WO2024033576A1

WO2024033576A1 - Biocontrol product using a co-product of leek

Info

Publication number: WO2024033576A1
Application number: PCT/FR2022/051581
Authority: WO
Inventors: Agnès SAINT-POL; Patrick Gonzalez; Valentina GLIGORIJEVIC
Original assignee: Institut Supérieur Des Biotechnologies (Supbiotech)
Priority date: 2022-08-12
Filing date: 2022-08-12
Publication date: 2024-02-15

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

BIOCONTROL PRODUCT FROM LEEK CO-PRODUCT

Field of the invention

The invention relates to the field of biocontrol molecules and the valorization of agricultural waste. The invention relates in particular to the valorization of the leek co-product as a biocontrol product, and its process for obtaining it.

State of the art

The cultivation of cereals, fruits and vegetables is subject to attacks by pests (insects, weeds, microorganisms, etc.) which affect crop yields and thus reduce producers' profits. In order to overcome these disadvantages, farmers use pesticides regularly, if not massively, 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 useful to plants can be affected. It should be noted that since their use, certain pesticides are now prohibited for sale.

To remedy the shortcomings of pesticides, farmers looked for 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, in application WO2022047396 it is demonstrated that microbial compositions have beneficial effects on the agricultural level. Although efficient, the production of natural products by microorganisms still remains low and the quantities that crops need to be protected may not be sufficient.

In application WO2021236799 reference is made to a biological control composition based on an intact minicell containing a nucleic acid which targets a transcription product encoding a polypeptide and a method for combating pests and pathogens with novel systems and compositions for delivering RNA molecules into cultures. Here again, the use of an intact minicell, although effective, may encounter problems of stability of use within the soil.

So there is an obvious need for an alternative to pesticides and biocontrol products are one of these alternatives solving 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 daily use of them, having a positive impact on crop yield while preserving them and the 'environment.

In particular, there is a need for effective biocontrol agents to deal with fungal diseases involved in many plant diseases, and causing significant production losses. For example, diseases such as gray rot, early blight, and fusarium wilt affect different plant production, from seed to post-harvest product.

Plant extracts make it possible to at least partially meet these needs. For example, the allium family, and particularly garlic, has been known for many years for its antibacterial effects, as well as its antifungal and insecticidal activity. Its effectiveness having been widely demonstrated, garlic extract is now recognized in France in the list of phytopharmaceutical biocontrol products, under Article L. 253-5 and L. 253-7 of the rural and maritime fishing code. Garlic extract is marketed in concentrations of 45%w/w as a biocontrol product.

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

Summary of the invention

An aim of the invention is therefore to propose a new biocontrol product effective against fungal diseases, producible in large volumes and at a moderate price.

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

Indeed, it is the merit of the inventors to have demonstrated that dry leek in powder form with a particle size <700 pm presents 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 the growth of mold.

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 in itself a biocontrol product, or it can be incorporated into a formulation comprising excipients, preservatives and/or other active ingredients, or be used in the form of extract.

The biocontrol product according to the invention comes 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 valued as food.

Advantageously, the biocontrol product of the invention comes 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 recycle plant waste whose source is perennial and abundant. Indeed, the annual production of leek in France is around 165,000 tonnes and it is estimated that 15% or 20% of production is lost as a co-product resulting from post-harvest peeling. The present invention therefore makes it possible to promote as a biocontrol product this part of the leek harvest which is currently unexploited.

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 has great 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.

In one embodiment, the invention provides an aqueous extract whose concentration of leek powder per volume of solvent is 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 powder leek for 1 L of water extracts approximately 60 to 90% of the antifungal activity of the powder. The extract can be used with leek powder suspended in water or as a filtrate to facilitate the use of the extract.

The pH of the simple aqueous extract is between 6 and 7 without pH adjustment, in particular 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. 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.

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.

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 proposes 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:

-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 their mixtures, and making it possible to envisage a reduction of at least by 2 the quantities conventionally used in the treatment of crops with copper;

- 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 by 5 of the use of leek co-product biomass and by 2 the quantity of detergent required;

- a lipopeptide type compound having 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 less by 5 the use of leek co-product biomass while reducing by at least 2 times the required concentrations of lipopeptides to achieve an antifungal effect.

- an organic acid chosen from acetic acid, propionic acid or mixtures thereof 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.

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

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 sole use of said compounds as biocontrol products. Effective antifungal concentrations can therefore be considered from 0.5% to 5% m/V (5 to 50g/L if V= 1 L), preferably 1% to 2.5% (10 g/L to 25 g/L if V= 1 L) for the leek green powder according to the invention or the aqueous extract resulting from said concentration. Leek powder can also be combined with surfactant compounds such as sodium lauryl sulfate at a quantity of between 0.01% to 0.1% of the mass of leek powder included in the biocontrol composition. Leek powder can also be associated with fungicidal compounds such as copper salts which may be present in a quantity between 5% to 20% of the mass of leek powder included in the biocontrol composition. Leek powder can also be associated to a mixture of fungicides and surfactants at the aforementioned concentrations.

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 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.

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

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

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

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

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

The invention also relates to a seed covered by film coating or spraying with a biocontrol product, an extract or a composition according to the invention.

The invention also relates to a process for obtaining a biocontrol product according to the invention, said process comprising the following steps:

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

2) Cutting of the parts according to 1);

3) Drying of said parts of the leek;

4) Grinding 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 the extract obtained in step 4.

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

-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.

- drying step 2) 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.

- drying step 2) is carried out until a moisture 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. the invention.

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.

Optionally, the process also includes 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.

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.

Definitions

In the present invention, powder means a form of material consisting of more or less fine dry solid particles, in a free or agglomerated state.

By white or lower part of the leek, or by leek white we mean the white leaves of Allium porrum also known as Allium ampeloprasum var. porrum.

By green or upper part of the leek, or by green of the leek we mean the green leaves of Allium porrum also known as Allium ampeloprasum var. porrum.

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

By aqueous extract we mean 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.

Figures

Fig. 1 Antifungal activity of 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).

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

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

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 then treated with leek green extract before germination and growth for 7 days. (B) Measurement of seedlings at 7 days

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.

Fig. 7 Antifungal activity of the aqueous extract in combination with a biocontrol product. (A) Extract + copper sulfate, (B) Extract 50g/L 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.

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

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

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 a strain of A. Alternata for incubation at room temperature. (A) Microscopy observation after 20 hours of treatment, (B) Macroscopic observations after 6 days of treatment.

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

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

Fig. 14 A and 14B Impact of maceration duration on the level of extraction of antifungal activity. (A) Quantification by measuring growth after staining with crystal violet. (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.

Detailed description of the invention

The aim of the present invention is the valorization of market garden co-products, in particular leek as a biocontrol product. In particular, the aim of the invention is to propose a form of leek ensuring that biocontrol effects are obtained, as well as to propose forms of preparation of leek making it possible to obtain said effect and facilitating its application to plant products such as crops, seeds and harvests.

For these purposes, the invention proposes a biocontrol product comprising or consisting of leek powder having a particle size less than 700 μm. Indeed, the inventors have noted that the use of leek powder with such a particle size makes it possible to obtain antifungal effects against a broad spectrum of phytopathogenic molds. The leek powder of the invention can therefore be directly used as a biocontrol product by sprinkling or spraying a product to be protected. By biocontrol product we mean a product of biological origin used as a treatment in the protection of plants against diseases of microbial origin and insects.

In particular, the invention proposes to valorize 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 having greater activity than the green part. In fact, white leek powder has an antifungal activity approximately 5 to 10 times greater than green leek 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 valorized. The experimental results presented below are based exclusively on leek green powder.

In order to facilitate the use of leek, the invention proposes to produce an aqueous extract of leek powder according to the invention. Surprisingly, a simple aqueous maceration makes it possible to obtain an extract with high antifungal effectiveness, 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. 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 for diluting 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.

The inventors have also demonstrated the synergistic effect of the combination of leek powder according to the invention and other control agents of which it is desired to reduce the doses used for environmental reasons. A synergy has notably been found with copper salts, detergents and lipopeptides exhibiting antimicrobial activity. The invention thus makes it possible to significantly reduce the amounts used of said agents when they are combined with the antifungal effects of leek powder or its extract.

The invention also relates to the optimization of the process for obtaining the leek powder and the aqueous extract of the invention. As has already been 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, that is to say a form of particles in the dry state, necessarily implies the drying of the fresh form of the leek before or after its grinding.

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

The invention proposes in particular a process for obtaining the biocontrol product comprising or consisting of leek powder with a particle size <700pm. This process proposes to optimize the drying stage to maintain the highest possible activity. The drying step can in particular be optimized by previously cutting the fresh parts of the leek into sections or strips, and drying said pre-cut parts to approximately 5% humidity, 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, simply aqueous maceration at room temperature for a period of 2 to 30 minutes of the leek powder thus obtained. The extract can be used with the suspended powder or filtered for ease of use.

The experimental part below demonstrates the numerous advantages of valorizing the leek co-product when it is put in the form of powder and a specific particle size, or of an extract obtained from this powder. The potential as a biocontrol product has been demonstrated through antifungal activity, but use as an insecticide is also considered.

EXPERIMENTAL PART

I. Leek processing

1. 1 Obtaining leek powder

The green leek leaves are separated and washed before being dried at low or medium temperature. Drying will be done in such a way as to preserve the activity of 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 obtaining a rate preferential humidity of the order of 5%.

The leek leaves are then crushed coarsely and then ground indifferently using a hammer mill or a blade mill until reduced to powder form. Preferably, this grinding is carried out by cycle (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 active ingredients, i.e. diffusion of active ingredients from the powder. towards 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 makes it possible to obtain faster drying at low temperature and leads to stabilization of the active forms. The strips or sections are then left to dry for 24 hours 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 crushed" parts of the leek because in this case the antifungal activity decreases more than significantly, or even risks disappearing.

I.2 Aqueous extract

The antifungal activity can be extracted by maceration in water at leek green powder concentrations varying from 10 to 50 g/L in order to obtain a fraction containing active ingredients sufficiently concentrated so as not to add an additional step. . Concentrations of 20 to 25g/L are generally used to consider subsequent dilutions while maintaining a high extraction yield (of the order of 90%) (Figure 4). The optimal maceration time to preserve antifungal activity is less than 1 hour, and preferably 2 minutes to 30 minutes.

Maceration is generally followed by a filtration step under vacuum or at atmospheric pressure in order to eliminate insoluble residues and facilitate the use of the extract. Generally speaking, it is advisable to use the extract quickly after its preparation.

II. Materials and methods

2.1 Determination of the antifungal activity of the dry form

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 the concentration of 10 ⁵ spores/ml.

The leek powder is weighed 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 μL 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 effectiveness is evaluated by measuring the growth diameter using the following formula:

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

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

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

The protocol implemented is adapted to 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 makes it possible to correlate the coloring intensity with the biomass produced. Leek extract is prepared according to the method described from a powder concentration of 25 g/L.

100 μl of spores suspended in Potato Dextrose Broth (PDB) medium at a concentration of 10 ⁵ spores/mL are distributed in a 96-well plate then placed 50 minutes at 25°C. The planktonic cells are then removed by washing with 100 μL of PBS then 100 μL of PDB2X are added and mixed with 100 μL of crude or diluted extract to obtain percentages of extract 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 a microscope and quantification with crystal violet. The latter can also be implemented after an incubation of 20 hours.

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

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

The negative control corresponds to a culture without treatment

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

2.3 Post-harvest fruit processing

• Treatment with the extract

Mandarins from organic cultivation underwent surface cleaning with clear water then a 1 cm incision was made with a scalpel in the skin of the fruit before inoculating it with 3pL of a suspension comprising 1000 spores of Penicillium Digitatum in a physiological solution containing 0.9% NaCl. Hydrated leek green powder (50 g/L) in a vehicle solution (physiological solution with 0.9% NaCl) or the vehicle solution was introduced into the wound then the fruit was incubated at room temperature in an enclosure. transparent airtight.

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

• Treatment with powder

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

2.4 Seed treatment

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 rot. The broccoli seeds are soaked for 3 minutes in a 2% bleach solution then rinsed 3 times in water. 500 μL of a solution of Alternaria or Botrytis spores at 10 ⁴ spores/mL diluted in sterile water are added to approximately 70 Broccoli seeds which are infected for 1 hour - 1 hour 30 minutes 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 then left for approximately 4 hours at room temperature. The water or extract is removed then the seeds are placed on the surface of a Petri dish containing 1.5% agar. They are then left to germinate in an enclosure away from light for 2 days. then in room light for another 5 days. After 7 days of cultivation, the seedlings are isolated then measured (stems and roots).

2.5 Evaluation of the antifungal effects of active substances in a solid medium

The lipopeptides were previously solubilized in the solvents recommended by the suppliers. Briefly, surfactin and Iturin A were solubilized in absolute ethanol at 10 g/L. Fengycin was dissolved at 1 g/L in a phosphate buffer comprising 0.1% DMSO. Caspofungin was solubilized at 10 g/L in distilled water. The growth control conditions were carried out in the presence of adequate quantities of solvents when necessary in order to exclude possible synergistic effects attributable to them. Copper sulfate was dissolved in distilled water at 200 g/L.

In order to evaluate the antifungal activities of substances of interest used in combination, the different agents were pre-mixed in the following order: water, extract, SDS (if necessary), solvent (if necessary) then lipopeptide. The whole is then mixed with nutrient agar (PDA) then placed in 24-well cell culture plates. After solidification, the media are seeded on the surface with 1000 spores of Alternaria alternata (in solution in a mixture of water and 0.05% Tween 80). The plates are incubated at room temperature for the different durations indicated and then photographed. Where possible, the growth diameter of the fungus was measured. For the evaluation of combined effects in a solid medium, the concentrations of leek extract, lipopeptide and lauryl sulfate selected are subtoxic when these substances are applied individually.

III. Results

3.1 Antifungal activity of dry powder

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

3.2 Effect of particle size

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 spore germination 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). Black arrows indicate germination.

3.3 Sporicidal and fungicidal effect of the aqueous extract

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 greater antifungal activity if added to spores and that this effectiveness decreases when the extract is added after germination and initiation of mold growth. Figure 3, panel B thus shows a complete inhibition of the germination of Alternaria spores 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% 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.

3.4 Post-harvest processing of fruits

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

3.5 Seed treatment

The infection of broccoli seeds by common infectious agents such as Alternaria or Botrytis cinerea, agents respectively causing alternaria and gray rot, can be strongly limited by treatment with a leek extract prepared from 25g/L of powder. in water. Figure 5 shows (panels A and B) the damage caused by the two infectious agents on the development of seedlings, in particular Botrytis which strongly 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 a seedling length distribution comparable to that of the control.

3.6 Potentiation of the antifungal effect with other products

To increase its effectiveness, the extract can be formulated in combination with different compounds which potentiate its activity. These may, for example, be 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

The extract can be used in reduced concentrations when combined with detergents. These may in particular be 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 the concentration needed to achieve the same effect by one treatment. consisting of using the extract alone (Figure 6, panel A).

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

Copper is the most widely used biocontrol treatment in agriculture. Although effective, this treatment causes environmental problems when it accumulates in the soil or is leached into groundwater. The use of this product is therefore subject to a strict regulatory limitation on the doses and frequencies of applications authorized in the fields (execution regulation (EU) no. 2018/1981 of 13/12/18) This Regulations reduce the possibility of farmers using this effective product. The results demonstrate that the aqueous extract of the invention can be exploited to reduce the doses of copper necessary to achieve a full antifungal effect. (Figure 7, panel A). ill) Combination with lipopeptides

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 that must be used to achieve a significant antifungal effect leading to excessive operating costs (Comont et al., 2021).

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

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.

The antifungal effect of leek green extract being positively correlated with the acidity of the pH (Figure 8, panel A), the potentiation of the activity of the extract by organic acids was evaluated. It has thus been shown, for example, that an extraction carried out with 0.012% (2 mM) acetic acid allows a gain in efficiency of the extract. 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 while 12.5% extract in the culture medium is necessary to achieve complete inhibition of germination of Alternaria spores if the extraction is carried out in water. An extraction carried out using acetic acid therefore makes it possible to increase the effectiveness of the leek extract by a factor of at least 2. v) Triple combination including leek extract, detergent and copper sulfate

The results show a synergy of antifungal effects on A. Alternata when leek extract, detergent and copper sulfate are combined. A sharp 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 5g/L or 10g/L. L. vi) Combination of leek extract, detergent and lipopeptides

A triple combination of leek extract, detergent and lipopeptides significantly reduces the concentrations of lipopeptides and extract required to produce an antifungal effect (Fig. 10 and 11). Indeed, these substances have been used at sub-toxic concentrations allowing some growth of A alternata when tested alone, but result in a significant reduction in the germination of the spores and the growth of their hyphae when they are combined.

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

3.6 Impact of the transformation process on antifungal activity i) Effect of cutting and drying temperature

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 quantification with crystal violet. The leek leaves are cut using the indicated approaches, then subjected to the following stages 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% extract in a culture medium. When crushing the fresh leaves (fig. 12, panel A, “fresh crushed”) of leek replaces cutting, the antifungal activity decreases more than significantly, or even risks disappearing. approximately 50% with a 50% extract, thus showing a very altered inhibitory effect. This drop 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 from allaceae and brassicas (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 drying, since the humidity level is 14% after 48 hours of drying.

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% extract while this inhibition is observable at from 25% extract prepared from sections dried at 60°C. Drying at a moderate temperature, such as 30° to 50° C, therefore leads to a humidity level of around 5%, ensuring optimal antifungal activity. Indeed, additional experiments (results not shown) 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 retained for months. In particular, almost no loss of activity was noted after storage at 4°C, 5% humidity for 36 months. ii) Effect of leek powder concentration

Figure 13 shows the impact of the concentration of the extract, in particular the concentration of dry mass used for the preparation of 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 making the extract. iii) Effect of the duration of maceration of the extract

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 to 30 minutes maceration of 25g/L powder. while partial germination (black arrows) is observed for prolonged maceration at 1 h and 2 h (Figure 14 B).

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

Claims

1. Biocontrol product comprising or consisting of leek powder whose average particle size is equal to or less than 700 μm, and preferably between 10 μm and 700 μm.

2. Biocontrol product according to claim 1 in which the powder comes from the green part of the leek, or from the green and white parts of the leek, and preferably comes 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 the form of powder 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 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 of 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 than acetic acid.

7. Biocontrol, insecticide 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 copper and expanded copper carbonate or their mixtures which could divide at least by 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 exhibiting 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 1 1 comprising an organic acid chosen from acetic acid, propionic acid or mixtures thereof. Composition according to any one of claims 7 to 12, said composition comprising a combination of 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 to biocontrol, insecticide or antifungal purposes. Use according to claim 12 for the treatment of seeds, crops or plants. Seed covered by filming, 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 the parts according to 1);

3) Drying of said parts of the leek;

4) Grinding the parts after drying to obtain an average particle size

5) Optionally sieving of the extract obtained in step 4. Method according to claim 16, in which said drying step is: -carried out at a temperature lower than 70°C, preferably lower than 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 24 hours to 72 hours, and/or until obtaining a moisture content of around 5% ± 1% or 2%. Process 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. Process according to claim 18, in which 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.