KR20200048417A - Composition for increasing resistance to plant disease comprising lignin phenolic polymer as effective component and uses thereof - Google Patents

Abstract

The present invention relates to a composition for promoting resistance to plant diseases, comprising a lignin phenol-based polymer as an active ingredient, and to a use thereof. A copolymer synthesized by adding a copolymer of catechol and vanillic acid or a mixture of catechol and the vanillic acid with L-3,4-dihydroxyphenylalanine can be usefully used as an active ingredient such as a composition for promoting plant growth, a fertilizer, and a soil improver due to an excellent effect of promoting disease resistance of plants.

Description

Composition for increasing resistance to plant disease comprising lignin phenolic polymer as effective component and uses thereof}

The present invention relates to a composition for promoting plant disease resistance containing a lignin phenol-based polymer as an active ingredient and its use.

Plants are exposed to attack by various microorganisms that cause disease throughout their growth. When a plant becomes ill, growth and growth are suppressed, and various forms of symptoms are exhibited. As a result, some or all of the plants die, resulting in a sharp decrease in productivity. Eco-friendly technologies for preventing or treating microbiologically plant diseases without using chemical synthetic pesticides include using microorganisms that produce antibiotics, using microorganisms that produce enzymes, and promoting plant growth. Varies. In addition, there is also a method of preventing a plant from having disease resistance by treating the plant with a microorganism that generates phenazine before the plant disease occurs. Besides phenazine, acibenzolar-S-Methyl, salicylic acid, probenazole, beta-aminobutyric acid, etc. are also known to induce resistance to plant diseases.

On the other hand, Korean Registered Patent No. 1834563 discloses 'a composition for promoting plant germination containing phenolic polymer as an active ingredient and its use', and Korean Registered Patent No. 959251 discloses a plant containing genetic material of bacteria. Compositions, methods for increasing resistance to pathogens, and plants produced by the methods. However, the composition for promoting plant disease resistance containing the lignin phenol-based polymer of the present invention as an active ingredient and its use have not been described.

The present invention has been derived by the above-mentioned needs, the inventors of the catechol and vanic acid copolymer; Or a copolymer synthesized by adding L-3,4-dihydroxyphenylalanine to a mixture of catechol and vanillaic acid; Pseudomonas syringae fib in Arabidopsis thaliana cultured in a containing MS medium. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) As a result of inoculating the bacteria, it was confirmed that the Arabidopsis thaliana cultured in the MS medium containing the copolymer showed resistance to plant pathogens compared to Arabidopsis thaliana cultured in the additive-free MS medium of the copolymer. Moreover, the resistance to phytopathogens was confirmed to be superior to the copolymer synthesized by adding L-3,4-dihydroxyphenylalanine to the mixture of catechol and vanic acid compared to the copolymer of catechol and vanic acid, The present invention was completed.

In order to solve the above problems, the present invention provides a composition for enhancing plant disease resistance containing a polymer of monomeric phenol as an active ingredient.

In addition, the present invention provides a method for enhancing disease resistance of a plant comprising the step of treating the composition to a plant, a plant seed or plant planting soil.

In addition, the present invention provides a fertilizer composition comprising the composition as an active ingredient.

Copolymer of catechol and vanic acid of the present invention; Alternatively, the copolymer synthesized by adding L-3,4-dihydroxyphenylalanine to the mixture of catechol and vanic acid has an excellent effect of enhancing the disease resistance of the plant, and is an active ingredient such as a composition for promoting plant growth, fertilizer, and soil improver. It is expected to be useful.

1 is a copolymer of catechol and vanic acid (CAVA) or a copolymer synthesized by adding L-3,4-dihydroxyphenylalanine to a mixture of catechol and vanic acid (L-DOPA) 8.6 mg / L And 86mg / L, showing the survival of Arabidopsis thaliana plant pathogens cultured in each MS medium. MS is a control with no treatment.
2 is a copolymer of catechol and vanic acid (CAVA) or a copolymer synthesized by adding L-3,4-dihydroxyphenylalanine to a mixture of catechol and vanic acid (L-DOPA) 8.6 mg / L After inoculating the plant pathogens in Arabidopsis thaliana cultured in MS medium containing the concentration of, the result is confirmed by Western blot expression of PR1 protein. MS is a control with no treatment.

In order to achieve the object of the present invention, the present invention provides a composition for enhancing plant disease resistance containing a polymer of monomeric phenol as an active ingredient.

In the composition for promoting plant disease resistance according to an embodiment of the present invention, the polymer of the monomeric phenol is a mixture of catechol and vanillic acid; Or a mixture of catechol, vanic acid, and L-3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalanine); may be a copolymer prepared by polymerizing with an oxidizing agent, but is not limited thereto.

In preparing the polymer of the monomeric phenol of the present invention, the catechol, vanic acid and L-3,4-dihydroxyphenylalanine are not limited to commercially synthesized / separated materials.

In the polymer production of the monomeric phenol of the present invention, the oxidizing agent may be at least one selected from the group consisting of copper (I) chloride, ammonium persulfate, sodium periodate, potassium chloride and oxidase, and the oxidase is peroxy Days, laccase or a mixture thereof, but is not limited thereto. Laccase is trametes versicolor ), Fomes fomentarius), sat hatred Thermo fillets (Chaetomium the four thermophile ), Neurospora crassa , Colorius versicol ), Botrytis cinerea , Rigidoporus lignosus , Ganoderma lucidum , Coriolus hirsutus , Russula delica , Pleurotos ostreatus ), Aspergillus nidulans ), etc., but is not limited thereto.

The copolymer of the present invention can be prepared by a conventional method for preparing an oxidized polymer of a phenolic compound known in the art.

In the composition according to an embodiment of the present invention, the plant disease may be a bacterial plant disease such as tomato spot bacterial disease, tomato ulcer disease, tomato foot blight disease, red pepper bacterial spot disease, cabbage rot disease, cabbage black rot disease or kiwi ulcer disease, preferably Pseudomonas Syringe Fifth. Tomato DC3000 ( Pseudomonas syringae pv. Tomato DC3000) may be a tomato spot bacterial disease caused by the strain, but is not limited thereto.

In order to achieve another object of the present invention, the present invention provides a method for enhancing disease resistance of a plant comprising the step of treating the composition of the present invention to a plant, a plant seed or a plant planting soil. As a method of enhancing the disease resistance of the plant, the composition containing the polymer of monomeric phenol as an active ingredient may be performed by immersing the plant in a plant or by irrigation, that is, spraying. In the case of the immersion method, the composition may be poured into the soil around the plant or the seeds may be immersed in the composition, but is not limited thereto.

The present invention also provides a fertilizer composition comprising the composition of the present invention as an active ingredient.

The term 'fertilizer' refers to all substances that supply one or more of the elements necessary for the plant to grow normally, and organic fertilizers (consisting of decomposing plant / animal substances) and inorganic fertilizers (consisting of chemical and inorganic substances) ).

The fertilizer composition according to the present invention contains a polymer of a monomeric phenol that enhances the disease resistance of plants, as an active ingredient, and thus has an excellent effect of enhancing plant disease resistance and growth, so that it is a plant nutrient, soil conditioner, and foliar spray Or it can be used as an irrigation spray.

The fertilizer composition of the present invention may further include an agriculturally acceptable carrier, the acceptable carrier being fillers, solvents, excipients, surfactants, suspending agents, spreaders ), Adhesives, antifoaming agents, dispersants, wetting agents, drift reducitng agents, auxiliary agents, adjuvants or mixtures thereof.

The fertilizer composition of the present invention can be formulated in the form of concentrates, solutions, sprays, aerosols, immersion baths, dips, emulsions, suspension concentrates, gels, granules, and the like.

The fertilizer composition of the present invention, the fertilizer composition alone or other agricultural agents, for example, pesticides (pesticides), pesticides (insecticides), acaricides (acaracides), fungicides (fungal agents harmless to the fungi of the present invention), flesh Bacteria, herbicides, antibiotics, antibacterials, nematocides, rodenticides, entomopaogens, pheromones, attractants, plant growth regulators, plant hormones plant hormones, insect growth regulators, chemosterilants, microbial pest control agents, repellents, viruses, phagostimulents, plant nutrients, plant fertilizers and biological It can be combined with a control agent or used sequentially. The pesticides include carbamate, organic phosphate, organic chlorine pesticide, phenylpyrazole, pyrethroid, neonicotinoid, spinosin, avermectin, milbemycin, larva hormone analogs, alkyl halides, organic Tin compounds, nereistoxin analogs, benzoylurea, diacylhydrazine, insecticides of the METI acaricides, chloropicrin, pymetrozine, flonicamid, clofenthezin, hexythiazox, ethoxazole , Diafenthiuron, propargite, tetradiphone, chlorfenapyr, DNOC, bupropezin, cyromazine, amitraz, hydramethylnon, acequinosyl, fluacrypyrim, rotenone or derivatives thereof Insecticides can be used. As the plant nutrient, fertilizers for supplying nutrients to plants that are commonly used may be used. In addition, as the fertilizer, organic fertilizer, complex fertilizer, nitrogen fertilizer, phosphate fertilizer, calcium fertilizer, lime fertilizer, lactic acid fertilizer, sulfate fertilizer, magnesium fertilizer, trace element fertilizer, organic fertilizer, manure fertilizer, and the like can be used. At this time, a specific example of the agricultural preparation is obvious to a person having ordinary knowledge in the art.

When fertilizing the fertilizer composition of the present invention to a plant, the fertilizing amount and fertilizing method can be applied within a range commonly known in the art.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Manufacturing example  1. With catechol Vanic acid  Copolymer ( CAVA ) Produce

1 g of catechol (sigma, USA) and 1 g of vanillic acid (sigma) were added to 192 mL of 100 mM sodium acetate buffer containing 48 mL of 100% ethanol and mixed. Trametes versicola laccase in the mixture 20 mg of versicolor laccase, sigma) was added and stirred for 24 hours under conditions of 36 ° C. and 400 rpm to induce oxidative polymer synthesis. The stirred solution was centrifuged under the conditions of 13,000 rpm to obtain a polymer precipitate, and then the obtained polymer precipitate was washed with distilled water, and the stirred solution from which the precipitate was removed is an amicon with a fractional molecular weight cut-off of 5 kda. The polymer in the stirring solution was separated again by performing a desalination process with sterile distilled water using an ultra filter (millipore, Germany). The polymer obtained by centrifugation and the polymer separated through an ultra filter were mixed, and then dried at 70 ° C. to obtain a homopolymer of polymer powder catechol and vanillaic acid (CAVA).

Manufacturing example  2. Catechol, Vanic acid  And L-3,4- Dihydroxyphenylalanine  Preparation of copolymers of mixtures (L-DOPA)

0.8 g of catechol, 1 g of vanic acid and 0.2 g of L-3,4-dihydroxyphenylalanine were added to 192 mL of 100 mM sodium acetate buffer containing 48 mL of 100% ethanol. Mixed. Thereafter, 20 mg of Trametes versicolor laccase was added to induce polymer synthesis, and then the synthesized polymer was separated. The separation and purification process of the polymer is the same as described above. Through the above manufacturing process, a copolymer of catechol, vanic acid, and L-3,4-dihydroxyphenylalanine (L-DOPA) was prepared.

Example  1. of phenolic polymer  Plant disease  Analysis of resistance enhancement effect

Arabidopsis thaliana Columbia (Col-0) was used as a plant material and cultured for 2 weeks in MS medium in a single cycle (8 hour-light / 16 hour-cancer) culture room. Homopolymer of catechol and vanic acid (CAVA); And phenols of different concentrations (8.6 or 86 mg / L) to analyze the effects of catechol, vanic acid and copolymers of L-3,4-dihydroxyphenylalanine (L-DOPA) on biological stress tolerance. After 2 weeks, seedlings were transferred to the medium containing the polymer and planted 3 days later, Pseudomonas syringae, a representative pathogenic bacterium of Arabidopsis thaliana. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) strain. The Pseudomonas syringe dog blood. Tomato DC3000 strain was grown in KB medium (King's medium B broth) containing antibiotics, and then resuspended in 10mM magnesium chloride (MgCl ₂ ) buffer for 5 × 10 ⁴ CFU / cm ² per plate. Diluted to levels and treated in Arabidopsis thaliana.

As a result, 3 days after the treatment of the pathogenic bacteria, when compared to Arabidopsis thaliana grown in MS medium without treatment, a copolymer of catechol and vanic acid (CAVA) at a high concentration of 86 mg / L; And in the case of Arabidopsis thaliana grown in MS medium containing a copolymer (L-DOPA) synthesized by adding L-3,4-dihydroxyphenylalanine to a mixture of catechol and vanic acid, the survival rate was significantly reduced, Low concentration of 8.6 mg / L catechol and vanic acid copolymer (CAVA); And a mixture of catechol and vanic acid, L-3,4-dihydroxyphenylalanine added to the synthesized synthesized in the MS medium containing the copolymer (L-DOPA) Arabidopsis thaliana was confirmed that a significant number of Arabidopsis surviving (Fig. 1). Through this, it was confirmed that the polymer of the monomeric phenol had a positive effect on enhancing disease resistance of the plant.

Example  2. of phenolic polymer Plant disease  Analysis of the effect on the expression of resistance protein PR1

A homopolymer of catechol and vanic acid of the present invention (CAVA); And the effects of catechol, vanic acid, and copolymers of L-3,4-dihydroxyphenylalanine (L-DOPA) on the expression of pathogenesis related protein 1 (PR1), a typical disease resistance related protein in plants. .

Pseudomonas Syringe FB. After treating the tomato DC3000 strain, the tissues of the plants collected at 12, 24 and 36 hours, respectively, were frozen with liquid nitrogen, crushed with a mortar, and 100 mg of the tissue was taken and mixed with 100 µl of protein extraction buffer. Suspended completely. The suspension was centrifuged at 20,000x g and 4 ° C to take a supernatant, 6X protein loading dye was added, heated at 95 ° C for 1 minute, and then loaded onto an acrylamide gel for electrophoresis. (Bio-Rad, USA). The protein separated by size from the gel was transferred to the membrane, and the transferred membrane was soaked in a blocking solution (5% skim milk) and slowly shaken for 1 hour to block. The primary antibody against PR1 was diluted with 5% skim milk in a 1: 2,000 ratio to the blocking solution and reacted overnight at 4 ° C or 2 hours at room temperature. After the primary antibody reaction was completed, the secondary antibody (Rabbit 1: 5,000) was reacted for 1 hour at room temperature, and then washed repeatedly with Tris-Buffered Saline with Tween (TBST) buffer for 3 times for 10 minutes. After the ECL solution (Thermo Fisher Scientific, USA) was treated for 5 minutes to emit light, the expression level of the expressed protein was checked over time.

As a result, as shown in FIG. 2, expression of PR1 protein was confirmed in all treatment groups after 24 hours after pathogen treatment, and CAVA or L-DOPA, a phenol polymer of the present invention, was added compared to MS medium that was not treated with anything. It was confirmed that the expression of PR1 protein was significantly increased at the same time in plants grown in the grown MS medium. In addition, it was confirmed that the expression level of PR1 protein was remarkable in the results after 12 hours of pathogen treatment in the case of plants grown in MS medium to which L-DOPA was added in comparison with CAVA among the phenol polymers of the present invention.

Through the expression of the PR1 protein, a homopolymer of catechol and vanic acid of the present invention (CAVA); And the copolymer (L-DOPA) treatment of catechol, vanic acid and L-3,4-dihydroxyphenylalanine was found to induce disease resistance of plants.

Claims (7)

A composition for enhancing plant disease resistance, comprising a polymer of monomeric phenol as an active ingredient. The method of claim 1, wherein the polymer of the monomeric phenol comprises a mixture of catechol and vanillic acid; Or a mixture of catechol, vanic acid and L-3,4-dihydroxyphenylalanine (L-3,4-dihydroxyphenylalanine); to improve the plant disease resistance, characterized in that the copolymer (polymer) prepared by polymerizing with an oxidizing agent Dragon composition. 3. The composition for promoting plant disease resistance according to claim 2, wherein the oxidizing agent is at least one selected from the group consisting of copper (I) chloride, ammonium persulfate, sodium periodate, potassium chloride and oxidase. According to claim 1, wherein the plant disease is a composition for promoting plant disease resistance, characterized in that a bacterial plant disease. The method of claim 4, wherein the bacterial plant disease, tomato spot bacterial disease, tomato ulcer disease, tomato foot disease, pepper bacterial spot disease, cabbage rot disease, cabbage black disease and kiwi ulcer disease, characterized in that at least one plant disease selected from the group consisting of Composition for enhancing resistance. A method for enhancing disease resistance of a plant, comprising the step of treating the plant disease resistance enhancing composition of any one of claims 1 to 5 with a plant, a plant seed or a plant planting soil. A fertilizer composition comprising the composition for promoting plant disease resistance according to any one of claims 1 to 5 as an active ingredient.

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