KR102209184B1 - Composition for increasing resistance to plant disease comprising lignin or conversion product of lignin as effective component and uses thereof - Google Patents

Abstract

The present invention relates to a composition for improving plant disease resistance containing lignin or a lignin transformant as an active ingredient, and to a use thereof, and the lignin transformant by the lignin or Fenton oxidation reaction of the present invention has an excellent effect of enhancing disease resistance of plants. It is expected to be useful as an active ingredient such as a composition for promoting plant growth, fertilizer, and soil improvement agent.

Description

Composition for increasing resistance to plant disease comprising lignin or conversion product of lignin as effective component and uses thereof, comprising lignin or lignin transformant as an active ingredient

The present invention relates to a composition for enhancing plant disease resistance containing lignin or a lignin transformant as an active ingredient, and a use thereof.

Lignin (lignin) is a fat-soluble phenolic polymer component among the constituents of woody parts such as conifers and hardwoods, and accounts for 20 to 35% by weight of dry wood. Lignin is the most abundant and readily available organic polymer after cellulose among the constituents of biomass, and is known to account for 30% of unfossilized organic carbon. Specifically, lignin consists of 63.4% of carbon, 5.9% of hydrogen, 30% of oxygen, and 0.7% of ash. Since it contains many phenyl groups, it can be an important raw material for producing aromatic compounds.

In the present invention, the Fenton oxidation reaction, which is one of the advanced oxidation processes, was performed on lignin. Fenton oxidation reaction is a reaction using the strong oxidation of OH radicals generated by the reaction of hydrogen peroxide and ferrous iron (Fe ²⁺ ), and various hydrophilic functional groups can be introduced on the lignin surface by Fenton oxidation treatment. The structure can be oxidized to introduce an aliphatic structure, and the molecular weight of the polymer can be reduced by breaking the covalent bond.

On the other hand, Korean Patent Application Publication No. 2012-0042118 discloses a'plant disease resistance inducing agent containing paromomycin', and Korean Patent No. 1285792 discloses'a fertilizer composition containing Qsan, chitosan, and humic acid, and its manufacture. Method' is disclosed, but there is no description of the composition for enhancing plant disease resistance and the use thereof containing the lignin or lignin transformant of the present invention as an active ingredient.

The present invention was derived from the above requirements, the inventors of the present invention Pseudomonas syringae fiv on Arabidopsis cultured in MS medium containing lignin or lignin transformant (lignin's Fenton reaction oxide). Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) strain or Erwinia carotovora subsp. Erwinia carotovora subsp. carotovora ) As a result of inoculation, Arabidopsis cultivated in MS medium without lignin or lignin transformants was completely killed by plant pathogen inoculation, whereas Arabidopsis cultivated in MS medium containing lignin or lignin transformants was plant pathogens. It was confirmed that it shows resistance to. Moreover, the present invention was completed by confirming that the lignin transformant was superior to lignin in resistance to plant pathogens.

In order to solve the above problems, the present invention provides a composition for enhancing plant disease resistance containing lignin or a lignin transformant as an active ingredient.

In addition, the present invention provides a method of improving the disease resistance of plants, including the step of treating the composition to plants, plant seeds or plant planting soil.

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

The lignin transformants of the present invention by the lignin or Fenton oxidation reaction are expected to be useful as active ingredients such as compositions for promoting plant growth, fertilizers, soil improving agents, etc., as they have excellent effects of enhancing disease resistance of plants.

Figure 1A is a Pseudomonas syringae fiV on Arabidopsis cultured in MS medium containing lignin or lignin transformant (lignin subjected to Fenton oxidation reaction). Tomato DC3000 ( Pseudomonas syringae pv.tomato DC3000) is a photograph showing the survival of Arabidopsis thaliana 7 days after treatment of the strain, and FIG. 1B is a lignin or lignin transformant (lignin undergoing a Fenton oxidation reaction) in Arabidopsis inoculated with the strain. Pseudomonas Syringe PB that remains on the leaves of Arabidopsis after treatment. It is a graph showing the number of tomato DC3000 strains measured. control; Lignin, HA; Commercial humic acid, S; Lignin + pickling water, 0.1; Lignin + ferrous sulfate. Heptahydrate 0.05 g, 0.5; Lignin + ferrous sulfate. Heptahydrate 0.25 g, 1; Lignin + ferrous sulfate, heptahydrate 0.5g, lignin, commercial humic acid and lignin transformants were all treated at 1,060 mg/ℓ based on MS medium.
Fig. 2 shows Erwinia carotovora subsp on Arabidopsis cultured in MS medium containing lignin or lignin transformants (lignin undergoing Fenton oxidation reaction). Erwinia carotovora subsp. carotovora ) is a photograph showing the survival of Arabidopsis after 3 days of treatment. MS; MS medium without treatment, 0.1; Lignin + ferrous sulfate. Heptahydrate 0.05 g, 0.5; Lignin + ferrous sulfate. Heptahydrate 0.25 g, 1; Lignin + ferrous sulfate. Heptahydrate 0.5 g, control; Lignin, HA; Commercial humic acid, S; Lignin + pickling water.
3 is a result of confirming the expression level of PR1 protein by Western blot after inoculating plant pathogens in Arabidopsis cultivated in MS medium containing lignin or lignin transformant (lignin subjected to Fenton oxidation reaction). MS; MS medium without treatment, 0.1; Lignin + ferrous sulfate. Heptahydrate 0.05 g, 0.5; Lignin + ferrous sulfate. Heptahydrate 0.25 g, 1; Lignin + ferrous sulfate. Heptahydrate 0.5 g, control; Lignin, HA; Commercial humic acid, S; Lignin + pickling water.

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

Lignin is a major component of the cell wall of plants, a material that plays an important role in hardening the tree structure, and is a major resource accounting for 22-29% (weight) after cellulose (40-43% by weight) in wood composition.

In the composition for enhancing plant disease resistance of the present invention, the lignin may include lignin or a derivative of lignin contained in the plant structure, lignin treated under strong alkali or strong acid conditions in the pulp manufacturing process, preferably kraft It may be lignin, but is not limited thereto.

In addition, in the composition for improving plant disease resistance of the present invention, the lignin transformant may preferably be a Fenton reaction oxide of lignin, but is not limited thereto. In addition, the lignin transformant may be a humic acid analog. The humic acid analog refers to a substance having the properties of humic acid.

The term "humic acid" refers to a humic substance that dissolves only in an acidity of 2.0 or higher as a brown or black acidic organic substance that remains unremoved from a solvent among organic substances other than living organisms and humus contained in the soil. Humic acid accounts for more than 50% of dissolved organic substances in nature, and the degree of dissolution affects the distribution of substances in many soils. Various functional groups such as carboxyl, alcohol, phenol and enol groups of humic acid undergo chemical reactions such as adsorption of hydrophobic and hydrophilic substances, redox reactions, and the like. Another name is called humic acid. Treatment of humic acid on crops or soil has the same effect as compost or organic fertilizers are immediately absorbed by the crop or administered in a form that the soil can use immediately.

The Fenton oxidation method uses a Fenton oxidation reaction and uses a mixed solution of _divalent iron (Fe ²⁺ ) salt and hydrogen peroxide (H ₂ O ₂ ) used as a catalyst (Fenton reagent) to generate OH radicals during the reaction. It is a method of oxidizing organic matter with the oxidizing power of (OH·). As described above, the Fenton oxidation method uses commonly available iron salts and hydrogen peroxide, which is a relatively economical oxidizing agent that is easy to handle and can be stored in the field, has high reactivity with various organic substances, and does not cause toxic substances or chromatic substances. It belongs to the oxidation method.

In the composition according to an embodiment of the present invention, the lignin transformant may be produced by reacting lignin with hydrogen peroxide (H ₂ O ₂ ) and a catalyst, and preferably, produced by reacting lignin with hydrogen peroxide and an iron salt. It may be, but is not limited thereto. The iron salt may be a divalent iron salt (ferrous ion) such as FeSO ₄ , FeO, Fe(OH) ₂ , FeCO ₃ , and FeCl ₂ , but is not limited thereto.

In the composition according to an embodiment of the present invention, the lignin transformant (Fenton reaction oxide of lignin) is specifically,

1) mixing and stirring hydrogen peroxide with lignin;

2) reacting by adding an iron salt to the stirred mixture of step 1); And

3) a step of obtaining a pellet after centrifuging the reactant of step 2); and more specifically,

1) mixing and stirring 15 to 25 ㎖ of 30 to 40% (w/v) hydrogen peroxide in 0.8 to 1.2 g of kraft lignin;

2) adding 0.01 to 1.0% (w/v) aqueous solution of iron II sulfate heptahydrate to the stirred mixture in step 1) and reacting for a total of 1 to 3 hours; And

3) a step of obtaining a pellet after centrifuging the reactant of step 2), but is not limited thereto.

In the composition according to an embodiment of the present invention, the ferrous sulfate hexahydrate aqueous solution, which is the iron salt catalyst in step 2), may be replaced with pickling acid water, a by-product obtained in the steel making process, but is not limited thereto. .

In the composition according to an embodiment of the present invention, the plant disease may be a bacterial plant disease such as tomato spot bacterium, tomato ulcer disease, tomato green blight, pepper bacterial spot blight, cabbage soft rot, cabbage black blotch or kiwi ulcer disease, and is preferably Hagi is Pseudomonas Syringe Pv. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) may be tomato spot bacterial disease or Erwinia carotovora ( Erwinia carotovora ) strain caused by a strain, but is not limited thereto.

In order to achieve another object of the present invention, the present invention provides a method of improving the disease resistance of plants comprising the step of treating the composition of the present invention to plants, plant seeds or plant planting soil. As a method of improving the disease resistance of the plant, the composition containing lignin or a lignin transformant as an active ingredient may be immersed in the plant or drenched, that is, sprayed. In the case of the immersion method, the composition may be poured into the soil around the plant or seeds may be immersed in the composition, but the present invention is not limited thereto.

The present invention also provides a fertilizer composition comprising the composition of the present invention as an active ingredient. The fertilizer composition according to the present invention contains lignin or a lignin transformant that improves disease resistance of plants as an active ingredient, and has excellent effect of improving plant disease resistance and growth promoting effect of plants, so that plant nutrients, soil conditioners, foliar cotton It can be used as a spraying agent or a drench spraying agent.

The fertilizer composition of the present invention may further include an agriculturally acceptable carrier, and the acceptable carrier includes fillers, solvents, excipients, surfactants, suspending agents, and spreaders. ), adhesives, antifoaming agents, dispersing agents, 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 preparations, for example, pesticides, insecticides, acaracides, fungicides (fungal agents harmless to the fungi of the present invention), sterilization Bacteria, herbicides, antibiotics, antibacterial agents, nematocides, rodenticides, entomopathogens, 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 agents. It may be combined with a control agent or used sequentially. Examples of the insecticide include carbamate, organic phosphate, organic chlorine insecticide, phenylpyrazole, pyrethroid, neonicotinoid, spinosine, avermectin, milbemycin, larval hormone analogue, alkyl halide, organic Tin compounds, nereistoxin analogues, benzoylurea, diacylhydrazine, METI insecticides such as acaricides, chloropicrin, pymetrozine, flonicamid, clopentezine, hexythiazox, ethoxazole , Diafenthiuron, proparzit, tetradifon, chlorfenapyr, DNOC, buprofezin, cyromazine, amitraz, hydramethylnon, acequinosyl, fluacrypyrim, rotenone or derivatives thereof Pesticides can be used. As the plant nutrient, a commonly used plant nutrient fertilizer may be used. In addition, as the fertilizer, organic fertilizer, complex fertilizer, nitrogen fertilizer, phosphoric acid fertilizer, calcium fertilizer, lime fertilizer, lactic acid fertilizer, sulfuric acid fertilizer, magnesium fertilizer, trace element fertilizer, organic fertilizer, manure fertilizer, and the like may be used. At this time, a specific example of the agricultural formulation is obvious to those of ordinary skill in the art.

When applying the fertilizer composition of the present invention to a plant, the amount and method of fertilization may 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 only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

Example One. Kraft Lignin Fenton Reactive oxide preparation

1 g of kraft lignin (Sigma, USA) powder pulverized with a mortar and mortar and 20 ml of 35% hydrogen peroxide (H ₂ O ₂ ) solution were put into a flask, mixed, and stirred at a speed of 130 rpm. Then, 0.05, 0.25, or 0.5 g of ferrous sulfate heptahydrate (FeSO ₄ 7H ₂ 0, iron (II) sulfate heptahydrate; Junsei, Japan) was added to 5 ml of distilled water, and stirred at room temperature to prepare iron salt solutions of different concentrations. Ready. Thereafter, the iron salt solution was injected 5 times in a flask containing a mixture of kraft lignin and hydrogen peroxide, 1 ml each at 10 minute intervals, and an oxidation reaction was induced for 2 hours. After the reaction was completed, the supernatant was removed by centrifugation at 3,000 rpm for 15 minutes, and each pellet was washed by centrifuging twice under the same conditions using distilled water. The washed pellets were freeze-dried and prepared in powder form, and then diluted in MS medium to be 1,060 mg/L, respectively, and used in the Arabidopsis plant test.

Commercial humic acid (Sigma, USA) was dissolved in distilled water, and kraft lignin without Fenton oxidation was centrifuged at 3,000 rpm for 15 minutes and washed to perform only lyophilization. Pickling water is a by-product obtained from the steelmaking process. After mixing pickling water and distilled water at a ratio of 1:10, injecting 1 ml each 5 times at 10 minute intervals into the flask containing the mixture of kraft lignin and hydrogen peroxide for 2 hours. After the reaction was performed, washing and lyophilization through centrifugation were performed in the same manner as above.

Example 2. Analysis of the effect of enhancing plant disease resistance of Fenton's reaction oxide of kraft lignin

2-1. Pseudomonas Syringe Pv . Tomato DC3000( Pseudomonas syringae pv. tomato DC3000 )for Analysis of resistance enhancement effect

Arabidopsis (Col-0) seeds were sown on MS solid medium and incubated for 2 weeks in a culture room under photoperiodic conditions for 16 hours/8 hours (light/dark). The cultured Arabidopsis seedlings were transferred to MS medium containing kraft lignin subjected to Fenton oxidation reaction, commercial humic acid (HA), kraft lignin treated with pickling water (S) or kraft lignin not treated with Fenton (control), respectively, After a 3-day adaptation period, Pseudomonas Syringe PB, a representative plant disease bacterium. Tomato DC3000 ( Pseudomonas syringae pv. tomato DC3000) strain was inoculated. Pseudomonas syringe pv. Tomato DC3000 strain was grown in KB medium containing antibiotics (King's medium B broth) and then resuspended in 10 mM magnesium chloride (MgCl ₂ ) buffer to 5×10 ⁴ CFU/cm ² per plate. Diluted to the level and treated on Arabidopsis.

As a result of treating the strain and incubating for 7 days, Arabidopsis grown in MS medium without any treatment completely died (no photo), whereas kraft lignin, commercial humic acid, pickled water treated kraft lignin or Fenton were not treated. In Arabidopsis cultivated in MS medium treated with kraft lignin, it was confirmed that a significant number of Arabidopsis survived. In particular, the kraft lignin treatment group subjected to the Fenton oxidation reaction showed superior survival rate of Arabidopsis thaliana than the treatment group of lignin that did not undergo the Fenton oxidation reaction (FIG. 1A).

In addition, Pseudomonas syringae PB that remain on the leaves of Arabidopsis after cultivation. As a result of measuring the amount of tomato DC3000 strain, the amount of strain remaining on the leaves decreased in the kraft lignin treated with Fenton, commercial humic acid or pickled water treated kraft lignin compared to the kraft lignin treatment without Fenton treatment, In particular, it was confirmed that the MS medium treated with 0.5 g of kraft lignin subjected to Fenton reaction decreased by approximately 50 times (y-axis log value) compared to the kraft lignin treated group not treated with Fenton (Fig. 1B).

2-2. Erwinia Karoto Bora ( Erwinia carotovora )on Analysis of the effect of increasing resistance to

Erwinia carotovora subsp. in Arabidopsis cultured in the same manner as described in Example 2-1 above. Erwinia carotovora subsp. carotovora ) strain was inoculated. Erwinia carotovora strain was grown in KB medium containing antibiotics, then resuspended in 10 mM magnesium chloride (MgCl ₂ ) buffer, and 5×10 ⁴ CFU/cm ² per plate. Diluted to the level and treated on Arabidopsis.

As a result of treating the strain and incubating for 3 days, commercial humic acid (HA) compared to Arabidopsis (MS) grown in MS medium without any treatment, kraft lignin without Fenton treatment (control) and kraft lignin (S) treated with pickling water And it was confirmed that Arabidopsis thaliana maintains a relatively healthy state in kraft lignin subjected to Fenton reaction. In particular, it was confirmed that the growth rate of Arabidopsis thaliana was increased in kraft lignin 0.5 (lignin + ferrous sulfate, heptahydrate 0.25 g) subjected to Fenton reaction (FIG. 2).

Through the above results, it was confirmed that kraft lignin subjected to the Fenton oxidation reaction had a positive effect on the improvement of disease resistance of plants.

Example 3. Analysis of Effect of Fenton Reactive Oxide of Kraft Lignin on Expression of Plant Disease Resistance Protein PR1

The effect of the Fenton reaction oxide of kraft lignin of the present invention on the expression of PR1 protein (pathogenesis related protein 1), a representative disease resistance related protein in plants, was analyzed.

Pseudomonas syringe pv. Plant tissues collected at 24 and 36 hours after treatment with tomato DC3000 strain, respectively, are frozen with liquid nitrogen, pulverized with a mortar, and 100 mg of tissue is taken, mixed with 100 µl of protein extraction buffer, and completely suspended. Made it. The suspension was centrifuged at 20,000x g at 4℃ to take a supernatant, heated at 95℃ for 1 minute by adding 6X protein loading dye, and then loaded on an acrylamide gel for electrophoresis. (Bio-Rad, USA) was implemented. Proteins separated by size from the gel were transferred to a membrane, and the transferred membrane was immersed in a blocking solution (5% skim milk) and then slowly shaken for 1 hour to block. The primary antibody against PR1 was diluted in the blocking solution at a ratio of 1:2,000 and reacted overnight at 4° C. or at room temperature for 2 hours. The membrane after the primary antibody reaction was reacted with a secondary antibody (Rabbit 1:5,000) at room temperature for 1 hour and then washed three times for 10 minutes with Tris-Buffered Saline with Tween (TBST) buffer, and the membrane was washed. The ECL solution (Thermo Fisher Scientific, USA) was treated for 5 minutes to emit light, and the expression level of the expressed protein was checked by time.

In samples after 24 hours of treatment with plant pathogens, expression of PR1 protein was observed in 0.5 Fenton reaction oxide of kraft lignin (: kraft lignin + ferrous sulfate hexahydrate 0.25 g) treatment and pickling treatment, especially 0.5 Fenton treated kraft lignin It was confirmed that the treatment group significantly increased the expression of the PR1 protein compared to other test groups. In addition, in the results 36 hours after treatment with plant pathogens, expression of the PR1 protein was observed in all groups except for the treatment group of kraft lignin Fenton's reaction oxide 0.1 (: kraft lignin + ferrous sulfate / heptahydrate 0.05 g) (Fig. 3).

Through the expression of the PR1 protein, it was found that the Fenton reaction oxide treatment of kraft lignin can induce disease resistance in plants, and compared to commercial humic acid, it was seen that the expression of the PR1 protein was induced in a short time in response to plant pathogens. , It was found that the effect of enhancing plant disease resistance to the Fenton oxidation reaction product of kraft lignin of the present invention is almost similar to that of commercial humic acid that has been used in the past. In addition, it could be determined that the oxide of kraft lignin caused by the Fenton oxidation reaction induces stronger disease resistance than the kraft lignin before the Fenton oxidation reaction.

Claims (5)

A composition for enhancing plant disease resistance by Erwinia carotovora containing Fenton's reaction oxide of kraft lignin produced by reacting kraft lignin with hydrogen peroxide and iron salt as an active ingredient. delete delete A method for enhancing plant disease resistance by Erwinia carotovora , comprising the step of treating the composition for enhancing plant disease resistance of claim 1 on plants, plant seeds or planting soil. A fertilizer composition comprising, as an active ingredient, a composition for enhancing plant disease resistance by Erwinia carotovora of claim 1.

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