FR3122178A1 - Plant treatment composition containing microbiota from millennial trees of the species Olea europaea L. for use in increasing bioactives and metabolites in plants. - Google Patents

Abstract

The subject of the present invention is a plant treatment composition containing the microbiota originating from ancient trees of the species Olea europaea L. and RNA extracted from this same microbiota, plant extracts rich in polyphenols for its use in the increase in vitamins, minerals in plants as well as for its use in the prevention of contamination by pathogenic bacteria, characterized in that the said composition contains at least one bacterium of the Bacteroidetes type. The vaporization or the application of the said composition on the plants increases the growth of the plants, increases the concentration of polyphenols in the leaves and the fruits and allows the appearance of new molecules resulting from the transformation of molecules already present in the plant by the present composition.

Description

Plant treatment composition containing microbiota from millennial trees of the species Olea europaea L. for its use in increasing bioactives and metabolites in plants.

The present invention relates to a plant treatment composition for its use in increasing the concentration of vitamins, minerals, fatty acids and polyphenols in plants and fruits as well as for its use in the prevention of contamination by bacteria. and pathogenic fungi, characterized in that it contains the microbiota from ancient trees of the species Olea europaea L., RNA extracted from this same microbiota and plant extracts rich in polyphenols.

We know that the microbiota is a group of microorganisms made up of bacteria, yeasts, fungi or viruses, living in a specific environment called the microbiome, in a host. Thus, humans and animals have several microbiota depending on the area of their body concerned. Plants also have a microbiota which consists in particular of soil micro-organisms, with which they live in symbiosis. The plant microbiota is made up of many and varied species of microorganisms, which perform several essential functions for the plant. The microbiota forms a symbiotic relationship with the roots of plants that allow them both to benefit from their environment but also to enrich it. The root system of plants explores the soil for nutrients and is therefore exposed to the various microorganisms present in the soil. Plants provide sugar to microorganisms, while microorganisms provide nitrogen and phosphorus, essential for the proper development of plants. Microorganisms also produce plant hormones that stimulate plant growth.

It is known that the microbial population can be modified by the environment, in particular by the nutrient content of the soil. Indeed, the environment from which the plants come is decisive for the micro-organisms. Each environment constitutes a unique local ecosystem. The weather, altitude, other species surrounding the plants of interest as well as the type and depth of the soil will influence the microbiota. These effects interact with each other and influence in particular the proliferation of certain microorganisms to the detriment of others. A unique ecosystem and adapted cultivation methods can therefore produce a unique microbiota.

Thus, several plant growth-promoting bacteria have been found in the rhizosphere of thousand-year-old trees of the species Olea europaea L. growing in plantations that do not use pesticides or fertilizers in the Alicante region, of the type Lactobacillus , Bifidobacterium, Pseudomonas Japonica, Pseudomonas fluorescens, archaeospora trappei or even Flavimonas . The rhizosphere is the soil region close to the roots, very rich in micro-organisms and biological substances. Given the longevity of thousand-year-old trees, the bacteria in the rhizosphere have undergone natural selection undisturbed by human action; thus the bacteria in the rhizosphere of these millennial trees are strong and beneficial bacteria. The rhizosphere of these millennial trees is therefore very rich and diverse, a direct consequence of their longevity and the unique conditions in which they have developed.

This microbiota also produces molecules (vitamins, metabolites) or dissolves minerals which are then absorbed in plants and incorporated into organic matter and in particular into fruits and seeds. A rich and balanced soil microbiota thus makes it possible to produce fruits, vegetables or seeds rich in bioactives with a beneficial effect on human health.

These exchanges between plants and micro-organisms are therefore essential in the health and quality of the soils of our crops since a diversified microbiota protects the plant and enriches it. But today because of the development of intensive agriculture, monoculture and the massive use of pesticides, the soils are generally greatly impoverished in microorganisms. The absence of symbiosis between microorganisms and plants reduces the production of metabolites by these bacteria, which directly influences not only plant growth but also the quality of harvests since the fruits, vegetables and seeds produced contain fewer bioactives. of interest, i.e. molecules that are beneficial to consumer health.

Fertilizer-based solutions containing bacteria of the genus Bacillus (WO2017105238A1) or even Bacillus , Pseudomonas , (WO2015118516A1) intended to fertilize the soil and allow faster growth of plants have been proposed. However, these solutions have a limited field of action, they make it possible to replace the action of chemical substances traditionally used to accelerate plant growth without improving the concentration of bioactives in the plants. In addition, the effectiveness of these solutions is often limited by the low persistence of bacteria in the soil.

It was by studying the effect of a fermented Olea europaea L. leaf extract on plant growth that the applicant discovered quite unexpectedly that these problems can be easily solved by the use of a composition intended to increase the concentration of polyphenols in plants containing a mixture of bacteria originating from the extraction of the microbiota of ancient trees of the species Olea europaea L. RNA extracted from bacteria of this same microbiota and plant extracts rich in polyphenols.

The object of the present invention therefore relates to a composition intended to increase the concentration of vitamins, minerals and polyphenols in plants, characterized in that it comprises a mixture of bacteria originating from the extraction of the microbiota from ancient trees of the species Olea europaea L., RNA extracted from bacteria of this same characterized microbiota and plant extracts rich in polyphenols.

It should be noted that the composition is characterized in that it is used in the increase of bioactives and metabolites in plants. The invention also relates to a composition characterized in that it is used in the prevention of the contamination of plants by pathogenic bacteria.

It should be noted that the composition is characterized in that the spraying or application of said composition on the plants or on the soil surrounding the plants increases the growth of the plants. The invention also relates to a composition characterized in that the vaporization or application of said composition to plants maintains the mycorrhizal network and reduces infections by pathogenic bacteria and fungi.

For the clarity of the following explanations we have decided to call OXYBIOTA a composition intended to increase the concentration of polyphenols in plants containing a mixture of bacteria from the extraction of the microbiota of ancient trees of the species Olea europaea L., RNA extracts of bacteria from this same characterized microbiota and plant extracts rich in polyphenols

The applicant describes below a composition intended to increase the concentration of polyphenols in plants containing a mixture of bacteria originating from the extraction of the microbiota from ancient trees of the species Olea europaea L., RNA extracted from bacteria of this same microbiota characterized and plant extracts rich in polyphenols

To do so, the applicant has developed a composition containing a mixture of bacteria originating from the extraction of the microbiota from thousand-year-old trees of the species Olea europaea L., RNA extracted from bacteria of this same characterized microbiota and plant extracts rich in polyphenols. Bacteria can act as catalysts in certain metabolisms, in particular the metabolism of polyphenols. Polyphenols are secondary metabolites that are found in large quantities in plants, from the root to the fruit. These polyphenols can be: catechins, flavonoids, anthocyanins, ellagic acid, resveratrol, coumarin. Secondary metabolites do not play a direct role in plant growth and reproduction mechanisms but can still play an important role. In the case of polyphenols, they allow the plant to protect itself against UV rays, parasites or even pathogens. They also have strong antioxidant power.

Polyphenols are a category of organic molecules very present in plants and characterized by the presence of several phenolic groups in their structure. The primary role of polyphenols is to help plants in their survival, because they allow the plant to defend itself against attacks from its environment. Polyphenols include molecules such as coumarin, as well as categories of molecules such as flavonoids or lignin, which has a structural effect. Lignin accumulates in the structure of the plant, giving it its rigidity and its capacity for unlimited growth. Other types of polyphenols, such as flavonoids, have varying effects on plants. These molecules notably give plants their color, aroma and fragrance, and make it possible to attract pollinating animals but also to protect against the harmful effects of UV rays. Coumarin participates in the defense mechanism against pathogens and herbivores. These polyphenols also influence the microbiota and soil fauna that break down organic matter in plants. Polyphenols therefore indirectly affect soil nutrient fluxes and ultimately carbon turnover and ecosystem functioning in soils. Polyphenols are also recognized as molecules of interest for human health. Indeed, they possess antioxidant and anti-inflammatory properties as well as recently recognized specific biological properties associated with the prevention of chronic diseases such as cardiovascular diseases, various cancers and type II diabetes. (Cory, Hannah et al. “The Role of Polyphenols in Human Health and Food Systems: A Mini-Review.” Frontiers in nutrition vol. 5 87. 21 Sep. 2018). The consumption of plants rich in polyphenols is important for the maintenance of human health.

The microbiome of millennial olive trees is unique. The longevity of thousand-year-old trees has enabled natural selection which has made it possible to select bacterial strains enabling thousand-year-old trees to resist abiotic stress (for example exposure to ultraviolet rays, soil erosion) and biotic stress (attacks by insects, nematodes and pathogenic bacteria). The strains of bacteria isolated from millennial olive trees are unique compared to strains of the same species isolated from other tree species. The applicant carried out her studies using 15 thousand-year-old olive trees. Species common to the microbiota of all trees are attributed as the species responsible for the health and longevity of patients. These species have the most effective properties stimulating plant growth and protecting trees against pathogenic bacteria. The bacteria are isolated in the rhizosphere, leaves and trunk of millennial olive trees. Species were identified by PCR followed by 16s rRNA sequencing. PCR or polymerase chain reaction is a technique for the amplification of DNA fragments in vitro. DNA amplification allows for easier sequencing and better species identification. 16s rRNA is an RNA fragment possessed by all species of bacteria with sequence variations characteristic of each species. The sequences obtained are compared to a ribosomal sequence database. The samples used therefore contain at least one or more strains of the type Pseudomonas japonica, Pseudomonas fluorescens, Archaeospora trappei, Bacillus methylotrophicus, Pseudomonas putida, Flavimonas oryzihabitans, Acinetobacter haemolyticus, Rhizobium radiobacter, Bacillus atrophaeus, Bacillus subtilis, Bacillus megaterium, Lactobacillus paracasei, Serratia marcescens, Arthrobacter globiformis .

The RNAs of the composition described in the present invention are isolated from the same bacteria described above. To do this, a culture of the bacteria is lysed and the RNAs are isolated either by selective binding of the nucleic acids on a silica membrane in a purification column, or by extraction with a mixture of aqueous solvent phenol and chloroform and by centrifugation.

All of the RNAs can be used or only the non-coding RNAs. In this case, the non-coding RNAs are purified by isolating the coding RNAs by chromatography using paramagnetic beads comprising a sequence of nucleotides which can bind to all the coding RNAs. RNAs of interest can be isolated by amplification using specific primers that will allow the targeted replication of certain RNAs. The RNAs are at a concentration in the present invention of at least 0.001% by mass and preferably at least 0.005% by mass relative to the total mass of said composition.

The object of the present invention is therefore described as a composition containing a mixture of bacteria originating from the extraction of the microbiota from ancient trees of the species Olea europaea L. and RNA extracted from bacteria of this same microbiota, characterized in that at least one bacterium either of the genus Pseudomonas japonica, Pseudomonas fluorescens, Bacillus methylotrophicus, Pseudomonas putida, Flavimonas oryzihabitans, Acinetobacter haemolyticus, Rhizobium radiobacter, Bacillus atrophaeus, Bacillus subtilis, Bacillus megaterium, Lactobacillus paracasei, Serratia marcescens, Arthrobacter globiformis .

The composition described in the present invention also contains plant extracts rich in polyphenols. The plants are taken from the following list without this being exhaustive: pomegranate, olive, rosemary, rose, lemon, cherry, blackcurrant, grapefruit, fig, loquât, strawberry tree, apple, peach, apricot, melon, watermelon, cactus, grape. These plant extracts can be in the form of plant powder which is dried beforehand and then pulverized, aqueous extracts, hydroalcoholic extracts, plant extracts which have undergone fermentation, plant extracts which have undergone supercritical CO2 extraction. Polyphenols can be: catechins, flavonoids, anthocyanins, ellagic acid, resveratrol, coumarin. For optimum effectiveness, the plant extracts are at a concentration in the present invention of at least 1% by mass and preferably at least 10% by mass relative to the total mass of said composition. The polyphenols are therefore at a concentration in the present invention of at least 0.5% and preferably of at least 5% by mass relative to the total mass of said composition.

Mycorrhizal networks are networks created by fungi that link trees together and increase the rate of plant growth, resistance to insects and pathogens and increase the survival rate of seedlings. The mycorrhizal network includes glomeromycetes and ectomycorrhizal fungi, e.g. Glomus mosseae, Glomus intraradices, Funneliformis mosseae

This network allows plants to transfer between them nutrients necessary for their survival, allowing them to colonize unfavorable places. This mycorrhizal network also helps to prevent surrounding plants from attack by insects or pathogens (Gorzelak, Monika A et al. “Inter-plant communication through mycorrhizal networks mediates complex adaptive behavior in plant communities.” AoB PLANTS vol. 7 plv050). Mycorrhizal networks also modulate gene expression in plants. The colonization of plants by the mycorrhizal network thus stimulates the expression of genes related to the biosynthesis of polyphenols.

Thus certain bacteria of the genus Parabacteroides such as the bacterium Parabacteroides distasonis , maintain the mycorrhizal network of the plant. The mycorrhizal network is essential in the mineral nutrition of the plant, it also allows a better tolerance or resistance of the plant vis-à-vis biotic stresses .

Bacteria and fungi are able to transfer nutrients to plants but also transmit RNA (Ribonucleic acid). RNAs are a sequence of nucleotides and are intermediate supports of genes to synthesize the proteins that the cell needs. Non-coding RNAs are small RNAs between a hundred nucleotides and several kilobases and which will not be transcribed into proteins. These non-coding RNAs can interfere with gene transcription by targeting coding RNAs. In particular, non-coding RNAs from bacteria can penetrate plant cells and modify gene expression which will impact plant growth and the immune system.

An interaction with a bacterium and a fungus is not always symbiotic; Plants only tolerate the invasion of these microorganisms under conditions of nitrogen or nutrient deficiency. To defend itself during an interaction with bacteria and fungi, the plant's immune system is activated through salicylic acid, SA is a hormone involved in the activation of plant defenses in response to pathogens.

RNAs from bacteria are able to increase the gene expression of auxin, a hormone that stimulates plant growth, but also inhibits the plant defense system. Camilo López, Boris Szurek and Álvaro L. Perez-Quintero (September 17th 2012). Small Non-Coding RNAs in Plant Immunity, Plant Science, Nabin Kumar Dhal and Sudam Charan Sahu, IntechOpen), which has the effect of improving plant growth and also the symbiosis between plants and bacteria from OXYBIOTA.

The object of the invention is therefore described as a composition characterized in that the vaporization or application of said composition to plants maintains the mycorrhizal network of the plant.

The present composition does not only allow the increase of polyphenols but also the increase of other molecules already present in the plant such as vitamins (Vitamin A, B, C, D, E) and minerals (iron, zinc, magnesium , calcium, phosphorus, selenium) as well as the appearance of new molecules, resulting from the transformation of molecules already present in the plant. The bacteria present in the composition produce new molecules, thus increasing the rate of these molecules at the level of the plant. For example, the bacterium Pseudomonas putida produces the enzyme superoxide dismutase (SOD).

Bacteria secrete metabolites including many enzymes. These enzymes can degrade the cell wall of plants and allow the release of various compounds, including polyphenols, in the plant.

The bacteria contained in OXYBIOTA will therefore increase the level of polyphenols released in the plants.

The bacteria of the plant microbiota playing a role in the metabolism and synthesis of several substances, they will also allow the appearance of new molecules beneficial to the plant. OXYBIOTA is therefore described as a composition characterized in that it is used in the increase of bioactives and metabolites in plants.

Plants take up minerals from the soil in the form of inorganic ions, mainly through the roots. Certain parameters modulate the efficiency of mineral absorption: mineral solubility, their complexation with a ligand, composition, pH, humidity, oxidation-reduction potential, humification (decomposition of plants into organic matter ) of the ground.

The microbiota plays an important role in the uptake of minerals by plant roots.

The Archaeospora trappei bacterium produces vitamin D. Its vaporization on the soil and/or the plant will therefore allow its proliferation and increase the quantity of vitamin D in or on the plant.

The bacterium Pseudomonas japonica solubilizes the zinc present in the soil facilitating its assimilation by the plant, while the bacterium Serratia marcescens solubilizes the magnesium.

Polyphenols also have a role in the bioavailability of minerals. Indeed, polyphenols have a modulating role on the bacterial communities of the soil, thus impacting the rate of decomposition and the cycling of nutrients.

OXYBIOTA is therefore described as a composition characterized in that it is used in the increase of vitamins, in particular vitamin D in plants and fruits, in the increase of minerals, in particular zinc and in the increase of acids bold.

As with humans and animals, plants can be infected by pathogenic bacteria and fungi. These bacteria can be of the type: Xanthomonas campestris, Clavibacter michiganensis, Pseudomonas spp., Xanthomonas campestris, Ralstonia solanacearum, Pseudomonas syringae . Phytophatogenic fungi can cause diseases like downy mildew caused by the fungus Phytophthora infestans, fruit rot caused by Aspergillus, fruit rot caused by Alternaria and Boytrytis. These infections have a certain ecological and economic cost. Indeed, they can lead to the rotting of fruits, thus leading to a reduction in harvests; they can also lead to the death of plants. Different strains of bacteria are able to communicate with each other through chemical signals and to modulate the growth and activity of other strains by quorum sensing. Bacteria strains are also able to modulate the growth of fungi. The bacteria that make up OXYBIOTA have been selected for their modulating effect on the microbial ecosystem. These bacteria inhibit the proliferation of bacteria and fungi harmful to plants by competition effect. The polyphenols, the quantity of which increases following the vaporization or the application of the present composition to the plant, also enable the plant to protect itself against pathogenic agents. OXYBIOTA, by promoting the proliferation of beneficial bacteria and the production of polyphenols by plants, will reduce the risk of proliferation and colonization of the plant by pathogenic bacteria. The object of the present invention is therefore described as a composition characterized in that it is used in the prevention of the contamination of plants by pathogenic bacteria.

By preventing the contamination of plants with harmful bacteria, the risk of disease is therefore reduced. The plant therefore has safer conditions to grow. In addition, the polyphenols, the presence of which increases after vaporization or application of the OXYBIOTA composition to the plant, bring numerous benefits to the plants. Indeed, polyphenols protect plants against parasites, predators and pathogens but also against the negative effects of UV rays or free radicals. The proliferation of bacteria beneficial to the plant ensures a more balanced microbiota. This balanced microbiota will provide it with nutrients essential to its growth, such as nitrogen and phosphorus, with greater efficiency.

Auxin is a plant hormone plays a key role in many growth and behavioral processes in plant life cycles and is essential for plant development. The bacterium Pseudomonas fluorescens produces auxin, so its application will directly stimulate plant growth.

The object of the present invention, by protecting the plant and its microbiota, therefore ensures better growth. OXYBIOTA is therefore described as a composition characterized in that the vaporization or application of said composition to plants increases plant growth.

This composition can be applied to the plants or the soil surrounding the plants as such or in a mixture of water, polysaccharides, fertilizers, earth, clay, etc. The composition can be applied by spray, irrigation, mixing with the soil, direct application to the tree. The composition is in the form of liquids, foams, pastes, emulsions, oils, gel, jellies, syrups, solids, powders, sprays, aerosol

The present invention will be better understood on reading the following examples which illustrate the invention without limitation. Remember that for the clarity of the explanations that follow we have decided to call OXYBIOTA a composition containing a mixture of bacteria from the extraction of microbiota from ancient trees of the species Olea europaea L. and RNA extracted from bacteria of this same microbiota characterized in that at least one bacterium is of the Parabacteroides genus.

Example 1: Increase in Omega-3, Zinc and Vitamin D levels

A study was carried out in order to evaluate the effect of the vaporization or application of the OXYBIOTA composition on fruits of the Punica granatum species, more precisely on their Omega-3 concentration.

The OXYBIOTA composition from the microbiota of millennial trees of the species Olea europaea L. was dispersed on young trees of the species Punica granatum .

The results obtained are shown in the following table:

Omega 3 Zinc Vitamin D Normal Punica granatum fruits 18.6% 0.4mg/100g 0.8ug/100g Fruits of Punica granatum from plants sprayed with a composition comprising OXYBIOTA 27.85% 1.2mg/100g 4.1ug/100g Percentage increase +149.7% + 300.0% 512.5+%

It is observed that the rate of Omega-3 is only 18.6% for normal fruits. For fruits enriched with the Olea europaea L. microbiota, the Omega-3 level is 27.85%, that is to say 49.7% higher than normal fruits.

Claims (10)

Plant treatment composition intended to increase the concentration of vitamins, minerals, fatty acids and polyphenols in plants and fruits, characterized in that it contains a mixture of bacteria and RNA originating from the extraction of the microbiota from ancient trees of the species Olea europaea L. and one or more total crude extracts or one or more extracts of a plant part rich in polyphenols. Plant treatment composition according to Claim 1, characterized in that the composition comprises one or more strains of bacteria and RNA extracted from these same bacteria taken from the following list, without this being limiting: Pseudomonas japonica, Pseudomonas fluorescens, archaeospora trappei , Bacillus methylotrophicus, Pseudomonas putida, Flavimonas oryzihabitans, Acinetobacter haemolyticus, Rhizobium radiobacter, Bacillus atrophaeus, Bacillus subtilis, Bacillus megaterium, Lactobacillus paracasei, Parabacteroides distasonis, Serratia marcescens, Arthrobacter globiformis. Plant treatment composition according to Claims 1 and 2, characterized in that the composition comprises extracts rich in plant polyphenols taken from the following list: pomegranate, olive, rosemary, rose, lemon, cherry, blackcurrant, grapefruit, fig, loquât, strawberry tree, apple, peach, apricot, melon, watermelon, cactus, grape. Plant treatment composition according to claims 1 to 3, for its use in maintaining the mycorrhizal network. Plant treatment composition according to claims 1 to 4, for its use in increasing bioactives and metabolites in plants. Plant treatment composition according to claims 1 to 5, for its use in increasing vitamins, in particular vitamin D in plants and fruits, increasing minerals, in particular zinc and increasing acids bold. Plant treatment composition according to claims 1 to 6, for its use in the prevention of contamination of plants by pathogenic bacteria and fungi. Plant treatment composition according to claims 1 to 7, for use in plant growth. Plant treatment composition for its use according to any one of Claims 1 to 8, characterized in that the composition is in the form of liquids, foams, pastes, emulsions, oils, gels, jellies, syrups, solids, powders, sprays, aerosol. Process for treating plants intended to increase the concentration of vitamins, minerals, fatty acids and polyphenols in plants and fruits according to any one of Claims 1 to 9, characterized in that the composition is sprinkled or spread on the ground and/or the plant.