Use of a strain of the bacillus siamensis species as a biosolution in tomato cultivation

Abstract

The present invention relates to the use of a particular bacterial species as a biosolution in the production and cultivation of tomatoes. More particularly, the invention relates to the use of a Bacillus siamensis strain, deposited with the National Collection of Cultures of Microorganisms (CNCM) under the accession number CNCM I-5921, in the stimulation of the development and growth of seeds and of tomato plants or the improvement of the yields of tomato crops, and in the improvement of the resistance and tolerance of seeds, tomato plants and tomato crops to abiotic stress.

Description

[0001] USE OF A STRAIN OF THE SPECIES BACILLUS SIAMENSIS AS A

[0002] BIOSOLUTION IN TOMATO CULTIVATION

[0003] The present invention relates to the use of a particular bacterial species as a biosolution in the production and cultivation of tomatoes. More specifically, the invention relates to the use of a strain of Bacillus siamensis, deposited with the National Collection of Microorganism Cultures (CNCM) under the order number CNCM 1-5921, in stimulating the development and growth of a tomato seed and plant or improving the yields of tomato crops, and in improving the resistance and tolerance to abiotic stress of a tomato seed, plant and tomato crops.

[0004] The tomato, or Solanum lycopersicum, is a member of the nightshade family, native to Mexico. Its global production is currently the largest among fruiting vegetables, with a market estimated at €192 billion in 2023. Two distinct production sectors exist: market tomatoes for fresh consumption and processing tomatoes for manufacturing. This rapidly expanding crop has spread to every continent, thanks in particular to soilless and greenhouse cultivation under highly controlled and automated conditions.

[0005] Sowing remains the most common propagation technique, but it is still a crucial step for cultivation and a particularly sensitive stage for the proper development of seedlings. Indeed, their quality and their ability to resist abiotic stresses are partly determined by the first few weeks of growth after sowing.

[0006] In response to consumer demand, farmers are now seeking effective and environmentally friendly biosolutions that can help them navigate the ongoing agroecological transition more smoothly. In this context, it has become essential for our societies to leverage the expertise of microorganisms naturally present in the environment to stimulate crop growth and increase yields, while preserving the health of harvests.

[0007] The use of biostimulants in agricultural production methods is rapidly expanding. Biostimulants are substances capable of stimulating a plant's metabolism and its natural nutrient uptake processes. More specifically, Regulation (EU) 2019 / 1009 of the European Parliament and of the Council of 5 June 2019, which entered into force on 22 July 2022, defines a biostimulant as "a product which stimulates plant nutrition processes independently of the nutrients it contains, with the aim of improving one or more of the following characteristics of plants or their rhizosphere: (a) nutrient use efficiency, (b) tolerance to abiotic stress, (c) qualitative characteristics, (d) the availability of nutrients confined in the soil or rhizosphere."

[0008] The aim of the present invention is thus to identify and propose an effective biostimulant suitable for tomato production and cultivation methods. In particular, a biostimulant according to the invention should stimulate the development and growth of tomato seeds and plants, or improve tomato crop yields, or at least prevent limiting development and yield loss, and improve the resistance and tolerance to abiotic stress of seeds, tomato plants, and tomato crops, or at least prevent lowering this resistance and tolerance to abiotic stress. Finally, the biostimulant according to the invention should be usable in conventional methods of substrate production and tomato production.It is for this purpose that the applicant company conducted its research and discovered that a species of Bacillus exhibited surprising biostimulation capabilities on the development and growth of tomato seeds and plants or on the improvement of tomato crop yields, as well as in improving the resistance and tolerance to abiotic stress of a seed, a tomato plant and tomato crops, particularly when added to their substrates or in seed soaking solution, and could therefore be used in tomato cultivation processes and methods.

[0009] For this purpose, the invention relates to an isolated strain of the species Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921.

[0010] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of the bacterial strain isolated from Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921, for the stimulation of the development and growth of a tomato seed and plant or in the improvement of tomato crop yields.

[0011] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of the bacterial strain isolated from Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921, for the improvement of the resistance and tolerance to abiotic stress of a seed, a tomato plant and tomato crops.

[0012] The bacterial strain isolated in France by the filing company was identified as Bacillus siamensis according to the NCBI (National Center for Biotechnology Information) database, after genotypic identification by Sanger sequencing.

[0013] This Gram-positive bacterium is aerobic and facultatively anaerobic, and capable of producing ellipsoidal endospores. It is highly motile with flagella surrounding the cell and peritrichous flagella. This species is distinguished by its ability to divide over a wide temperature range, from 4°C to 55°C, with an optimal growth temperature of 37°C. It forms creamy-white, mucous, semi-opaque, semi-domed colonies that grow 3 to 4 mm around the point of inoculation after 2 to 3 days of incubation at 25°C on PDA. The genus Bacillus is valued in biotechnology and widely used in industrial-scale microbial fermentation because its robustness and rapid growth in bioreactors allow it to withstand aggressive processes such as spray drying, which is much less expensive than freeze-drying for obtaining a dehydrated cell powder that is particularly stable over time.

[0014] The depositing company deposited the bacterial strain at the CNCM (National Collection of Microorganism Cultures) of the Pasteur Institute, which validated it and assigned it the order number CNCM 1-5921.

[0015] In tomato cultivation, the CNCM 1-5921 strain has been shown to significantly increase seed germination rates, seedling height, and average fresh and dry seedling biomass. After a single application at 2.5 x 10 7 cells per liter of substrate at sowing, the average dry biomass of the seedlings is 102% higher than that of the control treatment. By simply soaking the seeds at 1.10 8 cells per millilitre of solution, the average dry biomass of seedlings is increased by 48% compared to those of the control treatment.

[0016] Under abiotic stress conditions, the strain according to the invention improves the resistance and adaptation capacity of tomato crops when subjected to water stress. After a single application at 2.5 x 10 7 With a higher cell count per liter of substrate at sowing, the average fresh biomass of seedlings is significantly greater, by 34%, than that of the control treatment, and by 20% in dry biomass. The bacterial strain CNCM 1-5921 also leads to a significant 30% increase in the average leaf area of ​​seedlings, as well as an 8% increase in their average leaf circumference. These parameters highlight improved plant adaptation to water stress, by optimizing root tissue development and reducing evapotranspiration to limit water loss.

[0017] The numerous beneficial effects of the CNCM 1-5921 strain on tomato cultivation demonstrate its ability to produce phytohormones such as auxins like IAA (Indole-3-Acetyl Acid), gibberellins, and cytokinins. The presence of these phytohormones explains, in particular, the increase in seedling dry biomass and root system elongation. In parallel, certain bacteria, such as the CNCM 1-5921 strain, can produce ACC deaminase, an enzyme that degrades a precursor in the synthesis of ethylene, a plant hormone that induces oxidation and senescence of vegetative tissues, thereby mitigating the effects of stress and optimizing seedling development.

[0018] In nature, there exist bacteria known as PGPB (Plant-Growth-Promoting Bacteria) or FGPB (Fungal-GPB), and even, exceptionally, MGPB (Mycelium-GPB and / or Mushroom-GPB). The CNCM 1-5921 bacterial strain from the applicant company has been shown to be both PGPB and MGPB, through biological mechanisms, some of which have been elucidated by internal research. This dual capability is all the more interesting because Bacillus siamensis can also improve the yields of an agricultural crop amended with tomato post-harvest substrate, to which it had previously been applied.

[0019] The use of broad-spectrum microbial biostimulants found in the environment is a true innovation for tomato production. The use of biostimulant products has a major impact on tomato production. Indeed, the main limitation for farmers lies in their ability to quickly produce, store, and distribute their fruits and vegetables to their customers. The use of the microbial biostimulant according to the invention allows producers to shorten their production cycles by increasing germination vigor and seedling growth, thus strengthening their crops from the first weeks after sowing. Under abiotic stress conditions, the strain according to the invention prevents seedling weakness by promoting better development of vegetative tissues and elongation of the root system.These adaptations allow farmers to save water and ensure lower loss rates. With periods of drought and water restrictions becoming increasingly frequent worldwide, the ability for producers to preserve their crops under critical conditions, while maintaining sufficient yields, will quickly become vital.

[0020] The Bacillus siamensis species selected for the purposes of this invention has the particularity of exhibiting significantly positive effects on several parameters of tomato plant metabolism. The strain according to the invention is effective over a wide range of concentrations, whether applied by soaking seeds, watering, or spraying into the substrate or onto the surface of a tomato crop.

[0021] Thus, the present invention relates to the use of a bacterial strain of Bacillus siamensis, or of the CNCM 1-5921 strain, for the stimulation of the development and growth of a tomato seed and plant or the improvement of tomato crop yields, and in the improvement of the resistance and tolerance to abiotic stress of a seed, a tomato plant and tomato crops.

[0022] Furthermore, during tomato cultivation, the application of the CNCM 1-5921 strain according to the invention increases the seed germination rate and the average dry biomass of the seedlings. Under water stress conditions, the strain according to the invention also increases the average leaf area of ​​the seedlings, as well as their leaf circumference. The effects on the tomato sector are therefore accompanied by economic and practical benefits for producers at each stage:

[0023] Energy savings linked to the possibility of shortening production cycles, reducing the time required to obtain young tomato plants while improving their quality,

[0024] Savings in raw materials through better utilization of substrates linked to the increase in aboveground and root biomass of seedlings on the same unit of surface area,

[0025] Savings on tomato seeds, which are particularly costly, through the increased germination vigor induced by the strain according to the invention and the reduction in the loss rate resulting from delayed emergence.

[0026] Water and input savings through improved resistance and enhanced tolerance to water stress in seeds and crops,

[0027] Savings in time, labor, and space through the application of seed soaking prior to sowing.

[0028] Application by watering and spraying into the substrate mass or onto the surface of the crop, easily adaptable to all production methods and combinable with the use of other types of biostimulants such as mycorrhizal fungi.

[0029] Application of a sustainable and environmentally friendly biosolution to the cultivation of the most cultivated fruit-vegetable in the world.

[0030] The bacterial species Bacillus siamensis, formulated within the framework of the invention, can be used simply and effectively in conventional methods of growing tomato seeds or plants, without the need to complicate these methods.

[0031] Advantageously, the bacterial strain is in a solid form such as a powder or granules, or in a liquid form, preferably aqueous.

[0032] A product containing the bacterial strain according to the invention can thus be in liquid form, such as a concentrated suspension (CS), or in solid form, such as a dry powder obtained by fermentation in a bioreactor followed by spray-drying or by the addition of a cryoprotectant before freeze-drying. The dry powder can be in the form of a wettable powder (WP or WDP = Water Dispersible Powder) or soluble granules (WG or WDG = Water Dispersible Granules). The Bacillus siamensis species can optionally be produced in one of the forms described above and then encapsulated.

[0033] As an example, soluble powder containing dehydrated bacterial cells is prepared as follows:

[0034] Release of the bacterial strain from cryopreservation, Subculturing of the strain onto PDA culture media, Growth of the strain in an incubator at 37°C, Placement in a nutrient solution in a 5-liter bioreactor, Centrifugation of the solution after a 48-hour cycle, Collection and washing of the pellet, Spraying of the liquid input in an atomization chamber, Drying under reduced pressure, Obtaining a dry powder, Packaging. The invention further relates to a method for obtaining tomato seed, characterized in that it comprises the following steps: preparation of a soaking solution containing a bacterial strain of Bacillus siamensis, or an isolated Bacillus strain deposited with the CNCM under CNCM order number 1-5921, soaking a seed in the soaking solution, filtration and drying of the seed, obtaining a seed, packaging a seed.

[0035] Advantageously, according to this method, the bacterial strain is applied so that the concentration is between 10 5 at 10 10 cells / mL of soaking solution.

[0036] The invention further relates to a method of growing tomatoes from tomato seed, possibly obtained according to the seed production method described above, which is characterized in that it comprises the following steps:

[0037] (a) preparation of a substrate,

[0038] (b) inoculation of the substrate with tomato seed, possibly obtained using a seed production method described above,

[0039] (c) possibly, incorporation into the substrate of mycorrhizal fungi, amendments or mixtures of nutrients specific to tomato cultivation,

[0040] (d) germination,

[0041] (e) growth,

[0042] (f) transplanting,

[0043] (g) flowering,

[0044] (h) fruiting, (i) harvests,

[0045] (j) end of harvest, the method being characterized in that a bacterial strain of Bacillus siamensis, or an isolated strain of Bacillus deposited with the CNCM under CNCM order number 1-5921, or a composition comprising therein, is applied at least once during at least one of the steps (a), (b), (d), (e), (f) (i) and (j).

[0046] Advantageously, according to this method, the bacterial strain is applied in the mass of the substrate or on the surface of a tomato culture, so that the concentration is between 10 5 cells and 1O 10 cells / L of substrate or between 5.10 6 cells and 5.10 11 cells / m 2 .

[0047] Advantageously, according to this method, the bacterial strain is applied by soaking, watering, or spraying with a liquid solution.

[0048] The invention further relates to the use of a substrate from the end of the cultivation of a tomato obtained by a method described previously as an amendment or fertilizer for an agricultural crop.

[0049] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of an isolated strain of Bacillus deposited with the CNCM under the order number CNCM 1-5921, to prevent the synthesis of ethylene produced by a seed or by a tomato plant.

[0050] The invention further relates to the use of a bacterial strain of Bacillus siamensis, or of an isolated strain of Bacillus deposited with the CNCM under the order number CNCM 1-5921, to produce a phytohormone such as an auxin or a gibberellin or a cytokinin.

[0051] The features of the invention mentioned above, as well as others, will become clearer upon reading the following description of exemplary embodiments.

[0052] Figure 1 illustrates the impact of the bacterial strain CNCM 1-5921 on the growth and development of tomato seedlings, 4 weeks after sowing, applied at a concentration of 2.5 x 10⁷ cells / Liter of substrate. Figure 2 illustrates the impact of the bacterial strain CNCM 1-5921 on the growth and development of tomato seedlings, 4 weeks after sowing, applied at a concentration of 2.5 x 10⁷ cells / Liter of substrate.

[0053] Example 1: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants

[0054] 1.1 Preparation of a solution comprising the strain according to the invention

[0055] The solution is formulated based on the following elements:

[0056] Microorganism: Bacillus siamensis bacterial strain deposited with the CNCM under order number CNCM 1-5921 in the form of a dry powder, obtained by spray drying.

[0057] A dry powder is obtained as follows: multiplication of the bacterial strain by fermentation, drying by spray drying at the end of fermentation, packaging.

[0058] The resulting dry powder is intended to be dissolved in water before application. This will be referred to hereafter as "CNCM 1-5921 solution".

[0059] 1.2 Experimental protocol for application in substrate mixture of sowing i tomatoes

[0060] Prepare 2 liters of substrate, composed of 35% horticultural seed-starting mix and 65% vermiculite. Mix the CNCM 1-5921 solution (as described in section 1.1) into the substrate to obtain a concentration of 2.5 x 10⁻³ 7 cells dissolved in 200 mL of water, per litre of substrate.

[0061] Deposit of 1 litre of pre-mixed substrate per 50-cell seed tray, i.e. 20 mL of substrate per cell.

[0062] One tomato seed is placed in each cell of each tray.

[0063] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0064] Germination rate measurement at 6 and 7 days after sowing.

[0065] Measurement of seedling heights, from the collar to the base of the apex, 3 weeks after sowing.

[0066] Measurement of the leaf system coverage rate with image analysis software, 3 and 4 weeks after sowing.

[0067] Photographs taken of the growing trays.

[0068] Extraction of seedlings from substrate, cleaning of the root system with water and absorption of excess water.

[0069] Seedlings are dried in an oven at 80°C for 24 hours.

[0070] Measurement of the dry biomass of the root system and the aerial part for each seedling.

[0071] Each treatment described above was conducted on an identical 50-hole experimental plate, corresponding to a sample size of 50 seeds per treatment. The tomato variety used for all experimental trials was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd.

[0072] 1.3 Results Table 1 below presents the germination rate, expressed as a percentage of the number of germinated seeds, for each experimental treatment described previously. The germination rate is also expressed as a percentage, relative to the control treatment.

[0073] Table 1]

[0074] Table 2 below shows the average seedling height, expressed in centimeters and measured from the collar to the base of the apex, 3 weeks after sowing, for each experimental treatment described previously. The average height is also expressed as a percentage, relative to the control treatment.

[0075] [Ta b .2]

[0076] Student's t-test (threshold 5%)

[0077] Table 3 below presents the leaf system coverage rate, expressed as a percentage and calculated using image analysis software, for each experimental treatment described previously. The coverage rate is also expressed as a percentage, relative to the control treatment.

[0078] [Table 3] Table 4 below presents the average dry biomass of the root system, aerial parts, and cumulative seedlings, expressed in milligrams, for each experimental treatment described previously. The average dry mass is also expressed as a percentage, relative to the control treatment.

[0079] Table 4]

[0080] Student's t-test (threshold 5%)

[0081] Figures 1 and 2 illustrate the impact of the bacterial strain CNCM 1-5921 on the growth and development of tomato seedlings, 4 weeks after sowing, applied at a concentration of 2.5 x 10 7cells / Litre of substrate.

[0082] According to the results of the experimental trials of Example 1, multiple and lasting beneficial effects can be observed on tomato seedlings when the solution containing the CNCM 1-5921 strain is applied mixed with the substrate before sowing.

[0083] The solution according to the invention makes it possible to improve the germination rate of tomato seeds from the first week, to significantly increase the height of seedlings after 3 weeks (+11%), and to progressively improve their coverage rate after 3 and then 4 weeks (+71%).

[0084] The CNCM 1-5921 solution primarily allows for a significant increase in the average dry biomass (Student's t-test at the 5% significance level) of seedlings when simply pre-mixed with the substrate. After a single application at 2.5 x 10 7With a higher cell count per liter of substrate, the seedlings contain an average dry biomass 102% greater than that of the control group in this experimental trial. In conclusion, the strain according to the invention allows growers to improve the emergence rate and germination vigor of their seedlings, and to strengthen their crops from the first weeks of production, when the seedlings are most fragile. Indeed, seedlings that develop their foliage and root systems better will strengthen more rapidly through photosynthesis and improved absorption of water and nutrients from the substrate. Once transplanted into open ground, these same seedlings will be much more resistant to abiotic stresses and pest attacks, allowing farmers to save water and inputs, in addition to ensuring lower loss rates.

[0085] Example 2: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants

[0086] 2.1 Experimental protocol for application in substrate mixture

[0087] Application of the CNCM 1-5921 strain during the sowing phase of young tomato plants

[0088] Preparation of 4 litres of substrate, composed of 35% horticultural substrate for seedlings and 65% vermiculite.

[0089] Mix the CNCM 1-5921 solution (as described in point 1.1) with the substrate to obtain a concentration of 1.10 6 1.10 7 and 5.10 7 cells dissolved in 200 mL of water, per litre of substrate.

[0090] Deposit of 1 litre of pre-mixed substrate per 50-cell seed tray, i.e. 20 mL of substrate per cell.

[0091] One tomato seed is placed in each cell of each tray.

[0092] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0093] Germination rate measurement at 6 and 7 days after sowing.

[0094] Measurement of the leaf system coverage rate using image analysis software, 3 weeks after sowing. Removal of seedlings from the substrate, cleaning of the root system with water and absorption of excess water.

[0095] Seedlings are dried in an oven at 80°C for 24 hours.

[0096] Measurement of the root system length of each seedling.

[0097] Measurement of the dry biomass of the root system and the aerial part for each seedling.

[0098] Each treatment described above was conducted on an identical 50-hole experimental plate, corresponding to a sample size of 50 seeds per treatment. The tomato variety used for all experimental trials was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd.

[0099] 2.2 Results

[0100] Table 5 below presents the germination rate, expressed as a percentage of the number of germinated seeds, for each experimental treatment described previously. The germination rate is also expressed as a percentage, relative to the control treatment.

[0101] [Table 5]

[0102] Table 6 below presents the leaf system cover rate, expressed as a percentage and calculated using image analysis software, for each experimental treatment described previously. The cover rate is also expressed as a percentage, relative to the control treatment. [Ta b.6]

[0103] Table 7 below presents the average root system length after seedling drying, expressed in centimeters, for each experimental treatment described previously. The average length is also expressed as a percentage, relative to the control treatment.

[0104] Ta b .7]

[0105] Student's t-test (threshold 5%)

[0106] Table 8 below presents the average dry biomass of the root system, aerial parts, and cumulative seedlings, expressed in milligrams, for each experimental treatment described previously. The average dry mass is also expressed as a percentage, relative to the control treatment.

[0107] [Ta b .8]

[0108] Student's t-test (threshold 5%) According to the results of the experimental tests of Example 2, multiple and lasting beneficial effects can be observed on tomato seedlings when the CNCM 1-5921 solution is applied mixed with the substrate before sowing.

[0109] The strain according to the invention makes it possible to improve the germination rate of tomato seeds particularly 6 days after sowing, to improve the rate of seedling coverage after 3 weeks, and to significantly increase the length of the root system of the seedlings (+21%).

[0110] The CNCM 1-5921 strain significantly increases the average dry biomass (Student's t-test at the 5% risk threshold) of seedlings and proves effective over a wide range of concentrations. After a single application at 5.10 7 cells per liter of substrate, the seedlings contain on average 22% more dry biomass than those of the control treatment, in this experimental trial.

[0111] In conclusion, the strain according to the invention allows producers to adjust the application dose of the CNCM 1-5921 strain according to the needs of their plant crops, to adapt their development to the technical routes followed, while making savings on inputs.

[0112] Example 3: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants, with combinatorial application of mycorrhizal fungi

[0113] 3.1 Experimental protocol for application in substrate mixture

[0114] Application of the CNCM 1-5921 solution during the sowing phase of young tomato plants

[0115] Preparation of 2 litres of substrate, composed of 35% horticultural substrate for seedlings and 65% vermiculite.

[0116] Mix the CNCM 1-5921 solution (as described in point 1.1) with the substrate to obtain a concentration of 2.10 9 Cells are dissolved in 200 mL of water per liter of substrate. One liter of pre-mixed substrate is deposited per 50-cell seed tray, i.e., 20 mL of substrate per cell.

[0117] One tomato seed is placed in each cell of each tray.

[0118] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0119] Germination rate measurement at 5 and 6 days after sowing.

[0120] Measurement of seedling heights, from the collar to the base of the apex, 3 weeks after sowing.

[0121] Measurement of the leaf system coverage rate with image analysis software, 3 and 4 weeks after sowing.

[0122] Extraction of seedlings from substrate, cleaning of the root system with water and absorption of excess water.

[0123] Seedlings are dried in an oven at 80°C for 24 hours.

[0124] Measurement of the dry biomass of the root system and the aerial part for each seedling.

[0125] Each treatment described above was conducted on an identical 50-hole experimental plate, corresponding to a sample size of 50 seeds per treatment. The tomato variety used for all experiments was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd. The mycorrhizal fungus species used was Glomus intraradices in its commercial form, AGTIV SC-P brand, produced by the Canadian company Premier Tech.

[0126] 3.2 Results

[0127] Table 9 below presents the germination rate, expressed as a percentage of the number of germinated seeds, for each experimental treatment described previously. The germination rate is also expressed as a percentage, relative to the control treatment. [Table 9]

[0128] Table 10 below shows the average seedling height, expressed in centimeters and measured from the collar to the base of the apex, 3 weeks after sowing, for each experimental treatment described previously. The average height is also expressed as a percentage, relative to the control treatment.

[0129] Table 10]

[0130] Table 11 below presents the leaf system coverage rate, expressed as a percentage and calculated using image analysis software, for each experimental treatment described previously. The coverage rate is also expressed as a percentage, relative to the control treatment.

[0131] [Table 11]

[0132] Table 12 below presents the average dry biomass of the root system, aerial parts, and cumulative biomass of the seedlings, expressed in milligrams, for each experimental treatment described previously. The average dry mass is also expressed as a percentage, relative to the control treatment. [Table 12]

[0133] Student's t-test (threshold 5%)

[0134] According to the results of the experimental trials of Example 3, beneficial effects can be observed on tomato seedlings when the CNCM 1-5921 solution is applied mixed with the substrate before sowing, in combination with mycorrhizal fungi.

[0135] The strain according to the invention makes it possible to improve the germination rate of tomato seeds as early as 5 days after sowing (+31%), and to significantly improve their coverage rate after 4 weeks (+33%).

[0136] The CNCM 1-5921 strain, combined with the application of mycorrhizae, significantly increases the average dry biomass (Student's t-test at the 5% risk threshold) of seedlings, compared to the addition of mycorrhizae alone. The application of 2.10 9 cells per liter of substrate increases the amount of dry biomass of seedlings by an average of 51%, compared to those produced solely with mycorrhizal fungi.

[0137] In conclusion, the strain according to the invention allows producers to combine it with other biostimulant products to further improve and optimize the development and quality of their plant production.

[0138] Example 4: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants under abiotic stress conditions

[0139] 4.1 Experimental protocol for application in substrate mixture of young seedlings tomatoes

[0140] Preparation of 2 litres of substrate, composed of 35% horticultural substrate for seedlings and 65% vermiculite.

[0141] Mix the CNCM 1-5921 solution (as described in point 1.1) with the substrate to obtain a concentration of 2.5 x 10 7 cells dissolved in 200 mL of water, per litre of substrate.

[0142] Deposit of 1 litre of pre-mixed substrate per 40-cell seed tray, i.e. 25 mL of substrate per cell.

[0143] One tomato seed is placed in each cell of each tray.

[0144] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0145] Extraction of seedlings from the substrate and cleaning of the root system.

[0146] Transplanting seedlings into an inert substrate composed of perlite.

[0147] Grown in a greenhouse for 4 weeks, maintained at 25°C during the day and 16°C at night, under water stress conditions.

[0148] Extraction of seedlings from the inert substrate and cleaning of the root system.

[0149] Measurement of fresh biomass of the root system and aerial part for each seedling.

[0150] Measurement of leaf area and leaf perimeter of seedlings, using image analysis software.

[0151] Drying of the root system and aerial part of each seedling in an oven at 80°C for 24 hours.

[0152] Dry biomass of the root system and aboveground parts was measured for each seedling. Each treatment described above was conducted on an identical 40-hole experimental plate, corresponding to 40 seeds per treatment. The tomato variety used for all experimental trials was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd.

[0153] 4.2 Results

[0154] Table 13 below presents the average fresh biomass of the root system, the aerial parts, and the two cumulative seedling biomasses, expressed in milligrams, for each experimental treatment described previously. The average fresh mass is also expressed as a percentage, relative to the control treatment.

[0155] Table 13]

[0156] Student's t-test (threshold 5%)

[0157] Table 14 below presents the average dry biomass of the root system, the aerial parts, and the two cumulative seedling biomasses, expressed in milligrams, for each experimental treatment described previously. The average dry mass is also expressed as a percentage, relative to the control treatment.

[0158] Student's t-test (threshold 5%)

[0159] Table 15 below presents the average leaf area of ​​the seedlings, expressed in square centimeters, for each experimental treatment described previously. The average leaf area is also expressed as a percentage, relative to the control treatment.

[0160] Table 15]

[0161] Student's t-test (threshold 5%)

[0162] Table 16 below presents the average leaf circumference of the seedlings, expressed in centimeters, for each experimental treatment described previously. The average circumference is also expressed as a percentage, relative to the control treatment.

[0163] [Table 16]

[0164] Student's t-test (threshold 10%)

[0165] According to the results of the experimental trials of Example 4, multiple beneficial effects can be observed on tomato seedlings subjected to abiotic stress, when the CNCM 1-5921 solution is applied mixed with the substrate before sowing.

[0166] The strain according to the invention allows, under water stress conditions, a significant increase in fresh biomass and average dry biomass (Student's t-test at the 5% risk threshold) of seedlings when simply premixed with the substrate. After a single application at 2.5 x 10 7With 31% more cells per liter of substrate, the seedlings contained 19% more average fresh biomass and 31% more average dry biomass than the control group. Notably, the biomass increase was more pronounced in the root zone of the seedlings, with a 34% increase in fresh biomass and a 20% increase in dry biomass, reflecting their better adaptation to water stress compared to the control seedlings.

[0167] The CNCM 1-5921 bacterial solution also resulted in a significant 30% increase in the average leaf area of ​​seedlings, as well as an 8% increase in their average leaf circumference. These parameters further demonstrate improved plant adaptation to water stress by reducing evapotranspiration and thus limiting water loss.

[0168] In conclusion, the strain according to the invention allows growers to improve the resistance and adaptability of their crops when subjected to water stress. Indeed, seedlings that develop and strengthen their root systems more rapidly increase their chances of survival in the event of water deficiency. In this respect, the CNCM 1-5921 strain, applied as a substrate mix before sowing, allows farmers to save water and ensure lower loss rates. With periods of drought and water restrictions becoming increasingly frequent worldwide, the ability for growers to preserve their crops under critical conditions, while maintaining sufficient yields, will quickly become vital.

[0169] Example 5: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants

[0170] 5.1 Experimental protocol for applying CNCM 1-5921 solution to seed soaking in young seedlings Tomatoes: Preparation of 5 litres of substrate, composed of 35% horticultural substrate for seedlings and 65% vermiculite.

[0171] Deposit of 1 litre of prepared substrate per 50 cell seedling tray, i.e. 20 mL of substrate per cell.

[0172] Dissolving the CNCM 1-5921 solution (as described in point 1.1) to obtain a concentration of 1.10 7 and 1.10 8 cells per millilitre of water.

[0173] Soaking batches of 50 tomato seeds in 100 mL of solutions for 20 and 40 minutes.

[0174] Filtration and drying of seed batches.

[0175] One tomato seed is placed in each cell of each tray.

[0176] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0177] Measurement of seedling heights, from the collar to the base of the apex, 3 weeks after sowing.

[0178] Measurement of the leaf system coverage rate with image analysis software, 3 and 4 weeks after sowing.

[0179] Extraction of seedlings from substrate, cleaning of the root system with water and absorption of excess water.

[0180] Measurement of fresh biomass in seedling batches.

[0181] Seedlings are dried in an oven at 80°C for 24 hours.

[0182] Measurement of the root system length of each seedling.

[0183] Measurement of the dry biomass of the root system and the aerial part for each seedling.

[0184] Each treatment described above was conducted on an identical 50-hole experimental plate, corresponding to a sample size of 50 seeds per treatment. The tomato variety used for all experimental trials was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd.

[0185] 5.2 Results

[0186] Tables 17 and 18 below show the average seedling height, expressed in centimeters and measured from the collar to the base of the apex, 3 weeks after sowing, for each experimental treatment described previously. The average height is also expressed as a percentage, relative to the control treatment.

[0187] [Ta b.17]

[0188] Student's t-test (threshold 5%)

[0189] [Ta b.18]

[0190] Tables 19 and 20 below present the leaf system coverage rate, expressed as a percentage and calculated using image analysis software, for each experimental treatment described previously. The coverage rate is also expressed as a percentage, relative to the control treatment.

[0191] [Ta b.19] Ta b.20]

[0192] Tables 21 and 22 below present the average fresh biomass per seedling after substrate removal, root system cleaning, and excess water absorption, expressed in milligrams, for each experimental treatment described previously. The average fresh mass is also expressed as a percentage, relative to the control treatment.

[0193] [Table 21]

[0194] [Table 22]

[0195] Tables 23 and 24 below present the average root system length after seedling drying, expressed in centimeters, for each experimental treatment described previously. The average length is also expressed as a percentage, relative to the control treatment.

[0196] Ta b.24]

[0197] Tables 25 and 26 below present the average dry biomass of the root system, the aerial parts, and the two cumulative seedling biomasses, expressed in milligrams, for each experimental treatment described previously. The average dry mass is also expressed as a percentage, relative to the control treatment. [Table 25]

[0198] Table 26] Student's t-test (threshold 5%) According to the results of the experimental trials of Example 4, multiple and lasting beneficial effects can be observed on tomato seedlings when the CNCM 1-5921 solution is applied as a seed soaking solution before sowing.

[0199] The strain according to the invention makes it possible to significantly increase the height of the seedlings after 3 weeks (+11%), to progressively improve their coverage rate after 3 and then 4 weeks (+31%), and to significantly increase the length of the root system of the seedlings (+12%).

[0200] The CNCM 1-5921 strain increases fresh biomass (+29%) but, more importantly, significantly increases the average dry biomass (Student's t-test at the 5% significance level) of seedlings when applied as a seed dip. After a 40-minute soak at a concentration of 1.10 8cells per millilitre of solution, the seedlings contain an average dry biomass 48% higher than those of the control treatment, in the context of this experimental trial.

[0201] In conclusion, the strain according to the invention allows producers to treat their seeds in large quantities simply by soaking them before sowing. This particularly easy-to-implement practice saves time and increases efficiency, while significantly reducing the workload. The treated seeds can be sown immediately or stored for staggered sowing over time. Applying the treatment by soaking the seeds thus allows farmers to save on labor, space, and energy by reducing the equipment required for implementation.

[0202] Example 6: Evaluation of the impact of the CNCM 1-5921 bacterial strain applied according to the invention on the development of Solanum lycopersicum plants under abiotic stress conditions

[0203] 6.1 Experimental protocol for application in substrate mixture

[0204] Application of CNCM 1-5921 solution in the sowing phase of young tomato plants. Preparation of 4 liters of substrate, composed of 35% horticultural substrate for sowing and 65% vermiculite.

[0205] Deposit of 1 litre of prepared substrate per 40 cell seedling tray, i.e. 25 mL of substrate per cell.

[0206] Dissolving the CNCM 1-5921 strain (as described in point 1.1) to obtain a concentration of 2.10 8 cells per millilitre of water.

[0207] Soaking batches of 40 tomato seeds in 100 mL of solutions for 40 minutes.

[0208] Filtration and drying of seed batches.

[0209] One tomato seed is placed in each cell of each tray.

[0210] The plates were placed in a germination room at 20°C for 4 weeks, under controlled humidity and light conditions.

[0211] Extraction of seedlings from the substrate and cleaning of the root system.

[0212] Transplanting seedlings into an inert substrate composed of perlite.

[0213] Grown in a greenhouse for 4 weeks, maintained at 25°C during the day and 16°C at night, under normal irrigation conditions and under water stress conditions.

[0214] Extraction of seedlings from the inert substrate and cleaning of the root system.

[0215] Measurement of the leaf area of ​​seedlings, using image analysis software.

[0216] Drying of the root system and aerial part of each seedling in an oven at 80°C for 24 hours.

[0217] Measurement of the dry biomass of the root system and the aerial part for each seedling.

[0218] Each treatment described above was conducted on an identical 40-hole experimental plate, corresponding to a sample size of 40 seeds per treatment. The tomato variety used for all experimental trials was the commercial variety of Solanum lycopersicum, Sweetbaby brand, produced organically by the French company Girerd.

[0219] 6.2 Results Tables 27 and 28 below present the average dry biomass of the root system, the aerial parts, and the two cumulative seedling biomasses, expressed in milligrams, for each experimental treatment described above. The average dry mass is also expressed as a percentage, relative to the control treatment.

[0220] Table 27]

[0221] [Table 28] Table 29 below presents the average leaf area of ​​the seedlings, expressed in square centimeters, for each experimental treatment described previously, under water stress conditions. The average leaf area is also expressed as a percentage, relative to the control treatment.

[0222] [Table 29] According to the results of the experimental trials of Example 6, multiple beneficial effects can be observed on tomato seedlings subjected to abiotic stress, when the CNCM 1-5921 strain is applied in seed soaking solution before sowing.

[0223] The strain according to the invention increases the average dry biomass of seedlings when applied as a seed soak. After a 40-minute soak at a concentration of 2.10 8With a high cell count per milliliter of solution, the seedlings contained an average dry biomass 6% higher than the control group under normal conditions, and 8% higher under water stress conditions. The CNCM 1-5921 bacterial solution also resulted in a 7% increase in the average leaf area of ​​the seedlings. These parameters indicate improved plant adaptation to water scarcity through tissue strengthening via nutrient assimilation.

[0224] In conclusion, the strain according to the invention allows producers to improve the resistance and adaptability of their crops through a simple seed soaking treatment. This inexpensive and easy-to-implement technique will allow farmers to store their pre-treated seeds before sowing, while also saving labor and water. With droughts and water restrictions becoming increasingly frequent worldwide, the ability for producers to preserve their crops under critical conditions while maintaining sufficient yields will quickly become vital.

Claims

DEMANDS 1) Isolated strain of the species Bacillus siamensis deposited with the CNCM under the order number CNCM 1-5921. 2) Use of the bacterial strain according to claim 1, for the stimulation of the development and growth of a tomato seed and plant or the improvement of the yields of a tomato crop. 3) Use of the bacterial strain according to claim 1, for the improvement of resistance and tolerance to abiotic stress of a seed, a tomato plant and a tomato crop. 4) Use according to any one of claims 2 to 3, wherein the bacterial strain is in a solid form such as a powder or granules, or in a liquid form, preferably aqueous. 5) Method of obtaining a tomato seed characterized in that it comprises the following steps: preparation of a soaking solution containing a strain according to claim 1, soaking a seed in the soaking solution, filtration and drying of the seed, obtaining a seed, conditioning a seed. 6) Method according to claim 5, wherein the bacterial strain is applied such that the concentration is between 10 5 at 10 10 cells / mL of soaking solution. 7) Method for growing tomatoes from tomato seed, possibly obtained according to the conditions of claim 6, characterized in that it comprises the following steps: a) preparation of a substrate, b) sowing the substrate with tomato seed, possibly obtained according to a method according to claim 5 or 6, c) optionally, incorporation into the substrate of mycorrhizal fungi, amendments or mixtures of nutrients specific to tomato cultivation, d) germination, e) growth, f) transplanting, g) flowering, h) fruiting, i) harvesting, j) end of harvest, characterized in that the bacterial strain according to claim 1, or a composition comprising it, is applied at least once during at least one of the steps (a), (b), (d), (e), (f) (i) and (j). 8) Method according to claim 7, wherein the bacterial strain is applied in the mass of the substrate or on the surface of a tomato culture, such that the concentration is between 10 5 cells and 1O 10 cells / L of substrate or between 5.10 6 cells and 5.10 11 cells / m 2 . 9) Method according to any one of claims 7 to 8, wherein the bacterial strain is applied by soaking or watering or by spraying with a liquid solution. 10) Use of a tomato end-of-crop substrate obtained by a method according to claims 7 to 9 as an amendment or fertilizer for an agricultural crop. 11) Use of a bacterial strain according to claim 1, to prevent the synthesis of ethylene produced by a tomato seed or plant. 12) Use of a bacterial strain according to claim 1, to produce a phytohormone such as an auxin or a gibberellin or a cytokinin.

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