] Infection of broccoli seeds by common infectious agents such as Alternaria or Botrytis cinerea, agents causing alternaria and gray mold respectively, can be significantly limited by treatment with a leek extract prepared from 25g / L of powder in water. Figure 5 shows (panel A and B) the damage caused by the two infectious agents on seedling development, in particular Botrytis, which severely limits seed germination and seedling growth. A treatment of a few hours by soaking the seeds in the extract allows the seedlings to exhibit growth and seedling length distribution comparable to that of the control.

[0123] 3.6 Potentiation of the antifungal effect with other products

[0124] To increase its effectiveness, the extract can be formulated in combination with different compounds that potentiate its activity. These may include, for example, substances capable of destabilizing the cell wall of molds (Fait et al., 2019; Gonçalves et al., 2017; Jahan et al., 2020). These combinations can reduce the use of biomass and / or antifungal agents used for biocontrol in agriculture. i) Combination with detergents

[0125] The extract can be used at reduced concentrations when combined with detergents. These include anionic detergents. For example, when used with 0.05% sodium lauryl sulfate, the extract concentrations required to achieve maximum antifungal activity can be reduced by 10 times compared to the concentration required to achieve the same effect by treatment using the extract alone (Figure 6, panel A).

[0126] Cationic detergents can also be used to form effective antifungal treatment combinations with leek extract. This is the case, for example, of CTAB (cetyltrimethylammonium bromide), which in combination with leek extract can be used at lower doses while significantly reducing the quantities of leek by-product biomass used (Figure 6, panel B). The negative ecotoxic profile of CTAB can therefore be reduced thanks to the extract of the invention, in particular if the use of this detergent proves necessary for an application as a control agent. ii) Combination with copper salts

[0127] Copper is the most widely used biocontrol treatment in agriculture. Although effective, this treatment causes environmental problems when it accumulates in soils or leaches into groundwater. The use of this product is therefore subject to strict regulatory limitations on the doses and frequencies of applications authorized in fields (Implementing Regulation (EU) No. 2018 / 1981 of 13 / 12 / 18). This regulation reduces the possibility of farmers using this effective product. The results demonstrate that the aqueous extract of the invention can be used to reduce the doses of copper required to achieve a complete antifungal effect. (Figure 7, panel A). ill) Combination with lipopeptides

[0128] Lipopeptides are peptides with antimicrobial properties of interest in the field of biocontrol. However, their use is currently hampered by the high quantities of peptides required to achieve a significant antifungal effect, leading to excessive operating costs (Comont et al., 2021).

[0129] The aqueous extract of the invention can be used to potentiate the effects of lipopeptides with biocontrol properties. For example, the aqueous extract of leek powder potentiates the antifungal effects of surfactin. Surfactin used as a treatment alone at the concentrations studied does not produce any significant antifungal effect. In combination with aqueous extract, surfactin allows an additional gain in antifungal efficacy (radial growth of the mycelium reduced by 40%) compared to the combination of the aqueous extract (Figure 7, panel B). iv) Combination with organic acids

[0130] The antifungal properties of organic acids such as lactic acid, acetic acid, propionic acid have been shown (Chaves et al., 2021; Hassan et al., 2015) and some of them have been added to the list of biocontrol products authorized in agriculture.

[0131] Since the antifungal effect of leek green extract is positively correlated with pH acidity (Figure 8, panel A), the potentiation of the extract activity by organic acids was evaluated. It was shown, for example, that an extraction carried out with 0.012% (2 mM) acetic acid allows a gain in extract efficiency. Figure 8 (panel B) shows that with 6.25% of an aqueous extract prepared in 0.012% acetic acid, no development of Alternaria spores and growth of mold is detectable after 5 hours of incubation, whereas 12.5% ​​of extract in the culture medium is necessary to achieve complete inhibition of Alternaria spore germination if the extraction is carried out in water. An extraction carried out with acetic acid therefore makes it possible to increase the effectiveness of the leek extract by a factor of at least 2. v) Triple combination comprising a leek extract, a detergent and copper sulfate

[0132] The results show a synergy of antifungal effects on A. Alternata when leek extract, detergent and copper sulfate are combined. A strong reduction in the concentration of copper sulfate required to produce this antifungal effect (Figure 9). Indeed, no growth is observed from a copper sulfate concentration of 1 g / L when combined with 0.01% lauryl sulfate and the aqueous extract at 5 g / L or 10 g / L. vi) Combination of leek extract, detergent and lipopeptides

[0133] A triple combination comprising leek extract, detergent and lipopeptides significantly reduced the concentrations of lipopeptides and extract required to produce an antifungal effect (Figs. 10 and 11). Indeed, these substances were used at subtoxic concentrations allowing some growth of A alternata when tested alone, but resulted in a significant reduction in spore germination and hyphal growth when combined.

[0134] In conclusion, the aqueous extract of leek powder can be combined with biocontrol products such as copper salts or lipopeptides, detergents, particularly anionic detergents, organic acids, particularly acetic acid, allowing a gain in antifungal efficacy while reducing the doses of extract and combining agent.

[0135] 3.6 Impact of processing method on antifungal activity i) Effect of cutting and drying temperature

[0136] Figure 12 shows the impact of the cutting step (Panel A) and the drying temperature (Panel B) on the antifungal activity evaluated by measuring the growth of the phytopathogenic mold Alternaria by crystal violet quantification. The leek leaves are cut using the indicated approaches, then subjected to the following steps of the process (drying at 40°C, grinding and extraction). The results show that with an extract prepared from dried strips or sections, no growth is observable from 6.25% of extract in a culture medium. When grinding fresh leek leaves (Fig. 12, Panel A, "freshly ground") is substituted for cutting, the antifungal activity decreases more than significantly, or even risks disappearing. It is only about 50% with a 50% extract, thus showing a very altered inhibitory effect.This decrease in activity could be explained by oxidation, volatilization or degradation of certain active compounds potentially associated with the intervention of specific enzymes. This type of mechanism has been previously described for organosulfur compounds or glucosinolates of alliaceae and brassicaceae (Miekus et al., 2020; Putnik et al., 2019; Sikorska-Zimny ​​& Beneduce, 2021). A lack of cutting also leads to a notable loss of activity (50% growth observed with 50% extract prepared from whole leaves) which could result from difficulty in drying, since the humidity level is 14% after 48 hours of drying.

[0137] Regarding the drying temperature, the results show that the antifungal activity is affected from 60°C. For example, Figure 12 (panel B) shows an absence of growth of Alternaria treated with an extract prepared from sections dried at 30 and 40°C from 6.25% of extract while this inhibition is observable from 25% of extract prepared from sections dried at 60°C. Drying at moderate temperature, such as 30° to 50°C, therefore leads to a humidity level of around 5% to guarantee optimal antifungal activity. Indeed, additional experiments (results not shown) have highlighted the loss of antifungal activity of leek powder stored in a humidity-saturated environment. On the contrary, storing leek powder in a dry environment and at a humidity percentage of around 5% allows the antifungal activity to be maintained for months.In particular, almost no loss of activity was observed after storage at 4°C, 5% humidity for 36 months. ii) Effect of leek powder concentration.

[0138] Figure 13 shows the impact of extract concentration, including the dry mass concentration used to prepare the extract and the antifungal activity. Concentrations of 10 to 20g of leek powder per liter of solvent show an activity of around 90% and are therefore preferred for producing the extract. iii) Effect of extract maceration time

[0139] As illustrated in Figures 14A and 14B, complete inhibition of Alternaria spore germination can be observed when spores are incubated with 12.5% ​​extract obtained after 2-30 min maceration of 25g / L powder while partial germination (black arrows) is observed for prolonged maceration at 1 h and 2 h (Figure 14 B).

[0140] However, antifungal activity leading to complete growth inhibition is found at higher extract concentrations during 1 and 2 hour macerations (25% and 50% extract in the nutrient medium) (Figure 14A).

Claims

CLAIMS 1. Biocontrol product comprising or consisting of leek powder with an average particle size equal to or less than 700 pm, and preferably between 10 pm and 700 pm.

2. Biocontrol product according to claim 1 in which the powder is derived from the green part of the leek, or from the green and white parts of the leek, and preferably is derived mainly or exclusively from the green part of the leek.

3. Aqueous extract of leek, said extract being derived from the green part of the leek, or from the green and white parts of the leek in powder form having an average particle size equal to or less than 700 pm, and preferably between 10 pm and 700 pm, and such as an aqueous extract derived from a biocontrol product according to one of claims 1 or 2.

4. Aqueous extract according to claim 3 comprising a concentration of powder per volume of aqueous solvent of between 5 g / L and 50 g / L, preferably 10 g / L to 25 g / L.

5. Aqueous extract according to one of claims 3 or 4, in which said aqueous extract is a filtrate and / or has a pH between 6 and 7, such as 6.

5.

6. Aqueous extract according to any one of claims 3 or 4, the pH of which is between 3.5 and 5.5, preferably between 3.7 and 4.7, when it is acidified by adding an acid, preferably an organic acid such as acetic acid.

7. A biocontrol, insecticidal or antifungal composition comprising a biocontrol product or an aqueous extract according to any one of the preceding claims.

8. Composition according to claim 8 comprising a copper salt such as copper sulfate, cuprous oxide, copper hydroxide, copper oxychloride, tribasic copper sulfate, copper acetate, copper tallate and expanded copper carbonate or mixtures thereof which could divide by at least 2 the quantities conventionally used in the treatment of crops with copper.

9. Composition according to any one of claims 8 or 9, comprising at least one anionic antiseptic or disinfectant surfactant compound such as sodium lauryl sulfate or a cationic surfactant such as cetrimonium bromide.

10. Composition according to any one of claims 7 to 9 comprising at least one lipopeptide-type compound having antimicrobial properties, preferably antifungal activity, such as caspofungin, surfactins, iturins, and fengycins produced by different strains of Bacillus sp. Composition according to any one of claims 7 to 11 comprising an organic acid selected from acetic acid, propionic acid or mixtures thereof. Composition according to any one of claims 7 to 12, said composition comprising a combination of the compounds including said cationic or anionic surfactant compound, and said copper salt or said lipopeptide. Use of a biocontrol product according to one of claims 1 and 2, or of an aqueous extract according to one of claims 3 to 6, or of a composition according to any one of claims 7 to 12 for biocontrol, insecticide or antifungal purposes. Use according to claim 12 for the treatment of seeds, crops or plants.Seed covered by film-coating, soaking, contacting or spraying with a biocontrol product according to one of claims 1 and 2, or with an aqueous extract according to one of claims 3 to 6, or with a composition according to any one of claims 7 to 12. Process for obtaining a biocontrol product according to any one of claims 1 to 2 comprising the following steps:. 1) Harvesting the green or green and white parts of the leek; 2) Cutting of parts according to 1); 3) Drying of the said parts of the leek; 4) Grinding of the parts after drying to obtain an average particle size < 700 pm, and preferably between 10 pm and 700 pm, preferably 90 pm and 500 pm, even more preferably 90 pm and 180 pm. 5) Optionally Sieving of the extract obtained in step 4. Method according to claim 16, wherein said drying step is: -carried out at a temperature below 70°C, preferably below 60°C, and more preferably at a temperature between 30 and 50°C or between 30 and 40°C; and / or for a drying time varying from approximately 24h to 72h, and / or until a moisture content of the order of 5% ± 1% or 2% is obtained. Method according to claim 16 or 17 comprising an aqueous extraction step in order to obtain the aqueous extract according to one of claims 3 or 4. Method according to claim 18, wherein the extraction time is less than 60 minutes, and preferably is between 2 and 30 minutes. Method according to one of claims 16 to 19 comprising a step of adjusting the pH between 3.5 and 5.5, preferably 3.7 to 4.7.

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