Biological control composition

Abstract

Disclosed is a biological control composition comprising a known molar amount of bacilysin, or a known and high ratio of bacilysin to fengycin, uses of said composition, a method for purifying bacilysin and a method for absolute quantification of bacilysin.

Description

[0001] BIOCONTROL COMPOSITION

[0002] technical field

[0003] The present invention relates to a biocontrol composition, particularly for the protection of plants of interest. The composition comprises an effective and known amount of bacilysine.

[0004] Previous art

[0005] Many microorganisms belonging to the Bacillus subtilis group (in the broad sense) are known to have potentially interesting properties in biocontrol.

[0006] In practice, either a strain is added alone, or an extract of the supernatant from a culture of a strain is applied, see for example WO 2021 / 163810.

[0007] The most frequently highlighted biomolecules are fengycin, surfactin, and various molecules derived from iturine.

[0008] Even though these peptides are "non-ribosomal", their synthesis involves functional operons. Thus, at the level of a strain collection, the presence, or absence, of functional genes, or even their expression levels, will create a very large diversity of mixtures of these peptides, and many publications in this field highlight promising strains, which the inventors consider to be potentially secreting the desired biomolecules and probably even sometimes in effective levels, even accidentally.

[0009] Abbas Aqleem et al., "Antagonist effects of strains of Bacillus spp. Against Rhizoctonia solani for their protection against several plant diseases: Alternatives to chemical pesticides," in *Comptes Rendus Biologies*, vol. 342, no. 5, June 1, 2019, pages 124-135, describes the use of various formulations derived from Bacillus spp., including the reference commercial product Serenade®, as biofungicides for the management of Rhizoctonia solani. The article mentions various components, and bacilysin is listed among them. However, the article does not provide an explanation of the effect of bacilysin; in particular, no quantities or methods of measuring concentrations are disclosed.

[0010] However, even if the idea is appealing, the results obtained in practice are disappointing or associated with an empirical, case-by-case approach since such an approach is difficult to modulate: it is possible to prove that a commercial product works under given conditions against a given pathogen, but the inventors believe that this same product, if modified in light of the present invention, could work much better, including against other pathogens.

[0011] The inventors suspect that part of the difficulties encountered are related to too low a content of key metabolites and / or too high a content of unfavorable, or even harmful, co-products.

[0012] Indeed, it is not possible to concentrate such extracts at will. For example, WO2019 / 016492 describes a composition comprising a high content of mycosubtilin (an iturine-type lipopeptide) in association with other biomolecules, while reporting the great difficulty associated with concentrating the extracts beyond the critical micellar concentration of the biomolecules, which leads to aggregates or gels.

[0013] On the other hand, the purification of these antimicrobial peptides is delicate and expensive.

[0014] Lipopeptide purification methods including hydrochloric acid precipitation, methanol extraction and Cl 8 column chromatography are described in CN 106754489.

[0015] This document does not describe the purification of bacilysine.

[0016] Brief summary of the invention

[0017] A first aspect of the present invention relates to a liquid biocontrol composition, comprising a known amount of bacilysin, advantageously between 0.001 mmol / L and 20 mmol / L of bacilysin.

[0018] Preferably, the molar ratio between bacilysine and fengycin (potentially present in this composition) is greater than 5, preferably greater than 100.

[0019] Preferably, this composition further comprises a known molar amount of a siderophore, preferably bacillibactin, preferably between 0.005 and 0.2 millimoles / L.

[0020] Preferably, it is an aqueous liquid composition.

[0021] Advantageously, this composition is for phytosanitary use, preferably for the prevention or treatment of an infection selected from Gibberella sp., Ophiostoma sp., Plasmopara sp., Phytophthora sp., Pyrenophora sp., Pseudoperonospora sp., Rhizoctonia sp., Sclerotinia sp., and Xanthomonas sp., preferably Gibberella sp., Phytophthora sp., Pyrenophora sp., and Xanthomonas sp. Advantageously, this composition is an extract of a species from the Bacillus subtilis group (in the broad sense), preferably under culture conditions allowing overproduction of bacilysin, preferably the member of the Bacillus subtilis group is a Bacillus pumilus.

[0022] Advantageously, this extract also includes bacilibactin and / or pumilacidines.

[0023] A related aspect of the present invention relates to a process for the purification of bacilysin comprising the steps of taking a composition comprising bacilysin; performing chromatography on a nonpolar support and retaining the polar fraction; adding an acidic solution to obtain a pH between 2.0 and 4.0; performing a liquid-liquid extraction by adding an alcohol or a water-insoluble polyol and recovering the alcohol / polyol fraction.

[0024] Preferably, this process further comprises the step of ion-exchange chromatography of the butanolic fraction in the presence of an eluent of pH greater than 8.0, said chromatography being carried out on an acid resin, preferably followed by an ethanol extraction, followed by a second liquid-liquid extraction by addition of butanol and recovery of the butanolic fraction.

[0025] Another related aspect of the present invention relates to a method for the (absolute) quantification of bacilysin in a sample, comprising the step of carrying out the above purification process, so as to obtain a pure bacilysin composition, of preparing at least two dilutions of said pure bacilysin composition, of obtaining at least one standard of known quantity of bacilysin and of carrying out a (liquid chromatography followed by mass spectrometry (LC-MS)), so as to quantify the area under the curve of the bacilysin peak.

[0026] Another related aspect of the present invention relates to a substantially pure composition of bacilysine that can be obtained by the above purification process.

[0027] Another aspect of the present invention relates to a pharmaceutical or disinfection composition comprising bacilysine in a known content as described above or the substantially pure composition of bacilysine as described above, said pharmaceutical composition preferably being antifungal or anti-Gram negative.

[0028] Another aspect of the present invention relates to the use of this composition described above or of the extract described above for the biocontrol of Phytophthora infestons Phytophthora sojae and / or Gibberella equiseti.

[0029] Another aspect of the present invention relates to the use of this composition described above or of the extract described above for the biocontrol of Xanthomonas sp.

[0030] Another aspect of the present invention relates to the use of this composition described above or of the extract described above for the biocontrol of Gram negative bacteria, preferably Xanthomonas sp., Pseudomonas synringae, Ralstionia solanacearum, Candidatus liberibacter sp., Clavibacter michiganensis, Xylella fastidiosa, Brenneria sp., Erwinia sp., Pantoea sp. and Curtobacterium sp.

[0031] Brief description of the drawings

[0032] Figure 1 shows the effect of several concentrations of bacilysine on the growth of Phytophthora infestons.

[0033] Figure 2 shows the bacilysine and bacillibactin contents of a commercial product and two formulations.

[0034] Detailed description of an embodiment of the invention

[0035] The inventors had an intuition that a detailed characterization of microorganism extracts was necessary to better potentiate the effect of the secreted biomolecules. Unexpectedly, they identified a major role for bacilysin. However, they noted that, for some plant pathogens, the presence of fengycin reduced the beneficial effect of bacilysin.

[0036] Initially, the inventors favored strains producing lipopeptides such as iturins or fengycins. However, after testing purified fractions, they concluded that bacilysin was a major active agent. Bacilysin is suspected of inhibiting cellulose metabolism or plant cell wall structure by inhibiting glucosamine-6-phosphate synthase and mannoprotein synthesis, potentially posing a problematic application in plants. Therefore, the inventors researched and, where necessary, developed microorganisms and culture conditions for preferential bacilysin production.

[0037] Furthermore, the inventors observed that the methods for purifying and quantifying non-ribosomal antimicrobial peptides, including bacilysin, are inadequate and do not allow for proper purification of bacilysin, nor for precise or absolute quantification of its concentration. This prevents testing using this biomolecule alone, or in synergy with other biomolecules. However, the inventors believe that such tests are necessary to develop the technology—currently based on empirical trial and error without understanding the underlying mechanism—into a coherent system that determines the concentrations, and relative concentrations, of a biomolecule enabling the biocontrol of a plant pathogen.

[0038] Thus, a first aspect of the present invention relates to a biocontrol composition comprising a known amount (in moles) of bacilysin. Preferably, the composition is an agricultural composition. Alternatively, preferably, the composition is for cleaning a surface or is a pharmaceutical composition, for example, for application to the skin or mucous membranes.

[0039] In the context of the present invention, preferably, bacilysin, also called tetain, is a dipeptide (of 270 Da), NL-Alanyl-3-(5-oxo-7-oxabicyclo(4.1.0)hept-2-yl)-L-alanine, or the "dipeptide" L-alanine-L-anticapsin, as well as its active salts and peptides comprising this dipeptide. The structure of bacilysin is shown below (Formula 1).

[0040] Formula 1 The present invention also covers stereoisomers, protonated or basic forms, as well as salts and active derivatives, such as peptides comprising bacilysine.

[0041] Preferably, the biocontrol composition is an agronomic composition for the prevention or treatment of an attack on a plant of interest by a parasite such as a fungal infection or an attack by insects (aphids) or by bacteria, preferably Gram negative, preferably phytopathogenic bacteria, preferably selected from Xanthomonas sp., Pseudomonas synringae, Ralstionia solanacearum, Candidatus liberibacter sp., Clavibacter michiganensis, Xylella fastidiosa, Brenneria sp., Erwinia sp., Pantoea sp. and Curto bacterium sp.

[0042] Oomycetes (e.g., causing downy mildew), and also agents causing powdery mildew, are among the fungal infections preferably treated by the composition of the present invention.

[0043] The inventors have noted that the composition according to the invention gives very good results when the infectious agent (of fungal type) is chosen from Bremia sp., Gibberella sp., Ophiostoma sp., Plasmopara sp., Phytophthora sp., Pyrenophora sp., Pseudoperonospora sp., Rhizoctonia sp., Sclerotinia sp., Peronospora sp., Pseudomonas sp., Candidatus sp., Ralstonia sp., Erwinia sp., Clabvibactersp, Xylella sp. and Xanthomonas sp., preferably Gibberella sp., Phytophthora sp., Pyrenophora sp., and Xanthomonas sp.

[0044] Preferably, in this biocontrol composition, the molar ratio between bacilysine and fengycin (and / or plipastatin; see below) is greater than 5, preferably greater than 10, preferably greater than 100. For example, there is no fengycin in the biocontrol composition (possible content below the limit of detection).

[0045] In the context of the present invention, "fengycin," also called plipastatin, preferably means a series of cyclic lipopeptides (decapeptides) (fengycins A, B, G, D, E, with a molecular weight of 1463.7 Daltons, FenC), comprising a lactone linkage, a β-hydroxy fatty acid of 16 to 19 carbons, and having the chiral sequence LDDDLDLLLL, or LDLDLDDLLL (plipastatin). In the context of the present invention, all potentially present fengycins (FenA, FenB, FenC, FenD, FenE, plipastatins, and their biological variants) are considered a single "fengycin" entity, and for the calculation of the bacilysin:fengycin ratio, the molar quantities of the different fengycins are therefore added together.

[0046] This composition, having very little (no) fengycin, has given very good results in the biocontrol of infections of tomatoes, potatoes and peppers by Gibberella equiseti and / or by Phytophthora infestons, of trees by Ophiostoma piliferum (Ceratocystis ulmi), of cereals by Pyrenophora tritici-repentis and / or by Xanthomonas transluscens, of lemon trees by Xanthomonas citri, of rice by Xanthomonas oryzae and X. oryzicola, of brassicas by Xanthomonas campestris.

[0047] Preferably, the composition according to the invention further comprises a known molar amount of a siderophore, preferably bacillibactin.

[0048] An advantageous bacillibactin content is between 0.005 and 0.2 millimoles / L, preferably between 0.01 and 0.1 millimoles / L.

[0049] Advantageously, the biocontrol composition according to the invention is an aqueous liquid composition.

[0050] This is particularly useful since a supernatant from well-selected strains (and grown under optimal production conditions) can be partially purified and / or concentrated, and thus formulated at little cost, for example for an agronomic composition or surface treatment.

[0051] However, the inventors noted that it was not practically possible to produce this type of composition from extracts obtained from just any bacilysin-producing strain. Indeed, an excessive presence of lipopeptides, which have surfactant properties, will affect the physicochemical properties of the composition, especially during the concentration steps. The present invention is therefore associated with a selection of strains and / or culture conditions that provide a high production (relative to lipopeptides and surfactins or pumilacidins) of bacilysin.

[0052] Preferably, the (aqueous) biocontrol composition according to the invention is applied at a bacilysin concentration of at least 0.001 mol millimole of bacilysin / L, preferably at least 0.005 millimole / L, preferably at least 0.01 millimole / L, or even at least 0.03 millimole / L, and at most 100 millimole / L, preferably 50 millimole / L, preferably or 20 millimole / L, preferably 10 millimole / L, or even 5 millimole / L or 3 millimole / L.

[0053] Indeed, depending on the pathogens, a minimum concentration is highly preferred, often on the order of 0.01 millimole / L (bacilysin alone; therefore potentially less for synergistic combinations including bacilysin and other biomolecules).

[0054] On the other hand, the inventors noted during confidential greenhouse and field tests, at least for certain plants affected by fungal agents such as Bremia lactucae, that excessively high concentrations have a detrimental effect on growth (disruption of metabolism, even the onset of chlorosis), such that, at high doses, the benefit in terms of biocontrol of the pathogen(s) was masked by the interference with plant metabolism. Thus, depending on the plant species, the maximum bacilysine concentrations described above (e.g., 20 millimoles / L) are highly preferred.

[0055] Thus, an aqueous liquid composition (an extract) was successfully produced in the present invention by (i) selecting strains based on their production of bacilysin (relative production of bacilysin compared to the overall production of lipopeptides and / or non-ribosomal antimicrobial peptides) and / or (ii) applying mutations to reduce the production of other lipopeptides (and / or non-ribosomal antimicrobial peptides) and / or (iii) optimizing culture conditions so that a promising strain, under these optimized conditions, produces significantly more bacilysin; the inventors tested hypotonic culture media (without added NaCl), a pH maintained below 8, a controlled vitamin content twice that of standard conditions, and / or a constant dissolved oxygen content.

[0056] The inventors observed that several species within the Bacillus subtilis group (broadly defined) are capable of producing bacilysin. However, they noted a discrepancy between genomic and extract analyses, so only the latter are considered relevant.

[0057] Among the strains tested, B. aerius, B. altitudinis, B. amyloliquefaciens, B. mojavensis, B. nakamurai, B. pumilus, B. safensis, B. subtilis (narrow sense), B. subtilis sbsp. inaquosorum, B. subtilis sbsp. spizizenii, B. tequilensis, and B. velezensis produce bacilysin, while other strains of the Bacillus subtilis group (broad sense) do not, even though bioinformatic analysis might have suggested otherwise. For example, the species B. atrophaeus, B. glycinifermentas, B. goblensis, B. licheniformis, B. paralicheniformis, B. sonorensis, and B. xiamenensis do not produce bacilysin in useful or even measurable amounts.

[0058] B. pumilus, on the other hand, allowed for good bacilysin production. The choice of B. pumilus is not, a priori, preferred since this species does not produce iturins or fengycins, which are lipopeptides generally highlighted for their anti-pathogenic or plant growth-promoting activities.

[0059] A preferred extract includes, in addition to bacilysine (in known concentrations), bacillibactin.

[0060] The inventors noticed that the presence of this molecule increases the inhibitory effect of bacilysin, despite it being a siderophore, and therefore potentially beneficial also for pathogens.

[0061] A related aspect of the present invention relates to a process for the purification of bacilysin comprising the steps of: taking a composition comprising bacilysin, preferably an extract of Bacillus sp, such as Bacillus pumilus; performing chromatography on a nonpolar support and retaining the polar fraction; preferably adding methanol or ethanol and performing a liquid-liquid extraction in which the alcoholic fraction is taken; adding an acidic solution; performing a liquid-liquid extraction by adding butanol (and / or an alcohol or a polyol not soluble in water) and recovering the butanolic fraction (alcohol, polyol).

[0062] This process first allows the separation of nonpolar compounds, and then of polar compounds, with the "protonated" bacilysine migrating into the alcohol phase.

[0063] Preferably, the composition including the bacilysin to be extracted is an extract from a strain (see the species and subspecies of the Bacillus subtilis group described above) cultivated to produce, among other things, bacilysin.

[0064] Preferably, in this process, the chromatography on a nonpolar support is a derivatized resin with Cl 8 groups, the elution being carried out using water and then an amphiprotic nonpolar solvent acetonitrile / methanol, preferably via a gradient between water and the acetonitrile / methanol composition.

[0065] Preferably, in this process, the acid is a weak acid and / or an organic acid.

[0066] For the purposes of this invention, "weak acid" is preferably understood to mean an acid having a pKa between 3 and 5, preferably between 3.5 and 4.5, or even between 3.5 and 4.0. A preferred weak acid is a carboxylic acid, such as formic acid.

[0067] A weak acid can be an acidic salt.

[0068] Preferably, the weak acid is not an acidic chloride salt.

[0069] Such a weak acid (formic acid, acetic acid), if liquid, is preferably incorporated in a content of between 1 and 5% (v:v).

[0070] Preferably the pH of the solution after the addition of the weak acid is less than 4 (4.0), preferably less than 3.5, preferably less than 3 (3.0), but preferably greater than 2 (2.0).

[0071] This ensures that substantially all of the bacilysine is in "protonated" form.

[0072] Preferably butanol (and / or alcohol or polyol not miscible in water) is added to the composition comprising the acid in a volume content of 1:1, by adding pure butanol; other volume ratios, typically between 1:3 and 3:1, are also advantageous.

[0073] This extraction operation in water-immiscible alcohol (butanol) or water-immiscible polyol is advantageously repeated once or several times (e.g., twice), in order to increase the recovery of bacilysine.

[0074] Advantageously, this process may include a preliminary sterilizing filtration step (e.g., 0.22 m filter) to remove cellular debris and cells, and / or one or more water / ethanol or water / methanol extraction steps to separate the salts from bacilysin.

[0075] Preferably, this process further includes the step of ion-exchange chromatography of the butanol fraction in the presence of an eluent with a pH greater than 8.0 (preferably a pH greater than 9.0, greater than 10.0, greater than 11.0, or even greater than 12.0), said chromatography being carried out on a strong acidic resin. A preferred eluent is a strong base, for example, NaOH. This allows for rapid detachment of the bacilysin (abruptly deprotonated) from the chromatographic support.

[0076] In the context of the present invention, "acid resin" preferably means that the active groups of the resin are negatively charged, and "strong acid resin" means that the active groups of the resin have a negative charge, even in the presence of weak acid solutions, for example at a pH between 3.5 and 4.0.

[0077] Preferably, the (alkaline) eluate containing bacilysine is then treated by ethanol extraction, followed by a second liquid-liquid extraction by adding butanol (pure; in a 1:1 volume ratio) and recovering the butanol fraction.

[0078] A complementary aspect of the present invention relates to a method for quantifying bacilysin in a sample, comprising

[0079] - to carry out the above process,

[0080] - prepare at least two dilutions of the pure bacilysine composition obtained via the above process,

[0081] - obtain at least one standard of known quantity of bacilysine and

[0082] - to perform liquid chromatography coupled with mass spectrometry (LC-MS), preferably HPLC-MS or even UPLC-MS (ultra-high pressure coupled with mass spectrometry), in order to quantify the area under the curve of the bacilysine peak of the extract, and to relate it to that of the standard of known quantity.

[0083] This process advantageously allows the absolute quantification of bacilysine given (i) the level of purity obtained and (ii) the presence of standards of known concentration.

[0084] Thus, another associated aspect of the present invention relates to a substantially pure composition of bacilysine that can be obtained by this purification process.

[0085] By "substantially pure", we preferably mean that the mass quantity of bacilysin relative to all the biomolecules in the extract is greater than 90%, preferably greater than 95, 96, 97, 98, 99% (weight of bacilysin: weight (dry) of all the biomolecules in the extract after purification).

[0086] This (substantially) pure bacilysin composition is advantageously used in biocontrol compositions, for example, to enhance Bacillus sp. extracts that contain a promising panel of antimicrobial biomolecules but an insufficient bacilysin content (absolute quantity of bacilysin; bacilysin:fengycin molar ratio), or this substantially pure bacilysin composition is advantageously formulated in the presence of other biomolecules, such as surfactants and / or enzymes (exogenous to those of the starting extract). Alternatively, this (substantially) pure bacilysin composition is useful as a standard that can be used in the purification process described above.

[0087] Furthermore, this (substantially) pure bacilysin composition is advantageously incorporated into (non-therapeutic) compositions for surface disinfection, in which the bacilysin concentration is at least 0.0001 mol / L, preferably at least 0.001 mol / L, preferably at least 0.01 mol / L, preferably at least 0.02 mol / L, or even at least 0.03 mol / L. Preferably, the maximum concentration is less than 10 mol / L, preferably less than 1 mol / L. Since this composition is not normally intended for use in agriculture, any potential adverse effects at high concentrations are not encountered.

[0088] Preferably, this composition for surface disinfection includes at least one other biomolecule, such as one or more enzymatic activities (protease, polysaccharidase) and / or another surfactant molecule (in a weight content of at least 0.1%).

[0089] Finally, this (substantially) pure bacilysine composition is advantageously incorporated into a pharmaceutical composition in which the concentration of bacilysine is at least 0.0001 mol / L, preferably at least 0.001 mol / L, preferably at least 0.01 mol / L, preferably at least 0.02 mol / L, or even at least 0.03 mol / L.

[0090] This pharmaceutical composition is advantageously chosen from among an emulsion, a gel, a lotion.

[0091] Preferably, this pharmaceutical composition is for topical application (e.g., skin, mucous membranes).

[0092] Another aspect of the present invention relates to this (agronomic) composition described above, having a known quantity of bacilysine, for the biocontrol of Phytophthora infestons Phytophthora sojae and / or Gibberella equiseti.

[0093] Another aspect of the present invention relates to this (agronomic) composition described above, having a known quantity of bacilysine, for the biocontrol of Xanthomonas sp.

[0094] Other features and advantages of the present invention will be derived from the following non-limiting description, and with reference to the drawings and examples.

[0095] Examples.-

[0096] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications may be made to it without departing from the scope of the appended claims. Example 1:

[0097] The inventors purified and quantified bacilysin using the methods described above, and applied known quantities of this bacilysin to pathogen cultures on agar. The inventors further tested the effect of fengycin alone or in combination with bacilysin.

[0098] Figure 1 shows the test when Phytophthora infestons is cultured in the absence of bacilysin (left), or in the presence of 0.0068; 0.0137, 0.0684 or 0.1368 millimole / L of bacilysin (alone; from left to right).

[0099] Inhibition is already observed at the lowest concentration.

[0100] Other tested phytopathogens against which bacilysin showed an effect include Bremia lactucae, Gibberella equiseti, Ophiostoma piliferum, Plasmopara viticola, Phytophthora infestons, Phytophthora sojae, Pyrenophora tritici-repentis, Rhizoctonia solani (especially in synergy with fengycin), Rhizoctonia zeae, Sclerotinia sclerotium, and various Xanthomonas species (X. citri, X. oryzae, and X. transluscens). An effect was also observed against Alternaria brassicicola (especially in synergy with fengycin). The effects observed on Xanthomonas, Phytophthora, and Gibberella equiseti were obtained only with bacilysin.

[0101] On the other hand, bacilysin did not inhibit the growth of Aspergillus niger, Cercospora beticola, various species of Fusarium sp or Pythium sp, Trichoderma harzianum and Verticillium sp, various species of Alternaria (A. porri, A. radicina, A. solani), Botrytis cinerea, Cercospora beticola or Phoma betae.

[0102] Figure 2 shows the bacilysin and bacillibactin content of controlled (non-optimized) formulations according to the invention, compared to the contents of a commercial product which includes Bacillus spp spores and also biomolecules, which were secreted during culture under controlled conditions.

[0103] For comparison, the inventors selected this commercially available product and verified that it works effectively in the biocontrol of fungal pathogens.

[0104] Next, the inventors applied the purification protocol according to the invention and performed the absolute quantification from the undiluted formulation for use of this commercial product.

[0105] The bacilysin and bacillibactin content of the commercial product is particularly low, below the level required for these biomolecules to have a direct effect, yet the product is still functional. This is because the measurement was performed on the concentrated product, and the values ​​reported are multiples of the values ​​used in the field. The inventors calculated that an effective bacillysin concentration would require applying a volume far exceeding that recommended by the manufacturer. Therefore, the effect obtained with the commercial formulation is attributable to other biomolecules derived from the product's main active ingredient and / or to the action of the biocontrol microorganism, this time in an uncontrolled environment.

Claims

DEMANDS 1. A liquid biocontrol composition, comprising a known amount of bacilysine.

2. The biocontrol composition according to claim 1 comprising between 0.001 mmol / L and 20 mmol / L of bacilysin.

3. The biocontrol composition according to claim 1 or 2, wherein the molar ratio between bacilysin and fengycin is greater than 5, preferably greater than 100.

4. The biocontrol composition according to any of the preceding claims, further comprising a known molar amount of a siderophore, preferably bacillibactin, preferably between 0.005 and 0.2 millimoles / L.

5. The biocontrol composition according to any of the preceding claims being an aqueous liquid composition.

6. The biocontrol composition according to any of the preceding claims being a phytosanitary composition.

7. The phytosanitary composition of claim 6, being for the prevention or treatment of an infection selected from Gibberella sp., Ophiostoma sp., Plasmopara sp., Phytophthora sp., Pyrenophora sp., Pseudoperonospora sp., Rhizoctonia / sp., Sclerotinia sp., and Xanthomonas sp., preferably Gibberella sp., Phytophthora sp., Pyrenophora sp., and Xanthomonas sp.

8. The composition according to any one of the preceding claims being an extract of a species from the Bacillus subtilis group, preferably under culture conditions allowing overproduction of bacilysin, preferably the Bacillus subtilis is a Bacillus pumilus.

9. The composition according to claim 8 further comprising bacilibactin and / or pumilacidines.

10. A process for the purification of bacilysin comprising the steps of preparing a composition containing bacilysin; performing chromatography on a nonpolar support and retaining the polar fraction; adding an acidic solution to obtain a pH between 2.0 and We perform a liquid-liquid extraction by adding an alcohol or a polyol that is insoluble in water and recovering the alcohol / polyol fraction.

11. The process according to claim 10 further comprising the step of ion exchange chromatography of the butanolic fraction in the presence of an eluent of pH greater than 8.0, said chromatography being carried out on an acid resin, preferably followed by an ethanol extraction, followed by a second liquid-liquid extraction by addition of butanol and recovery of the butanolic fraction.

12. Method for quantifying bacilysin in a sample, comprising carrying out the method according to claim 10 or 11 so as to obtain a pure bacilysin composition, preparing at least two dilutions of said pure bacilysin composition, obtaining at least one standard of known quantity of bacilysin and carrying out a liquid chromatography followed by mass spectrometry (LC-MS) so as to quantify the area under the curve of the bacilysin peak.

13. The method according to claim 12 being for the absolute quantification of bacilysine.

14. Substantially pure composition of bacilysine capable of being obtained by the process according to claim 10 or 11.

15. Pharmaceutical or disinfection composition comprising bacilysine according to any one of claims 1 to 5 or the substantially pure bacilysine composition of claim 14, said pharmaceutical composition preferably being antifungal or anti-Gram negative.

16. Use of the composition according to any of the preceding claims 1 to 8 for the biocontrol of Phytophthora infestons Phytophthora sojae and / or Gibberella equiseti.

17. Use of the composition according to any of the preceding claims 1 to 8 for the biocontrol of Xanthomonas sp.

18. Use of the composition according to any of the preceding claims 1 to 8 for the biocontrol of Gram negative bacteria.

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