KR102057318B1 - Composition for improving immunity of plant comprising cultural filtrate of Chlorella sp. - Google Patents

Abstract

The present invention relates to a composition for enhancing immunity of a plant containing Chlorella sp. Microalgal culture filtrate as an active ingredient, and more specifically, the culture solution of the chlorella microalgae of the present invention or its culture filtrate is salicylic acid and jasmon Depending on the signaling mechanism of acid, it induces resistance to plant pathogens, and in particular, the culture filtrate of chlorella microalgae has a superior resistance inducing effect compared to the culture of microalgae of chlorella microorganisms, and thus, microalgae of chlorella genus, which incur much waste cost, are generated. The culture filtrate of has the advantage that can be used as an environmentally friendly crop protection agent.

Description

Composition for improving immunity of plant comprising cultural filtrate of Chlorella sp.}

The present invention relates to a composition for enhancing immunity of plants containing the culture filtrate of Chlorella sp. Microalgae as an active ingredient, and a method for controlling plant diseases using the composition.

Plant diseases are currently the most important obstacle to agricultural production. Five to 80 percent of crop yields are associated with reduced crop yields. In order to control such plant diseases, countries around the world have been manufacturing and using pesticides based on organic compounds, but recently, it has been reported that these organic compounds adversely affect the environment and the human body. We are strictly restricting the use of and attempting to significantly reduce the amount of use.

Since plants cannot move once settled, an immune system has been developed to combat various attacks in order to effectively respond to changes in the external environment. In particular, plants have induced resistance that activates a resistance reaction not only in the leaves of pathogens but also throughout the whole plant. BTH (benzo (1,2,3) -thiadiazole-7-carbothioic acid S-methyl ester, benzothiadiazole) is known as one of the most potent effects among various inductive resistance inducers. However, serious problems were also found in these inductively-resistant inducers, which seemed to overcome the problems of chemical pesticides. When the crops were treated in actual packaging, the growth of the plants was suppressed or the yield was significantly reduced compared to the untreated control. Therefore, there is a need for development or treatment of new inductively-resistant inductive materials to compensate for this.

On the other hand, Chlorella sp. Microalgae are green algae that can live independently through photosynthesis. They are the raw materials that produce proteins, carbohydrates, pigments, vitamins and minerals, which are nutrients necessary for the human body. , Animal feed, waste water purification, etc. are widely applied, and mass cultivation techniques and useful material extraction techniques are widely known.

However, mainly chlorella ocean cultivation for biodiesel production or dietary supplement production is mainly used to collect cells. After cell collection, the remaining dozen tons of filtrate must be disposed of after treatment such as purification treatment, which requires a lot of cost. Therefore, an environmentally friendly post treatment or recycling method of chlorella culture filtrate is required. It is true.

The inventors of the present invention, while conducting a study on the induction resistance to plant diseases, the culture filtrate of the microalgae of the chlorella shows a significant induction resistance induction effect against the pathogens of the plant, Chlorella which generates a large waste cost It was confirmed that the culture filtrate of the genus microalgae can be used as an environmentally friendly crop protection agent, to complete the present invention.

Korean Unexamined Patent Publication No. 2010-0134214

Kim, Hyun-Jin et al. "Physical activity of chlorella elliposidea CO20 ethanol extract" Korean Society for Biotechnology and Bioengineering (2008.04)

An object of the present invention is to provide an agent capable of enhancing the immunity of a plant.

In addition, an object of the present invention is to provide a method for controlling plant diseases using the agent for enhancing plant immunity as described above.

In order to achieve the above object, an aspect of the present invention provides a composition for enhancing immune system of a plant containing a culture solution of Chlorella sp. Microalgae or a culture filtrate thereof as an active ingredient.

In addition, in order to achieve the above object, another aspect of the present invention provides a method for controlling a plant disease containing a culture solution of the microalgae of the genus Chlorella or the culture filtrate thereof as an active ingredient.

The culture solution of Chlorella sp. Microalgae of the present invention or its culture filtrate enhances plant immunity by inducing resistance to plant pathogens depending on the signaling mechanisms of salicylic acid and jasmonic acid, and in particular, of the microalgae of Chlorella sp. Since the culture filtrate has a significantly superior resistance inducing effect compared to the microalgal algae culture medium, there is an advantage that the culture filtrate of the microalgae of chlorella, which generates a lot of waste costs, can be used as an environmentally friendly crop protection agent.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

Figure 1 is a schematic diagram showing the method of verifying the induction-resistance of tomato bacillus pathogen after treatment with Chlorella fusca ( Clorella fusca ) in the Arabidopsis.
Figure 2 is a diagram confirming the induction-resistance to the tomato bacillus pathogen of the Arabidopsis treatment by Chlorella pusca.
In FIG. 2A, 'Clorella pusca' is a Arabidopsis treated with mixed cultured chlorella pusca at a concentration of 10 ⁷ cells / ml, and 'control' is a Arabidopsis treated with BG-11 medium for chlorella pusca culture.
In FIG. 2B and FIG. 2C, 'chlorella' is a sphere treated with chlorella mixed culture culture at a concentration of 10 ⁷ cells / ml, 'Pol B' is an antibiotic polymyxin B treated group, and 'control' is treated with BG-11 medium. One phrase.
Figure 3 is a diagram confirming the optimum culture conditions and treatment concentration of Chlorella Pusca.
In Figure 3 'control' is a baby pole treated with BG-11 medium.
4 is a diagram confirming the molecular mechanism of induction-resistance by chlorella fusca treatment.
In FIG. 4, 'chlorella' is a sphere treated with chlorella pusca in a mixed nutrition culture at a concentration of 10 ⁷ cells / ml, and 'control' is a sphere treated with BG-11 medium.
5 is a diagram confirming the induction-resistance mechanism by Chlorella fusca using the Arabidopsis mutants of salicylic acid and jasmonic acid signaling mechanism.
In FIG. 5, 'chlorella' is a sphere treated with chlorella cultured in culture at 10 ⁷ cells / ml, and 'control' is a sphere treated with BG-11 medium.
6 is a diagram confirming the induction-resistance determinants of Chlorella pusca.
In FIG. 6, the 'culture filtrate' is a sphere treated with a culture filtrate of chlorella pusca, and the 'cell' is a cell separated from the culture solution of chlorella pusca, and the 'culture filtrate + cells' is treated with a culture solution of chlorella pusca. One, control, was treated with BG-11 medium.
Figure 7 is a diagram confirming the induction-resistance of the bacillus bacillus of cucumber in chlorella pusca culture filtrate.
In FIG. 7, 'culture filtrate' is a sphere treated with a culture filtrate of Chlorella pusca, and 'control' is a sphere treated with BG-11 medium.

Hereinafter, the present invention will be described in detail.

1. Composition for enhancing immunity of plants

One aspect of the present invention provides a composition for enhancing immunity of plants containing a culture solution of Chlorella sp. Microalgae or a culture filtrate thereof as an active ingredient.

The culture solution of the microalgae of the genus Chlorella or the culture filtrate thereof may enhance plant immunity by inducing resistance of the plant to pathogens.

The microalgae of the genus Chlorella of the present invention is Chlorella fusca , and the chlorella fusca is preferably cultured as follows:

1) adding chlorella fusca to the medium to which the carbon source is added;

2) culturing the medium to which chlorella fusca is added in step 1) while irradiating with light for 7 to 12 days.

The chlorella pusca culture medium is cultured under a mixed nutrient condition for irradiating a carbon source and light, and in step 1), the carbon source is preferably glucose, and 0.5 g / L to 2 g / L in BG-11 medium. It is preferable to add.

Light irradiation in step 2) may be Chlorella crispus car incubated with preferably one, various well-known mixing nutrient culture conditions to investigate the quantity of light and 24 hours of light from 80 to 120 μmol / m ² / s by using a fluorescent light source . In addition, the temperature conditions of step 2) is preferably incubated at 25 to 30 ℃ room temperature conditions.

Preferably, the culture solution contains chlorella fusca cells at a concentration of 10 ⁶ cells / ml to 10 ⁸ cells / ml, most preferably at a concentration of 10 ⁷ cells / ml, and the 10 ⁶ cells / ml When included below the concentration of, the resistance inducing effect of the present invention is significantly reduced, the culture medium should be contained in a concentration of 10 ⁶ cells / ml to 10 ⁸ cells / ml Chlorella fusca cells in order to improve the resistance induction effect .

In addition, the culture filtrate of the microalgae of the genus Chlorella may be obtained by centrifugation of the microalgae of the genus Chlorella, separating the cells and the culture filtrate, and filtering the cells.

When the cells and the culture filtrate were treated separately, the culture filtrate showed a significant resistance inducing effect, but it was confirmed that the suspension separated only cells did not differ from the control. Therefore, the composition for enhancing immunity can be provided by separating the culture filtrate of the genus Chlorella microalgae.

In addition, salicylic acid (salicylic acid) and jasmonic acid (jasmonic acid) is a plant hormone that delivers a disease-resistant response systemically, the resistance inducing effect of the composition of the present invention may be dependent on salicylic acid signaling or jasmonic signaling.

In addition, the plant of the present invention is a food crop including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, millet; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including lygras, redclover, orchardgrass, alfalfa, tolskew, perennial lygragrass.

In addition, the composition of the present invention enhances the immunity of the plant by inducing resistance to the pathogen of the plant. Plant disease caused by the pathogen may be selected from the group consisting of late blight, incurable disease, spot bacteria, wildfire disease, anthrax, mozzarella disease, wilting disease, root rot disease, gray mold disease, green blight disease, leaf blight, vine cutting disease, spots Most preferably it is a bacterial disease. The pathogen for the spot bacteria is Pseudomonas Pseudomonas syringae fib tomato DC3000 syringae pv. tomato DC3000) or one pseudomonas ache Blood V-Rock Creations Mens (Pseudomonas syringae pv. lacrylmans ).

In order to confirm the resistance induction and immune enhancement effect of the chlorella microalgae culture medium and its culture filtrate of the present invention, in a specific embodiment of the present invention, after producing a culture solution of Chlorella pusca and treating the Arabidopsis, inoculated phytopathogens As a result, it was confirmed that the resistance control effect similar to the BTH treatment, a positive control (see Fig. 1 and 2A).

In addition, in order to confirm the antagonistic action of the chlorella microalgal culture of the present invention against plant diseases, the culture of the chlorella microalgal culture with the pathogen, as a result of the direct pathogen inhibitory effect was confirmed (see Fig. 2B). .

In addition, in order to confirm the generation of free radicals used as a marker of the plant immune response by the chlorella microalgae culture of the present invention, after reacting the chlorella microalgae culture to the Arabidopsis leaf, and treated with flg22, active oxygen Was increased by treatment of chlorella microalgae culture (see FIG. 2C).

In addition, in order to establish the optimum culture and treatment conditions of the chlorella microalgae culture of the present invention, as a result of confirming the resistance induction effect in a variety of culture conditions and treatment conditions, cell concentration in the mixed nutrient culture conditions treated with both light and glucose 10 ^When treated with ⁷ cells / ml it was confirmed that a significant resistance induction effect compared to the control (see Figure 3).

In addition, in order to confirm the resistance induction mechanism by the Chlorella pusca of the present invention, after treatment with Chlorella pusca to Arabidopsis, transcriptome analysis using RNA-seq, as a result of various plants by treatment with Chlorella pusca culture solution It is confirmed that the immune mechanism is activated, in particular dependent on the resistance-related plant hormones salicylic acid and jasmonic acid signaling mechanisms (see FIGS. 4 and 5).

In addition, in order to confirm the determinants that induce resistance by chlorella fusca of the present invention, after treating chlorella pusca culture filtrate or chlorella pusca cells, respectively, in Arabidopsis, the resistance induction effect was confirmed, Chlorella pusca Since the culture medium of chlorella fusca cells reduced resistance inducing effect than chlorella pusca culture filtrate, it was confirmed that the determinant that induces resistance by chlorella fusca is present in the culture filtrate, not the cells (see FIG. 6). .

In addition, in order to confirm the cucumber immunity-promoting effect of the chlorella pusca culture filtrate of the present invention, after spraying the culture filtrate of chlorella pusca on the cucumber leaf, after confirming the effect of inducing resistance to cucumber spot bacterial disease, Chlorella pusca It was confirmed that the immune enhancing effect of the culture filtrate was shown not only in the model plant Arabidopsis but also in the crop cucumber (see Fig. 7).

Therefore, the culture solution of the Chlorella sp. Microalgae of the present invention or the culture filtrate thereof induces plant resistance depending on the signaling mechanisms of salicylic acid and jasmonic acid, and the culture filtrate of the chlorella microalgae is particularly a microalgae of Chlorella sp. Since the resistance inducing effect is remarkably superior compared to the culture solution, there is an advantage that the culture filtrate of the microalgae of Chlorella, which generates a lot of waste costs, can be used as an environmentally friendly crop protection agent.

On the other hand, the compositions of the present invention may include agrochemically acceptable buffers, carriers, adjuvants or excipients, which are well known in the art. The composition of the present invention can be dried by lyophilization, spray drying or spray cooling.

The 'buffer' refers to an aqueous solution containing an acid-base mixture for the purpose of stabilizing pH, and the buffering agent is tris, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, tartrate, cacodylate , Ethanolamine, glycine, imidazole, imidazolic acid and the like can be used.

The 'diluent (or carrier)' means an aqueous or non-aqueous solution for the purpose of diluting the culture or the filtrate of the present invention, the diluent is one or more saline, water, polyethylene glycol, propylene glycol, ethanol or oil ( Such as corn oil, peanut oil, cottonseed oil, or sesame oil.

The 'adjuvant' means a specific compound added to the formulation to increase the biological effect of the composition of the present invention, it may be used agrochemically acceptable compounds and the like that can enhance the biological effect.

The 'excipient' may be one or more carbohydrates, polymers, lipids and inorganics. Examples of carbohydrates may include, for example, lactose, sucrose, mannitol, and cyclodextrins added to the composition to facilitate lyophilization. Examples of polymers include starch, cellulose ether, cellulose carboxymethylcellulose, alginate, carrageenan, hyaluronic acid, polyacrylic acid, polysulfonate, polyethyleneglycol / polyethylene oxide, polyvinylalcohol / polyvinylacetate with different hydrolysis degrees, and polyvinyl Pyrrolidones (all with different molecular weights).

In addition, the composition of the present invention may be in the form of an emulsion, an oil solution, a wettable powder, a suspension concentrate, a powder, a granule, a tablet, an aerosol or an ointment. It may be prepared as, but is not limited thereto. If desired, emulsifiers, suspending agents, spreaders, penetrants, wetting agents, thickening agents, stabilizers and the like may be combined, and these formulations may be prepared by methods known in the art. Preferably, the composition for inducing resistance of a plant of the present invention or the composition for controlling plant diseases may be in the form of a wettable powder or a liquid concentrate, and more preferably in a liquid form. In addition, an extender may be added to form a powder powder or granulated by formulating it. However, the formulation is not particularly limited.

The hydrating agent of the present invention is a culture filtrate of chlorella microalgae as an active ingredient, white carbon as a moisture absorbent, sodium bis [2-ethylhexyl] sulfosuccinate as a humectant, sodium lignosulfo as a dispersant And a kaolin as extender, preferably a culture filtrate of 10% by weight Chlorella microalgae, 1% by weight white carbon, 1% by weight sodium bis [2-ethylhexyl] sulfosuccinate, 1 wt% sodium lignosulfonate and 87 wt% kaolin, but is not limited thereto.

Liquid hydrating agent of the present invention is MBSC (Nonylphenol, ethoxylated, monoether with sulfuric acid, sodium salt, Sodium bis [20 ethylhexyl] sulfosuccinate Polyoxyethylene nonylphenol), It may consist of isopropanol as excipient and water as extender. The liquid hydrating agent may preferably be composed of a culture filtrate of 50 wt% chlorella microalgae, 4 wt% MBSC, 30 wt% isopropanol and 16 wt% water, but is not limited thereto.

2. Plant diseases  Control method

Another aspect of the present invention provides a method for controlling plant diseases comprising a plant; treating the plant with a culture solution of the microalgae of the genus Chlorella or the culture filtrate thereof.

In the chlorella culture medium or a culture filtrate of microalgae is the same bar, a detailed description to that described in "1. immune enhancing composition of the plant, the entry distance" 1. immune enhancing composition of the plant "column and, in the following plants Only the specific composition of the bottle control method will be described.

The plant treatment may be plant foliar treatment or soil irrigation, more preferably foliar treatment.

In addition, through the treatment of the culture solution of the chlorella microalgae as described above or the culture filtrate thereof, the plant obtains the induction-resistance to the pathogens to enhance immunity, thereby controlling plant diseases of vegetable crops, including cucumbers.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for illustrating the present invention, and the content of the present invention is not limited by the following examples.

Example  One

Chlorella genus Chlorella sp .) Preparation of Microalgae Culture and Culture Filtrates

In order to prepare a culture solution and a culture filtrate of microalgae of the genus Chlorella, in the present invention, the chlorella is Chlorella ( Chlorella). fusca ) was used.

Specifically, after adding 1 g / L of glucose (glucose) as a carbon source to BG-11 (sigma-Aldrich, co. Ltd.), and incubated Chlorella Fusca in the medium for 7 to 10 days, using a fluorescent light source The culture was irradiated with light amount of 100 μmol / m ² / s and light for 24 hours. On the other hand, the culture was incubated in the conditions of the room temperature control room temperature controlled to 25 ℃, put a magnetic stir for stirring to incubate at 300 rpm to prepare a chlorella pusca culture. In addition, during the culture, 0.45 μm syringe filter was connected to the culture medium by supplying a hose connected to the culture gas.

In addition, in order to prepare a chlorella pusca culture filtrate, the prepared chlorella pusca culture solution was separated into cells and culture filtrates at 5000 rpm using a centrifugal separator, and the separated culture filtrates were separated using a 0.45 μm syringe filter. Filtering was performed, and the centrifuged cells were resuspended in BG-11 medium.

Example  2

Chlorella Puska  Identification of the effect of inducing resistance to pathogens on culture media

[2-1] Identification of Induction Effect of Plants Against Pathogens

In order to confirm the resistance-inducing effect of the chlorella pusca culture against the pathogen, after treating the chlorella pusca culture in the Arabidopsis, the inoculation of phytopathogens was confirmed (Fig. 1).

Specifically, Arabidopsis Colombia, Col-0), a wild type of thaliana L. Heynh.), was sown, transplanted with 7 days old seedlings in 9.5 cm diameter pots containing sterilized soil, and grown at 25 ° C. for 24 days. After transplantation, the chlorella pusca culture solution prepared in [Example 1] was sprayed at 20 days per pollen with a cell concentration of 10 ⁷ cells / ml using a hemocytometer on the 7th and 14th day using a spray. Pseudomonas , a tomato bacillus pathogen causing disease in the Arabidopsis 21 days after transplantation syringae pv. Tomato DC3000 was inoculated by spraying the leaf diluted with an absorbance OD ₆₀₀ value of 1, and then the number of colonies was measured by dilution plate culture method on Arabidopsis leaves on days 0 and 7 after inoculation. On the other hand, 0.33 mM BTH (benzothiadiazole) diluent was used as a positive control, and negative control was sprayed on foliar to the Arabidopsis by the same method using BG-11, a chlorella culture medium.

As a result, as shown in Fig. 2A, the treated chlorella pusca culture solution was confirmed that on the 7th day of inoculation, the number of tomato spot bacterial pathogens was 14.9 times lower than that of the negative control, and the leaf symptom was also reduced compared to the negative control. When compared with the BTH treatment, a positive control, it was confirmed that both the population and the symptoms of the pathogen were similar (FIG. 2A).

Thus, it can be seen that chlorella pusca culture can induce resistance to plant diseases.

[2-2] Chlorella Puska  Of culture In plant diseases  Antagonism

Chlorella tomato bacterial spot pathogen Fu ska culture (Pseudomonas syringae pv. tomato DC3000) showed direct antagonistic effects.

Specifically, the paper disk treated with the Chlorella pusca culture solution prepared in [Example 1] was placed on 1/10 King's B medium, and then cultured together with tomato spot bacteria. In addition, as a positive control, a paper disk treated with 1 mg / ml of the antibiotic polymyxin B, which targets Gram-negative bacteria, was incubated with tomato antibacterial bacterium, and a negative control was used as a paper disk treated with BG-11. It was.

As a result, as shown in FIG. 2B, the antimicrobial bacterium did not grow on the paper disc treated with polymyxin B, whereas the tomato antibacterial pathogen grew normally on the paper disc treated with chlorella pusca culture or negative control BG-11. It was confirmed that (Fig. 2B).

Therefore, it can be seen that the chlorella puska culture medium does not show a direct pathogen inhibitory effect against plant pathogens.

[2-3] Chlorella Puska  Defense of culture Priming  Check the effect

Induction of resistance of plants can help plant survival through defense priming, which responds quickly and strongly only upon invasion of pathogens in order to maximize plant energy efficiency. Accordingly, by measuring the reactive oxidative species (ROS) used as a marker of the plant immune response, defense priming by chlorella was measured using a Luminol assay.

Specifically, after reacting the Chlorella pusca culture solution or BG-11 (negative control) prepared in [Example 1] to the Arabidopsis leaves for 1 hour, the ROS was generated by treating flg22 to induce free radicals. Then, the luminescence value by luminol reaction was measured according to the amount of ROS generated.

As a result, as shown in FIG. 2C, after treatment with the chlorella pusca culture solution, it was confirmed that the active oxygen increased significantly compared to the negative control, the chlorella pusca culture solution alone or the media alone treatment group.

Thus, it can be seen that induction-resistant priming occurs by treatment of chlorella pusca culture.

Example  3

Chlorella Puska  Confirmation of Optimal Culture Conditions

In order to optimize the resistance-inducing ability of chlorella pusca, induction-resistance was evaluated by treating chlorella pusca with culture conditions and concentrations of cells.

Specifically, chlorella pusca was incubated in BG-11 medium for 10 days under the three culture conditions shown in Table 1 according to the presence of light and glucose, and then chlorella pusca was cultured in each culture condition using a hemocytometer. ⁷ , 10 ⁵ , 10 ³ cells / ml after the foliar sprayed on the plant, the resistance was induced in the same manner as in Example 2 above.

Culture condition With or without light With or without glucose Mixed Nutritional Cultivation Conditions 100 μmol / m ² / s light intensity, 24-hour light irradiation 1 g / L addition Taga Nutritional Cultivation Conditions × 1 g / L addition Self-nourishment condition 100 μmol / m ² / s light intensity, 24-hour light irradiation ×

As a result, as shown in FIG. 3, the chlorella pusca treatment of the concentration of 10 ⁵ , 10 ³ cells / ml in the mixed nutrient culture condition and the remaining value of the other nutrient and autotrophic culture conditions differed from the negative control and the number of pathogens. It was confirmed that there is no. However, when chlorella pusca in mixed nutrient culture conditions were treated at a concentration of 10 ⁷ cells / ml, it was confirmed that the number of disease and pathogens was significantly reduced compared to the negative control (FIG. 3).

Therefore, it can be seen that the induction-resistance by the treatment of chlorella pusca culture appears when the cell concentration is 10 ⁷ cells / ml under mixed light culture conditions in which both light and glucose are treated.

Example  4

Chlorella In Puska  Resistive Induction Mechanism

In order to confirm the resistance induction mechanism by Chlorella pusca, Chlorella pusca grown in the optimum condition identified in [Example 3] was treated with Arabidopsis, and then transcriptome analysis using RNA-seq was performed.

Specifically, after inoculating the tomato pole bacterial pathogen ( Pseudomonas syringae pv. Tomato DC3000) in the Arabidopsis, the Arabidopsis leaves were sampled at 0 and 12 hours after inoculation and stored in -70 ℃ ultra-low temperature refrigerator. RNA was extracted from the sampled Arabidopsis leaves and synthesized cDNA, RNA-seq was performed for transcript analysis, and qRT-PCR was verified. Primers used for qRT-PCR are described in the following [Table 2]. In addition, after treating Chlorella pusca grown in the optimum condition identified in Example 3 above to the Arabidopsis at 10 ⁷ cells / ml, and inoculating the tomato bacillus pathogen by the same method, at 0 hours and 12 hours. After performing transcript analysis on the sampled Arabidopsis leaves, GO onto gene biological biology (GO BP) analysis was performed on DEG (Differentially Expressed Genes) with different expressions using MAPMAN software.

Signaling


CRK4_F GAA AAG GCC GGA ACT CCT CT SEQ ID NO: 1 CRK4_R AGT GAG CCC GCA GTG GTA AT SEQ ID NO: 2 CRK7_F AAG ACC GAA TTG ACC GCA CT SEQ ID NO: 3 CRK7_R GTA CCA ATG GCG GTT TCG TT SEQ ID NO: 4 RLP23_F ATG CAG GGC TTT GTG GTC TT SEQ ID NO: 5 RLP23_R CCC TAT TGC CAC TGC TCT CC SEQ ID NO: 6

Defense response


AtPR1-F TTC CAC AAC CAG GCA CGA GGA G SEQ ID NO: 7 AtPR1-R CCA GAC AAG TCA CCG CTA CCC SEQ ID NO: 8 PDF1.2_F CAC CCT TAT CTT CGC TGC TC SEQ ID NO: 9 PDF1.2_R GTT GCA TGA TCC ATG TTT GG SEQ ID NO: 10 CHIB_F CTA CTA TGG CCG TGG TGC AA SEQ ID NO: 11 CHIB_R CCA GAA AGC GGT CTT GAA GG SEQ ID NO: 12

ROS burst


GSTF6_F CTT CGC AAC CCC TTT GGT AA SEQ ID NO: 13 GSTF6_R TAT GAT CGC CAT GTC CTT GC SEQ ID NO: 14 GSTF7_F ACA CAG GCT TGG TGA GTC CA SEQ ID NO: 15 GSTF7_R ACC CAA GCA CTG ACA TGT GG SEQ ID NO: 16 GSTU4_F TTG CAA TCA AGG AGG CTC AA SEQ ID NO: 17 GSTU4_R GCG ACC AAG TCC AAA AAT CC SEQ ID NO: 18

Transcription factor


WRKY30_F CGG AGC CAA ATT TCC AAG AG SEQ ID NO: 19 WRKY30_R TCT GGT TCG GTT CGA GGT TT SEQ ID NO: 20 WRKY58_F GAG CTT CCA CGG ATG CTA AG SEQ ID NO: 21 WRKY58_R CAT ACG ATG GTG GTC AGA CG SEQ ID NO: 22 WRKY70_F ACG CAG AAA CTC CCA AGA GC SEQ ID NO: 23 WRKY70_R AAA CCA TTT CTG GCC ACA CC SEQ ID NO: 24 AtActin2-F2 GAA GAA CTA TGA ATT ACC CGA TGG SEQ ID NO: 25 AtActin2-R2 TAC AGA TCC TTC CTG ATA TCC ACA SEQ ID NO: 26

As a result, as shown in FIG. 4, the genes expressed by chlorella fusca treatment were identified as genes that function as signaling, transcription factor, defense response, ROS busting, cell wall synthesis, secondary metabolite, and the like. In addition, it was confirmed that most of the signaling mechanisms related to plant immunity are activated by chlorella pusca pretreatment, and gene expression patterns were verified through qRT-PCR of representative genes belonging to the functional taxonomy. Although it was not seen, the expression was increased at 12 hours, and the expression of PR1 and PDF1.2, which are marker genes of salicylic acid and jasmonic acid, was about 15 and 2 times higher in chlorella pusca treatment than in the negative control, respectively. It was confirmed that (Fig. 4).

Example  5

Chlorella using Arabidopsis mutants In Puska  Resistive Induction Mechanism

Disease resistance evaluation by chlorella pusca treatment was performed using the Arabidopsis wild type Col-0 and the Arabidopsis mutants. The mutants used were npr1 , NahG, jar1 , mutants of salicylic acid and jasmonic acid, which are important hormones in plant immune response.

Specifically, after treating Chlorella pusca grown in the optimum conditions identified in the above [Example 3] to Col-0 or the mutant at 10 ⁷ cells / ml, respectively, the tomato bacillus pathogen in the same manner as in [Example 2] above After inoculation, 0 and 7 days after inoculation, the number of colonies in infected Arabidopsis leaves was measured.

As a result, as shown in Figure 5, compared with the wild type, all three Arabidopsis mutants of salicylic acid and jasmonic acid signaling mechanisms did not show a resistance induction effect in the Chlorella pusca treatment (Fig. 5).

 Therefore, it can be seen that various plant immune mechanisms are activated by treatment with chlorella pusca culture, and in particular, are dependent on the resistance-related plant hormones salicylic acid and jasmonic signaling.

Example  6

Chlorella Puska  Identify Induction-Resistant Determinants

In order to identify the determinants that induce resistance by chlorella pusca, the chlorella pusca culture filtrate or chlorella pusca cells prepared in [Example 1] were treated separately in the Arabidopsis; -1] resistance evaluation was performed in the same manner. In addition, the heat resistance of the determinant in the culture filtrate was evaluated by autoclave treatment to determine the characteristics of the determinant in the Chlorella pusca culture filtrate.

As a result, as shown in Fig. 6, the cell treatment of Chlorella pusca showed no resistance effect as in the negative control. However, the treatment of chlorella pusca culture filtrate of the present invention was confirmed to significantly reduce the pathogens compared to the culture of the positive control BTH or chlorella pusca. Therefore, it was confirmed that the culture solution of chlorella fusca reduced resistance inducing effect due to the chlorella fusca cell filtrate.

In addition, the Chlorella pusca culture filtrate was confirmed to induce resistance regardless of the heat treatment despite the Autoclave treatment (Fig. 6B).

Therefore, it was confirmed that the determinant that induces resistance by Chlorella pusca is present in the culture filtrate, not the cell, and the determinant has heat resistance.

Example  7

Chlorella Puska  Confirmation of Immunity Enhancing Effect of Cucumber by Culture Filtrate

In order to confirm whether the present invention has a chlorella fuchsia culture filtrate exhibits the immune enhancing effect on a variety of plants, after the foliar spray treatment of the culture filtrate of chlorella fuchsia, the effect of resistance induction to cucumber spot bacterial disease was confirmed .

Specifically, cucumbers were sown in 50 seedling plates containing sterilized top soil and grown for 5 days, and seedlings were transplanted into a 9.5 cm diameter pot containing sterilized top soil and grown at 25 ° C. for days. On day 5 and day 10 after transplantation, the chlorella pusca culture filtrate of the present invention was foliarly sprayed by 10 ml per individual. After 5 days of culture filtrate treatment, Pseudomonas , a cucumber bacillus pathogen, syringae pv. The lacrylmans were solid-cultured and inoculated by spraying the foliar with a suspension of absorbance OD ₆₀₀ diluted to 1, and 5 days after the inoculation, the number of lesions on the main leaf of the infected cucumber was measured.

As a result, as shown in Fig. 7, after spraying the chlorella fuchsia culture filtrate of the present invention, the number of lesions in the cucumber leaf infected with cucumber spot bacillus pathogen was 4 than the negative control treated with BG-11 as in the positive control BTH It was confirmed that the fold decreased (FIG. 7).

Therefore, the immunity enhancing effect of the culture filtrate of Chlorella pusca is shown not only in the Arabidopsis, which is a model plant, but also in the cucumber, which is a crop.

In the above described exemplary embodiments of the present invention by way of example, the scope of the present invention is not limited only to the specific embodiments as described above, those skilled in the art to the scope of the claims of the present invention It will be possible to change accordingly.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Composition for improving immunity of plant comprising cultural          filtrate of Chlorella sp. <130> 2017-DPA-2529 <160> 26 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRK4_F <400> 1 gaaaaggccg gaactcctct 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRK4_R <400> 2 agtgagcccg cagtggtaat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRK7_F <400> 3 aagaccgaat tgaccgcact 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CRK7_R <400> 4 gtaccaatgg cggtttcgtt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RLP23_F <400> 5 atgcagggct ttgtggtctt 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> RLP23_R <400> 6 ccctattgcc actgctctcc 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> AtPR1-F <400> 7 ttccacaacc aggcacgagg ag 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> AtPR1-R <400> 8 ccagacaagt caccgctacc c 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDF1.2_F <400> 9 cacccttatc ttcgctgctc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PDF1.2_R <400> 10 gttgcatgat ccatgtttgg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIB_F <400> 11 ctactatggc cgtggtgcaa 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CHIB_R <400> 12 ccagaaagcg gtcttgaagg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTF6_F <400> 13 cttcgcaacc cctttggtaa 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTF6_R <400> 14 tatgatcgcc atgtccttgc 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTF7_F <400> 15 acacaggctt ggtgagtcca 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTF7_R <400> 16 acccaagcac tgacatgtgg 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTU4_F <400> 17 ttgcaatcaa ggaggctcaa 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GSTU4_R <400> 18 gcgaccaagt ccaaaaatcc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY30_F <400> 19 cggagccaaa tttccaagag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY30_R <400> 20 tctggttcgg ttcgaggttt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY58_F <400> 21 gagcttccac ggatgctaag 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY58_R <400> 22 catacgatgg tggtcagacg 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY70_F <400> 23 acgcagaaac tcccaagagc 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WRKY70_R <400> 24 aaaccatttc tggccacacc 20 <210> 25 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AtActin2-F2 <400> 25 gaagaactat gaattacccg atgg 24 <210> 26 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> AtActin2-R2 <400> 26 tacagatcct tcctgatatc caca 24

Claims (12)

As a composition for enhancing the immunity of plants containing the culture filtrate of Chlorella sp. Microalgae as an active ingredient,
The culture filtrate is to remove the chlorella microalgae cells from the culture of the microalgae of the genus Chlorella, the composition for enhancing immunity of plants.
The method according to claim 1,
The microalgae of the genus Chlorella is Chlorella fusca ( Chlorella fusca ) composition for enhancing immunity of plants.
The method according to claim 1,
The culture of the microalgae of the genus Chlorella is a composition for enhancing immunity of plants, wherein the chlorella pusca cells are contained at a concentration of 10 ⁶ cells / ml to 10 ⁸ cells / ml.
The method according to claim 1,
The culture of the microalgae of the genus Chlorella is a composition for enhancing the immunity of the plant, which is cultured by chlorella pusca (mixotrophic growth).
delete The method according to claim 1,
The culture filtrate of the microalgae of the genus Chlorella is a heat-resistant composition for enhancing immunity of plants.
The method according to claim 1,
The immune enhancement is a composition for enhancing immunity of plants that are dependent on salicylic acid signaling or jasmonic signaling.
The method according to claim 1,
The plant is a food crop including rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, millet; Vegetable crops including Arabidopsis, Chinese cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, pumpkin, green onion, onion, carrot; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, sugar beet, perilla, peanut, rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, leeks, grapes, citrus fruits, persimmons, plums, apricots, bananas; Flowers, including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And fodder crops comprising lygras, red clover, orchardgrass, alfalfa, tolsque, and perennial lygras.
The method according to claim 1,
The composition is a composition for enhancing the immunity of the plant to induce resistance to spot bacteria.
The method according to claim 9,
The pathogen for the spot bacteria is Pseudomonas syringae pv. Tomato DC3000 or Pseudomonas syringae pv. Lacrylmans Immunity enhancement composition of the plant.
A plant disease control method of a plant, comprising the step of treating the plant with a culture filtrate of chlorella microalgae,
The culture filtrate is to remove chlorella microalgae cells from the culture of the microalgae of the genus Chlorella, plant disease control method of the plant.
The method according to claim 11,
The treatment is a plant disease control method for plants that are foliar spray or soil irrigation.

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