KR100961895B1 - Method of enhancing disease resistance and drought resistance of plant and plant produced by the same - Google Patents

Abstract

     The present invention relates to a method for increasing disease resistance and drought resistance of a plant, and to a plant according to the present invention. More specifically, the method includes treating an extracellular polysaccharide isolated from Pseudomonas crolorapis O6 strain to a plant. To increase disease resistance and drought tolerance of plants; Plants with increased disease resistance and drought resistance produced by the above method; Seeds of the plant; The present invention relates to a composition for increasing disease resistance and drought resistance of a plant comprising, as an active ingredient, an extracellular polysaccharide isolated from Pseudomonas crolorapis O6 strain.

     Drought, Disease, Extracellular Polysaccharides, Tolerance, Plants, Methods, Compositions

Description

     Method of enhancing disease resistance and drought resistance of plant and plant produced by the same}

     The present invention relates to a method for increasing disease resistance and drought resistance of a plant and to plants accordingly.

     Plants are not only invaded by various phytopathogens such as bacteria, viruses, and fungi, but also greatly reduce plant productivity due to various stresses such as drought and low temperature stress. There is no clear progress on how to protect plants from environmental stresses such as drought. In recent years, there is also a preventive method that makes plants resistant to disease by treating the plants with microorganisms that induce disease resistance to plants before the disease has come [Van Loon et al., 1998]. As benzobenzo-S-methyl [Kunz et al., 1997], 2,6-dichloro isonicotinic acid [Metraux et al., 1991], Salicylic acid [Sticher et al., 1997], Benazol [Watanabe et al., 1973], beta-amino butyric acid [Tosi et al., 1998] and cyclo propane carboxylic acid derivatives [Michael et al., 2001] are also known to induce plant disease resistance.

     Pseudomonas crolorapis O6 is a microorganism known to have a function of inducing plants to resist plant diseases, drought and high salts and to have a prevention effect against invasion or drought of phytopathogens [Patent No. 0521744]. Regardless of the generation of plant disease resistance inducers, volatile 2R and 3R-butanediol have been reported to induce plant disease resistance [Han et al., 2006]. However, no metabolites of Pseudomonas crolorapis O6 induce and induce plant disease resistance and plant drought tolerance.

     Korean Patent No. 0521744 discloses Pseudomonas crolorapis 06 strains and methods for controlling plant diseases and reducing drought damage using the same, but there is no disclosure of extracellular polysaccharides that increase plant disease resistance and drought resistance.

     The present invention has been made in accordance with the above requirements, the present invention is resistant to the disease and drought stress of plants when treated with plants the extracellular polysaccharide extracted from the culture medium of Pseudomonas Crolorapis O6 strain using an organic solvent. We want to find out if

     In order to solve the above problems, the present invention provides a method for increasing the disease resistance and drought resistance of plants comprising the step of treating the plants with extracellular polysaccharides isolated from Pseudomonas Crolorapis O6 strain. I would like to.

     The present invention also provides a plant with increased disease resistance and drought resistance produced by the above method.

     The present invention also provides a seed of the plant.

     The present invention also provides a composition for increasing disease resistance and drought resistance of a plant comprising an extracellular polysaccharide isolated from Pseudomonas crolorapis O6 strain as an active ingredient.

      When the method according to the present invention is applied to crop cultivation, it is possible to increase the productivity of crops by increasing resistance to drought and plant diseases of plants, which are biologically produced, not chemically synthesized plant disease resistance inducers. It can be applied to plant disease control and prevention technology. Although Pseudomonas crolorapis O6 strain is known to induce resistance to drought and high salt, it is highly valuable by newly confirming that it is caused by extracellular polysaccharide of O6 strain through the present invention.

     In order to achieve the object of the present invention, the present invention provides a method for increasing the disease resistance and drought resistance of a plant comprising the step of treating the plant with extracellular polysaccharides isolated from Pseudomonas crolorapis O6 strain. do. The microorganism-derived extracellular polysaccharide of the present invention is secreted around the root of the plant in the microorganism. These secreted extracellular polysaccharides are known to form a matrix of banks consisting of cells and enzymes, which are then coated on plant roots to protect plants from drying or toxic substances. After treatment with extracellular polysaccharides isolated from Pseudomonas chlorophyllaceae O6 strain of the present invention to the inoculation of pathogens and drought to obtain a plant with increased disease resistance and drought resistance.

     In the method according to an embodiment of the present invention, the extracellular polysaccharide is a step of centrifuging Pseudomonas crolorapis O6 cell culture broth, solvent extraction of the cell precipitate obtained after centrifugation with acetone, followed by centrifugation of the supernatant, and further generation. The precipitate may be separated by cutting into protein K, but is not limited by the above method. Preferably, the Pseudomonas crolorapis O6 strain was cultured in Luria LB (Luria-Bertani) liquid medium for 60 hours at 27 ° C. and 150 rpm for 60 hours, and the purified cell culture was centrifuged at 8000 rpm for 15 minutes. Cell precipitates were solvent-extracted with the same amount of acetone, and then the supernatant was taken and centrifuged again. The precipitate was cut with Proteinase K to recover extracellular polysaccharides and dissolved in sterile water.

     In the method according to an embodiment of the present invention, the plant has increased resistance to any one of the plant pathogenic diseases selected from pepper bacterial wilting disease, cucumber galvanic disease, bacterial spot pattern disease, cucumber mosaic virus, tobacco purifying disease, seedling disease, soil fungus. May be, but is not limited thereto.

     In the method according to an embodiment of the present invention, the treatment site of the extracellular polysaccharide for the plant is characterized in that the leaf or root.

     Drought resistance of the plant in the method according to an embodiment of the present invention is characterized in that the pores of the plant is closed to induce resistance to drought. This means that the extracellular polysaccharide treatment prevents evaporation of water in the plant by inhibiting the transpiration of the plant by causing pore closure in the event of drought, resulting in resistance to drought.

     In the method according to an embodiment of the present invention, the extracellular polysaccharide is characterized in that it is treated to plants before invading pathogens. Treatment of the extracellular polysaccharides with plants before invading pathogens can effectively enhance resistance to pathogens.

     In the method according to an embodiment of the present invention, the extracellular polysaccharide is characterized in that it is treated to plants before drought. The extracellular polysaccharide is treated before the drought to obtain a plant with enhanced resistance to drought.

     The present invention provides a plant with increased disease resistance and drought resistance produced by the above method. The present invention, after dissolving the extracellular polysaccharide of the O6 strain in water and pre-treatment in the Arabidopsis at 500 nanograms in advance, when the bacterial plant or drought treatment of the same plant serves to inhibit more than 80% of drought and bacterial bruise. That is, the present invention has a preventive function that makes plants resistant to drought and plant diseases.

     In the method according to an embodiment of the present invention, the plant may be tobacco or Arabidopsis, but is not limited thereto.

     The present invention provides seed of the plant.

     The present invention provides a composition for increasing disease resistance and drought resistance of a plant comprising an extracellular polysaccharide isolated from Pseudomonas crolorapis O6 strain as an active ingredient. The composition may further include substances known in the art for increasing disease resistance and drought resistance of plants in addition to the extracellular polysaccharides isolated from the Pseudomonas chromolapis O6 strain.

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

     However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Incubation of Microorganisms

     The extracellular polysaccharide of the O6 strain used in the present invention was purified after incubating the Pseudomonas crolorapis O6 strain in a Luria broth (LB) liquid medium for 60 hours at 27 ° C. and 150 rpm.

Example 2 Purification of Plant Disease Resistance and Drought Resistance Inducing Substances

The Pseudomonas crolorapis O6 strain culture obtained above was centrifuged at 8000 rpm for 15 minutes. The cell precipitate obtained after centrifugation was solvent-extracted with the same amount of acetone. The supernatant was collected and centrifuged again. Then, the precipitate was cut with Proteinase K to recover extracellular polysaccharides and dissolved in sterile water (Fig. 1) .

<Example 3. Bacterial Soft Disease Resistance Induction Bioassay Using Tobacco>

Tobacco seeds were surface sterilized with 70% ethanol and hypochlorous acid (HClO ₄ ), followed by incubation for 3 days in a culture dish containing vitamins, sugars, and nutrients. The culture composition is as follows.

      Murashige & Skoog medium including vitamin 4.4 g / L

      2-Morpholinoethansulfonic acid and monohydrate 0.5 g / L

      Sucrose 30 g / L

      Phyto gel agar 5 g / L

Germinated seeds were transferred to tissue culture dishes, and then, tobacco was incubated at a place where light and dark conditions were 14 hours and 10 hours, respectively. After about 2 weeks, when 5-6 leaves of tobacco were grown, the extracellular polysaccharides of O6 strain suspended in 100 ㎍ / ml of water were diluted 10-fold and treated with 5 μl of each of the leaves or roots. Tobacco bacteria were treated on tobacco leaves at Erwinia carotovora SCC1 1.0 X 10 ⁸ cell level. Two days after treatment with pathogens, we investigated the incidence of softworms caused by bacterial fungal bacteria in tobacco leaves. As a control, distilled water was used, and as a reference, Pseudomonas crolorapis suspension of 1 × 10 ⁸ viable cell / ml was used.

In the fraction which induces resistance to bacterial disease soft rot by the O6 strain of the present invention, extracellular polysaccharides of the O6 strain induced resistance to soft rot (FIG. 2) . Control-treated sterile water, most plants are then treated with bacterial soft rot fungus eoteuna mouth affected by soft rot, O6 strain suspension and a plant treated with extracellular polysaccharides O6 isolates were harmed in soft rot (Fig. 2) .

<Example 4. Drought tolerance induced bioassay using Arabidopsis pole>

For drought treatment, the Arabidopsis seeds germinated by the above method were transferred to a tissue culture dish containing a sterilized Whatman filter paper, and then cultured in a plant tissue culture room having light and dark conditions for 14 hours and 10 hours, respectively. After about two weeks, the extracellular polysaccharides of the O6 strain suspended in sterile water were treated with leaves or roots, and after 3 days of treatment, the plants were left in a sterile capless dish without moisture and subjected to drought treatment. Leaves killed by 6 hours after treatment were examined as drought damaged leaves. As a control, distilled water was used, and as a reference, 1 × ^{10 8} viable cell / ml Pseudomonas chromolapis suspension was used.

In the present invention, the ability to induce drought resistance by extracellular polysaccharides of O6 strain showed only about 20% of the sterile water-treated control group survived drought stress, whereas the O6 strain showed 80% survival rate in 500 nanograms of extracellular polysaccharides. , 250 nanograms, 100 nanograms, and even 50 nanograms treatment showed a survival rate of more than 60% ( FIG. 3 ). When the extracellular polysaccharides of 100 nanogram O6 strain were treated with leaves, the survival rate was 70%, which showed drought tolerance similar to 80% survival rate of Pseudomonas crolorapis O6 strain treated with reference, and used as a control. Compared to the survival rate of 20% in the sterilized water treated spheres showed a significant drought resistance inducing ability (Fig. 4) . In addition, when treated with roots, the survival rate of drought treatment of extracellular polysaccharide treatment of O6 strain and Pseudomonas crolorapis O6 treatment of O6 strain was significantly higher than that of control treatment (FIG. 5) .

<Example 5. Histological verification of drought using Arabidopsis pole>

After growing the Arabidopsis by the method of Example 3, and then treated with O6 strain and extracellular polysaccharides of O6 strain in the same way, the tissue of very strong light (100μE / m ² / s) to open the pores of the plant as much as possible After 3 hours of incubation, the leaves were stained with safranin O (0.5%) for 30 seconds to 1 minute and washed three times with sterile water. Samples were examined under an optical microscope (Zeiss, Model DE / AXIOLAB-POI) to determine whether the pores were opened or closed, or the pores were opened or closed under a scanning electron microscope (Hitachi).

In the present invention, the ability to induce drought resistance by extracellular polysaccharides of O6 strains has been shown to induce drought tolerance by closing the pores of the present material. The water-treated plants, which were controls, had about 20% of pores closed, but when 100 nanogram extracellular polysaccharides were treated in the roots, more than 50% of the pores were closed, and the Pseudomonas crolorapis O6 strain treated as a reference The treated plants had about 70% pore closure (FIG. 6) .

     Through the above results, the present invention means that extracellular polysaccharide of O6 strain induces resistance to bacterial bacterium in tobacco or Pseudomonas crolorapis O6 in Arabidopsis was a major metabolite that exhibited plant disease resistance and drought tolerance. Treatment of this material with O6 strains closed the pores, leading to drought tolerance.

references

Han, SH, Lee, SJ, Moon, JH, Park, KY, Yang, KY, Cho, BH, Kim, KY, Kim, YH, Lee, MC, Anderson, AJ, and Kim, YC (2006) Gacs-dependent production of 2R, 3R-butanediol by Pseudomonas chlororaphis O6 is a major determinant for eliciting systemic resistance against Erwinia carotovora but not against Pseudomonas syrngae pv. tabaci in tobacco. Mol Plant-Microbe Interact 19, 924-930.

Kunz W., Schurter R., and Maetzke T. (1997) The chemistry of benzothiadiazole plant activator. J Pestic Sci 50, 275-282.

Metraux J.P., Ahl-Goy P., Staub T., Speich J., Steinemann A., Ryals J., and Ward E (1991) Induced systemic resistance in cucumber in response to 2,6-dichloro-isonicotinic acid and pathogens. Mol Plant-Microbe Interact 1, 432-439

Sticher L., Mauch-mani B., and Metraux J.P. (1997) Systemic acquired resistance. Annu Rev Plant Pathol 35, 235-270.

Tosi L., Luigetti R., and Zazzerini A. (1998) Induced resistance against Plasmopora helianthi in sunflower plants by DL-beta-amino-n-butyric acid. J Phytopathol 146, 259-280.

Van Loon L.C., Bakker P.A.H.M., and Piertese C.M.J. (1998) Systemic resistance induced by rhizosphere bacteria. Annu Rev Phytopathol 36, 453-483.

Watanabe T (1997) Effects of probenazole (Oryzemate ^® ) on each stage of rice blast fungus ( Pyricularia oryzae Cavara ) in its life cycle. J Pestic Sci 2, 394-404

     1 is a diagram showing a method for separating extracellular polysaccharides of Pseudomonas chromolapis O6 strain.

     Figure 2 is a diagram showing the degree of inducing disease resistance to bacterial soft disease in tobacco of the extracellular polysaccharide of the isolated O6 strain.

     Figure 3 is a diagram showing the drought resistance induction when treated in the Arabidopsis root of the concentration of extracellular polysaccharide of the isolated O6 strain.

     Figure 4 is a chart showing the induction of drought resistance when treated on the leaves of Arabidopsis by the concentration of the extracellular polysaccharide of the isolated O6 strain.

     Figure 5 is a diagram showing the induction of drought resistance when treated in the Arabidopsis root of the concentration of extracellular polysaccharide of the isolated O6 strain.

     6 is a diagram inducing Arabidopsis leaf pore closure after treatment to the Arabidopsis root by concentration of the extracellular polysaccharide of the isolated O6 strain.

Claims (11)

     A method of increasing drought tolerance of Arabidopsis plants comprising treating extracellular polysaccharides isolated from Pseudomonas chlorophylla O6 strain to Arabidopsis plants.      The method of claim 1, wherein the extracellular polysaccharide comprises: centrifuging Pseudomonas crolorapis O6 cell culture fluid; Centrifuging the cell precipitate obtained after centrifugation with acetone and then taking the supernatant and centrifuging again; And cleaving the resultant precipitate with Protease K. delete      The method of claim 1, wherein the site for treatment of the extracellular polysaccharide for the Arabidopsis plant is a leaf or a root. delete delete      The method of claim 1, wherein the extracellular polysaccharide is treated in Arabidopsis plants before the drought.      A Arabidopsis plant with increased resistance to drought prepared by the method of any one of claims 1, 2, 4 and 7. delete delete delete

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