BIOCONTROL COMPOSITION

Abstract

An agronomic fungal biocontrol composition comprising one or more strains of Bacillus, selected from Bacillus amyloloiquefaciens, Bacillus licheniformis, Bacillus velezensis, Bacillus thuringiensis, Bacillus cereus, Bacillus subtilis, Bacillus megaterium and Bacillus pumilus and an extract of Limnospira sp., said one or more strains of Bacillus being capable of proliferating, use of this composition and use of an extract of Limnospira sp. for the preservation of cut plants, preferably fruits.

Description

1. BIOCONTROL COMPOSITION Technical Field The present invention relates to the use of a Bacillus strain in synergy with an extract of a cyanobacterium for biocontrol purposes, as well as to biocontrol methods. The prior art 5 Biocontrol of pathogens attacking a plant of interest is known. In particular, various strains of the Bacillus subtilis group in its broad sense (B. velezensis, B. amylolique faciens, B. thuringiensis, B. pumilus, B. subtilis, B. cereus, B. megaterium) are commercially available or known for this purpose. The production of cyclopeptides and / or lipopeptides or even of particular siderophores is often put forward to explain the superiority of one strain over another. The strains of the Bacillus amyloidosa group, such as FZB42, IT45, or QST 713 (Serenade®), are known for controlling pathogenic organisms. Similarly, the experimental strain GA1 has been tested for its efficacy in the biocontrol of pathogenic organisms, but it has not been commercialized (Arguelles-Arias et al., 2009). However, such strains are sometimes useful, sometimes not.Depending on field conditions, the plant type and the pathogen type, several development strategies have been proposed. Among these, the addition of other molecules, intended to potentiate the effect of the added Bacillus sp., has been suggested. There are potentially many compositions where a synthetic chemical molecule, whose mechanism of action is well known or predictable, is combined with a Bacillus sp. However, with the aim of reducing chemical inputs, the inventors believe that the formulation to be developed should combine only microorganisms or extracts thereof. 2025 / 5013 BE2025 / 5013 2 This requires (i) the identification of promising microorganisms, (ii) the selection of other microorganisms or extracts thereof that are synergistic, and (iii) verification that the synergistic effect is obtained under agronomic conditions. CN113508821 describes an antibacterial composition comprising a strain of Bacillus amylolic acid and Spirulina polysaccharides in a weight ratio of 10:0.5 to 0.5:2. However,This composition has not been tested for possible antifungal properties. Brief summary of the invention A first aspect of the present invention relates to a fungal biocontrol composition comprising a) one or more strains of Bacillus, selected from Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus velezensis, Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Bacillus pumilus and b) an extract of Limnospira, said one or more strains of Bacillus being capable of proliferating, said extract of Limnospira comprising a protein component and a polysaccharide component. Preferably, the Bacillus strain(s) present in the fungal biocontrol composition secrete(s) a cyclic peptide or a lipopeptide chosen from among sitruses, fungicides (and / or lipostatins), surfactins, and mixtures thereof. Alternatively, or in addition, preferably,the Bacillus strain(s) present in the fungal biocontrol composition comprises a plasmid having at least 95% identity with SEQ25 IDNO: 1 and / or the Bacillus strain has at least 90% identity with SEQIDNO: 5, said identity being calculated over the entire length of said SEQIDNO: 5 and / or 2025 / 5013 BE2025 / 5013 3 the Bacillus strain comprises a plasmid having at least 95% identity with SEQIDNO: 1 and said plasmid further encoding a peptide having 100% identity with SEQIDNO: 2, SEQIDNO: 3 and / or SEQIDNO: 4. Preferably, the Limnospira strain present in the fungal biocontrol composition is Limnospiraindica, preferably strain Limnospiraindica 5 PCC8005. Advantageously, the agronomic fungal biocontrol composition is in dry form, preferably having a residual moisture content of less than 10% (water weight: total weight of the composition), preferably measured by thermogravimetry. 10 Alternatively, preferably,The agronomic composition for fungal biocontrol is an oil-in-water emulsion in which the aqueous phase comprises the extract of Limnospirasp. and the oil phase comprises the proliferative Bacillus. Preferably, in this emulsion, the Limnospirasp. extract is at a concentration between 10 mg / ml and 500 mg / ml (weight of dry extract / volume of aqueous phase) and / or the proliferative Bacillus is at a concentration of at least 10⁷ CFU / ml (of the emulsion). Preferably, as an alternative, the sugar-to-protein ratio of the Limnospirasp. extract present in these fungal biocontrol compositions is greater than 1.5, preferably greater than 2. Preferably, according to the alternative, the protein-to-sugar ratio of the Limnospirasp. extract present in these fungal biocontrol compositions is greater than 1.5, preferably greater than 2. Advantageously, the Limnospirasp. extract present in these 25 fungal biocontrol compositions is the culture medium. Alternatively, preferably,The Limnospirasp extract present in these fungal biocontrol compositions consists essentially of lysed cells. Preferably, the agronomic fungal biocontrol composition 30 comprises, in addition to phosphite, preferably at least 1% phosphite 2025 / 5013 BE2025 / 5013 4 (volume:volumes if the composition is liquid, for example an emulsion, or weight:weight if the composition is dry). A related aspect of the present invention relates to a process for manufacturing the Limnospirasp extract to be incorporated into the fungal biocontrol composition, comprising the steps of culture (i) under nitrogen deficiency conditions, or (ii) under nitrogen-free conditions, separation of the cells from the environment, and either collection of the environment before drying, or lysis of the cells. In the context of the present invention, "nitrogen deficiency" is preferably understood to mean a culture condition reducing the peptide:sugar ratio, and / or in which the nitrogen content is less than 2.0g / lNaNO3 or an equivalent nitrogen source, preferably less than 1g / lNaNO3, preferably less than 0.5g / lNaNO3,even in the absence of mineral nitrogen. Conversely, in the context of the present invention, "nitrogen-free" preferably means a culture condition increasing the peptide-to-sugar ratio, and / or in which the nitrogen level is greater than 2.0 g / l NaNO3 or an equivalent nitrogen source, preferably greater than 2.5 g / l NaNO3. Preferably, according to an alternative to this process, the lysed cells are incorporated directly into the (dry) fungal biocontrol composition. According to the other alternative, the lysed cells are incorporated after dilution in water, preferably to form the aqueous phase of the fungal biocontrol composition in the form of an emulsion (the Bacillus being in the oil phase). Another aspect of the present invention relates to the use of an extract of Limnospirasp, preferably Limnospiraindica, preferably strain PCC8005, for the preservation of isolated plant structures.depréférencedesfruits. Another aspect of the present invention relates to an agronomic biocontrol composition comprising a Bacillus strain comprising a plasma having at least 95% identity with SEQIDNO:1 and / or a Bacillus strain having at least 90% identity with SEQIDNO:5, said identity being calculated over the entire length of said SEQIDNO:5 and / or a Bacillus strain comprising a plasma having at least 95% identity with SEQIDNO:1 and said plasma further encoding a peptide having 100% identity with SEQIDNO:2, SEQIDNO:3 and / or SEQIDNO:4, said liquid composition preferably comprising at least 10⁷ cfu / ml of said proliferative Bacillus and said composition being liquid and comprising between 1 and 5% by volume of potassium phosphate (volume potassium phosphate:volume of the fungal biocontrol composition), preferably between 2 and 4% by volume of potassium phosphate; or said composition being solid and comprising between 1 and 5% (weight of potassium phosphate:weight of the fungal biocontrol composition) of potassium phosphate,preferably between 2 and 4% in potassium phosphate. Detailed description of an embodiment of the invention The inventors have noted that the combination of a strain of a Bacillus producing slipopeptides and / or cyclic peptides, such as a strain 15 chosen from the species Bacillus myloliquefaciens, Bacillus licheniformis, Bacillus velezensis, Bacillus thuringiensis, Bacillus subtilis, Bacillus cereus, Bacillus megaterium and Bacillus pumilus with either a phosphite (e.g. between 1 and 3% by volume), or an extract of Limnospirasp. (or both) offers better protection to a plant of interest against fungal infections, even though in vitro tests show no interference effect 20 with fungal growth for the extracts of Limnospirasp. or for the addition of phosphite. Preferably, the vegetable is chosen from: Poaceae (grasses), for example maize, durum wheat, wheat, barley, rye, triticale, oats; Legumes, for example soybeans, beans, peas, broad beans, peanuts, lentils; Cucurbitaceae, for example squash, melon,Watermelon, pumpkin, butternut squash, cucumber; Asteraceae, for example, lettuce, chicory, sunflower, Jerusalem artichoke, artichoke, cardoon; Solanaceae, for example, tomato, potato, eggplant, chili pepper; Crucifera, for example, rapeseed or cabbage; Beetroot, carrot, strawberry, banana, kiwi; Horticultural plants (geranium, pelargonium, chrysanthemum), and Arboriculture plants (apple, pear, apricot, cherry, peach, grapevine). Turf (grasses, clover; forage or horticultural production) also benefits from the composition according to the invention, preferably by spraying. The fungal infections best prevented or treated by the formulations according to the invention are chosen from among the Ascomycetes (including the 10 fungi), the Oomycetes, and the Basidiomycetes. These are foliar fungi or fungi present in the soil. Among the fungal infections best treated by the formulation according to the invention are Fusarium (e.g., F. oxysporum or F. culmorum), Alternaria (e.g., A. solani),Sclerotina(ex.S.sclerotiorum),Pythium(ex.P.ultimum), Phytophthora(ex.P.infestans)etBotrytis(B.cinerea),maiségalementRhizoctonia15 (solani),Ceratocystispilifera,Cercosporabeticola,Gaeumannomycesgraminis, Verticiliumsp.,Zymoseptoriatritici,Graminopassaloragraminis,Atheliarolfsii,Aspergillus niger,etNeocamarosporiumbetae. Lesinventeursontremarquéquelaformulationselonl’inventionest particulièrementadaptéepourlecontrôled’infectionspardeschampignonsprésents20 danslesolmaisaégalementdeseffetssurdespathogènesfoliaires. Danslecontextedelaprésenteinvention,lasouchedeBacillus appartientaugroupeBacillussubtilisausenslarge.CeBacillussécrète avantageusementunouplusieurspeptidecycliqueet / ouunouplusieurslipopeptides choisisparmilesiturines,lesfengycines(et / ouplipastatines),lessurfactinesetles25 mélangesdeceux-ci. DessouchesdeBacillussubtilis(ausenslarge,ycomprisBacillus amyloliquefaciens,Bacillusvelezensis,Bacilluslicheniformis,Bacillusthuringiensis,Bacillus subtilis,Bacilluscereus,Bacillusmegaterium,Bacillus pumilus (and Bacillus pumilus) that do not secrete any of these cyclic peptides and / or slipopeptides is not useful; however, one of these strains that secretes at least one of these peptides is potentially useful. Seven strains, if their biocontrol capacity is uncertain, can be tested, first in vitro, for their biocontrol capacity, for example, for their ability to inhibit the growth of a problematic fungus. Preferably, this in vitro test is carried out in the presence of Limnospir extract (N+, N- or floating) in order to verify that the Limnospir extract does not interfere with the metabolism of the Bacillus being tested. The preferred strains are derived from Bacillus myloliquefaciens, Bacillus velezensis, and Bacillus licheniformis. Commercial strains such as Bacillus myloliquefaciens FZB42, IT45, or QST713 are advantageous. Preferably, the Bacillus strain comprises a plasma having at least 95% identity with SEQIDNO:1 and / or this Bacillus strain having at least 90% identity with SEQIDNO:5.said identity being calculated over the entire length of said SEQIDNO:5 and / or this Bacillus strain comprises a plasma having at least 95% identity with SEQIDNO:1 and this plasma also encodes a peptide having 100% identity with SEQIDNO:2, SEQIDNO:3 and / or SEQIDNO:4. Indeed, the inventors have noted that these characteristics of Bacillus strains (taken individually and potentially cumulatively) are associated with a particularly marked biocontrol effect. In the context of the present invention, the synergistic extract is preferably derived from a cyanobacterium, preferably Limnospira, formerly called Arthrospira, or Spirulina. Preferably, the Limnospirasp. is a Limnospiraindica, preferably Limnospiraindica PCC8005. This extract comprises a peptide component (active or potentially active) and a polysaccharide component (active or potentially active), preferably the weight ratio between the peptide (peptide component) and the sugars (polysaccharide component) of this extract is between 1:10 and 10:1, preferably between 1:5 and 5:1,preferably between 1:2 and 2:1. In the context of the present invention, preferably, the protein content is measured via the Bradford assay using bovine gamma globulin (BgG) as the standard. For example, the biomass is resuspended in an extraction buffer (guanidine HCl 6M, K2HPO450mM) and passed through a sonotrode at 4°C before centrifugation at 13,000 rpm for 10 minutes and recovery of the supernatant. The protein content is then measured on this supernatant by the Bradford assay with bovine gamma globulin (BgG) as the standard. In the context of the present invention, advantageously, sugars are quantified via the Yemmet Willis method (1954) on resuspended cell pellets. in 1 ml of water. 5 ml of Anthrone reagent (CAS 90-44-8) is added to the samples. This reagent was prepared by dissolving 0.2 g in 100 ml of H2SO4 obtained by mixing 500 ml of concentrated sulfuric acid (96%) with 200 ml of water. The 10 samples were vortexed and cooled. Then the tubes were sealed with cork stoppers and heated to 90°C for 10 minutes.The samples are then cooled in water for 5 minutes. The optical density of the samples is then measured at 620 nm. Several dilutions of the samples are performed and compared to standard D-glucose concentrations. The inventors have validated the methods described above, which offer robust results in the context of the present invention; in particular, the absence of phenol for sugar quantification is considered advantageous (interference with measurements). Other methods for quantifying sugars and / or peptides are well known and can be used instead of these, for example, by applying a conversion factor. According to one alternative, preferably, Limnospirasp. has been cultured in the presence of sufficient nitrogen (e.g., NaNO3), resulting in the Limnospirasp. extract having a protein (Bradford; see above):polysaccharides25 (sugars; see above) ratio greater than 1.5, preferably greater than 2. According to the other branch of the alternative, preferably, Limnospirasp. has been cultured in nitrogen deficiency,which means that the ratio of polysaccharides (sugars; measured above):proteins (Bradford; see above) of the extract of Limnospirasp. is greater than 1.5, preferably greater than 2.30 2025 / 5013 BE2025 / 5013 9 The inventors noted that, depending on the fungal pathogens and the plants to be protected, it was sometimes the (poly-)saccharide component that offered the most marked advantage, sometimes the protein component (e.g. siderophores). After culture, preferably, the cells are separated from the medium and subjected to one or more mechanical or physical stresses chosen from among drying, the application of pressure, freezing, and mixtures thereof. This advantageously allows for cell lysis; preferably, the Limnospirasp. extract consists essentially of lysed cells. Conversely, the medium conditioned by the culture is advantageously preserved, either as is or after at least partial drying, and can advantageously be applied to a culture to be protected, or even to a (harvested) fruit. In the context of the present invention,The extract of Limnospirasp is either the culture medium (dried), or the cells, or both the culture medium (dried) and the cells. According to a preferred alternative, the agronomic composition of the 15 fungal biocontrol agent is in dry form, preferably with a residual moisture content of less than 10% (water weight: total weight of the composition), preferably measured by thermogravimetry. This dry composition is advantageous for its stability over time. It can be advantageously applied at the same time as the sowing of the 20 seeds. This dry fungal biocontrol composition may be in the form of a hydrateable, dried powder, comprising a bulking agent and a surfactant (which may be a mixture of surfactants). Alternatively, preferably, this dry fungal biocontrol composition is in the form of granules, said granules comprising at least 40% by weight of calcium carbonate and / or magnesium carbonate (weight of calcium carbonates and / or magnesium carbonates: weight of granules free of Bacillus spet de Limnospira),Preferably, said granules having a diameter between 0.5 mm and 2 mm. Advantageously, a quantity of 0.1 to 20 kg / ha of the fungal biocontrol composition in granular form is applied per hectare, preferably 0.5 to 10 kg / ha, preferably 1 to 5 kg / ha. According to the other preferred branch of the alternative, this agronomic fungal biocontrol composition is an oil-in-water emulsion in which the aqueous phase comprises the extract of Limnospirasp. and the oily phase comprises the proliferative Bacillus. Preferably, in the case of this emulsion, the extract of Limnospirasp. is at a concentration between 10 mg / ml and 500 mg / ml (weight of extract: total weight of aqueous phase), preferably between 20 mg / ml and 200 mg / ml, preferably between 50 mg / ml and 150 mg / ml. The inventors noted that this emulsion offers several advantages, such as saving on the drying step, the plasticity of Bacillus, and ease of application. In other words, the formulation of the microorganism in oil is advantageous.This formulation is then emulsified in an aqueous phase 15 which includes the extract of Limnospirasp., thus saving on drying, or at least on thorough drying (partial drying to reach the above concentrations remains possible and even useful). Indeed, the application of the emulsion described above to the plant to be protected can advantageously be done by spraying, or even by coating the seed 20. In this fungal biocontrol composition, preferably the strain in the oil-in-water emulsion (see above) is applied to the plant (seed coating and / or spraying) at a concentration (in Bacillus) of at least 10⁷ CFU / mL, preferably at least 2*10⁷ CFU / mL, at least 5*10⁷²⁵ CFU / mL, for example at a concentration of approximately 10⁸ CFU / mL. This fungal biocontrol composition in liquid form (emulsion or rehydrated powder) or in solid form advantageously comprises between 1 and 5% (volume of the composition of phosphite (quasi-pure at 100%): total volume or total weight) by volume of phosphite (potassium).preferably between 2 and 4% by volume of 30 phosphite (potassium). 2025 / 5013 BE2025 / 5013 11 According to one option, the fungal biocontrol composition (dry powder or oil-in-water emulsion) is applied in or on the soil before the plant is planted or the plant seed is sown or, advantageously, substantially at the same time as the plant seed is sown, particularly for the solution in the form of a dry powder. 5 Preferably, alternatively or in addition, the fungal biocontrol composition is applied to the soil / or on the leaves after the plant has been planted or the plant seed has been sown. For example, the fungal biocontrol composition in the form of an oil-in-water emulsion can advantageously be sprayed on the leaves of a plant to be treated. 10 Another aspect of the present invention relates to the use of a strain chosen from Bacillussubtilis, Bacillusamyloliquefaciens, Bacillusvelezensis, Bacilluspumilus, Bacillusthuringiensis, Bacilluscereus, Bacillusmegaterium and Bacillus licheniformis, with an extract of Limnospirasp.,and or compositions described above, for the biocontrol of fungal infections, preferably chosen from 15 infections caused by Ascomycetes, Oomycetes or Basidiomycetes, preferably foliar or present in the soil, such as Alternaria (solani), Phytophthora (infestans),Sclerotina(sclerotiorum),Botrytis(cinerea),Fusariumsp.,Rhizoctonia(solani), Cercospora(beticola),Ceratocystispilifera,Gaeumannomycesgraminis,Verticilium sp.,Zymoseptoriatritici,Graminopassaloragraminis,Atheliarolfsii,Aspergillusniger,20 NeocamarosporiumbetaeandPythium(infestans). Other features and advantages of the present invention will be derived from the following non-exhaustive description,and referring to the drawings and examples. Examples.-25 It is understood that the present invention is in no way limited to the forms of embodiments described above and that many modifications may be made to it without departing from the scope of the annexed claims. Example 1: Culture of Limnospiraindica The inventors cultured Limnospiraindica PCC8005 in 30 type Erlenmeyer flasks of 150ml containing 50ml of modified Zarrouk medium (Cogne) in a phytotron incubator (MLR-352H-PE, Panasonic) at 30°C under a stirring of 2025 / 5013 BE2025 / 5013 12 120rpmet under a light intensity of 120µmol / m².s. After 10 days of culture, half the volume of culture medium is removed, and replaced by the same volume of culture medium. This medium is composed as follows: for 1l, 500ml of a solution A (10.5g / LNaHCO3; 7.6g / lNa2CO3; 0.5g / lK2HPO4; 2.6g / lNaNO3) 499ml of a solution B (1g / lK2SO4; 15 g / LNaCl;0.08g / LMgSO4.7H2O;0.03g / lCaCl2.2H2O;0.01g / LFeSO4.7H2O;0.08g / L EDTA) and 1ml of a solution of trace elements (0.23g / LMnCl2.4H2O;0.11g / L ZnSO4.7H2O;0,03g / LCuSO4.5H2O); the solutions were autoclaved separately. These are the culture conditions in the presence of nitrogen; hereafter N+. Alternatively, the same medium but without NaNO3 was used; these will be the N- conditions. Then, the cells were recovered and subjected to mechanical stress,They are then dried and used directly or frozen for later use. Example 2 – In vitro tests. The inventors tested the effect of different compositions on the in vitro growth of 7 pathogens representing the diversity of fungal pathogens: - Fusarium oxysporum; - Fusarium culmorum; - Alternaria solani; - Sclerotinia sclerotiorum; - Pythium ultimum; - Phytophthora infestans; - Botrytis cinerea. The filamentous fungi were cultured on PDA (Potato Dextrose Agar) medium at 22°C. Phytophthora infestans was cultured at 18°C. Next, the fungi are placed in a card in the middle of a Petri dish (PDA medium); the combinations of microorganisms to be tested for biocontrol are placed on both sides of the card. 2025 / 5013 BE2025 / 5013 13 Eight conditions were applied (one control, column 8, and seven test conditions: A, AG002 / 2 & 3% phosphite; B and C, biomass extracts obtained under nitrogen deficiency conditions: 100 and 10 mg / ml; D and E,idemculturesans deficientenazote; F phosphite3% by volume; Gbiomasssansdeficient100mg / ml&AGO2 / 2). The measured values ​​in triplicate were reported as a percentage of the control.5 - As can be seen, the conditions with AGO2 / 2 (columns A and G) show the best results, with particularly marked inhibition for Alternaria solani and Phytophthora infestans, significant effects for Sclerotina sclerotiorum and Botrytis cinerea, and intermediate effects for the two Fusarium sp. tested and Pythium infestans. The condition under 3% phosphites alone shows no inhibition. The addition of Limnospira indica extracts shows partial inhibition under certain conditions, but no in vitro synergy with AGO2 / 2. The inventors also obtained good results in inhibiting the in vitro growth of 15 pathogens: Rhizoctonia (solani) (up to 94% inhibition), Ceratocystispilifera (up to 84% inhibition), Cercosporabetica (up to 79% inhibition), Gaeumannomyces graminis (up to 94% inhibition), Verticillinum sp. (up to 74% inhibition).Zymoseptoriatritici (up to 90% inhibition), Graminopassaloragraminis (up to 90% inhibition), Atheliarolfsii (inhibition ranging from 20 to 52% and up to 92% inhibition), Aspergillus niger (up to 77% inhibition), and Neocamarosporium betae (up to 64% inhibition). Example 3 – Tests on plant portions. Salad leaves were inoculated and various protective formulations were tested (condition 8 is the control inoculated without application of a protective formulation): - 1 AG002 / 2 (108 Cfu / ml) - 2 a biomass extract of Limnospira N at a concentration (dry weight) of 100 mg / ml. Pathogen Conditions ABCDEFGCtrl 1 Fusarium oxysporum 60,681,388,587,082,382,763,7100 2 Fusarium culmorum 62,199,0105,096,173,8106,766,9100 3 Alternaria solani 74,093,068,071,168,5120,210,0100 4Sclerotiniasclerotiorum37,6106,392,2135,2119,6110,050,5100 5Pythiumultimum56,261,572,770,271,577,683,9100 6Phytophthorainfestans8,793,094,5104,1102,790,715,5100 7Botrytiscinerea36,666,872,368,863,994,055,5100 2025 / 5013 BE2025 / 5013 14 -3 Limnospira N+ biomass extract at a concentration (dry weight) of 100 mg / ml -4 N- culture medium at a concentration (dry weight) of 100 mg / ml -5 AG002 / 2 and Limnospira N- biomass extract at a concentration (dry weight) of 100 mg / ml -6 AG002 / 2 and Limnospira N+ biomass extract at a concentration (dry weight) of 1005 mg / ml -7 AG002 / 2 and the conditioned medium N- (100 mg / ml) The surface area colonized by the pathogen was measured 4, 7,11 and 15 days after inoculation. Botrytis on lettuce (plants) 10 8 Treatments x 5 replicates (40 lettuce plants / pathogen) including one untreated control. Production of plants necessary for the study (3 to 4 weeks). Preparation of the substrate and transplanting into pots. Culturing of the pathogen and multiplication a few weeks before application (1 to 2 weeks). Foliar application carried out in the treatment chamber 24 hours before inoculation and following the protocol: 200 L / ha, 1 L slurry volume. Artificial inoculation of the pathogen onto two leaves using a pipette; placement of a plastic cover to promote incubation of the disease. Observation of symptoms 4, 6, 10, and 14 days after application: diameter of lesions. The results are shown in the table below. 2025 / 5013 BE2025 / 5013 15 Condition 7 cannot be analyzed because the variations between conditions were too large. A similar test was carried out on picked tomatoes.Inoculated and treated as for salad. 5 Botrytis on tomatoes (fruits) 8 Treatments x 5 replicates (40 tomato fruits) including one untreated control. Culture of the pathogen and multiplication a few weeks before application (1 to 2 weeks). 10 Washing of tomato fruits before application. The fruits will be wounded using a pipette 5 mm in diameter and 5 mm deep, then the fruits will be immersed in the different solutions according to the protocol. Volume of slurry 0.5 to 1 L. Artificial inoculation of the pathogen using a pellet on the wounds and placement in a climate chamber. Observation of symptoms 4, 6, 10, 14 days after application: diameter of the lesions. The results are shown in the table below: 4j7j11j15j 12588.2 2014.54 30.2101210 424.46.510.5 51.49.21211.2 600.223.4 7xxxxxxxxxxxx 857.48,413 2025 / 5013 BE2025 / 5013 16 Example 4 – Other tests on plants or fruits Phytophthora on tomatoes (plants) 8 Treatments x 5 replicates (40 plants / tomatoes / pathogen) including one untreated control. 5 Production of plants necessary for the study (8 to 10 weeks). Preparation of the substrate and transplanting into pots. Cultivation of the pathogens and multiplication a few weeks before application (3 to 4 weeks). Foliar application carried out in the treatment chamber 24 hours before inoculation following the protocol 200 l / ha volume of 1 l. Artificial inoculation with the pathogen and placement in a climate-controlled chamber for 48 to 72 hours to promote disease incubation. Observation of symptoms 4, 6, 10,14 days after application: % of diseases according to the EPPO scale. 15 Alternaria on potato leaves in Petri dishes. 8 treatments x 5 replicates (40 plants / stomata / pathogen), including one untreated control. Production of plants necessary for the study (8 to 10 weeks). Preparation of the substrate and transplanting into pots. 20 Culture of pathogens and multiplication a few weeks before application (3 to 4 weeks). 4d7d11d15d 10.43.9513.2528.5 20.41.73.8511 30.21.42.67.4 40.151.63.210.95 50.33.659.420.75 60.33.37.1516.5 70,253,859,520.75 80,554,3511,2521,75 2025 / 5013 BE2025 / 5013 17 Foliar application carried out in the treatment chamber 24 hours before inoculation according to the protocol 200 l / ha, volume of 1 l of solution. Leaf samples treated according to the protocol will be taken. They will be placed in Petri dishes containing a culture medium. A pellet of the pathogen will be placed in the center of the leaf. All these operations will be carried out in a sterile environment. The dishes will be kept in an environment conducive to the development of the disease. Observations will be carried out 4, 6, 10, and 14 days after application: diameter of lesions. Sclerotinia on lettuce (plants) 10 8 Treatments x 5 replicates (40 lettuce plants) including an untreated control. Cultivation of the pathogen and multiplication a few weeks before application (4 to 6 weeks). Preparation of the substrate with a mixture of purée and cereal seeds contaminated by the pathogen and soaking. 15 Soaking of the plant in a solution 0,5 to 11 treatments carried out according to the protocol (root treatment). Transplanting of treated plants into pots. Placement in an environment favorable to the incubation of the pathogen. Observation of symptoms 4, 6, 10, 14 days after application: 20 diameter of lesions. 2025 / 5013 BE2025 / 5013,