KR20240069909A - Bacillus tequilensis PAMB00293 strain promoting plant growth and uses thereof - Google Patents

Abstract

The present invention relates to Bacillus tequilensis PAMB00293 strain deposited with accession number KCTC 15056BP; A composition for promoting plant growth containing the above strain or its culture; microbial preparations; A plant growth promotion kit containing the plant growth promotion composition; and a plant growth promotion method comprising treating soil or plants with the plant growth promotion composition.

Description

Bacillus tequlensis PAMB00293 strain promoting plant growth and uses thereof {Bacillus tequilensis PAMB00293 strain promoting plant growth and uses thereof}

The present invention relates to Bacillus tequilensis PAMB00293 strain deposited with accession number KCTC 15056BP; A composition for promoting plant growth containing the above strain or its culture; microbial preparations; A plant growth promotion kit containing the plant growth promotion composition; and a plant growth promotion method comprising treating soil or plants with the plant growth promotion composition.

The indiscriminate use of chemical pesticides during plant production has raised environmental pollution issues, further increasing the need for biological pesticides. In response to these demands, the recurrence of pesticides using microorganisms among biological pesticides has been happening for a long time. In order to be used as an agricultural useful microorganism, it must have crop useful activities such as promoting plant growth and solubilizing inorganic nutrients in the soil.

Currently, various agricultural microbial preparations are being distributed and sold by city/county agricultural technology centers or agriculture-related companies across the country. However, there is no or insufficient verification of the exact crop useful activity of agricultural useful microorganisms currently distributed or commercially available (KR 10-2204522 B1). Accordingly, the present inventors isolated microorganisms that can be used as agricultural useful microorganism preparations, so that they can be used as eco-friendly agricultural microorganisms.

Plants have a system that regulates growth and development to cope with and adapt to various environmental changes. In particular, the control of plant development mainly occurs through the interaction between the external environment and the plant's intrinsic developmental program, and is mainly controlled by plant hormones. It is known to perform essential functions for such interactions. Among these, auxin is known to be involved in plant germination and growth from seeds or in stem new growth.

In the present invention, Bacillus tequilensis strain is isolated and its siderophore, protease, amylase, auxin and catalase production ability and plant growth promotion ability are determined. By confirming this, it was possible to use it as an eco-friendly agricultural microorganism.

Currently, not much research has been done on the growth promotion ability of crops and model plants using Bacillus tequilensis, so the strain of the present invention is a strain with high utility as an eco-friendly biofertilizer and an additive to eco-friendly agricultural materials.

The purpose of the present invention is to provide Bacillus tequilensis PAMB00293 strain deposited with accession number KCTC 15056BP.

Another object of the present invention is to provide a composition for promoting plant growth comprising the Bacillus tequlensis PAMB00293 strain or a culture thereof. Another object of the present invention is to provide a composition for promoting plant growth containing the Bacillus tequlensis PAMB00293 strain or a culture thereof. To provide a microbial preparation containing.

Another object of the present invention is to provide a plant growth promotion kit containing the plant growth promotion composition.

Another object of the present invention is to provide a method for promoting plant growth, comprising the step of treating soil or plants with the composition for promoting plant growth.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.

Additionally, it is intended that those skilled in the art will understand that using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein are encompassed by the invention.

One aspect of the present invention provides Bacillus tequilensis PAMB00293 strain deposited with accession number KCTC 15056BP.

The present inventor developed a new strain belonging to Bacillus tequilensis , named it Bacillus tequlensis PAMB00293, and deposited it with the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, an international depository under the Budapest Treaty, in August 2022. As of the 18th, it was given the accession number KCTC15056BP.

“Strain” in this application refers to a collection of individuals originating from a single cell, proliferating through asexual reproduction and having the same genetic characteristics, and within one species, several strains with different genetic characteristics exist. . In this application, strain refers to Bacillus tequlensis, and may specifically be Bacillus tequlensis deposited under the accession number KCTC 15056BP, but is not limited thereto.

In the present invention, the Bacillus tequlensis PAMB00293 strain may have plant growth promoting ability.

In addition, in the present invention, the Bacillus tequlensis PAMB00293 strain may have the ability to produce one or more of siderophore, protease, amylase, auxin, and catalase, , plant growth can be promoted due to the action of the above substances.

Siderophore of the present invention is an important mechanism of rhizosphere bacteria that promotes plant growth, and refers to a peptide chelating substance secreted by microorganisms outside the body to absorb iron.

Protease of the present invention is also used interchangeably with proteolytic enzymes, and is an enzyme that hydrolyzes proteins and peptide bonds. It is widely present in plant tissues, cells, and microorganisms, and is largely divided into exopeptidases and endopeptidases according to the decomposition mode. I am not limited to this. In plants, it is known to act in many cases to regulate plant development or photosynthesis, but is not limited to this.

Amylase of the present invention, also used interchangeably with amylase, is an enzyme that hydrolyzes polysaccharides such as starch, and is widely distributed in nature, including higher plants. It is known to be used to decompose starch in the early stages of plant germination and use it as an energy source, but is not limited to this.

Auxin of the present invention is a type of plant hormone, an amino acid derivative synthesized from tryptophan, and is one of the hormones that regulate plant growth. Auxin is known to function in almost all plant development processes, such as promoting plant stem, leaf, and root growth, preventing fruit drop, and promoting fruit set, but is not limited to this. Types of auxins include indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 4-chloroindole-3-acetic acid (CIAA), and phenylacetic acid. (phenylacetic acid), and synthetic auxins include 2,4-D (2,4-dichlorophenoxyacetic acid) and naphthalene-1-acetic acid (NAA), but are not limited thereto.

Catalase of the present invention is an enzyme that catalyzes the reaction in which hydrogen peroxide is decomposed to produce water and oxygen, and is known to be widely distributed in living organisms.

Plants subject to growth promotion in the present invention include, for example, monocot plants such as rice, wheat, barley, bamboo shoots, corn, taro, asparagus, onions, garlic, green onions, chives, chives, yam, and ginger. Examples of dicotyledonous plants include, but are not limited to, ginseng, Arabidopsis thaliana, eggplant, tobacco, pepper, tomato, burdock, mugwort, lettuce, bellflower root, spinach, Swiss chard, sweet potato, celery, carrot, water parsley, parsley, Chinese cabbage, cabbage, It can be radish, watermelon, melon, cucumber pumpkin, gourd, strawberry, soybean, mung bean, kidney bean, bird's foot trefoil, potato, duckweed, perilla seed, pigeon pea and pea. Preferably, it may be Arabidopsis thaliana, cabbage, or tomato, but is not limited thereto.

In the present invention, “plant growth” may mean increasing leaf fresh weight, root fresh weight, number of leaves, number of lateral roots, root length, or chlorophyll content, but is not limited thereto. Additionally, in the present invention, plant growth may promote fruit coloration, but is not limited thereto.

Another aspect of the present invention provides a composition for promoting plant growth comprising Bacillus tequlensis PAMB00293 strain or a culture thereof.

The term “culture” in the present invention refers to a product obtained after culturing the above-mentioned strain, and may be a culture stock solution containing bacteria, or may be a culture strain or a culture supernatant obtained by removing or concentrating the bacteria. It also includes concentrates of the cultures, dried cultures, fermented products, and processed products derived from the culture itself.

The composition of the culture may include not only components necessary for cultivating conventional Bacillus strains, but also additional components that act synergistically on the growth of Bacillus strains, and the composition thereof may be determined by those skilled in the art. can be easily selected.

The medium and other culture conditions used for cultivating the strain of the present application can be any medium used for cultivating ordinary Bacillus genus microorganisms without particular limitation. Specifically, the strain of the present application can be cultured with an appropriate carbon source, nitrogen source, and personnel. , can be cultured under aerobic or anaerobic conditions in a conventional medium containing inorganic compounds, inorganic salts, amino acids, vitamins and/or nucleic acids, while controlling temperature, pH, etc.

The medium may be a natural medium or a synthetic medium, and may be a solid medium or a liquid medium. For example, glucose, sucrose, dextrin, glycerol, starch, etc. can be used as carbon sources of the medium, and peptone, meat extract, yeast extract, dried yeast, soybeans, ammonium salts, nitrates, and other organic or inorganic sources can be used as nitrogen sources. Nitrogen-containing compounds may be used, and the ingredients may include monopotassium phosphate, dipotassium phosphate, or corresponding sodium-containing salts, but are not limited to these ingredients. Sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, calcium carbonate, etc. can be used as inorganic compounds, and magnesium. Manganese, calcium. Iron, potassium, etc. may be used, but are not limited to these.

In the present invention, the composition may include other by-products produced in the process of cultivating the new strain of the present invention.

In the present invention, “plant growth promotion” may mean increasing one or more of the plant's leaf fresh weight, root fresh weight, number of leaves, number of lateral roots, root length, and chlorophyll content, and coloring of the fruit. It may be promoted, but is not limited to this.

Another aspect of the present invention provides a microbial preparation containing Bacillus tequlensis PAMB00293 strain or a culture thereof.

The microbial preparation can be formulated for plant growth promotion by conventional methods, and can also be prepared in the form of dry powder or liquid fertilizer, but the formulation is not particularly limited. Preferably, it can be formulated as a plant growth-promoting biological fertilizer to replace chemical fertilizers, and as a biological fertilizer to overcome this problem in eco-friendly organic farming where the supply of chemical fertilizers is limited.

In the present invention, the microbial preparation can be manufactured by adding additives such as additives, extenders, and nutrients to the strain or its culture. At this time, the additives include polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl. Aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyalkylone alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100 and Tween 80. One or more selected from may be used, and the extender and nutrient may be one selected from the group consisting of skim milk (medium), soybean flour, rice, wheat, red clay, diatomaceous earth, bentonite, dextrin, glucose, and starch. Or two or more are used, and the disintegrant may be one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate. .

In the present invention, the effective amount of microorganisms contained in the microbial preparation may be 1 to 1 × 10 ¹⁰⁰ microorganisms per cultivated land area (㎡). In addition, the effective amount of microorganisms contained in the preparation treated by spraying in the above method may be included at a concentration of 1 to 1 × 10 ¹⁰⁰ microorganisms per ml, and the effective amount of microorganisms contained in the composition treated by immersion may be It may be included at a microorganism concentration of 1 to 1×10 ¹⁰⁰ .

Another aspect of the present invention provides a kit for plant growth promotion including the plant growth promotion composition.

The kit is for promoting plant growth, and the kit may be provided so that the composition can be stored in a separate container and mixed at the time of use, but is not limited thereto.

In addition, the kit of the present application may include one or more other components, compositions, solutions, or devices to promote plant growth, but is not limited thereto. Examples of the other components may include, but are not limited to, suitable carriers, solubilizers, buffers, stabilizers, etc. The carrier may include a soluble carrier or an insoluble carrier, and may be, for example, a physiologically acceptable buffer known in the art (e.g., PBS), but is not limited thereto.

In addition, the kit of the present application may further include a user guide describing optimal reaction performance conditions. The guide is a printed material that explains how to use the kit, for example, reaction conditions, etc. Instructions include information leaflets in the form of pamphlets or leaflets, labels affixed to the kit, and instructions on the surface of the package containing the kit. Additionally, the guide includes information disclosed or provided through electronic media such as the Internet.

Another aspect of the present invention provides a method for promoting plant growth, comprising treating soil or plants with the composition for promoting plant growth.

In the present invention, plant growth can be promoted by treating soil, plants, or plant seeds with the composition. At this time, treatment methods include commonly used methods, namely spraying (e.g. spraying, misting, atomizing, powder spraying, granule spraying, surface application, permanent use, etc.), soil application (e.g. mixing, irrigation, etc.) , surface application (e.g., application, smearing, coating, etc.), immersion, poisoning, smoking application, etc. The amount of use can be appropriately determined depending on the formulation, damage situation, application method, application location, etc.

The composition of the present invention may be treated on plants at a concentration of generally 0.1 to 50 μg/mL, specifically 0.5 to 50 μg/mL, and more specifically 1 to 50 μg/mL. Not limited. When manufacturing, storing, and transporting the composition of the present invention, the concentration is generally 1 to 100,000 times the concentration when treated with plants, specifically 100 to 50,000 times the concentration, and more specifically 500 to 10,000 times the concentration. It can be manufactured, stored, and transported in any concentration, but is not limited to this.

The Bacillus tequlensis PAMB00293 strain of the present invention has the ability to produce siderophores, proteases, amylase, auxin, and catalase, and has an excellent effect in promoting plant growth, so it can be used as eco-friendly biological fertilizer, eco-friendly agricultural material additives, etc.

Figure 1 is a diagram showing the production of colonies as a result of an experiment on the production capacity of siderophore, protease, and amylase of the Bacillus tequilensis PAMB00293 strain.
Figure 2 is a diagram showing the results of comparing the growth promotion ability of Arabidopsis thaliana in the control group, the group treated with the culture medium of the Bacillus mobilis PAMB00009 strain, and the group treated with the culture medium of the Bacillus tequilensis PAMB00293 strain.
Figure 3 is a diagram showing the results of comparing the tomato growth promotion ability of the control group and the Bacillus tequilensis ( Bacillus tequilensis ) PAMB00293 strain culture medium treatment group.
Figure 4 is a diagram showing the results of comparing the cabbage growth promotion ability of the control group and the Bacillus tequilensis ( Bacillus tequilensis ) PAMB00293 strain culture medium treatment group.
Figure 5 is a diagram showing the results of comparing the tomato coloring promotion ability of the control group and the Bacillus tequilensis PAMB00293 strain culture medium treatment group.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1. Isolation and cultivation of Bacillus tequlensis PAMB00293 strain

In order to isolate endophytic bacteria from plants, plant tissues were divided into root, stem, and flower tissues from the seaweed collected from the beach in Goseong-gun, Gangwon-do (128 ^o 25'8.93“E, 38 ^o 30'47.94”N), and then washed with 1% NaOCl. The surface was sterilized for 20 minutes. After washing five times with sterile water, 10 mL of 1xPBS (phosphate-buffered saline) was added, finely crushed with a mortar, plated on LB medium (1.5% agar), and cultured at 25°C for 3 days. The Bacillus tequlensis strain isolated from the medium was named Bacillus tequlensis PAMB00293, and was deposited at the Biological Resources Center of the Korea Research Institute of Bioscience and Biotechnology, an international depository under the Budapest Treaty, and was given accession number KCTC15056BP on August 18, 2022.

Example 2. Analysis of siderophore and enzyme (protease, amylase, auxin, catalase) production ability of Bacillus tequlensis PAMB00293 strain

Example 2-1. Siderophore production capacity analysis

To analyze siderophore production ability, Bacillus tequlensis PAMB00293 strain was inoculated into CAS blue medium containing chrome azurol S (CAS) and cultured at 25°C for 3 to 5 days. Afterwards, it was confirmed whether colony halo zone was formed. Additionally, the production capacity was calculated as follows.

Siderophore productivity index (SPI) = (colony halo zone + colony diameter length) / colony diameter length

As a result, as shown in Figure 1, it was confirmed that colonies were generated, and as shown in Table 1 below, the siderophore production ability index was confirmed to be 2.84, indicating that the strain of the present invention has a siderophore production ability. It was confirmed that it exists.

Example 2-2. Protease production capacity analysis

To analyze protease production ability, the isolated Bacillus tequlensis PAMB00293 strain was inoculated into TSA medium containing 2.5% skim milk, cultured at 25°C for 3 to 5 days, and the diameter of the transparent ring was examined. Additionally, the production capacity was calculated as follows.

Enzyme activity (Ratio) = transparent ring diameter (mm)/colony diameter (mm)

As a result, as shown in Figure 1, it was confirmed that colonies were created, and as shown in Table 1, the enzyme activity index was confirmed to be 2.38, confirming that the strain of the present invention has the ability to produce siderophores. .

Example 2-3. Amylase production capacity analysis

To analyze amylase production ability, the isolated Bacillus tequlensis PAMB00293 strain was inoculated into TSA (Tryptic soy agar) medium containing 1% soluble starch, and then cultured at 25°C for 3 to 5 days. Afterwards, 1% Iodine was added and the activity was measured based on the size of the clear zone.

Enzyme activity (Ratio) = transparent ring diameter (mm)/colony diameter (mm)

As a result, as shown in Figure 1, it was confirmed that colonies were created, and as shown in Table 1 above, the enzyme activity index was confirmed to be 2.18, confirming that the strain of the present invention has the ability to produce amylase.

Example 2-4 Auxin production ability analysis

To analyze auxin (IAA) production ability, Bacillus tequlensis PAMB00293 strain was inoculated into TSB (Tryptic soy broth) medium supplemented with 0.1% tryptophan (L-tryptophan) and cultured at 25°C for 3 days. 800ul of Salkowski reagent (35%HCLO4 +0.5M FeCl3) was mixed with 400ul of culture supernatant, reacted in the dark for 30 minutes, and absorbance was measured at A539 nm with a spectrophotometer. Quantification was performed using a standard curve obtained using standard IAA (Sigma-aldrich, USA).

As a result, as shown in Table 1 above, it was confirmed that the Bacillus tequlensis PAMB00293 strain has the ability to produce auxin by converting L-tryptophan, an auxin (IAA) precursor, into auxin (IAA), a form of plant growth hormone. .

Example 2-5. Catalase production capacity analysis

Catalase activity was confirmed to have catalase production ability by confirming that bubbles were formed as shown in Table 1 when the Bacillus tequlensis PAMB00293 strain was reacted with 3% hydrogen peroxide (H ₂ O ₂ ).

Example 3. Plant growth promotion experiment of Bacillus tequlensis PAMB00293 strain

Example 3-1. Plant growth promotion experiment method

Arabidopsis and tomatoes were surface sterilized in 70% EtOH for 1 minute and 1% sodium hypochloride for 20 minutes, placed on 1/2 MS media (1/2 MS salt, 0.05% MES, 1% Sucrose, 1.5% agar), and placed in light. It was cultured in-flight for 9 days on a plant growth bed under 16-hour dark conditions and 8-hour dark conditions. Seedlings of similar size were selected, the plant seedlings were arranged in a line on new 1/2 MS media, and Bacillus tequlensis PAMB00293 strain was inoculated in a straight line at a distance of 4 cm from the root tip. Plant growth was observed after 7 days of co-culture for Arabidopsis thaliana and 10 days after co-culture for tomato.

Chinese cabbage was grown in soil, and 5 days after seed germination, 20 ml culture medium of Bacillus tequlensis PAMB00293 strain 1x10 ⁷ cell/ml (OD = 0.1) was irrigated twice at 7-day intervals. Change in leaf weight of Chinese cabbage 2 weeks after the last irrigation date. was investigated.

Example 3-2. Plant growth promotion experiment results

As a result of the Arabidopsis growth promotion experiment, as shown in Figure 2, after 7 days of co-culture, the fresh leaf weight of Arabidopsis increased by about 3.3 times, the root weight by about 2.8 times, and the chloroplast content increased by about 2.5 times compared to the control group. did. On the other hand, the Bacillus mobilis PAMB00009 strain belonging to the same Bacillus genus did not show significant differences in plant growth (leaf weight, root weight, and chloroplast content) from the control group.

As a result of the tomato growth promotion experiment, as shown in Figure 3, it was confirmed that after 10 days of co-cultivation, the fresh leaf weight of tomatoes increased by about 2.6 times, the root weight increased by about 2.7 times, and the chloroplast content increased by about 2.3 times compared to the control group.

As a result of the cabbage growth promotion experiment, as shown in Figure 4, it was confirmed that the fresh weight of cabbage leaves increased about 2.5 times compared to the untreated control group 2 weeks after irrigation.

Example 4. Fruit growth promotion experiment of Bacillus tequlensis PAMB00293 strain

For an experiment to promote tomato coloration, tomatoes were grown in soil, and 7 days after seed germination, 20 ml culture medium of PAMB00293 strain 1x10 ⁷ cell/ml (OD = 0.1) was irrigated twice at 7-day intervals. Two months after the last irrigation date, the tomato yield and coloring effect of the control group and the culture solution treatment group were compared.

As a result of the coloration promotion experiment, as shown in Figure 5, compared to the untreated control group, it was confirmed that the proportion of green fruits in tomatoes decreased and the proportion of red fruits increased by about 1.5 times.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should include the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof.

Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC) KCTC15056BP 20220818

Claims (10)

Bacillus tequilensis PAMB00293 strain deposited with accession number KCTC 15056BP.
The Bacillus tequlensis PAMB00293 strain according to claim 1, wherein the strain has plant growth promoting ability.
The method of claim 1, wherein the strain has the ability to produce one or more of siderophore, protease, amylase, auxin, and catalase. PAMB00293 strain.
The Bacillus tequlensis PAMB00293 strain according to claim 2, wherein the plant is Arabidopsis thaliana, tomato, or Chinese cabbage.
A composition for promoting plant growth, comprising the Bacillus tequlensis PAMB00293 strain or a culture thereof according to any one of claims 1 to 4.
The composition for promoting plant growth according to claim 5, wherein the composition increases one or more selected from the group consisting of leaf fresh weight, root fresh weight, number of leaves, number of lateral roots, root length, and chlorophyll content.
The composition for promoting plant growth according to claim 5, wherein the composition promotes coloring of fruit.
A microbial preparation comprising the Bacillus tequlensis PAMB00293 strain of any one of claims 1 to 4 or a culture thereof.
A kit for plant growth promotion, comprising the plant growth promotion composition of claim 5.
A method for promoting plant growth, comprising treating soil or plants with the composition for promoting plant growth of claim 5.