LU101518B1 - Orthosilicic acid-based complex - Google Patents

Abstract

The present invention relates to a solution comprising: between 20 and 50% by mass of said solution, of chitosan oligomers; - between 0.2 and 4% by mass of said solution, of ortho-silicic acid; - between 6 and 69.8% by mass of said solution, of a solvent; said chitosan oligomers having a degree of deacetylation greater than 50% and a degree of polymerization between 5 and 50.

Description

1 LU101518 /

Ortho-silicic acid-based complex /

TECHNICAL AREA :

The present invention relates to the cultivation of plants, and more particularly;

plant protection by biocontrol using ortho-acid complexes | silicic acid (H4SiO.). | STATE OF THE ART i

Biotic stress in plants is caused by living organisms, in | especially viruses, bacteria, fungi, insects, | arachnids and weeds.

Unlike abiotic stresses È caused by environmental factors such as drought and heat,;

the agents causing biotic stress directly deprive their host of its | nutrients, thus reducing its vigor, and in extreme cases resulting in their /

dead.

In agriculture, biotic stress is a major cause of losses} which can occur before or after harvest, and many pesticides are | employees to protect crops and minimize these losses.

Massive employment)

of pesticides induces pollution of soil, water and consumer products. |

In order to reduce the harmful impact of pesticides, other solutions such as; biocontrol can be considered.

Biocontrol is a set of i plant protection methods, based on the use of mechanisms | natural.

Alone or combined with other means of plant protection, biocontrol techniques are based on the mechanisms and interactions which | govern the relationships between species in the natural environment.

Thus, the principle of E biocontrol is based on managing the balance of populations of aggressors: rather than on their eradication. 7

Silicon (Si) is an important natural element which has several functions; in the metabolism of plant organisms.

By insulating and stiffening the |

/

2 LU101518 | wall of plant cells, silicon protects plants against excessive evaporation 1 of water through transpiration, thus helping to improve the supply of: plant nutrients.

The presence of silicon in the plant organism relieves it in the event of stress, for example during a period of drought.

From | moreover, the cell walls reinforced with silicon constitute a barrier; natural more resistant to fungal spores and parasites.

The walls ; cells also become more resistant to enzymes produced by | pathogens.

Silicon also reduces abiotic stress (stress: caused by external factors) for example by increasing the tolerance of /

plants at low temperatures, by intensifying their photosynthesis at low: light intensity, and by reducing the toxic effects of aluminum present in soils on root growth, silicon compounds being a | selective ligand for aluminum ions. / The use of water-soluble silicon compounds in the cultivation of | plants thus reduce their vulnerability to pests and diseases, as well as to; stress caused by adverse weather conditions.

Bring | silicon bio-available to crops reduces the use of pesticides,:

and constitutes a method of biocontrol. ;

The Most Easily Assimilated Form of Silicon by Living Organisms /

is orthosilicic acid (H4SiO4). It is known to those skilled in the art that the monomer of ortho-silicic acid (or MOSA: Monomer of Ortho-Silicic Acid) is not stable.

Even at low concentration (from |

2 mmol / L), the monomers react with each other and form dimers,] trimers, or even short chains called oligomers, linear.

This :

polymerization reaction decreases the bioavailability of silicon. ; Several compositions allowing the stabilization of ortho-silicic acid in the form of a monomer have been described. |

Application EP1 092 728 describes a composition comprising ortho- | silicic acid complexed with polypeptides.

The silicon content of this;

i

3 | LU101518 | composition is between 1 and 5%. Monomeric silicon presents itself! in stable but solid form, which prevents its assimilation by plants. :

US Pat. No. 7,915,198 describes a composition comprising bio- / silicon available in the form of silicic acid in the presence of boron in the form of boric acid, as well as the use of this composition to increase the resistance of plants to biotic or stress. abiotic.

Ortho-silicic acid;

is bio-available and such a composition allows an improvement in the / tolerance of the treated plants in the face of biotic stress.

However, the presence;

too much boron can cause the death of treated plants. ;

Application WO2019150382 describes a composition comprising silicic acid stabilized with fulvic acid, and also comprising acid / phosphonic.

This composition can be used in order to improve the resistance of plants to biotic stress.

However, the use of phosphorus i in agriculture is highly regulated, because too high a content of -

phosphorus in edible products can cause poisoning of | consumer. | This is why it remains interesting to develop new products | comprising stabilized orthosilicic acid in monomer form | thus guaranteeing the bioavailability of silicon to increase resistance:

plants under biotic and / or abiotic stress. OBJECT OF THE INVENTION |

An object of the present invention is to provide a solution allowing the | stabilization of ortho-silicic acid in monomeric form, increasing the bioavailability of silicon, such a solution being able to be used in biocontrol for plants. | GENERAL DESCRIPTION OF THE INVENTION]

In order to solve the above-mentioned problem, the present invention; proposes in a first aspect a new solution making it possible to stabilize Ë

|

4 LU101518 | orthosilicic acid in monomeric form, a form of silicon / easily assimilated by plants, by oligomers of chitosan. | Thus, there is proposed - according to the invention - a solution comprising: | between 20 and 50% by mass of said solution, of chitosan oligomers:;

between 0.2 and 4% by mass of said solution, of ortho-silicic acid; | between 6 and 69.8% by mass of said solution, of a solvent; ; said chitosan oligomers having a degree of deacetylation greater than | 50% and a degree of polymerization less than 50. In the context of the invention, a degree of deacetylation is understood as an average degree of deacetylation, and a degree of polymerization is understood as: an average degree of polymerization.

In the context of the invention, the notation%}

by mass means that reference is made to a percentage by mass, sometimes | also noted as% weight or% m. ;

The degree of deacetylation can be determined by titration /

potentiometric while the degree of polymerization of said oligomers of | Chitosan can be determined by thin layer chromatography. ;

Chitosan oligomers stabilize orthosilicic acid monomers;

through electrostatic interactions between the expressed positive charges}

by said chitosan oligomers and orthosilicic acid monomers.

AT

The chitosan oligomers and the orthosilicic acid monomers which they stabilize then form complexes.

A complex is preferably 'formed from a monomer of orthosilicic acid and several oligomers | of chitosan. ; ;

Chitosan (Formula 1b) is a polymer obtained by partial elimination of;

acetyl (deacetylation) groups of chitin (Formula 1a). Chitin is found abundantly in nature, and occurs primarily as; structural component in the cell walls of fungi, yeasts: as well as in the exoskeletons of insects and arthropods (eg |

:

LU101518 | crabs, lobsters and shrimps). Chitin is insoluble in water, that is | why its use in industry requires its transformation into. chitosan. |

Chitosan exhibits a variety of physicochemical properties and |

5 interesting biologics, which in combination with its non-toxicity, biocompatibility and biodegradability make it suitable for many applications, especially for agriculture, cosmetics, water treatment and medicine. ;

LQ NT qu bu: NA tor Poe] po I

Pc, + PCH, OH n;

(at) :

. oH Ong CH OH; NAT NA po 2;

Formula 1. a: Chitin; b: Chitosan: To use chitosan in agriculture, it is necessary to cut it into smaller units called chitosan oligomers (COS), since it thus exhibits interesting antifungal and eliciting properties.

These oligomers: are prepared from chitosan either chemically by acid hydrolysis or /

by enzymatic hydrolysis with glycosylhydrolases such as chitinases / or chitosanases. |

Preferably, orthosilicic acid is present as a monomer. According to a preferred embodiment, the ortho-silicic acid represents from 0.2 | at 4%, preferably from 0.5 to 2%, by weight of the solution.

Preferably, the ortho-silicic acid represents at least 0.2%; 0.5%; 0.8%; 1%; |

| LU101518 | 1.2%; 1.5%; 1.8%; 2%; 2.2%; 2.5%; 2.8%; 3%; 3.2%; 3.5%; 3.8% or 4% | by mass of the solution. / Preferably, the ortho-silicic acid represents at most 4%; 3.8%; | 3.5%; 3.2%; 3%; 2.8%; 2.5%; 2.2%; 2%; 1.8%; 1.5%; 1.2%; 1%; 0.8%: | 0.5% or 0.2% by mass of the solution. | Orthosilicic acid is stabilized by oligomers of chitosan. The | chitosan oligomers represent 20 to 50% by mass of the solution. / Preferably, the chitosan oligomers represent at least 20%; | 25%: 30%; 35%; 40%; 45% or 50% by mass of the solution. | Preferably, the chitosan oligomers represent at most 50%; | 45%; 40%; 35%; 30% ; 25% or 20% by mass of the solution.

The chitosan oligomers preferably have a degree of deacetylation | greater than 50%, 55%, 60%, 65%, 70%, 75%, or 80%. / The chitosan oligomers preferably have a degree of polymerization of | at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 20, 25 or 50. The chitosan oligomers preferably have a degree of polymerization of | plus 50, 25, 20, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6 or 5. / Their degree of polymerization is preferably between 5 and 17, | advantageously between 8 and 15. | The solvent is preferably chosen from water, ethanol or a mixture | water-ethanol. It represents between 6% and 69.8% by mass of the solution. | According to a preferred embodiment, the solution also comprises a | stabilization aid donor of hydrogen bonds, advantageously a | polyalcohol, preferably chosen from glycerol, sorbitol and maltitol, or | one of their mixtures. This stabilization aid represents | preferably 10 to 40% by mass of the solution. | Preferably, the stabilization aid represents at least 10%; | 15%; 20%; 25%; 30% ; 35% or 40% by mass of the solution. |

. LU101518 |

The stabilization aid preferably represents at most 40%; 35%; / 30% ; 25%; 20%; 15% or 10% by mass of the solution. |

A second object of the present invention is to provide a method of / preparation of a solution according to the invention based on ortho-silicic acid /

stabilized by chitosan oligomers. ] To obtain the solution according to the invention, the following protocol is implemented: implementation: /

- Solubilization of the chitosan oligomers in a solvent; |

- Solubilization of ortho-silicic acid in said solvent containing | said chitosan oligomers.

In the first step, the prepared chitosan oligomers are solubilized /

with a mineral acid, preferably chosen from hydrochloric acid | (HCD), sulfuric acid (H2SO4) and phosphoric acid (H:; PO,), or by a | organic acid, preferably chosen from citric acid, malic acid /

and succinic acid. / The ortho-silicic acid is added by a mineral source, preferably | in the form of potassium silicate (K; 0.Si0O,), or of sodium silicate (NazO.Si0 ,, or an organic source, preferably silicon tetrachloride (SiCly). The solubilization of ortho-silicic acid is preferably carried out with stirring of the solution. According to a preferred embodiment, the chitosan oligomers are prepared in two stages.

The first step consists of hydrolysis of | chitosan polymers chosen for their compatible degree of deacetylation | with the application, and the second step is to separate the oligomers as well | obtained according to their molecular weight and therefore their size. | The hydrolysis of chitosan can be chemical hydrolysis or hydrolysis | enzymatic, preferably enzymatic hydrolysis.

The latter | helps to minimize chemical changes that may take place during | hydrolysis. |

: P-FERTIX-002 / LU 8 LU101518 The invention also relates in particular to a solution according to the invention for use in a method of treatment by biocontrol of plants, preferably for combating biotic stress, said solution preferably being sprayed on the plants to be treated, in order to ensure a better | dispersion of the solution to all the plants to be treated. |

DESCRIPTION OF FIGURES Fig. 1: Comparison of the frequency of infection of leaf fragments infected then treated with a solution comprising ortho-silicic acid stabilized with chitosan oligomers, treated with a commercial antifungal or untreated. DESCRIPTION OF A PREFERRED EXECUTION The example below illustrates a formulation for the production of a solution comprising ortho-silicic acid stabilized with chitosan oligomers, as well as an application of said solution as a treatment for protection of plants against biotic stress. The solutions which are the subject of the invention and their use as a treatment for plants are not limited to this example. | Preparation of chitosan oligomers (COS) Different fractions of chitosan oligomers (COS) are prepared by enzymatic hydrolysis. 5g of commercial chitosan supplied by Jiangsu Aoxin Biotechnology Co., Ltd and chosen for its degree of deacetylation of 80% compatible within the scope of the invention are first of all mixed with 50 mL of a sodium acetate buffer containing l acetic acid in variable proportions (0.1 mol / L - 0.2 mol / L) to reach pH 5.0, then the chitosan is treated with a mixture of enzymes consisting of chitanases obtained from cultures of strains of Trichoderma and supplied by the Limoges Natural Substances Chemistry Laboratory (1% by weight relative to chitosan). The hydrolysis of the chitosan is carried out at 50 ° C. with stirring for 24 h. Once the reaction ee ee ee ee Te is completed, the pH of the solution is adjusted to 9.0 with a 0.2 mol / L solution of potassium hydroxide (KOH), then filtered. The solution is filtered by ultrafiltration (Mini Pellicon, Millipore Corporation, USA), the molecular weight of which cut-off / retention threshold of the molecules is 5000 Da. A first precipitation is obtained by adding six volumes of ethanol to the concentrated solution by evaporation under vacuum. After cenirifugation at 2700g, 60 ml of anhydrous ethanol was added in the supernatant, which was then centrifuged at 300g for 20 min to collect the chitosan oligomers (COS).

The degree of deacetylation of the initially employed chitosan polymers can be calculated by potentiometric titration. To do this, a fraction of the chitosan polymers are dissolved in excess acetic acid in order to form quaternary ammonium ions from the amine groups resulting from the deacetylation, then titrated with a solution of sodium hydroxide (basic). at a concentration of 0.1 mol / L. The volume of the basic solution used between the two inflection points observed on the titration curve is linked to the quantity of acid consumed for the salification of the amine groups resulting from the deacetylation and makes it possible to determine the degree of deacetylation of the chitosan.

The degree of polymerization and therefore the molecular weight of the chitosan oligomers obtained can be characterized by thin layer chromatography on gel plates sold by Merck (Merck 60. GF-254) using a mixture of n-propanol: water: concentrated ammonia 7: 2: 1 (v) as a solvent. After elution, the spots are revealed by carbonization using ethanol containing 10% by mass of HzSO 3 sulfuric acid. Each elution spot is identified by comparison with standard chitosan oligomers supplied by the Limoges Natural Substances Chemistry Laboratory. .

Preparation of an ortho-silicic acid solution stabilized with chitosan oligomers (MOSA-COS) -

300g of an aqueous solution of chitosan oligomers (COS) at 5% by mass and with a viscosity of less than 30 mPs are dissolved in a solution comprising 590 g of demineralized water, 50 g of succinic acid and 50 g of maltitol .

The mixture is maintained at a temperature between 0 ° C and 8 ° C in a refrigerated tank.

With vigorous stirring of this mixture using an Ultra Turrax type apparatus, 80mL of a solution of Si02, K; 0 of grade 30 | (SiO molar ratio, on K2O: 2.2) is added dropwise.

The complexes thus obtained are stable in solution and the solution containing these complexes retains a viscosity of less than 30 mPs at 25 ° C. for 2 years.

Preparation of the fungicidal treatments Solutions of MOSA-COS and MOSA at three different concentrations (respectively 1.7; 3 and 6 mmol / L) are prepared in Tween 20 at 0.1%. Solutions are also prepared from commercial fungicides by dilution in 0.1% Tween 20 (Bion, 0.3 mmol / L and Actiol, 10 L / ha). Sufficient volumes of solutions are prepared to be able to subsequently treat the samples with a volume corresponding to 200L / ha.

Application of fungicide treatments 3 cm long fragments of the 1st leaf are taken from 6-row winter barley seedlings (Champie variety, supplier Florimond Desprez) at the 2-leaf stage (BBCH development stage 12). These leaf fragments are then plugged into agar water in Petri dishes.

The agar water contains an anti-senescence hormone in order to keep the leaf fragments green throughout the duration of the study.

Each 120 x 120 cm Petri dish contains 22 leaf fragments and is then treated with 0.1% Tween 20 (control) or one of the fungicide treatments previously prepared, using a hand sprayer set to 2 bars.

For each modality tested, 2 repetitions (Petri dishes) of 22 leaf fragments are analyzed. ee

"LU101518 After treatment, the Petri dishes are placed in incubation in a climatic chamber regulated as follows: photoperiod: 16h of light / 8h of darkness, 20 ° C during the day / 17 ° C at night, and a relative humidity of 70%.

The leaf fragments not treated or treated with the various products are then inoculated with B. graminis f. sp. hordei 3 or 5 days after treatment. To do this, the Petri dishes are opened and placed for% h in an inoculation tower above which barley seedlings will be shaken | heavily contaminated with B. graminis f. sp. Hordei.

After inoculation, the Petri dishes are closed and then placed in incubation | in a climatic chamber set as follows: photoperiod: 16h light / 8h | darkness, 20 ° C during the day / 17 ° C at night, and relative humidity | by 70%.

Evaluation of the fungicidal activity of the treatments The leaf fragments are observed after 11 days of incubation in order to determine the frequency of infection (number of leaf fragments showing symptoms of barley powdery mildew) and the intensity of infection (leaf area covered by pustules of powdery mildew compared to the total leaf area).

; The effectiveness of the various fungicide treatments is calculated with respect to; 20 control treated only with 0.1% Tween 20.

; The results of the frequencies and intensities of infection obtained for each; modalities tested are compared using the Newman-Keuls test (XL-Stat | software. Addinsoft Lid.) and are presented in Tables 1 (inoculation 3; days after treatment) and 2 (inoculation 5 days after treatment). In these / 25 tables, the values of the same column followed by the same letter are not | not significantly different according to the Newman-Keuls test (p <0.05), and the | values in brackets correspond to fungicidal efficiencies in percent; of the untreated witness. ———— ra RE AND AND

Said Tables 1 and 2, as well as Figure 1, indicate that after 11 days, the frequency and intensity of infection are lower for the plants treated with the MOSA-COS solution (containing the ortho -silicicum stabilized by chitosan oligomers) than for plants treated with MOS or Bion solutions. oon Pom | rss | Table 1 Table 1 shows the comparison of the frequency of infection and the intensity of infection of leaf fragments of barley untreated or treated with [different products and then inoculated 3 days after treatment with B. graminis f. sp.

Hordei under controlled conditions.

The values of the same column followed by the same letter are not significantly different according to the Newman-Keuls test (P <0.05). The values in brackets correspond to the fungicidal efficiencies in percent of the untreated control _û…

[Umar | wien | 880526 | Table 2 Table 2 shows the comparison of the frequency of infection and the intensity of infection of leaf fragments of barley untreated or treated with different products and then inoculated 5 days after treatment with B. graminis f. sp.

Hordei under controlled conditions. The values of the same column followed by the same letter are not significantly different according to the Newman-Keuls test (P <0.05). The values in brackets correspond to the fungicidal efficiencies in percent of the untreated control TESTER ER]

Claims (15)

| lu101518 Claims 1. Solution comprising: - between 20 and 50% by mass of said solution, of chitosan oligomers; - between 0.2 and 4% by mass of said solution, of ortho-silicic acid; - between 6 and 69.8% by mass of said solution, of a solvent; said chitosan oligomers having a degree of deacetylation ‘greater than 50% and a degree of polymerization between 5 and 50.: 2. Solution according to Claim 1, characterized in that the orthosilicic acid is present in the form of a monomer. ; 10 3. Solution according to Claims 1 and 2, characterized in that the ortho-silicic acid represents between 0.5 and 2%, by mass of the said solution. . AT. Solution according to any one of the preceding claims, characterized in that the chitosan oligomers have a degree of deacetylation greater than 80%. : 15 5. Solution according to any one of the preceding claims, characterized in that the chitosan oligomers have a degree of; polymerization between 5 and 17, advantageously between 8 and 15. | 6. Solution according to any one of the preceding claims; characterized in that the chitosan oligomers are obtained by! Chemical hydrolysis or enzymatic hydrolysis of chitosan. ; 7. Solution according to any one of the preceding claims, | characterized in that the solvent is selected from the group consisting of | water, ethanol and water-ethanol mixtures. | 8. Solution according to any one of the preceding claims, ‘25 characterized in that it further contains a stabilization aid, ee | lu101518 advantageously a polyalcohol, preferably chosen from the group consisting of glycerol, sorbitol, maltitol and mixtures thereof. 9. Solution according to claim 8, characterized in that the stabilization aid represents between 10 and 40% by mass of said solution. 10.Process for preparing a solution according to one of claims 1 to 9 comprising the following steps: - Solubilization of chitosan oligomers in a solvent; - Solubilization of ortho-silicic acid in a solution formed by said chitosan oligomers and said solvent. 11.Procédé according to claim 10, characterized in that the chitosan oligomers are solubilized with an acid selected from the group consisting of hydrochloric acid (HCl), sulfuric acid (H2SQ4), phosphoric acid ( H3: PO4), citric acid, malic acid, succinic acid, alone or in mixtures. 12.Procédé according to claim 10 or 11, characterized in that the ortho-silicic acid is provided by a source selected from the group consisting of potassium silicate (K2O.Si0 "), sodium silicate (Na: O. Si0>), and silicon tetrachloride (SiCls). 13. A method according to any one of claims 10 to 12 comprising a step of adding a stabilization aid, advantageously a | polyalcohol, preferably chosen from the group consisting of glycerol, sorbitol, maltitol or mixtures thereof. 14. A solution according to any one of claims 1 to 9 for use in a method of biocontrol treatment of plants. | 15. Solution according to any one of claims 1 to 9 for use according to claim 14 characterized in that said solution is sprayed on the plants to be treated. | TTT ee SE