Biostimulant microorganism

Abstract

The invention relates to a biostimulant composition comprising a Bacillus amyloliquefaciens strain having incorporated a plasmid having at least 95% sequence identity to SEQ ID NO: 1, to its use in agriculture, to a method for enhancing the growth of a plant of interest and to a method for increasing the production of lipid cyclopeptides such as surfactin, iturin, and / or fengycin by a microorganism.

Description

[0001] BIOSTIMULATING MICROORGANISM

[0002] Technical field

[0003] The present invention relates to the use of a strain of Bacillus amyloliquefaciens for biostimulation purposes, as well as methods of biostimulation.

[0004] Prior art

[0005] Biostimulation of the growth of a plant of interest is known. In particular, various strains of the Bacillus subtilis group are marketed for this purpose.

[0006] The strains of the Bacillus amyloliquefaciens group FZB42, IT45 or QST 713 (Serenade®) are known, the first two mainly for the control of pathogenic organisms. Similarly, the experimental strain GAI has been tested for its effectiveness in biocontrol of pathogenic organisms, but without it being commercialized (Arguelles-Arias et al., 2009).

[0007] In addition, the product RhiZoBas is a strain of Bacillus amyloliquefaciens marketed for biostimulation purposes.

[0008] On the other hand, patent application WO 2018 / 122541 describes the interest of a lipopeptide isolate for biostimulation, in particular to free itself from the metabolism of the microorganism, which risks varying according to the culture conditions, which are difficult to control.

[0009] Thus, the use of more controlled extracts of a particular strain in biostimulation reflects the difficulties encountered: depending on the agronomic conditions, a strain may have a biostimulant character, or not, or even have deleterious effects on the plant of interest. Certainly, some of the conditions are controllable or measurable before a possible application of the microorganism, such as the physicochemical parameters of the field: type of soil, abundance of nitrogen or other nutrients, presence or absence of pathogens in the soil.

[0010] However, other parameters are more random, such as weather conditions.

[0011] This will influence both the metabolism of the "biostimulant" microorganism and that of the plant of interest, so that there are many parameters which risk affecting the biostimulant properties of a microorganism, or reducing their scope.

[0012] A microorganism whose biostimulant effect is more robust and less dependent on agronomic conditions therefore represents a major interest.

[0013] Brief summary of the invention

[0014] A first aspect of the present invention relates to a biostimulant composition comprising a strain of Bacillus amyloliquefaciens having incorporated a plasmid having at least 95% identity with SEQ ID NO:1.

[0015] Preferably, this strain of Bacillus amyloliquefaciens has at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:5.

[0016] Advantageously, this strain of Bacillus amyloliquefaciens has incorporated a single copy of the plasmid having at least 95% identity with SEQ ID NO:1.

[0017] Preferably this strain of Baciiius amyloliquefaciens having incorporated the plasmid having at least 95% identity with SEQ ID NO:1, this plasmid further encoding a peptide having 100% identity with SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4.

[0018] Preferably, this biostimulant composition is a vegetable oil comprising the strain of Bacillus amyloliquefaciens or an oil-in-water emulsion comprising the strain of Bacillus amyloliquefaciens.

[0019] A related aspect of the present invention relates to a seed of a plant of interest coated with this biostimulant composition.

[0020] Alternatively, preferably, this biostimulant 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 and / or magnesium carbonates: weight of granules free from Bacillus amyloliquefaciens), preferably said granules having a diameter of between 0.5 mm and 2 mm.

[0021] Alternatively, preferably, this biostimulant composition is in the form of a rehydratable dry powder, comprising a bulking agent and a surfactant (which may be a mixture of surfactants).

[0022] Another related aspect of the present invention relates to a method for increasing the growth of plants comprising the step of administering to said plants this biostimulant composition.

[0023] Preferably, in this method, the biostimulant composition is applied in or onto the soil before said plant is planted or the seed of said plant is sown or, advantageously, substantially at the same time as the seed of said plant is sown. Preferably, alternatively or additionally, in this method, the biostimulant composition is applied to the soil after said plant has been planted or the seed of said plant has been sown.

[0024] Preferably, in this process, the plant is chosen from Poaceae, Legumes, Cucurbitaceae, Asteraceae, Solanaceae, Rapeseed, Beetroot, Carrots, Strawberries, Bananas, Kiwi, Geranium, Pelargonium, Chrysanthemum, Apple, Pear, Apricot, Peach and Vine, preferably Tomato, Corn, Wheat, Chicory or Cucumber.

[0025] Advantageously, in this method, the increased growth is that of the roots, the stem and / or the weight of the plant, preferably this increased growth being measured at least 40, 45, 50, 55, 60, 65 days after the administration of the Bacillus strain.

[0026] Another aspect of the present invention relates to a method for increasing the production, by a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, of lipid cyclopeptides, preferably selected from surfactin, iturin, fengycin and mixtures thereof, comprising the step of transfecting said strain with a plasmid having at least 95% identity with SEQ ID NO: 1.

[0027] Preferably, in this method, the strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, comprises at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:5.

[0028] Another aspect of the present invention relates to a use of a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, having incorporated a plosmid having at least 95% identity with SEQ ID NO: 1 for increasing plant growth.

[0029] Preferably, in this use, the plasmid encodes SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4.

[0030] Preferably, in this use, the strain has at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:5.

[0031] Brief description of the drawings

[0032] Figures 1-4 are a comparison between the AG002 / 2 strain and the SERENADE product (plant height: corn, wheat, tomato, cucumber).

[0033] Figures 5-8 are a comparison between strain AG002 / 2 and the RhiZoBas product (plant height: corn, wheat, tomato, cucumber).

[0034] Figure 9 shows the effect of AG002 / 2 on the root system of tomatoes.

[0035] Detailed description of an embodiment of the invention

[0036] The inventors noticed that their strain AG002 / 2 possesses superior biostimulant properties. This encouraged them to further characterize this strain of Bacillus amyloliquefaciens: genetics, metabolism and interaction with the plant of interest.

[0037] Thus, a first aspect of the present invention relates to a biostimulant composition comprising a live strain of Bacillus amyloliquefaciens having incorporated a plasmid having at least 95% identity with SEQ ID NO: 1. Advantageously, it is the strain of Bacillus having at least 90% identity with SEQ ID NO: 5, preferably at least 91, 92, 93, 94, 95, 96, 97, 98 or even at least 99% identity with SEQ ID NO: 5.

[0038] In the context of the present invention, the measurement of this sequence identity is preferably carried out by comparing a sequence to be tested with respect to complete SEQ ID NO: 1 and / or SEQ ID NO: 5.

[0039] Thus, sequence comparison or global alignment algorithms between two sequences can advantageously be used. Preferably, in this comparison, a "gap" will be counted as an absence of identity for the nucleotide in question. Similarly, with regard to the comparison with SEQ ID NO: 5, in the context of the present invention, preferably, a reorganization of operons at the level of the bacterial chromosome will be counted as an absence of identity, even if the genes in question are highly similar. This implies that a value of 90%, at the level of a comparison between two nucleotide sequences, is very high.

[0040] In the context of the present invention, preferably, the nomenclature of nucleotides is that established by IUPAC. Thus, "R" means "A" or "G"; "Y" means "C" or "T"; "S" means "G" or "G"; "W" means "A" or "T"; "K" means "G" or "T"; "M" means "A" or "C",... Thus, preferably, in the context of the present invention, the identity at a given position between a nucleotide of a target sequence, with respect to a character providing several possibilities for the corresponding position (e.g. of SEQ ID NO:5), will be considered as verified if one of the nucleotides covered by the character in question (of SEQ ID NO:5) corresponds to that of the target sequence (e.g. "Y" at the position in question of SEQ ID NO:5 and "T" at the corresponding position of a target sequence). The inventors noticed significant homologies with the genome of other bacteria of the Bacillus sp. group.(i.e. grouping Bacillus amyloliquefaciens, or Bacillus velezensis) However, these other bacteria provide a lesser, or erratic (varying according to conditions) biostimulant effect. The inventors therefore attribute this to the synergy between the genome of the strain in question, including very closely related strains, with the plasmid in question.

[0041] Preferably, this Bacillus strain has incorporated a single copy of the plasmid having at least 95% identity with SEQ ID NO:1. This is advantageously a stable incorporation of the plasmid, but not its integration into the bacterial chromosome.

[0042] Furthermore, or alternatively, advantageously, this plasmid having at least 95% identity with SEQ ID NO: 1 allows the expression of a peptide having at least 95%, at least 96%, 97, 98, 99, or even 100% identity with SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4.

[0043] Preferably, or alternatively, a protein that is not completely identical to these SEQ ID NO: 2, SEQ ID NO: 3 and / or SEQ ID NO: 4 remains advantageous if the protein in question retains the activity thereof. For example, so-called "conservative" substitutions are tolerated, such as aspartic acid with glutamic acid, asparagine with glutamine, phenylalanine with tyrosine, serine with threonine or cysteine, isoleucine with leucine, arginine with lysine, ... An advantageous way to determine if a substitution is conservative is to perform the comparison between the sequences on the basis of an alignment using the BLASTp algorithm with default parameters such as a BLOSUM62 matrix, a "word size" of 5 and a "Gap" penalty of 10, for a Gap extension penalty of 1.Residues marked with a “+” symbol, following such alignment, are preferably considered to correspond to a conservative substitution.

[0044] According to an advantageous alternative, the biostimulant composition is a rehydratable dry powder comprising the microorganism according to the invention (in the form of spores), a bulking agent or a mixture of bulking agents, and one or more surfactant(s).

[0045] The surfactant(s) allow easy dissolution in water.

[0046] Preferably, at least one "wetting" surfactant and / or at least one "dispersing" surfactant, preferably an anionic surfactant, is present.

[0047] In the context of the present invention, the term “wetting” surfactant preferably means a molecule having a value according to the HLB scale of between 7 and 9.

[0048] Typically, the surfactant(s) are incorporated in a weight content of between 1 and 10%, preferably between 2 and 9%, preferably between 5 and 8% by weight (weight of surfactants: total weight of the rehydratable dry powder).

[0049] Advantageously, the rehydratable dry powder has a water content (by weight) of less than 10%, preferably less than 5%, preferably less than 2%.

[0050] A preferred bulking agent is a silicate, an aluminosilicate and / or a polysaccharide, for example talc, kaolin or a maltodextrin.

[0051] An advantageous maltodextrin has a dextrose equivalent content (DE) of between 15 and 25. Advantageously, the microorganism according to the invention is present in this rehydratable dry powder in a content of at least 10 8 CFU / g, preferably at least 10 9 CFU / g, preferably at least 5*10 9 CFU / g, preferably at least 1010 CFU / g.

[0052] Typically, 100 grams of this rehydratable dry powder are applied to an area of ​​1 hectare.

[0053] In other words, this implies an amount of the microorganism according to the invention of at least 10 10 CFU / Ha, preferably less than 10 1 1 CFU / Ha, preferably at least 5*10 11 CFU / Ha, preferably at least 10 12 CFU / Ha.

[0054] According to another advantageous alternative, in the biostimulant composition according to the invention, but also in the biostimulation methods (which will be described later), the spores of the microorganism of the invention are formulated in an oily dispersion (vegetable oil), preferably in anhydrous conditions (less than 10% by weight of water, preferably less than 2% by weight of water).

[0055] This allows the maintenance of the microorganism according to the invention in sporulated form, which increases the shelf life, preferably at least 6 months.

[0056] This also allows for a homogeneous formulation for the product to be applied, without having to vary the concentration: depending on the needs, a more or less strong dilution of the oily composition comprising the microorganism according to the invention will be carried out before application. In addition, this allows for the application of realistic volumes, compatible with agricultural or horticultural instruments, per unit of surface.

[0057] Preferably, in this oily formulation, the microorganism according to the invention is present in a content of at least 10 8 CFU / ml, preferably at least 2*10 8 CFU / ml, preferably at least 5*10 8 CFU / ml, preferably at least 10 9 CFU / ml.

[0058] The inventors noticed that applying 100 ml of this suspension (after dilution) to an area of ​​one hectare provided a biostimulant effect.

[0059] In other words, this implies a quantity of at least 10 10 CFU / Ha, preferably at least 2* 10 10 CFU / Ha, preferably at least 5*10 10 CFU / Ha, preferably at least 10 1 1 CFU / Ha.

[0060] Thus, an advantageous form of the biostimulant composition is a suspension of the microorganism in oil in the presence of surfactants, so as to allow dilution in water in the form of an emulsion (oil in water), and a volume of this emulsion, which is the multiple of the dilution achieved, will be applied to the field (dilution factor *100 ml / Ha).

[0061] According to a variation of this alternative, the oily formulation (suspension) is applied directly for coating seeds or tubers, either as such or as an emulsion in water.

[0062] The inventors noticed that contents of the microorganism according to the invention in the coating of at least 10 9 CFU / Ha work well, preferably levels between 10 9 CFU / Ha and 10 12 CFU / Ha, preferably between 10 10 CFU / Ha and 10 11 CFU / Ha.

[0063] Advantageously, this content is defined per unit of surface area. Thus, in this context, the quantity of microorganism per seed of the plant to be sown is adapted, according to the density of seeds per hectare.

[0064] Indeed, the inventors have noticed that, depending on the plants (e.g. chicory), excessively high levels of the microorganism according to the invention may slow down germination. The biostimulant composition is thus advantageously absorbed into the seed of the plant of interest, or applied to the tuber of the plant of interest (potato, Jerusalem artichoke), which makes it possible both to ensure proximity between the biostimulant microorganism according to the invention and the plant of interest, but also not to duplicate the agricultural work (i) of applying the biostimulant composition and (ii) of sowing. Indeed, coating the seeds of a plant of interest is a widespread practice. The addition of the microorganism according to the invention will therefore be advantageously done during this step, while ensuring that this technique is carried out under conditions compatible with the survival of the microorganism.A preferred coating comprises the application of an oily formulation comprising the microorganism according to the invention (see above). Preferably, the coating increases the resistance of the spores, even under extreme conditions (acidity, alkalinity, prolonged water stress, toxicity at soil level), while allowing the transition to vegetative form as soon as the conditions are favorable for germination.

[0065] Thus, advantageously, the microorganism according to the invention is incorporated into the coating of Poaceae (grass) seeds, for example corn, durum wheat, soft wheat, barley, rye, triticale, oats; in the coating of legume seeds, for example soybeans, beans, peas, broad beans, peanuts, lentils; in the coating of Cucurbitaceae seeds, for example squash, melon, watermelon, pumpkin, squash; in the coating of Asteraceae seeds, for example lettuce, chicory, sunflower, artichoke, cardoon; in the coating of Solanaceae seeds, for example tomato, eggplant, chili pepper; in the coating of cruciferous seeds, for example rapeseed or cabbage; or in the coating of beet, carrot and strawberry seeds.Furthermore, in a similar manner, tubers, for example potato or Jerusalem artichoke, are advantageously combined with the microorganism according to the invention when they are placed in the ground, for example by direct sprinkling in the furrows.

[0066] According to the other alternative, the microorganism according to the invention is advantageously formulated into granules, in particular microgranules, where spores of the microorganism according to the invention are mixed and / or incorporated into particles comprising at least 40% by weight of calcium and / or magnesium carbonate (e.g. calcite and / or dolomite), preferably at least 60% by weight of calcium and / or magnesium carbonate. An advantageous content by weight of calcium and / or magnesium carbonate is thus between 40 and 80% by weight. In the context of the present invention, the content by weight of calcium and / or magnesium carbonate in the granules preferably means the ratio between the weight of calcium and / or magnesium carbonate in the granules divided by the total weight of the constituents of the granules, excluding the microorganism according to the invention.

[0067] The granules (microgranules) according to the invention advantageously have a diameter of between 0.5 and 2 mm, preferably between 1 and 1.5 mm.

[0068] This advantageously allows the application of microorganisms directly in the sowing furrow: the granules according to the invention can be applied at the same time as the seeds of the plant of interest.

[0069] Preferably these granules allow the application of a quantity of at least 10 10 CFU / Ha, preferably at least 10 11 CFU / Ha, preferably at least 5*10 11 CFU / Ha, preferably at least 10 12 CFU / Ha of microorganisms according to the invention. Thus, these granules have a content of microorganisms according to the invention, advantageously, of approximately 10 8 CFU / gram of granule.

[0070] Thus, as will be described later, the application of at least 1, at least 2, at least 5 kg of these granules per Ha allows a sufficient content of microorganisms to obtain the biostimulant effect, for example a quantity of between 1 and 20 kg, preferably between 2 and 10 kg is advantageously applied to one Ha of culture.

[0071] Furthermore, preferably, the products incorporated via the granules are essentially natural (calcium and / or magnesium carbonate as described above and at a content of at least 40% by weight, see potassium hydrogen carbonate, aluminosilicates, natural polymers such as polysaccharides (e.g. maltodextrins, starch derivatives, chitin derivatives, arabinoxylans, glucosaminoglycans, beta glucans), lignin derivatives, polyols (e.g. mannitol), disaccharides, trisaccharides, natural biostimulants, trace elements, amino acids), which is fully compatible with soil biology.

[0072] In addition, these bacteria remain in spore form until the water content in the environment exceeds a certain threshold, which will correspond with the growth of the roots of the plant of interest and therefore ensure good synergy, given the temporal concordance.

[0073] Advantageously, thanks to this formulation, the viability of the spores is long, preferably at least 9 months.

[0074] Finally, the spores, which are part of the granules, will be activated progressively, due to the resorption of these granules over time.

[0075] All of this allows for robust, long-lasting activation at a time that synergizes with the metabolism of the plant of interest. Another related aspect of the present invention relates to a method for increasing the growth of a plant comprising the step of administering the biostimulant composition described above (having a plasmid having at least 95%, 96, 97, 98, 99% identity with SEQ ID NO: 1, or even 100% identity).

[0076] In this method, advantageously, the strain has at least 90% (preferably at least 91, 92, 93, 94, 95, 96, 97, 98, 99%) identity with SEQ ID NO: 5, or even 100% identity.

[0077] Advantageously, the increased growth is that of the roots, the stem and / or the weight of the plant.

[0078] Preferably, in this method, the increased growth is measured at least 40, 45, 50, 55, 60, 65 days after administration of the biostimulant composition to the plant.

[0079] Advantageously, according to an alternative to this process, the biostimulant composition is applied to the soil during sowing (via coating the seeds or via the addition of granules).

[0080] Advantageously, in addition to the concomitant application, or according to the other alternative of this method, the biostimulant composition is applied to the soil after said plant has been planted or the seed of said plant has been sown.

[0081] Thus, as described above, the microorganism according to the invention is applied in the coating of the seed of the plant, or by sprinkling the tuber and / or is applied during sowing via the granules described above, and / or is then applied to the plant, post-emergence, via spraying of the oily suspension or the emulsion comprising the oily suspension described above, or even via irrigation. The inventors have noted that this makes it possible to reduce the load of microorganism according to the invention which is applied to the coating of the seed (or tuber), while administering a sufficient quantity of microorganisms. Thus, this makes it possible to offer the maximum biostimulant effect.

[0082] Preferably, the plant is chosen from:

[0083] Poaceae (grasses), for example corn, durum wheat, soft wheat, barley, rye, triticale, oats;

[0084] Legumes, for example soybeans, beans, peas, broad beans, peanuts, lentils;

[0085] Cucurbits, for example squash, melon, watermelon, pumpkin, squash, cucumber;

[0086] Asteraceae, for example, lettuce, chicory, sunflower, Jerusalem artichoke, artichoke, cardoon;

[0087] Solanaceae, for example tomato, potato, eggplant, chili pepper;

[0088] Cruciferous vegetables, for example rapeseed or cabbages;

[0089] Beetroot, carrot, strawberry, banana, kiwi;

[0090] Horticultural plants (geranium, pelargonium, chrysanthemum), and

[0091] Arboriculture plants (apple, pear, apricot, cherry, peach, vine). Turf (grasses, clover; fodder or horticultural production) also benefits from the composition according to the invention, preferably via spraying. Thus, in this method, the application of the liquid formulation, therefore of the oily suspension or emulsion described above, can advantageously be done via spraying or via irrigation. As described above, the formulation of the microorganism in oil is advantageous, and this formulation can then be diluted in water (including to form an emulsion therein).

[0092] In this method, preferably, the strain suspended in oil (see above) or diluted in oil-in-water emulsion (see above) is applied to the plant (seed coating and / or spraying) at a concentration of at least 10 9 CFU / milliliter, preferably at least 10 10CFU / milliliter, at least 10 1 1 CFU / milliliter, for example at a concentration of about 10 12 CFU / military.

[0093] Advantageously, alternatively, or in addition, an amount of 0.1 to 20 kg / Ha of the biostimulant composition in the form of granules (see above) is applied per hectare, preferably 0.5 to 10 kg / Ha, preferably 1 to 5 kg / Ha.

[0094] Another related aspect of the present invention relates to a use of a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, having incorporated a plasmid having at least 95% identity with SEQ ID NO: 1 for increasing plant growth.

[0095] Another related aspect of the present invention relates to a use of a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, having incorporated a plasmid encoding SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4 for increasing plant growth.

[0096] Another related aspect of the present invention relates to a method for increasing the production, by a strain selected from Bacillus subfilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, of lipid cyclopeptides, preferably selected from surfactin, iturin, fengycin and mixtures thereof, comprising the step of transfecting this strain with a plasmid having at least 95% identity with SEQ ID NO: 1.

[0097] Other features and advantages of the present invention will be drawn from the following non-limiting description, and with reference to the drawings and examples.

[0098] Examples. -

[0099] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications may be made thereto without departing from the scope of the appended claims.

[0100] Example 1:

[0101] The inventors identified a strain of Bacillus amyloliquefaciens which exhibits interesting properties in terms of biostimulation and named it AG002 / 2.

[0102] The inventors performed a genetic analysis. This strain contains the plasmid of SEQ ID NO: 1, and this plasmid is stable. The inventors consider this to be a rare event, as plasmids can instead either be incorporated into the genome or be lost, which is more common when there are only a low number of copies per cell.

[0103] Furthermore, the inventors demonstrated that there was only one plasmid per cell. Example 2:

[0104] The inventors formulated the strain according to the invention at 10 12 CFU / kg. Then, between 0.25 and 4 kg of this formulation were applied per hectare, and compared to the effect of 1 L / Ha of the Serenade® product (also 10 12 CFU / I), which is another specific strain of Bacillus amyloliquefaciens.

[0105] The plants tested were corn (variety LG30258), wheat (variety Quintus), cucumber (variety Sonja) and tomato (variety Prestige Fl).

[0106] Various plant growth parameters were measured, such as height, vigor or chlorophyll content.

[0107] The results are shown in Figures 1-4 compared to the SERENADE product, and 5-8 compared to the RhiZoBas product (dose, 1 kg / Ha; plant height in cm).

[0108] The composition according to the invention caused an increase in the size of the plants tested (corn, Figures 1 and 5; wheat, Figures 2 and 6, tomato, Figures 3 and 7; cucumber Figures 4, 8), from 10 to 50% for the 4% dose (less for the lower doses). This increase is more marked for the AG002 / 2 strain than for the Serenade® product.

[0109] Furthermore, plant vigor was measured and found to be significantly increased by strain AG002 / 2, with only a marginal increase observed in the control where the biostimulant product is Serenade®.

[0110] The same formulations were tested in synergy with a mineral fertilizer composition comprising nitrogen, phosphorus, and potassium. The control is the Rhizobas formulation (at 1 kg / Ha). The beneficial effect on growth is found for the tested plants, but the specific effect of strain AG002 / 2 is reduced. There is also a beneficial effect of strain AG002 / 2 on wheat vigor. However, the dose-response effect is less evident.

[0111] Example 3:

[0112] A real-life test was carried out on chicory by measuring emergence (%) and growth (expressed as a % of desired growth). Again, the AG002 / 2-based composition allowed an increase in emergence (for example from 51.5 to 56% at 22 days, then from 54 to 59% at 29 days), as well as growth (from 42 to 46% at 22 days and from 39 to 47% at 29 days).

[0113] Example 4:

[0114] Field tests were carried out on soybean, winter wheat, potato, corn, cucumber, chrysanthemum and tomato crops. Compared to controls, the inventors tested conditions supplemented with a powder comprising AG002 / 2 (5*10 9 CFU / g), which will be suspended in water (0.1% by weight) before application at a rate of 100 ml / Ha.

[0115] In the case of soybeans, the inventors observed a very significant increase in the number of nodules, up to 3 times more. A significant increase in yields was observed (approximately 1.17% under normal conditions and 1.25% under reduced input conditions, compared to the control, 100%).

[0116] Yields have also increased significantly in the case of wheat.

[0117] Similarly, potato crops benefited from the composition comprising AG002 / 2 according to the invention with an increase at a level of 117% compared to the untreated control (100%), under “low” fertilization conditions without phosphate, for a yield greater than 54 T / Ha. The effect on corn crops is low in the presence of complete fertilization (4% increase), but becomes significant when fertilization is low (16% increase). On the other hand, the inventors noted that the vigor of the plants is improved, even under complete fertilization conditions.

[0118] For chrysanthemums, the treatment resulted in an increase in yield of 4 to 5.2 kg / plot, but also an increase in the number of flowers / plant and stem height. In this case, the application was carried out during transplantation and at 3-week intervals.

[0119] For cucumber, according to the tests, yields increased from 24 to 26 T / Ha (27 T / Ha for the condition with the highest fertilization) without the composition including AG002 / 2 (depending on the other fertilizers applied) to 26, 28 and even 30 T / Ha. This is, for each condition, an increase of approximately 10%.

[0120] Regarding tomatoes, for round tomatoes, the inventors noted an increase in yields to 1.12% (control=100%; control yield of 40 T / Ha) and, in another test, a similar increase for cherry tomatoes (control yield 22.5 T / Ha, test yield = 25.4 T / Ha). Given the quantities, and the fact that the costs for the farmer are fixed, this translates into a strongly stimulated profit and even allows a reduction in inputs such as phosphorus, a second environmental benefit.

[0121] The inventors analyzed the root system of tomatoes: Figure 9 shows that the root system developed better in the presence of AG002 / 2 (the left part of Figure 9, compared to the right part). More generally, out of the total number of field tests (18), 56% are associated with a visible effect, and 90% with a positive effect taking into account all the parameters. None of the tests were associated with a negative effect, which is remarkable.

Claims

CLAIMS 1. A biostimulant composition comprising a strain of Bacillus amyloliquefaciens having incorporated a plasmid having at least 95% identity with SEQ ID NO:

1.

2. The biostimulant composition according to claim 1 wherein the Bacillus strain has at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:

5.

3. The biostimulant composition according to claim 1 or 2 wherein the Bacillus strain has incorporated a single copy of the plasmid having at least 95% identity with SEQ ID NO:

1.

4. The biostimulant composition according to any one of the preceding claims, wherein the Bacillus strain has incorporated the plasmid having at least 95% identity with SEQ ID NO:1 and said plasmid further encoding a peptide having 100% identity with SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:

4.

5. The biostimulant composition according to any one of the preceding claims, being a vegetable oil comprising the strain of Bacillus amyloliquefaciens or an oil-in-water emulsion comprising the strain of Bacillus amyloliquefaciens.

6. The biostimulant composition according to any one of the preceding claims 1 to 4 being in the form of granules, said granules comprising at least 40% by weight of calcium carbonate and / or magnesium carbonate (weight of calcium and / or magnesium carbonates: weight of granules free from Bacillus amyloliquefaciens), preferably said granules having a diameter of between 0.5 mm and 2 mm.

7. The biostimulant composition according to any one of the preceding claims 1 to 4 being in the form of a powder rehydratable dryer, further comprising a bulking agent and a surfactant.

8. The biostimulant composition according to claim 7 in which the bulking agent is chosen from a silicate, preferably talc or kaolin, and / or a polysaccharide, preferably a maltodextrin.

9. A seed of a plant of interest coated with the biostimulant composition according to any one of the preceding claims 1 to 5.

10. A method for increasing the growth of plants comprising the step of administering to said plants the biostimulant composition according to any one of the preceding claims 1 to 8, or the coated seed according to claim 9. 1 1. The method of claim 10 wherein the biostimulant composition is applied in or onto the soil before said plant is planted or the seed of said plant is sown or, advantageously, substantially at the same time as the seed of said plant is sown.

12. The method of claim 10 or 11 wherein the biostimulant composition is applied to the soil after said plant has been planted or the seed of said plant has been sown.

13. The method according to any one of claims 10 to 12 in which the plant is chosen from Poaceae, Legumes, Cucurbitaceae, Solanaceae, Asteraceae, Cruciferae, Beetroot, Carrots, Tomatoes, Strawberries, Bananas, Kiwis, Geraniums, Pelargoniums, Chrysanthemums, Apple trees, Pear trees, Apricot trees, Peach trees and Vines, preferably Tomatoes, Corn, Wheat, Chicory or Cucumbers.

14. The method according to any one of claims 10 to 13 wherein the increased growth is that of the roots, the stem and / or the weight of the plant.

15. The method according to any one of claims 10 to 14 wherein the increased growth is measured at least 40, 45, 50, 55, 60, 65 days after administration of the Bacillus strain.

16. A method for increasing the production, by a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, of lipid cyclopeptides, preferably selected from surfactin, iturin, fengycin and mixtures thereof, comprising the step of transfecting said strain with a plasmid having at least 95% identity with SEQ ID NO:

1.

17. The method of claim 16 wherein the strain comprises at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:

5.

18. Use of a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, having incorporated a plasmid having at least 95% identity with SEQ ID NO: 1 to increase the growth of plants.

19. Use of a strain selected from Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus licheniformis, having incorporated a plasmid encoding SEQ ID NO:2, SEQ ID NO:3 and / or SEQ ID NO:4 to increase plant growth.

20. Use of a strain according to claim 18 or 19 wherein the strain has at least 90% identity with SEQ ID NO:5, said identity being calculated over the entire length of said SEQ ID NO:

5.

21. Use according to any one of the preceding claims. 18 to 20, in which the plant is chosen from Poaceae, Legumes, Cucurbitaceae, Solanaceae, Asteraceae, cruciferous vegetables, beetroot, carrot, tomato, strawberry, banana, kiwi, geranium, pelargonium, chrysanthemum, apple tree, pear tree, apricot tree, peach tree and vine, preferably tomato, corn, wheat, chicory or cucumber.

Images