Biological control composition

Abstract

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

Description

[0001]BIOCONTROL COMPOSITION Technical Field The present invention relates to a biocontrol composition, particularly for the protection of plants of interest. The composition comprises a known effective amount of bacilysin and an effective amount of fengycin. Prior Art Many microorganisms belonging to the Bacillus subtilis group (in the broad sense) are known to have potentially interesting properties for biocontrol. 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. The most frequently cited biomolecules are fengycin, surfactin, and various molecules derived from iturin. Even though these peptides are "non-ribosomal," their synthesis involves functional operons.Thus, within a strain collection, the presence or absence of functional genes, and even their expression levels, will create a very wide diversity of peptide mixtures. Numerous publications in this field highlight promising strains that inventors consider to be potentially secreting the desired biomolecules, perhaps even in effective concentrations, even accidentally. However, while 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 adjust: it is possible to prove that a commercial product works under given conditions against a particular pathogen, but inventors believe that this same product, if modified in light of the present invention, could work much better, including against other pathogens.The inventors suspect that some of the difficulties encountered are related to insufficient levels of key metabolites and / or excessive levels of undesirable, or even harmful, co-products. Indeed, it is not possible to concentrate such extracts at will. For example, WO2019 / 016492 describes a composition containing a high level of mycosubtilin (an iturin-type lipopeptide) in combination with other biomolecules, while reporting the significant difficulty associated with concentrating the extracts beyond the critical micellar concentration of the biomolecules, which leads to aggregates or gels. Furthermore, the purification of these antimicrobial peptides is delicate and expensive. Lipopeptide purification methods, including hydrochloric acid precipitation, methanol extraction, and C18 column chromatography, are described in CN106754489. This document does not describe the purification of bacilysin.Brief summary of the invention: A first aspect of the present invention relates to a liquid biocontrol composition comprising a known amount of bacilysin and a known amount of fengycin, preferably comprising between 0.001 mmol / L and 20 mmol / L of bacilysin, and / or between 1 and 10 mg / L of fengycin. Advantageously, the fengycin is a plipastatin, preferably SEQ ID NO: 1 or SEQ ID NO: 2. Preferably, this composition further comprises a known molar amount of a siderophore, preferably bacillibactin, preferably between 0.005 and 0.2 millimoles / L. Preferably, it is an aqueous liquid composition. Advantageously, this aqueous liquid composition is concentrated and comprises 25 to 3000 mg / l of fengycin and / or between 0.025 mmol / L and 6000 mmol / l of bacilysin, preferably 100 to 1000 mg / l of fengycin and / or 0.1 to 1000 mmol of bacilysin.This composition is advantageously for phytosanitary use, preferably for the prevention or treatment of an infection selected from Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp., Ophiostoma sp., Phoma sp., Plasmopara sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp. Advantageously, this composition is an extract of a species of Bacillus subtilis, preferably under culture conditions allowing overproduction of bacilysin and / or fengycin, preferably under conditions for overproduction of fengycin (plipastatin).A related aspect of the present invention relates to 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 4.0; and performing a liquid-liquid extraction by adding a water-insoluble alcohol or polyol and recovering the alcohol / polyol fraction. This process preferably further comprises the step of ion-exchange chromatography of the butanol fraction in the presence of an eluent with a pH greater than 8.0, said chromatography being performed on an acidic resin, preferably followed by ethanol extraction, followed by a second liquid-liquid extraction by adding butanol and recovering the butanol fraction.Another related aspect of the present invention relates to a method for quantifying bacilysin in a sample, comprising performing the above purification process 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 performing liquid chromatography followed by mass spectrometry (LC-MS) to quantify the area under the curve of the bacilysin peak. Advantageously, this method allows for the absolute quantification of bacilysin. Another related aspect of the present invention relates to the use of the above composition for the biocontrol of Alternaria sp., Botrytis sp., Cercospora sp., Fusarium sp., Phoma sp., and Zymoseptoria sp., preferably for the biocontrol of Alternaria brasicola, Bremia lactucae, Ophiostoma piliferum, Plasmopara viticola, Rhizoctonia solani, Rhizoctonia zeae, and Sclerotinia sclerotium. Another aspect of the present invention relates to the use of a plipastatin selected from SEQ ID NO: 1 or SEQ ID NO: 2 for the biocontrol of an infection selected from Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp., Ophiostoma sp., Phoma sp., Plasmopara sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp., preferably Alternaria sp., Bremia sp., Ophiostoma sp., Plasmopara sp., Rhizoctonia sp., Venturia sp., and Sclerotinia sp.A related aspect of the present invention relates to an aqueous liquid agronomic composition comprising from 1 to 3000 mg / l of SEQ ID NO:1 or SEQ ID NO:2, preferably, from 10 to 2000 mg / l of SEQ ID NO:1 or SEQ ID NO:2, preferably, from 50 to 1000 mg / l of SEQ ID NO:1 or SEQ ID NO:2, preferably from 100 to 500 mg / l of SEQ ID NO:1 or SEQ ID NO:2, preferably comprising from 1 to 1000 mg / m of SEQ ID NO:1 or SEQ ID NO:2 in which the fatty acid chain grafted to the peptide is at C. 15 and / or in C 17Brief description of the drawings: Figure 1 shows the effect of fengycin and bacilysin, or bacilysin alone, on the growth of the fungal pathogen Alternaria brassisicola. Figure 2 shows the effect of fengycin and bacilysin, or bacilysin alone, on the growth of the fungal pathogen Phoma betae. Figure 3 shows the bacilysin and bacillibactin contents of a commercial product and two formulations. Detailed description of an embodiment of the invention: The inventors had the intuition that a detailed characterization of microorganism extracts was necessary to better potentiate the effect of the secreted biomolecules. The inventors unexpectedly identified a major role for bacilysin. However, the inventors noted that, for some plant pathogens, the presence of fengycin was necessary and sometimes synergistic with the effect of bacilysin.Initially, the inventors favored lipopeptide-producing strains also capable of producing iturins. However, through testing purified fractions, they concluded that bacilysin was a major active agent, that fengycin alone was also a promising agent, and that, for some pathogens (not all; sometimes the combination loses its effectiveness), the combination of bacilysin and fengycin is synergistic. Bacilysin is nevertheless suspected of inhibiting cellulose metabolism or plant cell wall function via an inhibitory effect on glucosamine-6-phosphate synthase and mannoprotein synthesis, thus potentially posing a problematic application to plants. Based on this, the inventors researched and, where necessary, developed microorganisms and culture conditions for the preferential production of bacilysin and fengycin.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 or precise or absolute quantification of bacilysin. This prevents testing using this biomolecule alone or in synergy with other biomolecules such as fengycin. 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 or a combination of biomolecules (e.g., synergy or antagonism?) enabling the biocontrol of a plant pathogen.Thus, a first aspect of the present invention relates to a biocontrol composition comprising a known quantity (in moles) of bacilysin and a known quantity (in grams) of fengycin. Preferably, the composition is an agronomic composition. In the context of the present invention, bacilysin, also called tetain, is preferably 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-anticapsisin, as well as its active salts and peptides comprising this dipeptide. The structure of bacilysin is shown below (Formula 1). Formula 1 The present invention also covers stereoisomers, protonated or basic forms, as well as salts and active derivatives, such as peptides including bacilysin. In the context of the present invention, "fengycin", also called plipastatin, preferably means a series of cyclic lipopeptides (decapeptides) (fengycins A, B, C, D, E, of a molecular weight of 1463.7 Daltons, FenC), comprising a lactone linkage, a β-hydroxy fatty acid of 16 to 19 carbons (14 to 19 carbon atoms for plipastatin) and having the chiral sequence LDDDLDLLLL, or LDLDLDDLLL (plipastatin; e.g. SEQ ID NOs: 1 and 2). In the context of the present invention, the word "fengycin" covers both fengycins and plipastatins, whereas the word "plipastatin" is restricted to these chemical entities only, and is therefore a specific component within the generic group "fengycins".The generic group "fengycins" therefore does not cover just any stereoisomer, but only the two diastereomers described above. Thus, in the context of the present invention, all the fengycins (FenA, FenB, FenC, FenD, FenE, plipastatins and their biological variants) potentially present are considered as a single "fengycin" entity and, for the calculation of the bacilysin:fengycin ratio, the molar quantities of the different fengycins are therefore added together. Preferably, the fengycin of the present invention is a plipastatin having the amino acid sequence: L-Glu-D-Orn-L-Tyr-D-Thr-L-Glu-D-Val-L-Pro-L-Glu-D-Tyr-Ile (SEQ ID NO: 1); Alternatively, it is the sequence L-Glu-D-Orn-L-Tyr-D-Thr-L-Glu-D-Ala-L-Pro-L-Glu-D-Tyr-Ile (SEQ ID NO: 2). In the preferred plipastatin, this amino acid sequence is linked (at the terminal glutamate) to a 3-hydroxy fatty acid having 14–19 carbon atoms (saturated or unsaturated chain).Cyclization is produced via Tyr at position 3 and Ile at position 10 through a lactone linkage. Thus, an ancillary aspect of the present patent application relates to an agronomic composition comprising an effective active amount of plipastatin (SEQ ID NO. 1 or SEQ ID NO. 2). 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. Oomycetes (e.g., those causing downy mildew) and also agents causing powdery mildew are among the fungal infections preferably treated by the composition of the present invention. The inventors have noted that the composition according to the invention gives very good results when the infectious agent is chosen from Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp., Ophiostoma sp., Perenospora sp., Plasmopara sp., Phoma sp., Pyrenophora sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp.Preferably, the composition according to the invention further comprises a known molar quantity of a siderophore, preferably bacillibactin. Preferably, the bacillibactin is present (in the composition applied to the plant) at a concentration of between 0.005 and 0.2 millimoles / L, preferably between 0.01 and 0.1 millimoles / L, and preferably between 0.02 and 0.5 millimoles / L. Advantageously, the composition according to the invention comprises an additional molecule selected from a surfactin (lipopeptide), subtilosin A, pulcherriminic acid, and a mixture thereof. Advantageously, the biocontrol composition according to the invention is an aqueous liquid composition. This is particularly useful since a supernatant from well-selected strains (cultivated under optimal production conditions) can be partially purified and / or concentrated, and thus formulated at low cost for an agronomic composition.However, the inventors noted that it was not practically possible to produce this type of composition from extracts obtained from just any strain producing bacilysin and fengycin. 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 high production (relative to lipopeptides and surfactins) of bacilysin and fengycin.Preferably, the (aqueous) biocontrol composition according to the invention is applied at a bacilysin concentration of at least 0.001 millimole 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, or preferably 20 millimole / L, preferably 10 millimole / L, or even 5 millimole / L or 3 millimole / L. Furthermore, preferably, in this biocontrol composition, the effective content of fengycin (and / or plipastatin; see above) is between 1 and 10 mg / L. However, concentrated formulations, which are diluted before use, are advantageous.In this case, fengycin (plipastatin) concentrations between 25 and 3000 mg / L (preferably between 50 and 2500, preferably between 100 and 2000, preferably between 500 and 1000) are advantageous (i.e., a concentration factor of 25 to 300 times, which also applies to the bacilysin concentrations mentioned above). It should be noted that the concentrations for fengycin (plipastatin) are given in mg / L and not in mol / L for practical reasons of quantification. However, the conversion can be carried out with good accuracy by considering molecular weights (for fengycin in general) ranging from 1419 to 1532 g / mol for an average molecular weight of 1476 g / mol (in case of doubt, this value will be used for the conversion from g / L to mol / L).This diversity, in the preferred case of plipastatin according to the invention, is distributed (essentially) over three chromatographic peaks, each corresponding to an isovariant whose fatty acid chain is more or less long, oscillating between C. 14 etc 19 In practice, the majority of C isovariants 15 , C 16 etc 17Indeed, depending on the pathogen, a minimal concentration is highly preferred, often on the order of 0.01 millimole / L (bacilysin alone; therefore potentially less for synergistic combinations including bacilysin and fengycin / plipastatin). Furthermore, the inventors noted during confidential greenhouse tests, at least for certain plants, that excessively high levels of bacilysin have a detrimental effect on growth (disruption of metabolism, even the onset of chlorosis), such that, at high doses, the benefit in terms of pathogen biocontrol was masked by the interference with plant metabolism. Thus, depending on the plant, the maximum bacilysin levels described above (e.g., 20 millimole / L) are highly preferred.Thus, an aqueous liquid composition (an extract) has been successfully produced in the present invention, (i) by selecting strains based on their production of bacilysin and fengycin / plipastatin (relative production of bacilysin and fengycin / plipastatin compared to the overall production of lipopeptides and / or non-ribosomal antimicrobial peptides) and / or (ii) by applying mutations to reduce the production of other lipopeptides (and / or non-ribosomal antimicrobial peptides other than bacilysin and fengycin) and / or (iii) by optimizing culture conditions so that a promising strain, under these optimized conditions, produces significantly more bacilysin and fengycin (especially fengycin / plipastatin); The inventors tested hypotonic culture media (without added NaCl), a pH maintained below 8, a controlled vitamin content twice the standard conditions, and / or a constant dissolved oxygen content.The inventors observed that several subspecies / strains of Bacillus subtilis (in the narrow sense) are capable of producing bacilysin and fengycin (or plipastatin). However, they noted a discrepancy between genomic and extract analyses, such that only the latter are considered relevant. Among the strains tested, B. aerius, B. altitudinis, B. amyloliquefaciens, B. mojavensis, B. nakamurai, B. pumilus, B. safensis, B. subtilis (in the 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 (in the broad sense) do not, even though bioinformatic analysis would have suggested otherwise. For example, the species B. atrophaeus, B. glycinifermentas, B. goblensis, B. licheniformis, B. paralicheniformis, B. sonorensis and B.*B. xiamenensis* does not produce bacilysin in useful or even measurable amounts. The choice of *B. subtilis* is not preferred a priori since this species does not produce iturins, a lipopeptide highlighted, among other things, for its anti-pathogenic activity. A preferred extract includes, in addition to bacilysin and fengycin (in known concentrations), bacillibactin and / or, as stated above, an additional molecule chosen from among a surfactin (lipopeptide), subtilosin A, pulcherriminic acid, and a mixture thereof. The inventors observed that the presence of these molecules increases the inhibitory effect of bacilysin and fengycin / plipastatin, including for bacillibactin despite it being a siderophore.A related aspect of the present invention relates to a process for the purification of bacilysin comprising the steps of: - preparing a composition containing 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 collected; - adding an acidic solution; - performing a liquid-liquid extraction by adding butanol (and / or an alcohol or a polyol insoluble in water) and recovering the butanolic fraction (alcohol, polyol). This process allows first the separation of the nonpolar compounds, and then of the polar compounds, with the protonated bacilysin migrating in the alcohol phase.Preferably, the composition comprising 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. Preferably, in this process, the chromatography on a nonpolar support is a derivatized resin with C18 groups, the elution being carried out using water followed by a nonpolar amphiprotic acetonitrile / methanol solvent, preferably via a gradient between water and the acetonitrile / methanol composition. Preferably, in this process, the acid is a weak acid and / or an organic acid. By "weak acid," in the context of the present invention, 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. A weak acid can be an acidic salt.Preferably, the weak acid is not an acidic chloride salt. Such a weak acid (formic acid, acetic acid), if liquid, is preferably incorporated at a concentration of 1 to 5% (v:v). 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). This ensures that substantially all of the bacilysin is in protonated form. Preferably, butanol (and / or the water-immiscible alcohol or polyol) is added to the composition comprising the acid at a volume concentration of 1:1, by adding pure butanol; other volume ratios, typically between 1:3 and 3:1, are also advantageous. 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.Advantageously, this process may include a preliminary sterilizing filtration step (e.g., a 0.22 µm filter) to remove cell debris and cells, and / or one or more water / ethanol or water / methanol extraction steps to separate the salts from the bacilysin. Preferably, this process further includes an ion-exchange chromatography step of the butanol fraction in the presence of an eluent with a pH greater than 8.0 (preferably greater than 9.0, 10.0, 11.0, or even 12.0), said chromatography being performed on a strong acidic resin. A preferred eluent is a strong base, for example, 1M NaOH. This allows for rapid detachment of the bacilysin (abruptly deprotonated) from the chromatographic support.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. Preferably, the (alkaline) eluate comprising bacilysin is then treated by an ethanol extraction, and followed by a second liquid-liquid extraction by the addition of butanol (pure; in a 1:1 volume ratio) and recovery of the butanol fraction.A further aspect of the present invention relates to a method for quantifying bacilysin in a sample, comprising: performing the above method; preparing at least two dilutions of the pure bacilysin composition obtained via the above method; obtaining at least one standard of known quantity of bacilysin; and performing liquid chromatography coupled with mass spectrometry (LC-MS), preferably HPLC-MS or even UPLC-MS (ultra-high pressure chromatography coupled with mass spectrometry), so as to quantify the area under the curve of the bacilysin peak in the extract and relate it to that of the standard of known quantity. This method advantageously allows for the absolute quantification of bacilysin given (i) the level of purity obtained and (ii) the presence of standards of known concentration.Another aspect of the present invention relates to this (agronomic) composition described above, having a known quantity of bacilysin and fengycin / plipastatin, for the biocontrol of Alternaria sp., Bremia sp., Botrytis sp., Cercospora sp., Fusarium sp., Ophiostoma sp., Perenospora sp., Plasmopara sp., Phoma sp., Pyrenophora sp., Rhizoctonia sp., Sclerotinia sp. and Zymoseptoria sp. With regard to Alternaria brassicicola, Bremia sp., Ophiostoma sp., Perenospora sp., Plasmopara sp., Rhizoctonia sp., Sclerotinia sp., the inventors have observed a synergy between bacilysin and fengycin / plipastatin. On the other hand, fengycin / plipastatin alone (in the absence of bacilysin) gives good results in the biocontrol of other Alternaria species (A. porri, A. radicina and A. solani), against Cercospora sp., against Fusarium (F. culmorim and F. graminearum), against Phoma betae and against Zymoseptoria tritici.Other features and advantages of the present invention will be derived from the following non-limiting description, with reference to the drawings and examples. Examples: It is understood that the present invention is in no way limited to the embodiments described above and that many modifications can be made to it without departing from the scope of the appended claims. Example 1: The inventors purified and quantified bacilysin according to 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. Figure 1 shows the test, compared to a negative control of the fungal pathogen Alternaria brassisicola (left), the effect on mycelial growth of bacilysin combined with plipastatin (center), and the lesser effect of bacilysin without plipastatin (right).The appearance of irregular white spots indicates sporulation, and therefore stress on the microorganism. Similarly, as shown in Figure 2, the growth of Phoma betae is not well inhibited by bacilysin alone (control on the left, compared to bacilysin alone on the right), but is well inhibited by fengycin / piplastatin according to the invention (center column: bacilysin + piplastatin). The other phytopathogens tested for which fengycin (piplastatin) alone, or in synergy with bacilysin, showed an effect are Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp. (F. culmorum and F. graminearum), Ophiostoma sp., Plasmopara sp., Phoma sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp. The effect measured on Alternaria sp (A. brassicicola), Bremia sp., Ophiostoma sp., Plasmopara sp., Rhizoctonia sp., and Sclerotinia sp. showed a synergy between bacilysine and fengycin (piplastatin).On the other hand, fengycin (piplastatin) alone showed an effect on Botrytis, Cercospora sp., Fusarium sp. (F. culmorum and F. graminearum), Phoma sp., and Zymoseptoria sp. However, fengycin combined with bacilysin did not inhibit the growth of Aspergillus niger, some Fusarium species (F. oxysporum, F. rodolens, F. solani), Pythium sp., Trichoderma harzianum, and Verticillium sp. Example 2 Figure 3 shows the bacilysin and bacillibactin content of controlled (non-optimized) formulations according to the invention, compared to the content of a commercial product containing Bacillus spp. spores and also biomolecules, which were secreted during culture under controlled conditions. For comparison, the inventors selected this commercially available product and verified that it works effectively in the biocontrol of fungal pathogens.Next, the inventors applied the purification protocol according to the invention and performed absolute quantification using the undiluted formulation of this commercial product. The bacilysin and bacillibactin content of the commercial product is particularly low, below the level required for a direct effect of these biomolecules, yet the product is still functional. Indeed, since the measurement was performed on the concentrated product, the values ​​reported are multiples of the values ​​used in the field. The inventors calculated that an effective bacillysin concentration would require the application of a volume far exceeding that recommended by the manufacturer. Thus, 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

CLAIMS 1. A liquid biocontrol composition comprising a known amount of bacilysin and a known amount of fengycin.

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

3. The biocontrol composition of claim 1 or 2, wherein the fengycin content is between 1 and 10 mg / L.

4. The biocontrol composition of any one of the preceding claims, wherein the fengycin is a plipastatin.

5. The biocontrol composition of any one of the preceding claims, wherein the fengycin (plipastatin) is SEQ ID NO: 1 or SEQ ID NO:

2.

6. The biocontrol composition of any one of the preceding claims, further comprising a known molar amount of a siderophore, preferably bacillibactin, preferably between 0.005 and 0.2 mmol / L. 7.The biocontrol composition according to any one of the preceding claims being an aqueous liquid composition.

8. A concentrated aqueous composition corresponding to the aqueous composition according to claim 7, comprising from 25 to 3000 mg / L of fengycin and / or from 0.025 mmol / L to 6000 mmol / L of bacilysin, preferably from 100 to 1000 mg / L of fengycin and / or from 0.1 to 1000 mmol of bacilysin.

9. The biocontrol composition according to any one of the preceding claims being a plant protection product composition.

10. The phytosanitary composition of claim 9, being for the prevention or treatment of an infection selected from Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp., Ophiostoma sp., Phoma sp., Plasmopara sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp., preferably Alternaria sp., Bremia sp., Ophiostoma sp., Plasmopara sp., Rhizoctonia sp., and Sclerotinia sp.

11. The composition according to any one of the preceding claims being an extract of a species of Bacillus subtilis, preferably under culture conditions allowing for overproduction of bacilysin and / or fengycin, preferably fengycin.

12. 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 4.0; - performing a liquid-liquid extraction by adding a water-insoluble alcohol or polyol and recovering the alcohol / polyol fraction.

13. The process according to claim 12 further comprising the step of ion-exchange chromatography of the butanol fraction in the presence of an eluent with a 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.

14. A method for quantifying bacilysin in a sample, comprising carrying out the method according to claim 12 or 13 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 performing liquid chromatography followed by mass spectrometry (LC-MS) to quantify the area under the peak curve of bacilysin.

15. The method according to claim 14 being for the absolute quantification of bacilysin.

16. Use of the composition according to any one of the preceding claims 1 to 11 for the biocontrol of Alternaria sp., Botrytis sp., Cercospora sp., Fusarium sp., and Phoma sp., and Zymoseptoria sp.

17. Use of the composition according to any one of the preceding claims 1 to 11 for the biocontrol of Alternaria brasicola, Bremia lactucae, Ophiostoma piliferum, Plasmopara viticola, Rhizoctonia solani, Rhizoctonia zeae, and Sclerotinia sclerotium.

18. Use of a plipastatin selected from SEQ ID NO: 1 or SEQ ID NO: 2 for the biocontrol of an infection selected from Alternaria sp., Bremia sp., Botrytis, Cercospora sp., Fusarium sp., Ophiostoma sp., Phoma sp., Plasmopara sp., Rhizoctonia sp., Sclerotinia sp., and Zymoseptoria sp., preferably Alternaria sp., Bremia sp., Ophiostoma sp., Plasmopara sp., Rhizoctonia sp., and Sclerotinia sp.

19. An aqueous liquid agronomic composition comprising 1 to 3000 mg / l of SEQ ID NO:1 or SEQ ID NO:2, preferably 10 to 2000 mg / l of SEQ ID NO:1 or SEQ ID NO:

2. 20.The composition according to claim 19, comprising from 1 to 1000 mg / l of SEQ ID NO:1 or SEQ ID NO:2, wherein the fatty acid chain grafted to the peptide is at C15 and / or C17.

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