Novel bacillus amyloliquefaciens strain having activity of promoting plant resistance against drought stress and uses thereof

Abstract

The present application relates to a novel Bacillus amyloliquefaciens strain, which enhances the stress resistance of plants and promotes the growth of plants. The Bacillus amyloliquefaciens N474 CP02-1004 strain, the Bacillus amyloliquefaciens N481 CP02-1005 strain, or a culture thereof of the present application can effectively mitigate the reductions in fresh weight of plants and water amount in plants caused by drought stress.

Description

Novel Bacillus amyloliquefaciens strain having activity for enhancing drought stress resistance of plants and use thereof

[0001] [Cross-citation with related application]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0003017, filed January 8, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present application relates to a novel Bacillus amyloliquefaciens strain that enhances drought stress resistance of plants and its use.

[0004]

[0005] Climate change exposes crops to a variety of unpredictable environmental stresses, leading to significant declines in productivity. In particular, climate change-induced abnormalities, such as heat waves, droughts, and the expansion of subtropical deserts, are significantly impacting crop production worldwide each year. Plants are significantly less able to avoid the various environmental stresses they face during their survival than animals. Therefore, environmental stresses such as low temperature, high temperature, salt, and drought significantly impact plant survival, growth, development, and yield. Drought stress is the most significant global problem, and its associated decline in crop yields is a pressing issue that must be addressed.

[0006] Water is an essential element for plant growth, and has a significant impact on the overall plant growth and development process, including photosynthesis, respiration, nutrient absorption, and movement, and is thus closely related to crop productivity.

[0007] Meanwhile, with growing public concern over the adverse effects of pesticides, demand is growing for eco-friendly products that can replace chemical fertilizers and pesticides. Therefore, there is a pressing need to develop eco-friendly products that can enhance plant resistance to drought stress and promote plant production.

[0008] Korean Patent No. 10-2239389 provides a Bacillus butanolivorans strain that has the activity of reducing drought stress in plants or promoting plant growth, and Korean Patent No. 10-2324981 provides a Pseudomonas fluorescens strain that induces resistance to drought stress in plants.

[0009]

[0010] [Prior Art Literature]

[0011] [Patent Document]

[0012] Korean Public Notice No. 2023-0109295

[0013] Korean Patent No. 10-2239389

[0014] Korean Patent No. 10-2324981

[0015]

[0016] The present inventors have conducted research efforts to discover a novel strain that enhances stress resistance in plants and promotes plant growth, and as a result, have experimentally proven that the strain of the present invention has the activity of increasing fresh weight and moisture content that are reduced under drought stress conditions, thereby completing the present invention.

[0017] Accordingly, the purpose of the present invention is to provide a novel strain that enhances stress resistance of plants and promotes plant growth.

[0018] Another object of the present invention is to provide a composition for improving stress resistance of plants or promoting growth of plants.

[0019] Another object of the present invention is to provide a method for enhancing stress resistance of a plant or a method for promoting plant growth.

[0020]

[0021] Each description and embodiment disclosed in this application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions set forth below. Furthermore, those skilled in the art will recognize or be able to ascertain, through routine experimentation alone, numerous equivalents to the specific embodiments of this application described in this application. Furthermore, such equivalents are intended to be encompassed by this application.

[0022] In order to achieve the above purpose, one aspect of the present invention provides a Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P.

[0023] Another aspect of the present invention provides a Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P.

[0024] Another aspect of the present invention provides a composition for enhancing stress resistance of a plant, comprising at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under accession number KCCM13393P or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under accession number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0025] Another aspect of the present invention provides a composition for promoting plant growth, comprising at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0026] Another aspect of the present invention provides a method for enhancing stress resistance of a plant, comprising the step of treating a plant, an area around the plant, or both, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under accession number KCCM13393P or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under accession number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0027] Another aspect of the present invention provides a method for promoting plant growth, comprising the step of treating a plant, an area around a plant, or both, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0028]

[0029] The present application is described in detail below.

[0030]

[0031] 1. New strains and their activity

[0032] One aspect of the present application provides Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under accession number KCCM13393P.

[0033] In addition, another aspect of the present application provides Bacillus amyloliquefaciens strain N481 CP02-1005 deposited under accession number KCCM13394P.

[0034] The Bacillus amyloliquefaciens N474 CP02-1004 strain deposited with the above-mentioned accession number KCCM13393P was deposited internationally with the Korea Microbiological Conservation Center, an international depository under the Budapest Treaty, and was assigned the accession number KCCM13393P on September 21, 2023.

[0035] The Bacillus amyloliquefaciens strain deposited in this application with the accession number KCCM13393P is named as strain N474 CP02-1004, and thus, in this specification, the terms 'Bacillus amyloliquefaciens N474 CP02-1004 microorganism', 'Bacillus amyloliquefaciens N474 CP02-1004 strain', 'Bacillus amyloliquefaciens N474 strain', 'N474 CP02-1004 strain', 'N474 microorganism' and 'N474 strain' may be used interchangeably. The base sequence of the 16s rRNA gene of the N474 strain is shown in SEQ ID NO: 1.

[0036] The Bacillus amyloliquefaciens N474 CP02-1004 strain deposited with the above-mentioned accession number KCCM13393P may include a base sequence of a 16S rRNA gene having a sequence identity of about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.9% or more with the base sequence of SEQ ID NO: 1. Specifically, the N474 CP02-1004 strain may include a base sequence of a 16S rRNA gene including the base sequence of SEQ ID NO: 1.

[0037] The Bacillus amyloliquefaciens N481 CP02-1005 strain deposited with the above-mentioned accession number KCCM13394P was deposited internationally with the Korea Microbiological Conservation Center, an international depository under the Budapest Treaty, and was assigned the accession number KCCM13394P on September 21, 2023.

[0038] The Bacillus amyloliquefaciens strain deposited in this application with the accession number KCCM13394P is named as strain N481 CP02-1005, and thus may be used interchangeably with 'Bacillus amyloliquefaciens N481 CP02-1005 microorganism', 'Bacillus amyloliquefaciens N481 CP02-1005 strain', 'Bacillus amyloliquefaciens N481 strain', 'N481 CP02-1005 strain', 'N481 microorganism' and 'N481 strain' in this specification. The base sequence of the 16s rRNA gene of the N481 strain is shown in SEQ ID NO: 2.

[0039] The Bacillus amyloliquefaciens N481 CP02-1005 strain deposited with the above-mentioned accession number KCCM13394P may include a base sequence of a 16S rRNA gene having a sequence identity of about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.9% or more with the base sequence of SEQ ID NO: 2. Specifically, the N481 CP02-1005 strain may include a base sequence of a 16S rRNA gene including the base sequence of SEQ ID NO: 2.

[0040] In one embodiment, the N474 CP02-1004 strain and / or the N481 CP02-1005 strain can enhance stress resistance of a plant and / or promote plant growth.

[0041] In one embodiment, the plant stress may be drought stress.

[0042] In one embodiment, the drought stress includes loss of moisture due to drought, reduction in plant fresh weight, reduction in crop yield, inhibition of normal plant growth, and vulnerability to pests and diseases.

[0043] In one embodiment, the “enhancement of stress resistance of plants” may be reduction of drought stress.

[0044] “Reduction of drought stress” in this specification may mean, but is not limited to, recovery of reduced plant moisture content induced by drought stress, preservation of fresh weight, preservation of crop yield, promotion of growth, and resistance to pests and diseases.

[0045] In the present application, the term “plant growth” refers to the process in which a plant seed germinates and produces roots, stems, leaves, and flowers, or thereafter gradually increases in size, weight, and quantity, and the term “promotion of plant growth” refers to promoting the germination of a plant seed or an increase in the size and / or weight of a germinated plant.

[0046] In this application, the term “plant” includes organisms belonging to the plant kingdom that perform photosynthesis, and is not taxonomically limited to a specific plant.

[0047] In the present application, the "plant" may be at least one selected from the group consisting of Cucurbitaceae, Brassicaceae, Amaranthaceae, Amaryllidaceae, Apiaceae, Asparagusaceae, Asteraceae, Pinaceae, Convolvulaceae, Leguminosae, Malvaceae, Muscariaceae, Poaceae, Polygonaceae, Rosaceae, Rubiaceae, Solanaceae, and Grapeaceae.

[0048] The above "Cucurbitaceae" plants may include, for example, cucumbers, melons, watermelons, cantaloupes, honeydews, melons, squashes, zucchinis, and pumpkins, and specifically, the above cucurbitaceae plants may be, but are not limited to, cucumbers, melons, watermelons, or melons.

[0049] The above "Brassicaceae" plants may include, for example, cabbage, arugula, broccoli, bok choy, Brussels sprouts, cabbage, cauliflower, canola, collard greens, white radish, Chinese cabbage, horseradish, kale, mustard, radish, rapeseed, rutabaga, turnip, wasabi, watercress, and Arabidopsis thaliana, and specifically, the above cruciferous plants may be, but are not limited to, cabbage, radish, mustard, rapeseed, or Arabidopsis thaliana.

[0050] The above "Amaranthaceae" plants may include, but are not limited to, for example, sorghum, spinach, sugar beets, and quinoa.

[0051] The above "Amaryllidaceae" plants may include, but are not limited to, chives, onions, garlic, chives, leeks, and shallots.

[0052] The above "Apiaceae" plants may include, but are not limited to, anise, caraway, carrot, celery, chervil, coriander, cumin, dill, fennel, parsley, and parsnips.

[0053] The above "Asparagaceae" plants may include, but are not limited to, agave and asparagus.

[0054] The above "Asteraceae" plants may include, but are not limited to, artichokes, aster, chamomile, chicory, chrysanthemums, dahlias, daisies, echinacea, chrysanthemums, guajul, lettuce, marigolds, safflowers, sunflowers, and zinnias.

[0055] The above "Bromeliaceae" plants may include, but are not limited to, pineapples, etc.

[0056] The above "Convolvulaceae" plants may include, but are not limited to, morning glory, and sweet potato.

[0057] The above "Fabaceae" plants may include, but are not limited to, alfalfa, beans, carob, clover, guar, lentils, mesquite, peas, peanuts, soybeans, tamarind, tragacanth, and vetch.

[0058] The above "Malvaceae" plants may include, but are not limited to, cacao, cotton, durian, hibiscus, kenaf, cola, and okra.

[0059] The above "Musaceae" plants may include, but are not limited to, bananas and plantains.

[0060] The above "Poaceae" plants may include, but are not limited to, bamboo, barley, corn, fonio, millet, oats, ornamental grasses, rice, rye, sorghum, sugarcane, triticale, wheat, and grasses such as bahia grass, umbrella grass, bluegrass, buffalo grass, centipede grass, fescue, or zoysia.

[0061] The above "Polygonaceae" plants may include, but are not limited to, buckwheat, etc.

[0062] The above "Rosaceae" plants may include, but are not limited to, almonds, apples, apricots, blackberries, blueberries, cherries, peaches, plums, quinces, raspberries, roses, and strawberries.

[0063] The above "Rubiaceae" plants may include, but are not limited to, coffee, etc.

[0064] The above "Solanaceae" plants may include, but are not limited to, bell peppers, peppers, eggplants, petunias, potatoes, tobacco, and tomatoes.

[0065] The above "Vitaceae" plants may include, but are not limited to, grapes, for example.

[0066] According to one embodiment, the N474 CP02-1004 strain and / or the N481 CP02-1005 strain may have an activity of enhancing stress resistance of a plant and / or promoting growth of a plant. Specifically, when cucumber (Cucumis sativus) is grown under drought stress conditions, the fresh weight of the cucumber and the moisture content within the plant decrease, but when the N474 CP02-1004 strain or the N481 CP02-1005 strain is treated to the cucumber, the fresh weight of the cucumber and the moisture content within the plant, which were reduced due to drought stress, can both be recovered.

[0067] In one embodiment, the N474 CP02-1004 strain or the N481 CP02-1005 strain can increase the fresh weight of cucumbers reduced by drought stress by 5 to 50%, 10 to 49%, 15 to 48%, 20 to 47%, or 25 to 45%.

[0068] In one embodiment, the N474 CP02-1004 strain or the N481 CP02-1005 strain can increase the moisture content in the cucumber plant reduced by drought stress by 5 to 40%, 7 to 38%, 9 to 36%, 10 to 34%, 11 to 32%, 12 to 30%, 13 to 28%, 14 to 26%, or 15 to 25%.

[0069] That is, the N474 CP02-1004 strain or the N481 CP02-1005 strain can enhance plant resistance to stress and promote plant growth, and thus can be usefully utilized for the purpose of enhancing plant stress resistance and promoting plant growth.

[0070]

[0071] 2. Use of the novel strain of this application

[0072] According to another aspect of the present application, a bacterial composition is provided comprising Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the above-mentioned deposit number KCCM13393P or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the above-mentioned deposit number KCCM13394P as an active ingredient.

[0073] In one embodiment, the composition may further comprise a cryoprotectant.

[0074] The Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P of the present application or the Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P can be prepared in the form of a lyophilized composition to preserve the activity of the microbial cells, and specifically, can be prepared in the form of a lyophilized powder, and the composition can further include a cryoprotectant to prevent damage to the cells.

[0075] In one embodiment, the cryoprotectant may include, but is not limited to, one or more selected from the group consisting of glycerol, ethylene glycol, propylene glycol, trehalose, sucrose, glucose, maltodextrin, sorbitol, skimmed milk powder, and starch.

[0076] In one embodiment, the cryoprotectant may be present in an amount of 0.01 to 30 wt% relative to the total composition, specifically 0.01 to 30 wt%, 0.01 to 25 wt%, 0.01 to 20 wt%, 0.01 to 15 wt%, 0.1 to 30 wt%, 0.1 to 25 wt%, 0.1 to 20 wt%, 0.1 to 15 wt%, 1 to 30 wt%, 1 to 25 wt%, 1 to 20 wt%, or 1 to 15 wt% relative to the total composition.

[0077] In one embodiment, the N474 CP02-1004 strain or the N481 CP02-1005 strain is included as an active ingredient in the composition, and its concentration is 1 x 10 5 CFU / mL to 1 x 10 12 CFU / mL. For example, the concentration of the strain may be 1 x 10 5 CFU / mL to 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 7 CFU / mL, 1 x 106 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 10 CFU / mL 내지 1 x 10 11CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 12 CFU / mL, or 1 x 10 11 CFU / mL to 1 x 10 12 It may be, but is not limited to, CFU / mL.

[0078]

[0079] Another aspect of the present application provides a composition for enhancing stress resistance of plants.

[0080] Specifically, the composition for enhancing stress resistance of the plant of the present application may include, as an active ingredient, at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0081] In addition, the composition for enhancing stress resistance of the plant of the present application may include, as an active ingredient, at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0082] The content of one aspect of the composition for enhancing stress resistance of the above plant and the content described in '1. Novel strain and its activity' overlap with the content described above, and therefore, they are cited and are not described repeatedly to avoid excessive complexity of the specification.

[0083] In this application, the "culture solution" refers to a product produced by culturing the strain, and the culture solution may include the culture solution itself in which the strain is cultured, the culture filtrate obtained by filtering or centrifuging the culture solution to remove the strain, and the supernatant obtained by centrifuging the culture solution or culture filtrate.

[0084] In the present application, the "concentrate" increases the solid concentration of the culture medium, and may be a concentrate of a culture medium containing cells of the strain of the present invention, or a concentrate of a culture supernatant from which cells of the strain of the present invention have been removed. The concentrate may be concentrated by vacuum concentration, plate concentration, thin film concentration, etc., but is not limited thereto, and may be performed at a temperature of 40°C to 60°C using a known concentrator, for example. Depending on the concentration of the concentrate, the content of the culture medium included in the composition of the present invention can be appropriately adjusted.

[0085] In this application, the "extract" refers to a product obtained by extracting the culture medium using an appropriate solvent according to a known method. The extract refers to a product extracted from the culture medium or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, a crude purified product or purified product thereof, or a fraction obtained by fractionating the extract.

[0086] The above "dry product" refers to a product from which moisture has been removed from the culture medium or concentrate, and may be, for example, a dried cell form of the strain, but is not limited thereto. The above dried product includes, but is not limited to, a product dried through a method such as freeze drying, vacuum drying, hot air drying, spray drying, reduced pressure drying, spray drying, foam drying, high-frequency drying, or infrared drying.

[0087] In addition, the above "pulverized material" means the result of pulverizing the dried material from which the moisture of the culture medium or the cells of the strain has been removed, and may be, for example, a dried cell powder of the strain, but is not limited thereto.

[0088] The culture solution of the above strain can be prepared by inoculating the strain into a culture medium and according to a culture method known in the art, and the concentrate, extract, dried product or pulverized product can be prepared according to a treatment or drying method of the strain or its culture solution known in the art.

[0089] In one embodiment, the stress in the composition for enhancing stress resistance of the plant may be drought stress.

[0090] In one embodiment, in the composition for enhancing stress resistance of the plant, the plant may be at least one selected from the group consisting of Cucurbitaceae, Brassicaceae, Amaranthaceae, Amaryllidaceae, Apiaceae, Asparagusaceae, Asteraceae, Pinaceae, Convolvulaceae, Leguminosae, Malvaceae, Muscariaceae, Poaceae, Polygonaceae, Rosaceae, Rubiaceae, Solanaceae, and Grapeaceae.

[0091] According to one embodiment, at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product can effectively alleviate a decrease in plant fresh weight and moisture content in a plant caused by drought stress, and thus can be usefully utilized for the purpose of enhancing stress resistance of plants.

[0092] In addition, according to one embodiment, at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product can effectively alleviate a decrease in the fresh weight and moisture content of a plant caused by drought stress, and thus can be usefully utilized for the purpose of enhancing stress resistance of a plant.

[0093] In one embodiment, the composition comprises 0.00001 wt% to 80 wt%, for example, 0.00001 wt% to 60 wt%, 0.00001 wt% to 40 wt%, 0.00001 wt% to 30 wt%, 0.00001 wt% to 20 wt%, 0.00001 wt% to 10 wt%, 0.00001 wt% to 5 wt%, 0.05 wt% to 60 wt%, 0.05 wt% to 40 wt%, 0.05 wt% to 30 wt%, 0.05 wt% to 20 wt%, 0.05 wt% to 10 wt%, 0.05 wt% to 5 wt%, 0.1 wt% to 60 wt%, 0.1 wt% to 40 It may include a strain, a culture solution of the N474 or N481 strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product, in an amount of 0.1 wt% to 30 wt%, 0.1 wt% to 20 wt%, 0.1 wt% to 10 wt%, or 0.1 wt% to 5 wt%.

[0094] In one embodiment, the concentration of the N474 CP02-1004 strain in the composition for enhancing stress resistance of the plant is 1 x 10 5 CFU / mL to 1 x 10 12CFU / mL. For example, the concentration of the strain may be 1 x 10 5 CFU / mL to 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 8CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 12 CFU / mL, or 1 x 10 11 CFU / mL to 1 x 10 12 It can be CFU / mL.

[0095] Additionally, in one embodiment, the concentration of the N481 CP02-1005 strain in the composition for enhancing stress resistance of the plant is 1 x 10 5 CFU / mL to 1 x 10 12 CFU / mL. For example, the concentration of the strain may be 1 x 10 5 CFU / mL to 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 10CFU / mL, 1 x 10 5 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 7 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 1011 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 12 CFU / mL, or 1 x 10 11 CFU / mL to 1 x 10 12 It can be CFU / mL.

[0096] In one embodiment, when the composition for enhancing stress resistance of a plant is in a solid state rather than a liquid state, the unit "CFU / mL" used to describe the concentration of the strain may be changed to "CFU / g".

[0097] In the present application, the composition for enhancing stress resistance of the plant may be prepared in the form of a directly sprayable solution, powder, and suspension, or as a highly concentrated aqueous, oily, or other suspension, dispersion, emulsion, oily dispersion, paste, dust, dustable material, or granule, but is not limited thereto.

[0098] The composition for enhancing stress resistance of plants in the present application can be formulated in various forms. For example, the formulation can be prepared by adding a solvent and / or carrier, and inert additives and surface-active substances, such as emulsifiers or dispersants, can be mixed into the formulation. Suitable surface-active substances are aromatic sulfonic acids (e.g. lignosulfonic acid, phenolsulfonic acid, naphthalene- and dibutylnaphthalenesulfonic acid), fatty acids, alkyl- and alkylarylsulfonates, alkyl lauryl ethers, alkali metal, alkaline earth metal and ammonium salts of fatty alcohol sulfates, sulfated hexa-, hepta- and octadecanol, salts of fatty alcohol glycol ethers, sulfonated naphthalene and derivatives thereof, condensates with formaldehyde, condensates of naphthalene or naphthalenesulfonic acid, phenol and formaldehyde, polyoxyethyleneoctyl phenol ethers, ethoxylated isooctyl-, octyl- or nonylphenols, alkylphenyl or tributylphenyl polyglycol ethers, alkylarylpolyether alcohols, isotridecyl alcohol, fatty alcohol / ethylene oxide condensates, ethoxylated castor oil, It may be, but is not limited to, polyoxyethylene alkyl ether or polyoxypropylene, lauryl alcohol polyglycol ether acetate, sorbitol ester, lignin-sulfite waste liquor or methylcellulose.

[0099] The carrier may be, but is not limited to, an agriculturally acceptable carrier, such as mineral earth (e.g., silica, silica gel, silicate, talc, kaolin, limestone, lime, chalk, boll, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic material), fertilizer (e.g., ammonium sulfate, ammonium phosphate, ammonium nitrate, urea), plant products (e.g., grain flour, bark flour, wood meal, and nut shell flour), or cellulose powder, and the carrier may be used alone or in combination of two or more.

[0100] The composition for enhancing stress resistance in plants in the present application may additionally include any other ingredient used in the art to enhance stress resistance in plants. For example, the composition may additionally include at least one selected from the group consisting of plant growth regulators, plant growth stimulants, and plant nutrients.

[0101] The plant growth regulator, plant growth stimulant or plant nutrient may be another strain of a chemical or bacteria, and may be a herbicide, fungicide, insecticide, fertilizer or any combination thereof.

[0102] In the present application, the composition for enhancing stress resistance of the plant may additionally include at least one selected from the group consisting of other beneficial microorganisms, microbial activators, organic fertilizers, and chemical fertilizers.

[0103] Other beneficial microorganisms may include Bacillus spp., Azotobacter spp., Trichoderma spp., and Saccharomyces spp., which are known to enhance stress resistance in plants.

[0104] The above microbial activators may include enzyme precursors, microbial metabolites, organic acids, carbohydrates, enzymes, and trace elements. For example, the microbial activators may include processed enzyme products such as yeast autolysates, humic materials, seaweed extracts, starch, amino acids, and trace elements such as zinc, iron, copper, manganese, bromine, and molybdenum.

[0105] The above chemical fertilizers may include various chemicals that can provide nitrogen, phosphorus, and potassium nutrients to help increase plant stress resistance. For example, chemical fertilizers may include urea, calcium phosphate, potassium phosphate, and mixed nitrogen-phosphate-potassium (NPK) fertilizers, as well as other ingredients known in the art.

[0106] In the present application, the composition for enhancing stress resistance of the plant may further include a dispersing agent and a penetrating agent, or a surfactant, to increase absorption and effect of the plant.

[0107] In the present application, the composition for enhancing stress resistance of the plant may additionally include a preservative, stabilizer, wetting agent, emulsifier, cryoprotectant, or excipient.

[0108] For example, the composition for enhancing stress resistance of the plant may include an effective amount of a preservative, stabilizer, or excipient sufficient to reduce quality deterioration of the composition, thereby allowing the N474 CP02-1004 strain and / or the N481 CP02-1005 strain to survive or remain active for a longer period of time in the composition, unlike the N474 CP02-1004 strain and / or the N481 CP02-1005 strain that exist in nature.

[0109] The preservative, stabilizer, or excipient may be any one commonly used in the art without limitation, as long as it can reduce the deterioration of the quality of the composition for enhancing stress resistance of the plant. For example, the stabilizer may be at least one selected from the group consisting of alginate, sodium alginate, casein, sodium caseinate, cellulose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, pectin, gelatin, gum arabic, xanthan gum, dextran, cyclodextrin, and starch. The excipient may include a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, solid diluent, or other auxiliary agent, for example, a surfactant such as an emulsifier, a dispersant, or a foaming agent.

[0110] The above cryoprotectant can reduce or prevent damage to the N474 CP02-1004 strain and / or N481 CP02-1005 strain existing in a natural state by freezing during the freeze-drying process.

[0111] The cryoprotectant may be included in the composition in an effective amount sufficient to reduce quality deterioration of the composition when the composition for enhancing stress resistance of the plant is in a freeze-dried state. For example, unlike the N474 CP02-1004 strain and / or the N481 CP02-1005 strain existing in a natural state, the composition may include an effective amount of the cryoprotectant sufficient to reduce quality deterioration of the composition or strain in a freeze-dried state, thereby allowing the N474 CP02-1004 strain and / or the N481 CP02-1005 strain to survive or remain active for a longer period of time in the freeze-dried composition.

[0112] The cryoprotectant may be any agent commonly used in the art without limitation, as long as it is intended to reduce quality deterioration of a freeze-dried composition or strain. For example, the cryoprotectant may be at least one selected from the group consisting of glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), glucose, trehalose, maltodextrin, dextrin, skim milk, and starch. In addition, the cryoprotectant may be mixed with a preservation carrier, adsorbed, dried, and used as a solid, and the preservation carrier may be diatomaceous earth, activated carbon, and / or defatted rice bran.

[0113] In one embodiment, the composition for enhancing stress resistance of the plant may be in the form of a freeze-dried powder.

[0114] The description of the strain, carrier, excipient, and additive is as described above. When using a cryoprotectant as the additive, the novel Bacillus strain of the present invention and the cryoprotectant may be mixed, the mixture may be frozen at -45°C to -30°C, dried at 30°C to 40°C, ground in a mixer, and manufactured in the form of a freeze-dried powder. Specifically, the freezing process may be a vacuum freezing process at a temperature of -45°C to -30°C and a pressure of 5 to 50 mTorr for 65 to 75 hours.

[0115] When a cryoprotectant is further included in the composition of the present invention, damage or death of the strain can be suppressed during the freeze-drying process of the composition, and the composition in freeze-dried form has advantageous advantages during the storage, distribution, and preservation processes. Furthermore, the composition in freeze-dried form can be distributed in powder form, and in this case, the strain in the composition can exhibit its activity while growing or metabolizing in a subject under drought stress.

[0116] In the present invention, the amount of the composition to be used can be appropriately determined depending on the formulation, damage situation, application method, application location, etc.

[0117] In one embodiment, the composition for enhancing stress resistance of a plant can be added to a plant fertilizer by immersion, spraying, or mixing.

[0118] The above plant fertilizer can be manufactured by a manufacturing method commonly used in the art. For example, the plant fertilizer can further include excipients or carriers commonly used in the art, and can be manufactured in a formulation of a conventional plant fertilizer known in the art, such as a wettable powder, granule, powder, emulsion, spray, mist, capsule, or gel.

[0119] In one embodiment, the plant fertilizer may be an eco-friendly fertilizer that can replace chemical fertilizers, and the plant fertilizer may be used to enhance stress resistance of plants.

[0120]

[0121] Another aspect of the present application provides a method for enhancing stress resistance of plants.

[0122] The method for enhancing stress resistance of the plant of the present application comprises a step of treating a plant, an area around the plant, or all of them, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0123] In addition, the method for enhancing stress resistance of the plant of the present application includes a step of treating a plant, an area around the plant, or all of them, with at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0124] In one embodiment, the stress of the plant may be drought stress.

[0125] In the present application, the plant may include any plant part or type, including roots, stems, leaves, seeds, fruits, or any part or combination thereof. Specifically, the plant may be a plant capable of growing under stress conditions or may be a plant individual grown under stress conditions, but is not limited thereto.

[0126] In this application, the "area surrounding a plant" refers to the range within which the active ingredient of this application can exert its effect, and includes both above-ground and underground areas. For example, it may be an area within about 2 m, about 1 m, about 70 cm, about 50 cm, about 25 cm, about 10 cm, or about 5 cm around a plant, plant part, or fruit.

[0127] According to one embodiment, the method for enhancing stress resistance of a plant can effectively restore the plant's fresh weight and water content reduction caused by drought stress, thereby effectively enhancing the plant's stress resistance.

[0128] At least one treatment selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the extract of the culture solution, the dried product of the culture solution, and the pulverized product of the dried product may be applied to the plant by a method such as immersion, irrigation, spraying, injection, infusion, mixing, or mixing with soil, but is not limited thereto as long as it can directly or indirectly contact the inside or outside of the plant. In addition, the treatment may be performed simultaneously or at the same time as the plant grows under stress conditions, but is not limited thereto. For example, the treatment may be performed once or twice or more, but is not limited thereto. In addition, when the treatment is performed twice or more, the treatment may be performed at intervals of 3 to 15 days, 3 to 10 days, 3 to 7 days, 4 to 7 days, 5 to 7 days, or 6 days, but is not limited thereto.

[0129] Another aspect of the present application provides a use for enhancing stress resistance of a plant, comprising a composition comprising at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0130] In one embodiment, the stress of the plant may be drought stress.

[0131] In addition, another aspect of the present application provides a use for enhancing stress resistance of a plant, comprising a composition comprising at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0132] In one embodiment, the stress of the plant may be drought stress.

[0133]

[0134] Another aspect of the present application provides a composition for promoting plant growth.

[0135] The composition for promoting plant growth of the present application comprises, as an active ingredient, at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0136] In addition, the composition for promoting plant growth of the present application comprises, as an active ingredient, at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0137] The content of one aspect of the composition for promoting the growth of the plant and the content described in '1. Novel strain and its activity' and '2. Use of the novel strain of the present application' above overlap with the content described above, and therefore, they are cited and are not described repeatedly to avoid excessive complexity of the specification.

[0138] In one embodiment, in the composition for promoting plant growth, the plant may be at least one selected from the group consisting of Cucurbitaceae, Brassicaceae, Amaranthaceae, Amaryllidaceae, Apiaceae, Asparagusaceae, Asteraceae, Pinaceae, Convolvulaceae, Leguminosae, Malvaceae, Muscariaceae, Poaceae, Polygonaceae, Rosaceae, Rubiaceae, Solanaceae, and Grapeaceae.

[0139] According to one embodiment, at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product can effectively increase a decrease in the fresh weight of a plant and a water content in the plant caused by drought stress, and thus can be usefully utilized for the purpose of promoting plant growth.

[0140] In addition, according to one embodiment, at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product can effectively increase a decrease in the fresh weight of a plant and a moisture content in the plant caused by drought stress, and thus can be usefully utilized for the purpose of promoting plant growth.

[0141] In one embodiment, the concentration of the N474 CP02-1004 strain in the composition for promoting plant growth is 1 x 10 5 CFU / mL to 1 x 10 12 CFU / mL. For example, the concentration of the strain may be 1 x 10 5 CFU / mL to 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 10CFU / mL, 1 x 10 5 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 7 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 8 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 7 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 9 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 11 CFU / mL, 1 x 10 8 CFU / mL 내지 1 x 10 12 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 10 10 CFU / mL, 1 x 10 9 CFU / mL 내지 1 x 1011 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 12 CFU / mL, or 1 x 10 11 CFU / mL to 1 x 10 12 It can be CFU / mL.

[0142] Additionally, in one embodiment, the concentration of the N481 CP02-1005 strain in the composition for promoting the growth of the plant is 1 x 10 5 CFU / mL to 1 x 10 12 CFU / mL. For example, the concentration of the strain may be 1 x 10 5 CFU / mL to 1 x 10 6 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 5 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 7 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 6CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 6 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 8 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 7 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 9 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 8 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 10 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 9 CFU / mL to 1 x 10 12 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 11 CFU / mL, 1 x 10 10 CFU / mL to 1 x 10 12 CFU / mL, or 1 x 10 11 CFU / mL to 1 x 10 12 It can be CFU / mL.

[0143] In one embodiment, when the composition for promoting plant growth is in a solid state rather than a liquid state, the unit "CFU / mL" used to describe the concentration of the strain may be changed to "CFU / g".

[0144] In the present application, the composition for promoting plant growth may be prepared in the form of a directly sprayable solution, powder, and suspension, or a highly concentrated aqueous, oily, or other suspension, dispersion, emulsion, oily dispersion, paste, dust, sprinkling material, or granule, but is not limited thereto, and may be formulated in various forms.

[0145] The plant growth promoting composition in the present application may additionally include any other ingredient used in the art to enhance stress resistance in plants. For example, the composition may additionally include at least one selected from the group consisting of plant growth regulators, plant growth stimulants, and plant nutrients.

[0146] In the present application, the composition for promoting plant growth may additionally include at least one selected from the group consisting of other beneficial microorganisms, microbial activators, organic fertilizers, and chemical fertilizers.

[0147] In the present application, the composition for promoting plant growth may further include a dispersing agent and a penetrating agent, or a surfactant, to increase absorption and effectiveness of the plant.

[0148] In the present application, the composition for promoting plant growth may additionally include a preservative, stabilizer, wetting agent, emulsifier, cryoprotectant, or excipient.

[0149] In one embodiment, the plant growth promoting composition can be added to plant fertilizer by immersion, spraying or mixing.

[0150] In one embodiment, the plant fertilizer may be an environmentally friendly fertilizer that can replace chemical fertilizers, and the plant fertilizer may be used to promote plant growth.

[0151]

[0152] Another aspect of the present application provides a method for promoting plant growth.

[0153] The method for promoting plant growth of the present application includes a step of treating a plant, an area around the plant, or all of them, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0154] In addition, the method for promoting plant growth of the present application includes a step of treating a plant, an area around the plant, or all of them, with at least one selected from the group consisting of Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0155] In the present application, the plant may include any plant part or type, such as roots, stems, leaves, seeds, fruits, or parts or combinations thereof, without limitation. Specifically, the plant may be a plant individual requiring growth promotion, but is not limited thereto. According to one embodiment, the method for promoting plant growth can effectively increase the fresh weight of the plant and reduce the water content within the plant, thereby effectively promoting plant growth.

[0156] In one embodiment, the decrease in the fresh weight of the plant and the decrease in the water content within the plant may be induced by drought stress.

[0157] In one embodiment, the growth promotion of the plant may be promoting the growth of a plant that has been reduced by drought stress.

[0158] At least one treatment selected from the group consisting of the strain, the culture solution of the strain, the concentrate of the culture solution, the extract of the culture solution, the dried product of the culture solution, and the pulverized product of the dried product may be performed by immersing, irrigating, spraying, injecting, pouring, mixing, or mixing with soil, but is not limited thereto as long as it can directly or indirectly contact the inside or outside of the plant. For example, the treatment may be performed once or twice or more, but is not limited thereto. In addition, when the treatment is performed twice or more, the treatment may be performed at intervals of 3 to 15 days, 3 to 10 days, 3 to 7 days, 4 to 7 days, 5 to 7 days, or 6 days, but is not limited thereto.

[0159] Another aspect of the present application provides a use for promoting plant growth of a composition comprising at least one selected from the group consisting of N474 CP02-1004 strain, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0160] In addition, another aspect of the present application provides a use for promoting plant growth of a composition comprising at least one selected from the group consisting of N481 CP02-1005 strain, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

[0161]

[0162] The Bacillus amyloliquefaciens N474 CP02-1004 strain, the Bacillus amyloliquefaciens N481 CP02-1005 strain, or a culture thereof of the present application can effectively increase the decrease in fresh weight and water content in plants caused by drought stress. Therefore, the strain of the present application or the culture thereof can be usefully utilized as an eco-friendly product that can promote plant production by enhancing plant resistance to drought stress.

[0163] However, the effects of the present application are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0164]

[0165] Figure 1 shows the results of measuring the fresh weight of cucumbers after treating them with a culture of Bacillus amyloliquefaciens strain under drought stress conditions.

[0166] Figure 2 shows the results of comparing fresh weight (A) and relative water content (RWC) after treating cucumbers with a culture solution of Bacillus amyloliquefaciens N474 CP02-1004 strain or a culture solution of Bacillus amyloliquefaciens N481 CP02-1005 strain under drought stress conditions.

[0167]

[0168] Hereinafter, the present application will be described in detail with examples. However, the following examples are intended to specifically illustrate the present application, and the contents of the present application are not limited by the following examples.

[0169]

[0170] Example 1: Isolation and cultivation of Bacillus amyloliquefaciens strains

[0171] The strains used in this experiment were obtained from field soil (field soil in Yeongtong-gu, Suwon-si, Gyeonggi-do). Approximately 50 g of soil was collected from a depth of 5-10 cm from the ground, and the collected soil was sieved through a 2-mm mesh sieve to remove large stones and foreign substances. Five grams of each sieved soil sample was cultured in flasks, and the cultured solution was diluted 1 / 10 in sterile distilled water to isolate the microorganisms.

[0172] Among the isolated microorganisms, six strains with high antagonism against the representative plant pathogens Rhizoctonia solani (R. s), Pythium ultimum (P. u), and Colletotrichum truncatum (C. t) were selected. To confirm the bactericidal effect against plant pathogens, four paper discs were placed in an 8.5 cm diameter petri dish containing PDA (potato dextrose agarose) medium, and three paper discs were inoculated with the standard strain Bacillus amyloliquefaciens KACC 10116 (B. amyloliquefaciens KACC 10116), Serenade® (Bayer) (B. velezensis QST713), and the strains to be evaluated. The remaining one paper disk was used as an untreated control. For the three plant pathogens (Rs, Pu, Ct), the pathogen colonies were cultured on PDA (potato dextrose agarose) medium at 28℃ for 5 to 8 days, and then the pathogen colonies were placed in the center of the petri dish using a 5 mm diameter cork borer. After culturing in an incubator at 25℃ for 7 to 20 days, the diameter of the zone of inhibition was examined to compare the antibacterial effects of the Bacillus strains. Finally, six strains (N311, N474, N481, N484, N485, N490) with antibacterial effects equal to or greater than those of Serenade were selected (Table 1).

[0173] Antibacterial effect (unit: mm) Rhizoctonia solani Phythium ultium Colletotrichum trucatum N3 1 19.7 3.7 12.7 N4 7 4 10.33.3 11 N4 8 19.35.0 12.3 N4 8 4 8.7 1.0 11.7 N4 8 5 4.34.7 6.7 N4 9 0 9.74.0 10.7 serenade 9.7 4.0 11.7

[0174] Specifically, the identification of the six selected strains was performed by analyzing the identity of the 16S rRNA gene sequence with the previously reported Bacillus amyloliquefaciens standard strain KACC 10116 using the CLC genomics workbench 21 program (Qiagen). As a result of the analysis, each of the six strains was confirmed to have 99% sequence homology, and was finally identified as a Bacillus amyloliquefaciens strain: Bacillus amyloliquefaciens strains were used: Bacillus amyloliquefaciens N474 CP02-1004 strain (accession number KCCM13393P), Bacillus amyloliquefaciens N481 CP02-1005 strain (accession number KCCM13394P), Bacillus amyloliquefaciens N484 CP02-1006 strain (accession number KCCM13395P), Bacillus amyloliquefaciens N485 CP02-1007 strain (accession number KCCM13396P), Bacillus amyloliquefaciens N490, Bacillus amyloliquefaciens N311. Each identified strain was suspended in 20% glycerol and stored.

[0175] Among the six strains finally selected, strains N474 and N481 were found to have the best drought stress alleviation efficacy in follow-up experiments.

[0176] The above N474 strain was named Bacillus amyloliquefaciens N474 CP02-1004 strain and was internationally deposited at the Korea Microbiological Conservation Institute, an international depository under the Budapest Treaty, and was assigned the accession number KCCM13393P on September 21, 2023. In addition, the above N481 strain was named Bacillus amyloliquefaciens N481 CP02-1005 strain and was internationally deposited at the Korea Microbiological Conservation Institute, an international depository under the Budapest Treaty, and was assigned the accession number KCCM13394P on September 21, 2023.

[0177] The base sequences of the 16s rRNA genes of the N474 and N481 strains are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0178] The six Bacillus amyloliquefaciens strains selected and identified as described above and the Bacillus amyloliquefaciens strain isolated from Serenade (Bayer) as a control were cultured. One colony of the Bacillus amyloliquefaciens strain was streaked on an LB (Luria-Bertani broth) medium and cultured for primary culture in an incubator at 28°C for 20 hours. A colony was picked from the primary culture medium and streaked on an LB medium for a second time, followed by secondary culture in an incubator at 28°C for 20 hours. Afterwards, 10 mL of sterilized distilled water was added to the secondary culture medium to perform cell resuspension, and the cells were recovered using a spreader. After measuring the OD (optical density) value of each strain, it was diluted to OD = 1.0 and used in the experiment.

[0179]

[0180] Example 2: Selection of Bacillus strains with excellent drought stress alleviation efficacy.

[0181] An experiment was conducted to confirm the effect of the six Bacillus amyloliquefaciens strains cultured in Example 1 above on alleviating drought stress in plants.

[0182]

[0183] 2-1. Experimental method

[0184] Cucumber (Cucumis sativus) was used as the plant, and cucumber seeds were sown in a 7:3 mixture of horticultural soil and vermiculite. Normal cucumber growth conditions were set as temperature 25℃, humidity 60-65%, normal irrigation cycle, and photoperiod 16h / 8h (day / night). Drought stress growth conditions were set as short-water conditions for 14 days, temperature 25℃, humidity 60-65%, and photoperiod 16h / 8h (day / night). Control groups were normal condition + DW treatment group, drought stress condition + DW treatment group, and drought stress condition + product control group, and the product control group was set as Bacillus amyloliquefaciens strain culture treatment group isolated from Serenade (Bayer) (hereinafter referred to as 'Serenade treatment group'). The test area was set as a drought stress condition + a treatment area with the culture solution of six strains of Bacillus amyloliquefaciens (N474, N481, N484, N485, N490, N311) cultured in Example 1 above.

[0185] Ten days after sowing cucumbers, the Bacillus amyloliquefaciens strain culture (OD = 1.0, 1 × 10 8 CFU / mL) was applied to cucumber leaves at a rate of 5 mL / plant twice at 7-day intervals. After the first application, drought stress was induced by starting watering only. After growing under drought stress conditions for 7 days, the total weight of the above-ground part of the cucumbers was measured, which was used as the fresh weight.

[0186]

[0187] 2-2. Live weight measurement results

[0188] As a result of measuring fresh weight, when drought stress was induced, the fresh weight of cucumbers was significantly reduced compared to normal conditions, but the fresh weight reduction caused by drought stress was recovered in the Bacillus amyloliquefaciens N474 strain treatment group and the N481 strain treatment group (Fig. 1).

[0189] Specifically, based on the final live weight, the N474 strain treatment group and the N481 strain treatment group showed an increase in live weight of approximately 32.2% and 25.8%, respectively, compared to the drought stress condition + DW treatment group. In addition, compared to the Serenade treatment group, the N474 strain treatment group and the N481 strain treatment group showed an increase in live weight of approximately 17.1% and 11.4%, respectively. That is, in the case of the commercial product Serenade treatment group, the live weight increased by approximately 12.9% compared to the drought stress condition + DW treatment group, whereas in the case of treatment with the N474 strain and the N481 strain, the increases were approximately 32.2% and 25.8%, respectively, compared to the drought stress condition + DW treatment group, showing that it showed an excellent effect of more than twice that of the commercial product in recovering the live weight loss caused by drought stress. In addition, the N484, N485, N490, and N331 strain treatment groups showed live weights that were lower or equal to those of the untreated group or the Serenade treatment group.

[0190] Accordingly, additional experiments were conducted to confirm that the N474 and N481 strains had drought stress alleviation effects.

[0191]

[0192] Example 3: Verification of the efficacy of a strain with excellent drought stress mitigation efficacy.

[0193] The drought stress alleviation efficacy of the N474 and N481 strains selected in Example 2 above was verified.

[0194]

[0195] 3-1. Experimental method

[0196] Other conditions were the same as in Example 2-1, but in order to maximize the biological treatment effect, Bacillus culture solution was applied to cucumbers by irrigation at a concentration of 30 mL / pot. As in Example 2, drought stress was induced by starting watering only after one application of fertilizer, and after inducing drought stress, Bacillus culture solution was applied by irrigation once, and then cucumber fresh weight was measured and relative water content (RWC), an index that best represents the degree of drought stress, was also measured.

[0197]

[0198] 3-2. Live weight measurement results

[0199] As a result of measuring fresh weight, when drought stress was induced, the fresh weight of cucumbers was significantly reduced compared to normal conditions, but the fresh weight reduction due to drought stress was recovered in the N474 strain treatment group and the N481 strain treatment group (Panel A of Figure 2).

[0200] Specifically, based on the final live weight, the N474 strain treatment and the N481 strain treatment showed increases of approximately 43.3% and 30%, respectively, compared to the drought stress condition + DW treatment. In addition, compared to the Serenade treatment, the N474 strain treatment and the N481 strain treatment showed increases of approximately 53.5% and 39.2%, respectively. It was confirmed that the irrigation treatment showed a better recovery effect from the loss of live weight due to drought stress than the Serenade treatment.

[0201]

[0202] 3-3. RWC (Relative Water Content) Measurement Results

[0203] RWC is an indicator of the water content within a plant. Normally growing plants have an RWC of approximately 80%. A low RWC indicates low water content within the plant, while a high RWC indicates high water content within the plant. RWC values ​​were calculated by measuring fresh weight (FW), turgid weight (TW), and dry weight (DW).

[0204] First, after measuring fresh weight (FW), cucumber plants were soaked in water for 24 hours to saturate the moisture content in the plant tissue to 100%, and the weight of the plant with saturated moisture content (Turgid weight, TW) was measured. After measuring TW, the sample was dried in an oven at 70℃ for approximately 48 hours, and then the dry weight (DW) was measured. Using the FW, TW, and DW values ​​measured in this way, the RWC was calculated using the following formula.

[0205] RWC(%) = [(FW-DW) / (TW-DW)] x 100

[0206] As a result of RWC measurement, when drought stress was induced, the RWC value of cucumbers was significantly reduced compared to normal conditions, but the decrease in RWC due to drought stress was recovered in the N474 strain treatment group and the N481 strain treatment group (Panel B of Fig. 2).

[0207] Specifically, the RWC values ​​were confirmed to increase by approximately 22.1% and 16.4% in the N474 and N481 strain treatment groups, respectively, compared to the drought stress condition + DW treatment group. In addition, compared to the Serenade treatment group, the N474 and N481 strain treatment groups showed increases in the moisture content within the plant body by approximately 21.2% and 15.5%, respectively. Through this, it was confirmed that the N471 and N481 strain treatment groups were more effective in recovering the moisture content decrease within the plant body due to drought stress than the Serenade treatment group.

[0208]

[0209] Through the above experiments, we confirmed that Bacillus amyloliquefaciens strains N474 and N481 can effectively alleviate drought stress by preventing water loss within plants and efficiently using water when drought-stressed plants are treated. Therefore, these strains are expected to be useful for enhancing drought stress resistance and promoting growth in plants.

[0210]

[0211] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

[0212]

[0213] [Accession number]

[0214] Name of depositor: Korea Microbiological Conservation Center

[0215] Accession number: KCCM13393P

[0216] Date of acceptance: 20230921

[0217]

[0218] Name of depositor: Korea Microbiological Conservation Center

[0219] Accession number: KCCM13394P

[0220] Date of acceptance: 20230921

[0221] [Correction pursuant to Rule 91, February 19, 2025]

[0222] [Correction pursuant to Rule 91, February 19, 2025]

Claims

1. Bacillus amyloliquefaciens strain N474 CP02-1004 deposited under the deposit number KCCM13393P or Bacillus amyloliquefaciens strain N481 CP02-1005 deposited under the deposit number KCCM13394P.

2. In claim 1, The above strain is a strain that enhances stress resistance of plants.

3. In claim 2, The above stress is drought stress, strain.

4. In claim 1, The above strain is a strain that promotes plant growth.

5. A composition for enhancing stress resistance of a plant, comprising as an effective ingredient at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under deposit number KCCM13393P, or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

6. In claim 5, A composition for enhancing stress resistance of plants, wherein the above stress is drought stress.

7. In claim 5, A composition for enhancing stress resistance of a plant, wherein the plant is at least one selected from the group consisting of Cucurbitaceae, Brassicaceae, Amaranthaceae, Amaryllidaceae, Apiaceae, Asparagusaceae, Asteraceae, Pinaceae, Convolvulaceae, Legumeaceae, Malvaceae, Muscariaceae, Gramineae, Rosaceae, Rubiaceae, Solanaceae, and Grapeaceae.

8. A composition for promoting plant growth, comprising at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under deposit number KCCM13393P, or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

9. In claim 8, A composition for promoting plant growth, wherein the plant is at least one selected from the group consisting of Cucurbitaceae, Brassicaceae, Amaranthaceae, Amaryllidaceae, Apiaceae, Asparagusaceae, Asteraceae, Pinaceae, Convolvulaceae, Legumeaceae, Malvaceae, Muscariaceae, Gramineae, Rosaceae, Rubiaceae, Solanaceae, and Grapeaceae.

10. A method for enhancing stress resistance in a plant, comprising a step of treating a plant, an area around a plant, or both, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under deposit number KCCM13393P, or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

11. A method for promoting plant growth, comprising the step of treating a plant, an area around a plant, or both, with at least one selected from the group consisting of Bacillus amyloliquefaciens N474 CP02-1004 strain deposited under the deposit number KCCM13393P, or Bacillus amyloliquefaciens N481 CP02-1005 strain deposited under the deposit number KCCM13394P, a culture solution of the strain, a concentrate of the culture solution, an extract of the culture solution, a dried product of the culture solution, and a pulverized product of the dried product.

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