NEW BIOSTIMULATION AGENT AND ITS USES

Abstract

TITLE: NEW BIOSTIMULATION AGENT AND ITS USES The invention relates to the use of an isolated strain of Chitinophage sp. and falls more broadly within the field of agriculture. In particular, it relates to the use of said strain as a biostimulation agent, for example, as an agent to combat water stress and / or as a biofertilizer. In so doing, the invention relates to plant growth stimulant compositions comprising said isolated strain of Chitinophage sp., processes using them, and said strain itself. (no figure)

Description

Title of the invention: NEW BIOSTIMULATION AGENT AND ITS USES FIELD OF INVENTION

[0001] The invention relates to the use of an isolated strain of Chitinophage sp. and is more broadly relevant to the field of agriculture. In particular, it relates to the use of said strain as a biostimulant, for example, as an agent to combat water stress and / or as a biofertilizer. In so doing, the invention relates to plant growth stimulant compositions comprising said isolated strain of Chitinophage sp., processes using them, and said strain itself. EARLIER ART

[0002] Climate change and the increase in the human population are leading humankind to face numerous challenges, particularly in agriculture. Indeed, agricultural production must increase while growing conditions tend to deteriorate. Drought is, in fact, one of the main limitations to crop yields, if not the major problem. Its impact affects 1 to 3% of all land, resulting in limited crop growth and productivity. This phenomenon is also set to worsen because, in the coming years, the availability of water for agriculture will also be a major issue.

[0003] To cope with adverse water deficit conditions, plants use sophisticated and complex mechanisms that help them perceive stress signals and achieve optimal yield. Among these mechanisms, some rely on the presence of rhizobacteria in the soil and root tissue of plants. In doing so, these bacteria can enable better growth and higher yield through the production of phytohormones, osmolytes, antioxidants, volatile compounds, exopolysaccharides, etc. (Camaille M.; Fabre N.; Clément C.; Ait Barka E. Plant Growth Promoting Rhizobacteria: a sustainable way to trigger drought tolerance in wheat. Microorganisms 2021, 9, x.).

[0004] Among these rhizobacteria, and without being exhaustive, the following may be cited: Bacillus subtilis (Lastochkina O. (2019) Bacillus subtilis-Mediated Abiotic Stress Tolerance in Plants. In: Islam M., Rahman M., Pandey P., Boehme M., Haesaert G. (eds) Bacilli and Agrobiotechnology: Phytostimulation and Biocontrol. Bacilli in Climate Resilient Agriculture and Bioprospecting. Springer, Cham.) and Ensiler adhaerens (Zhou GC, Wang Y, Zhai S, Ge F, Liu ZH, Dai YJ, Yuan S, Hou JY. Biodegradation of the neoni- cotinoid insecticide thiamethoxam by the nitrogen-fixing and plant-growth-promoting rhizobacterium Ensifer adhaerens strain TMX-23. Appl Microbiol Biotechnol. 2013 May;97(9):4065-74).

[0005] However, although described as effective agents for combating water stress, Bacillus subtilis and Ensifer adhaerens cannot, on their own, solve all agricultural problems. Indeed, the diversity of agricultural crops and agricultural territories necessitates a diversification of tools for combating water stress. BRIEF OVERVIEW

[0006] In this context of improving agricultural yields and combating water stress in crops, a first objective of the invention relates to making available an isolated strain of Chitinophaga pinensis and its use as a biostimulant. A second objective aims to provide phytostimulant compositions, particularly for implementing processes to either prevent and / or combat water stress in plants, or to stimulate mineral assimilation by plants. Another objective of the invention relates to coated seeds combining plant seeds and the isolated strain of Chitinophaga pinensis. DETAILED DESCRIPTION

[0007] According to a first aspect of the invention, the object of this invention is the use of an isolated strain of Chitinophaga sp. as a biostimulation agent.

[0008] The term "biostimulating agent" or biostimulant means substances and / or microorganisms whose function, when applied to plants or the rhizosphere, is to stimulate natural processes that promote / improve:

[0009] - the absorption or utilization of nutrients / minerals; - tolerance to abiotic stresses, including tolerance to water stress; and / or - the quality or yield of the crop, regardless of the presence of nutrients.

[0010] Surprisingly, it has been identified that the species C. pinensis, when used, perfectly meets this definition and, in fact, constitutes a tool of choice that can be readily made available to agriculture. In this way, the use proposed above encompasses obtaining biofertilizing effects (improved mineral assimilation and / or crop quality or yield) and / or tolerance to abiotic stresses (including water stress).

[0011] In other words, another embodiment relates to the use of an isolated strain of Chitinophaga pinensis as a biostimulation agent, in particular as an agent for combating water stress and / or as a biofertilizing agent.

[0012] By "water stress control agent," it is understood that the isolated strain of Chitinophaga pinensis, once applied to a plant, is capable of increasing the plant's tolerance to water stress (lack of water). It can be used either preventively, i.e., in anticipation of a water stress episode, or curatively, i.e., after the onset of water stress and as a treatment thereof. Another embodiment therefore concerns the use of an isolated strain of Chitinophaga pinensis as described above in the prevention and / or treatment of water stress experienced by a plant.

[0013] in particular said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0014] In particular, another embodiment relates to the use of an isolated strain of Chitinophaga pinensis as described above in the treatment of water stress suffered by a plant,

[0015] in particular said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0016] In particular, another embodiment relates to the use of an isolated strain of Chitinophaga pinensis as described above in the prevention of water stress suffered by a plant,

[0017] in particular said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0018] By "biofertilizing agent," it is understood that the isolated strain of Chitinophaga pinensis, once applied to a plant, is capable of promoting the plant's growth and development, in particular by facilitating the plant's assimilation of minerals. Another embodiment therefore relates to the use of an isolated strain of Chitinophaga pinensis as described above to stimulate the assimilation of minerals by a plant.

[0019] in particular said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0020] That being said, it should be noted that the roles of water stress control agent and biofertilizer are not mutually exclusive and can be combined. Indeed, if the isolated strain of Chitinophaga pinensis is initially used to promote the growth of an agricultural crop, it can, during a sudden drought, prevent its potentially disastrous effects. The reverse is also true. The initial use of an isolated strain of Chitinophaga pinensis to prevent and / or treat a drought (water stress) can, afterward, prove to promote the recovery and growth of the agricultural crop, resulting in an excellent final yield. In other words, another embodiment involves using an isolated strain of Chitinophaga pinensis as a control agent. against water stress and as a biofertilizing agent.

[0021] Furthermore, and according to another embodiment, the invention relates to the use as described above, said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame. In particular, the invention relates to the use as described above, said plant being wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0022] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0023] By "nucleic acid sequence that codes for it," we mean that said nucleic acid sequence corresponds to an reading frame comprising a start codon and a stop codon, and whose translation by a ribosome results in a protein, i.e., a chain of amino acids. In this regard, it should be noted that:

[0024] - the nucleic acid sequence SEQ ID NO: 63 codes for the enzyme with sequence SEQ ID NO: 71, which corresponds to Tryptophan 2-3 oxygenase capable of catalyzing the following enzymatic reaction:

[0025] L-tryptophan + O2 = N-formyl-L-kynurenine

[0026] said enzyme being involved in the synthesis of indole-3-acetic acid (IAA) and whose activity can be easily tested (Yuzuru Ishimura,

[52] L-tryptophan 2,3-dioxygenase (tryptophan pyrrolase) (pseudomonas fluorescens), Methods in Enzymology, Academie Press, Volume 17, Part A, 1970, Pages 429-434);

[0027] - the nucleic acid sequence SEQ ID NO: 64 codes for the enzyme with sequence SEQ ID NO: 72, which corresponds to the beta subunit of Tryptophan synthase capable of catalyzing the following enzymatic reaction:

[0028] (lS,2R)-lC-(indol-3-yl)glycerol 3-phosphate + L-Serine = D-glyceraldehyde 3-phosphate + H2O + L-tryptophan

[0029] said enzyme being involved in the synthesis of tryptophan and whose activity can be easily tested (Buller, Andrew R et al. “Directed evolution of the tryptophan synthase beta-subunit for stand-alone function recapitulates allosteric activation.’1’ Proc Natl Acad Sci USA. 2015 Nov 24; 112(47): 14599-14604.) ;

[0030] - the nucleic acid sequence SEQ ID NO: 65 codes for the enzyme with sequence SEQ ID NO: 73, which corresponds to the alpha subunit of Tryptophan synthase capable of catalyzing the following enzymatic reaction:

[0031] (lS,2R)-lC-(indol-3-yl)glycerol 3-phosphate + L-Serine = D-glyceraldehyde 3-phosphate + H20 + L-tryptophan

[0032] said enzyme being involved in the synthesis of tryptophan and whose activity can be easily tested (RD Mosteller, Improved method for measuring the catalytic activity of tryptophan synthase a subunit in cell extracts. Biochimie, Volume 72, Issue 12, 1990, Pages 881-884);

[0033] - the nucleic acid sequence SEQ ID NO: 66 codes for the enzyme with sequence SEQ ID NO: 74, which corresponds to Indole-3-glycerol phosphate synthase TrpC capable of catalyzing the following enzymatic reaction:

[0034] l-(2-carboxyphenylamino)-l-deoxy-D-ribulose 5-phosphate + H+ = (lS,2R)-lC-(indol-3-yl)glycerol 3-phosphate + CO2 + H2O

[0035] said enzyme being involved in the synthesis of tryptophan and whose activity can be easily tested (Sôderholm, Annika et al. “Structure and kinetics of indole-3-glycerol phosphate synthase from Pseudomonas aeruginosa: Decarboxylation is not essential for indole formation.” The Journal of biological chemistry vol. 295,47 (2020): 15948-15956.);

[0036] - the nucleic acid sequence SEQ ID NO: 67 codes for the enzyme with sequence SEQ ID NO: 75, which corresponds to Phosphoglucomutase capable of catalyzing the following enzymatic reaction:

[0037] beta-D-glucose 1-phosphate = beta-D-glucose 6-phosphate

[0038] said enzyme being involved in glycolysis and probably the synthesis of exopolysaccharides (EPS), and whose activity can be easily tested (HU Bergmeyer (Ed.), Methods of Enzymatic Analysis (Second edition). Academie Press (1974), pp. 798-801);

[0039] - the nucleic acid sequence SEQ ID NO: 68 codes for the enzyme with sequence SEQ ID NO: 76, which corresponds to Phosphomannomutase / Phosphoglucomutase capable of catalyzing the following enzymatic reaction:

[0040] aD-mannose 1-phosphate = D-mannose 6-phosphate

[0041] said enzyme being involved in the synthesis of exopolysaccharides (EPS) and whose activity can be easily tested (Kômer C, Lehle L, von Figura K. Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency. Gly-cobiology. 1998 Feb;8(2):165-71. doi: 10.1093 / glycob / 8.2.165. PMID: 9451026.);

[0042] - the nucleic acid sequence SEQ ID NO: 69 codes for the enzyme with sequence SEQ ID NO: 77, which corresponds to dTDP-glucose 4,6-dehydratase capable of catalyzing the following enzymatic reaction:

[0043] dTDP-aD-glucose = dTDP-4-dehydro-6-deoxy-aD-glucose + H2O

[0044] said enzyme being involved in the synthesis of exopolysaccharides (EPS) and of which The activity can be easily tested (Okazaki R, Okazakit, Strominger JL, Michelson AM. Thymidine diphosphate 4-keto-6-deoxy-d-glucose, an intermediate in thymidine diphosphate L-rhamnose synthesis in Escherichia coli strains. J Biol Chem. 1962 Oct;237:3014-26. PMID: 13939805.); and

[0045] - the nucleic acid sequence SEQ ID NO: 70 codes for the protein with sequence SEQ ID NO: 78, which corresponds to YAlginate biosynthesis protein AlgA capable of catalyzing the following enzymatic reaction:

[0046] D-mannose 6-phosphate = D-fructose 6-phosphate

[0047] said enzyme being involved in the synthesis of exopolysaccharides (EPS) and whose activity can be easily tested (Shinabarger D, Berry A, May TB, Rothmel R, Fialho A, Chakrabarty AM. Purification and characterization of phosphomannose isomerase-guanosine diphospho-D-mannose pyrophosphorylase. A bifunctional enzyme in the alginate biosynthetic pathway of Pseudomonas aeruginosa. J Biol Chem. 1991 Feb 5;266(4):2080-8. PMID: 1846611.).

[0048] By "percentage of identity" is meant the percentage determined by direct comparison of two sequences of nucleic acid (or amino acid) molecules, by determining the number of identical nucleic acid residues between the two sequences, then dividing it by the number of nucleic acid residues in the longer of the two sequences, and multiplying the result by 100. Sequence comparison tools such as the algorithms of the BLAST platform or, in particular, the MatGat2.01 program (Campanella, JJ, Bitincka, L., & Smalley, J. (2003). MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences. BMC Bioinformatics, 4, 29.) can help to achieve this.Furthermore, by "at least 84% identity with a reference sequence," we mean that the identity between a sequence of interest and a reference sequence (chosen from SEQ ID NOs 63 to 70) can be at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, provided that said sequence of interest retains the enzymatic activity of the enzyme encoded by the aforementioned reference sequence. This therefore means that the sequence of interest (or the enzyme of interest) is functional and capable of catalyzing the same enzymatic reaction as the reference sequence (or reference enzyme).

[0049] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from SEQ ID NOs sequences 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0050] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs: 63 to 70 sequences. In particular, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence corresponds to the SEQ ID NO: 69 sequence, which codes for the enzyme of sequence SEQ ID NO: 77.

[0051] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs: 63 to 70 sequences. In particular, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence corresponds to the SEQ ID NO: 69 sequence, which codes for the enzyme of sequence SEQ ID NO: 77.

[0052] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least two enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0053] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0054] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least three enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0055] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0056] According to another embodiment, the invention relates to the use, as described above, of said isolated strain of Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least four enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0057] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0058] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least five enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0059] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0060] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least six enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0061] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0062] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least seven enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0063] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0064] According to another embodiment, the invention relates to the use such as described above, said strain isolated from Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them have at least 84% identity with a reference sequence chosen from the SEQ ID NOs: 63 to 70 sequences, said at least eight enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0065] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them correspond to the SEQ ID NOs sequences: 63 to 70.

[0066] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 95% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 22 to 61.

[0067] As before, the "percentage of identity" refers to the percentage determined by direct comparison of two sequences of nucleic acid (or amino acid) molecules, by determining the number of identical nucleic acid residues between the two sequences, then dividing it by the number of nucleic acid residues in the longer of the two sequences, and multiplying the result by 100. Sequence comparison tools such as the algorithms of the BLAST platform or, in particular, the MatGat2.01 program (Campanella, JJ, Bitincka, L., & Smalley, J. (2003). MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences. BMC Bioinformatics, 4, 29.) can help to achieve this.Furthermore, and by "an identity of at least 95% with a reference sequence", it is understood that the identity between a sequence of interest and a reference contig (chosen from the SEQ ID NOs sequences: 22 to 61) can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0068] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 22 to 61.

[0069] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 80% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 1 to 21.

[0070] As before, by "percentage of identity" is meant the percentage determined by direct comparison of two sequences of nucleic acid (or amino acid) molecules, by determining the number of identical nucleic acid residues between the two sequences, and then dividing it by the number of nucleic acid residues. Key elements of the longer of the two sequences, and multiplying the result by 100. Sequence comparison tools such as the algorithms of the BLAST platform or in particular the MatGat2.01 program (Campanella, JJ, Bitincka, L., & Smalley, J. (2003). MatGAT: an application that generates similarity / identity matrices using protein or DNA sequences. BMC Bioinformatics, 4, 29.) can help to achieve this. Furthermore, and by "an identity of at least 80% with a reference sequence", it is understood that the identity between a sequence of interest and a reference contig (chosen from the SEQ ID NOs sequences: 1 to 21) can be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0071] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 1 to 21.

[0072] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence has an identity of at least 99% with the SEQ ID NO: 62 sequence.

[0073] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence is the sequence SEQ ID NO: 62.

[0074] According to another embodiment, the invention relates to the use as described above, said strain isolated from Chitinophaga pinensis being strain ARDCPP14 deposited on April 23, 2021 at the National Collection of Microorganism Cultures (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under number CNCM 1-5676.

[0075] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis being the ARDCPP14 strain filed on April 23, 2021 with the CNCM under number CNCM 1-5676 and / or one of its (functional) mutants.

[0076] According to another embodiment, the invention relates to the use as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the coded enzyme by the aforementioned reference sequence,

[0077] in particular, said strain isolated from Chitinophaga pinensis being strain ARDCPP14 filed on April 23, 2021 with the CNCM under number CNCM 1-5676.

[0078] According to a second aspect, the invention relates to a phytostimulant composition comprising:

[0079] - an isolated strain of Chitinophaga pinensis as an active ingredient; or - an isolated strain of Chitinophaga pinensis as the first active ingredient in combination with at least one other active ingredient chosen from: • another biostimulation agent; • a biocontrol plant protection product; • a fertilizer; and • their mixtures,

[0080] in particular, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0081] By "active ingredient" is meant the (co-)formulant or (co-)formulants which in a composition has the desired effect (improvement of mineral assimilation and / or tolerance to water stress).

[0082] By "another biostimulating agent," it is understood that this other biostimulating agent is not the isolated Chitinophaga pinensis strain according to the invention. For example, it may be a Bacillus spp. strain, an Ensifer spp. strain, or a Chitinophaga spp. strain (different from that of the invention).

[0083] By "biocontrol plant protection product", we mean a chemical and / or biological (e.g. bacteria, fungus) (co-)formulant that can interfere with the growth and / or survival of plant pathogens.

[0084] The term "fertilizer" refers to chemical fertilizers, or mineral fertilizers, which are applied to (agricultural) crops, most often by spreading, and are intended to improve the quantity and quality of agricultural, horticultural, and forestry yields. The term "fertilizer" also includes organic fertilizers derived from living matter (animal, plant, fungal, bacterial, etc.), such as manure.

[0085] By "their mixtures", it is understood that said at least one other active ingredient may be a combination of:

[0086] - another biostimulant and a biocontrol phytopharmaceutical product; - another biostimulant and a fertilizer; - a biocontrol plant protection product and a fertilizer; or - another biostimulant, a biocontrol phytopharmaceutical product and a fertilizer.

[0087] In other words, another embodiment relates to the phytostimulant composition as described above comprising:

[0088] - an isolated strain of Chitinophaga pinensis as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one another biostimulant agent as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one biocontrol plant protection product as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one fertilizer as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one other biostimulant and with at least one biocontrol plant protection product as an active ingredient; or - an isolated strain of Chitinophaga pinensis in combination with at least one other biostimulant and with at least one fertilizer as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one biocontrol plant protection product and with at least one fertilizer as an active ingredient; or - an isolated strain of Chitinophaga pinensis in association with at least one other biostimulation agent, with at least one biocontrol plant protection product and with at least one fertilizer as an active ingredient.

[0089] Furthermore, and insofar as reference is made to "at least one other biostimulant", "at least one biocontrol plant protection product" and "at least one fertilizer", it is understood that the phytostimulant composition as described above may include at least two, at least three or even at least four other biostimulants and / or biocontrol plant protection products and / or fertilizers.

[0090] According to another embodiment, the invention also relates to the phytostimulant composition as described above, comprising an effective quantity of the isolated strain of Chitinophaga pinensis of 10⁶ to 10¹² CFU / g, more particularly of 10⁸ to 10¹⁰ CFU / g,

[0091] said phytostimulant composition possibly being obtained after a preparation step (e.g. dilution by suspension or mixing) making it ready for spreading.

[0092] By "effective quantity [...] between 10⁶ and 10¹² CFU / g", we mean the number of bacteria capable of forming a colony on culture medium (CFU = colony-forming unit). ionie) per g (gram) of product. In addition, the expression "from 106 to 1012 CFU / g" can also mean from 106 to 1011 CFU / g, from 106 to 1010 CFU / g, from 106 to 109 CFU / g, from 106 to 108 CFU / g, from 106 to 107 CFU / g, from 107 to 1012 CFU / g, from 108 to 1012 CFU / g, from 109 to 1012 CFU / g, from 1010 to 1012 CFU / g, from 1011 to 1012 CFU / g, from 107 to 1011 CFU / g, or from 108 to 1010 CFU / g.

[0093] Furthermore, since the phytostimulant composition as described above can be obtained after a preparation step, it is understood that an object of the invention is a concentrated phytostimulant composition. Indeed, the phytostimulant composition as described above can be presented initially (at the point of purchase) in the form of a concentrated solid or concentrated liquid composition, which can, for example, be obtained through a particular formulation of one of the active ingredients (see above). Therefore, by "concentrated composition," we mean a phytostimulant composition in which at least the concentration of the isolated strain of Chitinophaga pinensis is greater than an effective amount of 10¹² CFU / g.In other words, a particular aspect of the invention relates to the phytostimulant composition as described above comprising at least the isolated strain of Chitinophaga pinensis as the active ingredient, said phytostimulant composition being in the form of a concentrated solid or concentrated liquid composition in which the concentration of the isolated strain of Chitinophaga pinensis is greater than an effective amount of 10¹² CFU / g. By "concentration [...] greater than an effective amount of 10¹² CFU / g", is meant a concentration strictly greater than 10¹² CFU / g, which may also be (strictly) greater than 10¹³ CFU / g, 10¹⁴ CFU / g, 10¹⁵ CFU / g or even 10¹⁶ CFU / g.

[0094] Furthermore, since the phytostimulant composition of the invention comprises at least the isolated strain of Chitinophaga pinensis of the invention as an active ingredient, the particular characteristics thereof may apply thereto. In other words, and according to another embodiment, the invention relates to the phytostimulant composition as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0095] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0096] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs: 63 to 70 sequences. In particular, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence corresponds to the SEQ ID NO: 69 sequence, which codes for the enzyme of sequence SEQ ID NO: 77.

[0097] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence coding it is selected from the sequences SEQ ID NOs: 63 to 70. In particular, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence corresponds to the sequence SEQ ID NO: 69, which codes for the enzyme of sequence SEQ ID NO: 77.

[0098] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least two enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0099] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0100] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least three enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0101] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0102] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them have at least 84% identity with a reference sequence chosen from the SEQ ID NOs: 63 to 70 sequences, said at least four enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0103] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0104] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least five enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0105] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0106] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least six enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0107] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0108] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least seven enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0109] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0110] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least eight enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0111] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them correspond to the SEQ ID NOs sequences: 63 to 70.

[0112] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 95% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 22 to 61.

[0113] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 22 to 61.

[0114] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 80% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 1 to 21.

[0115] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 1 to 21.

[0116] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence has an identity of at least 99% with the SEQ ID NO: 62 sequence.

[0117] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence is the sequence SEQ ID NO: 62.

[0118] According to another embodiment, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis being the ARDCPP14 strain deposited on April 23, 2021 at the National Collection of Microorganism Cultures (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under the number CNCM 1-5676.

[0119] According to another embodiment, the invention relates to the phyto-stimulant composition as described above, said strain isolated from Chitinophaga pinensis being the ARDCPP14 strain filed on April 23, 2021 with the CNCM under number CNCM 1-5676 and / or one of its (functional) mutants.

[0120] Furthermore, and in view of the foregoing, it is understood that an object of the invention relates to the phytostimulant composition as described above, comprising:

[0121] - an isolated strain of Chitinophaga pinensis as an active ingredient; or - an isolated strain of Chitinophaga pinensis as the first active ingredient in combination with at least one other active ingredient chosen from: • another biostimulation agent; • a biocontrol plant protection product; • a fertilizer; and • their mixtures,

[0122] in particular, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from SEQ ID NOs sequences 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence,

[0123] in particular, said strain isolated from Chitinophaga pinensis being strain ARDCPP14 filed on April 23, 2021 with the CNCM under number CNCM 1-5676.

[0124] According to another aspect, the invention relates to a phytostimulant coating composition comprising an isolated strain of Chitinophaga pinensis and optionally at least one fixing agent.

[0125] The term “fixing agent” means a chemical or natural agent that allows the plant growth compound to adhere to a plant seed so as to coat the plant seed. Among the available fixing agents are carboxymethyl cellulose (CMC) and gum arabic.

[0126] By "an isolated strain of Chitinophaga pinensis", one means in particular the strain as described above. In fact, it is understood that another object of the invention relates to the phytostimulant coating composition as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence chosen from the SEQ ID NOs: 63 to 70 sequences, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0127] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0128] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence coding it is selected from the sequences SEQ ID NOs: 63 to 70. In particular, the invention relates to the phytostimulant composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence corresponds to the sequence SEQ ID NO: 69, which codes for the enzyme of sequence SEQ ID NO: 77.

[0129] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs: 63 to 70 sequences. In particular, the invention relates to the phytostimulant composition as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence corresponds to the SEQ ID NO: 69 sequence, which codes for the enzyme of sequence SEQ ID NO: 77.

[0130] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least two enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0131] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0132] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them have at least 84% identity with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least three enzymes preserving the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0133] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0134] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least four enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0135] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0136] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least five enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0137] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0138] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least six enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0139] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0140] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least seven enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0141] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0142] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least eight enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0143] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them correspond to the SEQ ID NOs sequences: 63 to 70.

[0144] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 95% identity with a sequence selected from the contigs of SEQ ID NOs sequences: 22 to 61.

[0145] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 22 to 61.

[0146] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 80% identity with a sequence selected from the contigs of SEQ ID NOs sequences: 1 to 21.

[0147] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 1 to 21.

[0148] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence has at least 99% identity with the SEQ ID NO: 62 sequence.

[0149] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence is the sequence SEQ ID NO: 62.

[0150] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis being strain ARDCPP14 deposited on April 23, 2021 at the National Collection of Microorganism Cultures (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under number CNCM 1-5676.

[0151] According to another embodiment, the invention relates to the phytostimulant coating composition as described above, said strain isolated from Chitinophaga pinensis being the ARDCPP14 strain filed on April 23, 2021 with the CNCM under number CNCM 1-5676 and / or one of its (functional) mutants.

[0152] Furthermore, and in view of the foregoing, it is understood that an object of the invention relates to the phytostimulant coating composition as described above comprising an isolated strain of Chitinophaga pinensis and optionally at least one fixing agent,

[0153] in particular, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from SEQ ID NOs sequences 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence,

[0154] in particular, said strain isolated from Chitinophaga pinensis being strain ARDCPP14 filed on April 23, 2021 with the CNCM under number CNCM 1-5676.

[0155] According to another embodiment, the invention also relates to the phytostimulant coating composition as described above, further comprising at least one co-formulant selected from:

[0156] - glycerol; - gum arabic; - polyvinylpyrrolidone (PVP); - ascorbic acid; - skimmed milk; and - their mixtures.

[0157] By "their mixtures" it is meant that said at least one co-formulant may be, for example, a combination of:

[0158] - glycerol and gum arabic and / or polyvinylpyrrolidone (PVP); - acacia gum and glycerol and / or polyvinylpyrrolidone (PVP); - polyvinylpyrrolidone (PVP) and gum arabic and / or glycerol; - ascorbic acid and skimmed milk; - glycerol and gum arabic and / or polyvinylpyrrolidone (PVP), and ascorbic acid and / or skimmed milk; - acacia gum and glycerol and / or polyvinylpyrrolidone (PVP), and ascorbic acid and / or skimmed milk; - polyvinylpyrrolidone (PVP) and gum arabic and / or glycerol; and ascorbic acid and / or skimmed milk; or - glycerol, gum arabic, polyvinylpyrrolidone (PVP), ascorbic acid and skimmed milk.

[0159] In other words, another embodiment relates to the phytostimulant coating composition as described above, further comprising a co-formulant selected from:

[0160] - the mixture comprising glycerol, gum arabic and / or polyvinylpyrrolidone (PVP); - the mixture comprising gum arabic, glycerol and / or polyvinylpyrrolidone (PVP); - the mixture comprising the mixture comprising polyvinylpyrrolidone (PVP), gum arabic and / or glycerol; - the mixture comprising ascorbic acid and skimmed milk; - the mixture comprising glycerol, gum arabic and / or polyvinylpyrrolidone (PVP), and ascorbic acid and / or skimmed milk; - the mixture comprising gum arabic, glycerol and / or polyvinylpyrrolidone (PVP), and ascorbic acid and / or skimmed milk; - the mixture comprising polyvinylpyrrolidone (PVP), gum arabic and / or glycerol, and ascorbic acid and / or skimmed milk; and - the mixture comprising glycerol, gum arabic, polyvinylpyrrolidone (PVP), ascorbic acid and skimmed milk.

[0161] In particular, another embodiment relates to the phytostimulant coating composition as described above, in which:

[0162] - the glycerol concentration is between 1% w / w and 4% w / w; and / or - the concentration of gum arabic is between 2.5% w / w and 10% w / w; and / or - the concentration of polyvinylpyrrolidone (PVP) is between 1% w / w and 4% w / w; and / or - the concentration of ascorbic acid is between 0.01% w / w and 0.04% w / w; and / or - the concentration of skimmed milk is between 2% w / w and 5% w / w.

[0163] In particular, another embodiment relates to the phytostimulant coating composition as described above, in which:

[0164] - the glycerol concentration is 2% w / w; and / or - the concentration of gum arabic is 5% w / w; and / or - the concentration of polyvinylpyrrolidone (PVP) is 2% w / w; and / or - the concentration of ascorbic acid is 0.02% w / w; and / or - the concentration of skimmed milk is 3% w / w.

[0165] In this respect, it should be noted that these co-formulants include: protective agents (ascorbic acid is an antioxidant and polyvinylpyrrolidone (PVP) protects against heat), carriers, and nutrients (e.g., talc, vermiculite, peat, skimmed milk, glycerol, sucrose). However, some elements can have a dual role. For example, glycerol is both a nutrient and a protective agent against drying, and gum arabic is often considered a fixing agent but also sometimes a carrier.

[0166] In view of the foregoing, it is understood that another aspect of the invention relates to a coated seed comprising a plant seed, said plant seed being coated by the phytostimulant coating composition as described above,

[0167] in particular said plant seed belonging to a plant of the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0168] According to another embodiment, the invention relates to coated seed as described above, said seed being a plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn, or sesame. In particular, the invention relates to coated seed as described above, said seed being a plant belonging to a plant selected from wheat, rice, maize, barley, millet, rye, spelt, einkorn, and sesame.

[0169] According to another embodiment, the invention also relates to the coated seed as described above, in which the concentration of the isolated strain of Chitinophaga pinensis is from 106 to 108 CFU / seed.

[0170] Furthermore, since the coated seed of the invention comprises the isolated strain of Chitinophaga pinensis of the invention via the coating which is made from / thanks to the The phytostimulant coating composition as described above may have its specific characteristics applicable (see above). In other words, and according to another embodiment, the invention relates to the coated seed as described above, in which said isolated strain of Chitinophaga pinensis comprises at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from SEQ ID NOs 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence. Etc.

[0171] According to another aspect of the invention, it relates to a method for protecting and / or treating water stress suffered by a plant, comprising applying the phytostimulant composition as described above to at least a part of said plant or of the soil near said plant,

[0172] in particular said part of said plant being a leaf, a fruit or a seed, and

[0173] in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0174] According to another embodiment, the invention relates to a method for protecting a plant from water stress, comprising applying the phytostimulant composition as described above to at least a part of said plant or to the soil near said plant,

[0175] in particular said part of said plant being a leaf, a fruit or a seed, and

[0176] in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0177] According to another embodiment, the invention relates to a method for treating water stress suffered by a plant comprising applying the phytostimulant composition as described above to at least a part of said plant or of the soil near said plant,

[0178] in particular said part of said plant being a leaf, a fruit or a seed, and

[0179] in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0180] According to another embodiment, the invention relates to a method for protecting and treating water stress suffered by a plant, comprising applying the phytostimulant composition as described above to at least a part of said plant or of the soil near said plant,

[0181] in particular said part of said plant being a leaf, a fruit or a seed, and

[0182] in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0183] By "process for protecting against water stress suffered by a plant" is meant a process which, once implemented, reduces the negative impact of water stress on the plant growth and development.

[0184] By "process for treating water stress suffered by a plant" is meant a process which, once implemented, allows the plant to better overcome the consequences of a lack of water.

[0185] Furthermore, and according to another embodiment, the invention relates to the process as described above, said part of said plant being a leaf, a fruit or a seed.

[0186] According to another embodiment, the invention also relates to the process as described above, said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, or sesame. In particular, the invention relates to the process as described above, said plant being selected from wheat, rice, maize, barley, millet, rye, spelt, einkorn or small spelt, and sesame.

[0187] According to another aspect of the invention, it relates to a method for stimulating the assimilation of minerals by a plant comprising the application of the phytostimulant composition as described above to at least a part of said plant or of the soil near said plant,

[0188] in particular said part of said plant being a leaf, a fruit or a seed, and

[0189] in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

[0190] By "process for stimulating the assimilation of minerals by a plant" is meant a process which, once implemented, promotes (stimulates) the growth and development of the plant.

[0191] Furthermore, and according to another embodiment, the invention relates to the process as described above, said part of said plant being a leaf, a fruit or a seed.

[0192] According to another embodiment, the invention also relates to the process as described above, said plant belonging to the Poaceae family such as wheat, rice, maize, barley, millet, rye, spelt, einkorn, or sesame. In particular, the invention relates to the process as described above, said plant being selected from wheat, rice, maize, barley, millet, rye, spelt, einkorn, and sesame.

[0193] Furthermore, it should be noted that in particular the invention relates to one of the methods as described above, in which said phytostimulant composition is spread

[0194] at a rate of 50 to 200 L / ha, particularly 80 to 100 L / ha, especially for aerial spraying; or at a rate of an effective quantity of the isolated strain of Chitinophaga pinensis including 1010 to 1014 UFC / ha, in particular including 1011 to 1013 UFC / ha, especially for aerial applications of the foliar spray type; or - at an effective quantity of the isolated strain of Chitinophaga pinensis including 105 to 109 UFC / plant, in particular including 106 to 108 UFC / plant, especially for aerial applications of the foliar spray type.

[0195] By "at a rate of 50 to 200 L / ha", it is understood that the plant growth stimulant composition of the invention is applied at a rate of 50 to 200 L / ha. This also means that this rate can be 50 to 150 L / ha, 100 to 200 L / ha, 80 to 100 L / ha, 50 to 100 L / ha or even 100 to 150 L / ha.

[0196] By "between 1010 and 1014 CFU / ha", it is meant that the Chitinophaga pinensis strain is applied at a dose of between 1010 and 1014 CFU / ha. This also means that this dose may be between 1010 and 1013 CFU / ha, between 1010 and 1012 CFU / ha, between 1010 and 1011 CFU / ha, between 1011 and 1014 CFU / ha, between 1012 and 1014 CFU / ha, between 1013 and 1014 CFU / ha or even between 1011 and 1013 CFU / ha.

[0197] By "between 105 and 109 CFU / plant", it is meant that the Chitinophaga pinensis strain is applied at a dose of between 105 and 109 CFU / plant. This also means that this dose may be between 105 and 108 CFU / plant, 105 and 107 CFU / plant, 105 and 106 CFU / plant, 106 and 109 CFU / plant, 107 and 109 CFU / plant, 108 and 109 CFU / plant, 106 and 108 CFU / plant or even 106 and 107 CFU / plant.

[0198] By way of illustration, the density ranges from 10⁵ plants / ha (e.g., maize) (100,000 plants per hectare) to 2,10⁶ plants / ha (e.g., wheat) (2,000,000 plants per hectare), given that the concentrated bacterial product (see the concentrated phytostimulant composition of the invention – see above) is used at a rate of 1 kg / ha dissolved in 50 to 100 L of water. Furthermore, since the concentrated phytostimulant composition contains up to 10¹² CFU / g of Chitinophaga pinensis, this means that there are at least 10¹⁵ CFU per kilogram of concentrated product. If this kilogram is taken up in 100 L of water, which 100 L are spread over one hectare, then this corresponds to a concentration of Chitinophaga pinensis of 1015 CFU / ha or 1010 CFU / plant if it is maize and 5.108 CFU / plant if it is wheat.That being said, and given that there is some product loss due to successive handling and spreading techniques, it is likely that ultimately the effective quantity spread of the Chitinophaga pinensis strain will be less and correspond to that previously described.

[0199] According to another embodiment, the invention also relates to one of the methods as described above, in which

[0200] - the stimulation of said plant includes spraying said phytostimulant composition at the time of sowing or germination of said plant; and / or

[0201] - the protection of said plant includes spraying said phytostimulant composition at the time of sowing or germination of said plant; and / or - the treatment of said plant includes spraying said phytostimulant composition during different phenological stages of the plant.

[0202] Furthermore, insofar as the methods of the invention include the use of the isolated Chitinophaga pinensis strain of the invention via the application of the phytostimulant composition as described above, the particular characteristics thereof may apply thereto (see above). In other words, and according to another embodiment, the invention relates to one of the methods as described above, in which said isolated Chitinophaga pinensis strain comprises at least one enzyme whose coding nucleic acid sequence has at least 84% identity with a reference sequence selected from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence. Etc.

[0203] In view of the foregoing, it is also understood that another aspect of the invention relates to a new isolated strain of Chitinophaga pinensis, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs: 63 to 70 sequences, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0204] By "isolated strain" is meant the culture of a single microorganism that has been isolated from different microorganisms present on and / or in a sample.

[0205] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence encoding it has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0206] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs: 63 to 70 sequences. In particular, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence corresponds to the SEQ ID NO: 69 sequence, which codes for the enzyme of sequence SEQ ID NO: 77.

[0207] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose coding nucleic acid sequence is selected from the SEQ ID NOs sequences: 63 to 70. In particular, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising an enzyme whose nucleic acid sequence corresponds to the SEQ ID NO sequence: 69, which codes for the enzyme of sequence SEQ ID NO: 77.

[0208] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least two enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0209] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least two enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0210] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least three enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0211] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least three enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0212] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least four enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0213] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least four enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0214] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them have at least 84% identity with a reference sequence selected from the SEQ ID sequences NOs: 63 to 70, the said at least five enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0215] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least five enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0216] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least six enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0217] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least six enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0218] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least seven enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0219] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least seven enzymes whose nucleic acid sequences encoding them are chosen from the SEQ ID NOs sequences: 63 to 70.

[0220] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them have an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least eight enzymes retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence.

[0221] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least eight enzymes whose nucleic acid sequences encoding them correspond to the SEQ ID NOs sequences: 63 to 70.

[0222] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at a genomic nucleic acid sequence having at least 95% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 22 to 61.

[0223] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 22 to 61.

[0224] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence having at least 80% identity with a sequence chosen from the contigs of SEQ ID NOs sequences: 1 to 21.

[0225] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising at least one genomic nucleic acid sequence selected from the contigs of SEQ ID NOs sequences: 1 to 21.

[0226] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence has at least 99% identity with the SEQ ID NO: 62 sequence.

[0227] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis comprising a 16S ribosomal DNA (rDNA) whose nucleic acid sequence is the sequence SEQ ID NO: 62.

[0228] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis being strain ARDCPP14 deposited on April 23, 2021 at the National Collection of Microorganism Cultures (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under number CNCM 1-5676.

[0229] According to another embodiment, the invention relates to the isolated strain as described above, said isolated strain of Chitinophaga pinensis being the ARDCPP14 strain filed on April 23, 2021 with the CNCM under number CNCM 1-5676 and / or one of its (functional) mutants.

[0230] By "one of its mutants" is meant functional mutant strains obtained by mutations or genetic manipulations of a reference strain, which in the context of the invention is the isolated Chitinophaga pinensis strain, being strain ARDCPP14, filed on April 23, 2021, with the CNCM under number CNCM 1-5676. By "functional mutant strains" is meant that these mutants retain the same physiological properties as the reference strain (i.e., CNCM 1-5676) or may even exhibit improved physiological properties. Also, one of the objects of The invention relates to an isolated strain of Chitinophaga pinensis, said isolated strain being a mutant strain of the isolated strain of Chitinophaga pinensis being strain ARDCPP14 filed on April 23, 2021 with the CNCM under number CNCM 1-5676.

[0231] Furthermore, and in view of the foregoing, it is understood that an object of the invention relates to the isolated strain of Chitinophaga pinensis as described above, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose coding nucleic acid sequence has an identity of at least 84% with a reference sequence chosen from the SEQ ID NOs sequences: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the aforementioned reference sequence,

[0232] said isolated strain of Chitinophaga pinensis being in particular strain ARDCPP14 filed on April 23, 2021 with the CNCM under number CNCM 1-5676.

[0233] In all respects, it should be noted that the various aspects of the invention, as well as the various embodiments thereof, are interdependent. These can therefore be combined in numerous ways to obtain preferred aspects and / or embodiments of the invention not explicitly described. This also applies to all the definitions provided in this description, which apply to all aspects of the invention and its embodiments.

[0234] In addition, the present invention is illustrated, but not limited to, by the following Figures and Examples. LIST OF FIGURES

[0235] [Fig.1]

[0236] Fig. 1 represents the production of indole-3-acetic acid (IAA) by the ARDCPP14 strain.

[0237] Concentration measured by spectrophotometry after addition of Salkowski reagent to the culture supernatant of the ARDCPP14 strain. The concentration was calculated after setting up an IAA standard curve, then reduced to the dry matter of bacteria contained in the initial liquid culture.

[0238] [Fig.2]

[0239] Fig. 2 represents the macroscopic visual aspect of exopolysaccharide (EPS) production by the ARDCPP14 strain.

[0240] [Fig.3]

[0241] Fig. 3 represents the appearance of a precipitate (indicated by an arrow) after the addition of 96% glacial ethanol, which demonstrates the production of exopolysaccharide (EPS) by the ARDCPP14 strain.

[0242] [Fig.4]

[0243] Fig. 4 represents the effects of the ARDCPP14 strain on the dry weight of grains, of wheat under water stress or not.

[0244] Each bar represents an increase (positive value) in grain dry weight in the presence of strain ARDCPP14, compared to the uninoculated control under the same watering or water stress conditions.

[0245] [Fig.5]

[0246] Fig. 5 represents the dry weight of wheat grains under water stress conditions or not, in the presence of the ARDCPP14 strain or not (NI: not inoculated), during two different trials (A (No. 3) and B (No. 4)).

[0247] [Fig.6]

[0248] Fig. 6 represents the pH, sucrose concentration and optical density at 600 nm measured during the growth of Chitinophaga pinensis ARDCPP14 strain in a 30 L fermenter. EXAMPLES

[0249] (I) Isolation and identification of the Chitinophasa pinensis strain ARDCPP14

[0250] Bacterial isolation

[0251] Soil samples were collected from the rhizosphere of various field crops in the Champagne-Ardenne region of France. Bacterial extraction was performed, and the bacterial strains were isolated and stored in cryotubes at -80°C.

[0252] Molecular identification of bacterial strains

[0253] After their isolation, the isolated bacterial strains were sent to Mi-crobesNG for sequencing. This was performed on the Illumina® NovaSeq 6000 instrument, and 16S ribosomal DNA (rDNA) identification was carried out using an in silico approach with the Barmap (BAsic Rapid Ribosomal RNA Predictor) tool. The resulting sequences were then aligned in the SINA rRNA database (Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucl. Acids Res. 41 (Dl): D590-D596.) to identify the isolated bacterial species.

[0254] Among these strains, the Chitinophaga pinensis ARDCPP14 strain was identified, and its sequencing allowed for the characterization of:

[0255] - the contigs of SEQ ID NOs sequences: 1 to 61; - 16S rDNA with sequence SEQ ID NO: 62; and - eight particular enzymes of SEQ ID NOs sequences: 71 to 78 whose open reading frame (ORF) includes SEQ ID NOs sequences: 63 to 70.

[0256] Furthermore, this was deposited on April 23, 2021 at the National Collection of Microorganism Cultures (CNCM; Institut Pasteur, 25-28 rue du Docteur Roux, F-75724 PARIS CEDEX 15) under the number CNCM 1-5676.

[0257] (II) Production of indole-3-acetate (IAA) by strain ARDCPP14

[0258] Materials & Methods

[0259] Bacterial strains were inoculated with 100 µL of bacterial solution into a culture tube containing 7 mL of TSB (Tryptic Soy Broth) rich medium, supplemented with tryptophan (Trp, 0.5 g L⁻¹). After overnight incubation at 110 rpm at 30°C, five milliliters of this pre-culture were transferred to 45 mL of TSB-Trp in a 500 mL Erlenmeyer flask. The cultures were incubated at 110 rpm at 30°C for 72 hours and then centrifuged for 10 minutes at 6000 g. The pellet was then used to obtain the dry mass, while the supernatant was used for quantitative IAA analysis. The determination of the IAA concentration was based on the method described by Gordon and Weber, 1951 (Gordon, SA, and Weber, RP (1951). Colorimetry Estimation of Inodoleacetic Acid. Plant Physiol. 26, 192-195). One milliliter of the supernatant was mixed with two milliliters of Salkowski reagent and then incubated at room temperature for 25 minutes.The optical density at 530 nm was then measured using a spectrophotometer. The concentration of IAA was determined using a calibration curve ranging from 0 to 100 qg.mL 1 and was correlated with the measured bacterial dry mass.

[0260] Results

[0261] As illustrated in [Fig.1], strain ARDCPP14 has been shown to be capable of producing IAA: 5.20 pg.mL 1 of IAA was measured in the supernatant, which corresponds to 2.27 pg of IAA per mg of dry biomass.

[0262] (III) Production of exopolysaccharide (EPS) by the ARDCPP14 strain

[0263] Materials & Methods

[0264] The bacterial strains were inoculated onto a Petri dish containing R2A solid medium, a mineral medium suitable for soil bacteria. The plates were then incubated at 30°C for 48 hours. Macroscopic observation allows for initial detection of EPS production. EPS-producing strains form smooth, convex, and mucoid colonies.

[0265] A validation of EPS production was then carried out on bacteria after growth at 30°C for 45 hours:

[0266] Composition of the culture medium:

[0267] - Sucrose 10 g / L - Soybean peptones 10 g / L KH2PO4 2.5 g / L NaCl 1 g / L

[0268] Growing conditions:

[0269] - 500 mL baffle-type vials - Agitation: 180 rpm - Temperature: 30°C - Sampling time: 45 hours

[0270] The bacterial culture was centrifuged at 16,000 RPM for 30 min and the supernatant was collected. Then, a volume of 96% glacial ethanol (stored at -20 °C) was added to a volume of the collected supernatant, and the mixture was kept refrigerated for 24 h, the time required for the reaction. If a white precipitate appears, this indicates that EPS produced by the bacterial strain is present in the supernatant.

[0271] Results

[0272] As illustrated in [Fig. 2], it has been demonstrated that the ARDCPP14 strain is capable of producing EPS, which was further confirmed by a precipitation test in 96% glacial ethanol. Indeed, the formation of a precipitate was observed, confirming the production of EPS by the ARDCPP14 strain (see [Fig. 3]).

[0273] (IV) Water stress of wheat - Effect of strain ARDCPP14

[0274] Materials & Methods

[0275] Bacterial culture and inoculation

[0276] The bacterial strains were inoculated with 100 qL of bacterial solution in a 500 mL Erlenmeyer flask containing 100 mL of TSB (Tryptic Soy Broth) rich medium. After 48 hours of incubation at 110 rpm at 30°C, the cultures were transferred to 50 mL tubes and centrifuged at 10,000 rpm for 10 minutes. The pellet was resuspended in a volume of sterile physiological saline to obtain a final concentration of 4 x 10⁸ CFU / mL (colony-forming units per milliliter). Each seed was inoculated with 250 qL of bacterial suspension, i.e., 10⁸ CFU / mL per seed.

[0277] Plant culture and stress conditions

[0278] Pots were filled with potting soil and 15 seeds were sown at a depth of 2–3 cm and inoculated with a bacterial culture (10⁸ CFU / seed). For each bacterial strain, 8 pots were prepared: 4 pots were well-watered throughout the experiment and 4 pots were subjected to drought stress. Uninoculated controls were also prepared, and the seeds were inoculated with 250 qL of physiological saline instead of the bacterial solution. The growing conditions were 14 hours of daylight (23°C) and 10 hours of darkness (18°C). The relative humidity was 60% and the light intensity was 25 klux per day. The plants were watered daily by sub-irrigation. Ten days after sowing, the number of seedlings per pot was reduced to 12 to homogenize the pots. When the wheat plants reached the flowering stage, drought stress was induced by completely stopping watering for 10 days.

[0279] Determination of plant growth promotion activity by yield measurements

[0280] Several parameters were measured and calculated to compare the yields of the different experimental conditions. First, the weight of the aerial parts was The stalk and ear were then separated. The kernels were harvested from the ears, dried for 48 hours at 50°C, weighed, and counted. For logistical reasons, all these measurements were taken for two ears, resulting in six measurements per jar. From these measurements, the weight of a thousand kernels was calculated.

[0281] Among these parameters, the dry weight of the grains is important since it reflects the improvement in crop production. Also, at the field level, the dry weight of the grains reflects the yield.

[0282] Statistical analysis

[0283] The collected data were analyzed using a one-way ANOVA. Means and standard errors for the performance parameters were calculated for 18 replicate values. Significant differences between treatments were analyzed based on ANOVA and Student's t-test at a significance level of p < 0.05.

[0284] Results

[0285] Treatment Water stress Non-inoculated ARDCPP14 Difference [%] ...................................... 'T. œ et c» ® CO * * * ■* * —s, > MM WM «4» r; © cm to mF io w> c*> G) « O) does not have m tri cri 4.55 5.15 12.5 !2 ® S3 citri tri ® L 8 “ 3 g X œ cm cry o © a ® *r cm b~ r- «O © 0> © © CM o tri fry » O æ N ® ri K. O © V htto MT CM CM<o c- r-~ o" tri uri r- T- ® C4 -f » * * 40 > MT gy G ® r- O c eo * « - CMr-<O o o> en cm xr « œ b~ CM t~* o“ Q cri CM CM © to m cm in ■■ v- j< o" rJ ” v- CM CM CM * b* 2 L. 2 ® fi® ~ en - CM « « CM O CN CM CM V CM S LO CM Cm O co S Rose Condition Not inoculated ARDCPP14 Difference (%) œ w <q <D cri ® m es £. b- « e>cm < co © to' <£> *other v- co ri in ri 5.53 6.06 CM O co S ©< / q>CM CM CM M' M © W « o œ k r- ri ri ri ri ri o œ œ c» » o © as O CO *" œ o sn co cm v-” O tri ri ci © NO) ® æ P'O 0378 Z&P8 tO xc O '”.<d CM CM" CM CM © Wî G> «0 os ob* © œ CM v" CM CM 'ri <O CD © <o CM CM 31,97 33,77** 5.6 27,44 29,78 8,5 29,32 30,99** 5,7 30,04 29.90 -0,5 23,91 24,07 0,7 28,54 29,70 4,0 Paramètres mesurés Essai No. Poids plante entière 1 2 3 4 _________5 <& 1 O 5 Nombre de grains 1 2 3 4 ________§ Moyenne Poids sec des grains 1 2 3 4 5 Mayenne Poids de mille grains 1 2 3 4 5 0) c c: £ O S . Table 1. Effect of the ARDCPP14 strain on wheat development in

[0286] normal watering conditions or conditions of water stress

[0287] Numbers in bold indicate that the results obtained with the ARDCPP14 strain are superior to those obtained with the uninoculated control under the same conditions. Numbers in italics refer to the mean value for each of the parameters measured over five trials. * p < 0.05; ** p < 0.01 and *** p < 0.001.

[0288] As illustrated above and in Figures 4 and 5, the presence of the ARDCPP14 strain has been shown to significantly improve wheat characteristics when subjected to water stress. This is also true when the wheat has been irrigated normally and is not suffering from any water stress.

[0289] Furthermore, it should be noted that similar results are obtained when the plant treated is barley.

[0290] In conclusion, the ARDCPP14 strain has demonstrated a role in the biostimulation of wheat and barley, on the one hand as a biofertilizing agent (i.e. improvement of grain dry weight (= yield) under normal conditions) and on the other hand as an agent for combating water stress (i.e. improvement of grain dry weight (= yield) under water stress conditions). (V) Phytostimulant coating composition

[0291] Materials & Methods

[0292] Bacterial must

[0293] Bacterial musts were supplied by the Biotechnology department of ARD.

[0294] The selected strain was Chitinophaga pinensis ARDCPP14. This strain was grown in a semi-defined medium, with sucrose as the main carbon source. Harvesting took place after a culture time ranging from 48 to 72 hours.

[0295] In detail:

[0296] A pre-culture was carried out under the conditions shown in Table 2, then a continuous-discontinuous culture, known as fed-batch culture, was carried out under the conditions shown in Table 3 and in a 30 L fermenter. During this fermenter culture, the pH, sucrose concentration and optical density at 600 nm were measured, which indicates the growth of the Chitinophaga pinensis ARDCPP14 strain (see [Fig.6]).

[0297] [Tables2] Pre-culture: 500 mL Erlenmeyer flask, 500 mL flasks, 1 baffle, culture medium: Sucrose 15 g / L, Soybean Peptones 10 g / L, KH2PO4 2.5 g / L, NaCl 1 g / L, MgSO4 1 g / L, medium volume 100 mL (x 3 flasks), temperature 30°C, initial pH 7.5, stirring 180 rpm on a 25 mm orbital shaker, inoculum quantity 800 pL, duration 20-24 h

[0298] Table 2. Culture conditions of the pre-culture in a 500 mL Erlenmeyer flask

[0299] [Tables3] Main culture: 30 L Fermenter - Fed-Batch Tank 30 L Fermenter Sartorius Biostat® Cplus Batch culture medium Sucrose 10 g / L, Soybean Peptones 5 g / L, (NH4)2SO4 2.0 g / L, KH2PO4 2.5 g / L, NaCl 1 g / L, MgSO4 1 g / L, trace element solution 0.1% (v / v) Fed-batch medium Batch medium concentrated 15 times Fed-batch type Flows Medium volume 15 L batch + 5 L fed-batch Temperature 30°C pH 7; Regulation with 15% NaOH + 10% H2SO4, pO2 maintenance >30%, pO2 cascade: increase agitation then aeration. Agitation: 400-800 rpm (1 Rushton propeller and 2 three-bladed propellers). Cover pressure: 1 bar. Aeration: 0.5 vvm (7.5 L / min) to 1 vvm (15 L / min). Inoculation rate: 300 mL (2% v / v). Batch duration: 24 h. Fed-batch duration: 24-48 h. Target sucrose: 10-15 g / kg.

[0300] Table 3. Culture conditions in a 30 L fermenter

[0301] Centrifugation: obtaining a bacterial cream

[0302] The bacterial musts were separated by centrifugation at 4°C. The centrifuge used was a Thermofisher® RC 12BP+ batch centrifuge. The centrifugation time of the musts was set at 1 hour and the acceleration speed at 6000 G.

[0303] At the end of this step, a bacterial pellet was obtained, which was called "bacterial cream".

[0304] Preparation of the phytostimulant coating composition

[0305] Since bacteria are very sensitive to drying, various co-formulants (including protective agents) were added to the bacterial cream to maintain better bacterial viability during drying and storage. These include glycerol, gum arabic, and polyvinylpyrrolidone (PVP). Also added was Ascorbic acid was added for its antioxidant role at the membrane level and protection of membrane integrity.

[0306] In order to facilitate the mixing of the co-formulants with the bacterial cream, these co-formulants were previously dissolved in the nutrient medium used for the production of the Chitinophaga pinensis ARDCPP14 strain to ensure maximum viability for storage and use.

[0307] The co-formulant solution was sterilized in an autoclave (121°C / 20 min) before use, and skimmed milk and ascorbic acid were added to prevent degradation. This solution was then added to the bacterial cream. To prepare this "bacterial cream / co-formulant solution" mixture, the bacterial cream obtained by centrifugation was placed in a glass bottle and the co-formulant solution was added at a 50 / 50 (w / w) ratio, then stirred on a stirring plate at 20°C for 30 minutes.

[0308] The list of co-formulants used and their doses are presented in Table 4 below.

[0309] [Tables4] Additive Co-formulant solution (concentration in % w / w) Bacterial cream with co-formulants (concentration in % w / w) Glycerol 4.2 PVP 4.2 Gum arabic 10.5 Skimmed milk 6.3 Ascorbic acid 0.04 0.02

[0310] Table 4. List of co-formulants used and their doses

[0311] (VI) Seed coating - coating by fluidized bed spray

[0312] Fluidization consists of passing air through a bed of supported particles The particles are suspended through a grid. Air ensures heat transfer to the surface of the particles and cyclic movement of the particles from the spray zone to the drying zone. Energy transfer efficiency is related to the height of the particle bed (dependent on the quantity and density of the support), the fluidizing air flow rate, and the humidity of the fluidizing air.

[0313] This type of technology allows for drying, coating, granulation or encapsulation.

[0314] For example, the Glatt GPCG3 apparatus can be used for the fluidized bed.

[0315] Materials & Methods

[0316] The fluidization coating process allows for continuous coating of the particles. This process was carried out in three stages:

[0317] - Fluidization of the support (seeds = seeds); - Spraying of the active ingredient (bacteria in cream form with added co-formulants = phytostimulant composition) coating the substrate; and - Drying and film-forming of the coating.

[0318] The coating material (i.e., plant growth stimulant composition) in liquid form was sprayed onto the substrate (seeds) using a spray nozzle. The bi-fluid nozzle ensured the passage of the plant growth stimulant composition (i.e., cream with added co-formulants) supplied by a pump and dispersed it into fine droplets using a flow of compressed air. The droplets came into contact with the surface of the substrate (i.e., seeds) and were deposited there, forming a coating film at the chosen drying temperature.

[0319] Throughout the coating process, parameters such as the fluidization air flow rate, the inlet and outlet air temperature and the phytostimulant composition incorporation rate were monitored and modified according to the behavior of the coated seeds in fluidization (see table 5).

[0320] [Tables5] Parameters Instructions Quantity of seeds processed per batch (kg) 1.5 Air inlet temperature (°C) 40 Product temperature (°C) 45 Air flow rate at start (m³ / h) 130 Air flow rate during coating (m³ / h) From 140 to 160 Air flow rate during drying (m³ / h) From 140 to 160 Spraying rate (kg / h) 0.8 Spraying pressure (bar) 1.5

[0321] Table 5. Selected fluidization and spraying parameters

[0322] For 1.5 kg of seeds, 300 g of phytostimulant composition were sprayed at a a flow rate of 0.3 kg / h on the seeds was followed by a drying phase at 30°C for a duration of 10 minutes to obtain a dry matter > 95%.

Claims

Claims

1. Use of an isolated strain of Chitinophaga pinensis as a biostimulation agent, in particular as an agent for combating water stress and / or as a biofertilizing agent.

2. Use of an isolated strain of Chitinophaga pinensis according to claim 1 in the prevention and / or treatment of water stress suffered by a plant, in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

3. Use of an isolated strain of Chitinophaga pinensis according to claim 1 for stimulating the assimilation of minerals by a plant, in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

4. Use according to any one of claims 1 to 3, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence which encodes it has an identity of at least 84% with a reference sequence chosen from the sequences SEQ ID NOs: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the above-mentioned reference sequence, in particular, said isolated strain of Chitinophaga pinensis being the strain ARDCPP14 deposited on April 23, 2021 at the CNCM under the number CNCM 1-5676.

5. Phytostimulant composition comprising: - an isolated strain of Chitinophaga pinensis as active ingredient; or - an isolated strain of Chitinophaga pinensis as first active ingredient in association with at least one other active ingredient chosen from: • another biostimulation agent; • a biocontrol phytopharmaceutical product; • a fertilizer; and • mixtures thereof, in particular, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence which codes it has an identity of at least 84% with a reference sequence chosen from the sequences SEQ ID NOs: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme coded by the above-mentioned reference sequence, in particular, said isolated strain of Chitinophaga pinensis being the strain ARDCPP14 deposited on April 23, 2021 at the CNCM under the number CNCM 1-5676.

6. Phytostimulant composition according to claim 5, comprising an effective amount of the isolated strain of Chitinophaga pinensis of from 106 to 1012 CFU / g, more particularly of from 108 to 1010 CFU / g, said phytostimulant composition optionally being obtained after a preparation step making it ready for spreading.

7. Phytostimulant coating composition comprising an isolated strain of Chitinophaga pinensis and optionally at least one fixing agent, in particular, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence which encodes it has an identity of at least 84% with a reference sequence chosen from the sequences SEQ ID NOs: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the above-mentioned reference sequence, in particular, said isolated strain of Chitinophaga pinensis being the strain ARDCPP14 deposited on April 23, 2021 at the CNCM under the number CNCM 1-5676.

8. Coated seed comprising a plant seed, said plant seed being coated with the phytostimulant coating composition according to claim 7, in particular said plant seed belonging to a plant of the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

9. A method of protecting and / or treating water stress suffered by a plant comprising the application of the phytostimulant composition according to claim 5 or 6 to at least a part of said plant or of the soil near said plant, in particular said part of said plant being a leaf, a fruit or a seed, and in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

10. A method for stimulating the assimilation of minerals by a plant comprising the application of the phytostimulant composition according to claim 5 or 6 to at least a part of said plant or the soil in the vicinity of said plant, in particular said part of said plant being a leaf, a fruit or a seed, and in particular said plant belonging to the Poaceae family such as wheat, rice, corn, barley, millet, rye, spelt, einkorn or small spelt, or sesame.

11. Method according to claim 9 or 10, wherein said phytostimulant composition is spread - at a rate of 50 to 200 L / ha, in particular 80 to 100 L / ha, in particular for aerial spraying; or - at a rate of an effective amount of the isolated strain of Chitinophaga pinensis of 1010 to 1014 CFU / ha, in particular 1011 to 1013 CFU / ha, in particular for aerial applications of the foliar spray type; or - at a rate of an effective amount of the isolated strain of Chitinophaga pinensis of 105 to 109 CFU / plant, in particular 106 to 108 CFU / plant, in particular for aerial applications of the foliar spray type.

12. A method according to any one of claims 9 to 11, wherein - stimulating said plant comprises spraying said phytostimulant composition at the time of sowing or at the germination of said plant; and / or - protecting said plant comprises spraying said phytostimulant composition at the time of sowing or at the germination of said plant; and / or - treating said plant comprises spraying said phytostimulant composition during different phenological stages of the plant.

13. Isolated strain of Chitinophaga pinensis, said isolated strain of Chitinophaga pinensis comprising at least one enzyme whose nucleic acid sequence which encodes it has an identity of at least 84% with a reference sequence chosen from the sequences SEQ ID NOs: 63 to 70, said at least one enzyme retaining the enzymatic activity of the enzyme encoded by the above-mentioned reference sequence, said isolated strain of Chitinophaga pinensis being in particular the strain ARDCPP14 deposited on April 23, 2021 at the CNCM under the number CNCM 1-5676.

Images