KR102239389B1 - Alleviation of drought stress in pepper plants by Bacillus butanolivorans KJ40 and uses thereof - Google Patents

Abstract

The present invention provides a microbial preparation comprising Bacillus butanolivorans KJ40 strain (KACC 92262P), spores of the strain, a culture solution of the strain, or a mixture thereof as an active ingredient and an effect of reducing drought stress of plants or enhancing the growth of plants using the microbial preparation.

Description

{Alleviation of drought stress in pepper plants by Bacillus butanolivorans KJ40 and uses thereof}

The present invention comprises Bacillus butanoliborans KJ40 strain (Accession No.: KACC 92262P) having an effect of reducing drought stress of plants or promoting plant growth, spores of the strain, culture medium of the strain, or a mixture thereof as an active ingredient. It relates to a microbial preparation, a method of reducing drought stress of plants, and a method of promoting plant growth.

Currently commercially available agricultural useful microorganisms include microbial preparations that have an antagonistic effect on plant pathogenic microorganisms, solubilization of inorganic nutrients in the soil, and promote the growth of various plants, and the number of chemical pesticides and fertilizers currently available is also decreasing. Therefore, a microbial agent that can replace it is also needed.

The technical problem to be achieved in the present invention is to use a microbial preparation and the microbial preparation containing Bacillus butanoliborance KJ40 strain (KACC 92262P), spores of the strain, a culture medium of the strain, or a mixture thereof as an active ingredient, It is to provide the effect of reducing the drought stress of the plant or promoting the growth of the plant.

The present invention is an aspect for solving the above problems, the present invention Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P), characterized in that it provides an effect of reducing drought stress of plants or promoting plant growth Provides.

As an embodiment of the present invention, the plant may be a pepper.

As an embodiment of the present invention, the strain may include the 16s rRNA gene nucleotide sequence of SEQ ID NO: 1.

The present invention provides a microbial preparation comprising the strain, the spores of the strain, the culture medium of the strain, or a mixture thereof as an active ingredient.

As an embodiment of the present invention, the microbial preparation may be administered in an irrigation treatment method.

As an embodiment of the present invention, the microbial preparation may further include any one or more selected from the group consisting of a plant growth regulator, a plant growth stimulator, and a plant nutrient.

As an embodiment of the present invention, the plant may be a pepper.

The present invention, Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P), any one or more plants selected from the group consisting of a spore of the strain, a culture medium of the strain, or a microbial preparation containing a mixture thereof as an active ingredient It provides a method for reducing drought stress of plants comprising the step of irrigation treatment.

As an embodiment of the present invention, the plant may be a pepper.

The present invention, Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P), any one or more plants selected from the group consisting of a spore of the strain, a culture medium of the strain, or a microbial preparation containing a mixture thereof as an active ingredient It provides a method for promoting plant growth comprising the step of irrigation treatment.

As an embodiment of the present invention, the plant may be a pepper.

When Bacillus butanoliborance KJ40 according to the present invention is applied to a plant, the growth of the plant is increased and the damage caused by drought stress is reduced, so that other microorganisms according to the present invention are used as a plant activity enhancer, a plant growth regulator, a plant growth stimulator and a plant. It can be used as a nutritional supplement.

1 shows the nucleotide sequence of a gene encoding 16S rRNA of Bacillus butanolivorans KJ40 strain (KACC 92262P).
Figure 2 shows the results of phylogenetic analysis of Bacillus butanolivorans KJ40 strain (KACC 92262P).
Figure 3a shows the red pepper in an environment without drought stress.
Figure 3b shows the red pepper in an environment subjected to drought stress for 5 days.
4A is a bar graph showing the weight of the above-ground parts (leaves and stems) of the peppers tested in a greenhouse.
Figure 4b is a bar graph showing the measurement of the pore conductivity of the peppers experimented in the greenhouse.
Figure 4c is a bar graph showing the MDA (malondialdehyde) level of the pepper that was experimented in a greenhouse.
5 is a view showing the physiological activity of microorganisms in the green house packaging in which the actual crops are grown in a red pepper subjected to drought stress for 5 days.
Figure 6a is a bar graph showing the pore conductivity of the red pepper irrigated (1ml/g of soil) with Bacillus butanolivorans KJ40 strain (KACC 92262P) in a green house.
6B is a bar graph showing the moisture content of the soil in which the peppers were planted in a green house by irrigation treatment (1 ml/g of soil) of Bacillus butanolivorans KJ40 strain (KACC 92262P).
7A is a bar graph showing the root growth of pepper in a stress-free environment.
7B is a bar graph showing the stem growth of pepper in a stress-free environment.

In the present invention, various modifications may be made and various embodiments may be provided, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific embodiment, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, when it is determined that a detailed description of a related known technology may obscure the subject matter of the present invention, a detailed description thereof will be omitted.

Hereinafter, embodiments disclosed in the present specification will be described in detail with reference to the drawings. The exemplary embodiments described above in the detailed description, drawings, and claims are not intended to be limiting, and other embodiments may be used, and other modifications may be made without departing from the spirit or scope of the technology disclosed herein.

Since the description of the disclosed technology is merely an embodiment for structural or functional description, the scope of the rights of the disclosed technology should not be construed as being limited by the embodiments described in the text. That is, since the embodiments can be variously changed and have various forms, the scope of the rights of the disclosed technology should be understood to include equivalents capable of realizing the technical idea.

All terms used herein have the same meaning as commonly understood by one of ordinary skill in the field to which the disclosed technology belongs, unless otherwise defined. As defined in a commonly used dictionary, terms should be construed as having the meaning of the related technology in context, and cannot be construed as having an ideal or excessively formal meaning unless explicitly defined in the present application.

Due to excessive use of organic substances and chemical fertilizers, problems such as soil pollution, continuous cultivation disorders, imbalance of soil nutrients, and the occurrence of pests have occurred, leading to side effects such as deterioration of product quality and lower income of farmers. Nitrogen (N), an important plant nutrient, has been supplied by the use of these chemical fertilizers, but the excessive use of chemical fertilizers to maintain higher yields is a problem against the principle of sustainable agriculture that requires high productivity with low input. , There was an environmental problem.

In order to solve such a problem, as a means to replace chemical fertilizers, a biological method of solving safe food while minimizing the burden on the agricultural environment and ecosystem is required. Accordingly, the present invention provides a microbial preparation comprising a strain capable of promoting plant growth by the biological method, a spore of the strain, a culture medium of the strain, or a mixture thereof as an active ingredient. In particular, the Bacillus butanoliborance KJ40 strain according to an embodiment of the present invention acts on plants to improve drought stress reduction method and plant growth.

As used herein, "plant" refers to any living organism belonging to the plant kingdom (ie, any genus/species within the plant kingdom), such as, but not limited to, trees, herbs, shrubs, grasses, vines, ferns, moss and green algae. Does not. Therefore, representative plants to which microbial agents can be applied are peppers, brassicas, bulbous vegetables, grains, citrus, cotton, gourds, fruit vegetables, leafy vegetables, legumes, oil seed plants, peanuts, root vegetables, tuber vegetables, and caliber vegetables. , Stone fruits, tobacco, strawberries and other berries, and various ornamental plants. As an example, it can be applied to the pepper of the present invention.

In the present specification, “culture solution” is a result obtained by inoculating a strain into a medium and culturing it for a certain period of time, and the culture solution itself obtained by culturing the strain according to the present invention in a suitable liquid medium, and the filtrate from which the strain is removed by filtration or centrifugation (filtration Solution or centrifuged supernatant), cell lysate obtained by sonicating the culture solution or treating the culture solution with lysozyme, a concentrate obtained by concentrating the culture solution, filtrate, cell lysis solution, etc. It is not limited. In addition, the probiotic or antibacterial composition may contain a solid medium culture, or a culture such as a dried product or an extract of the culture solution, but is not limited thereto, and further, a suitable excipient or carrier (recessive gene in a heterozygous state You can also include additional objects).

The microbial preparation of the present invention may be prepared in the form of a liquid fertilizer and may be used in the form of powdered powder by adding an extender thereto, or may be formulated and granulated. However, it is not particularly limited to the formulation.

In the present specification, “stress” may be an abiotic stress on a plant, and specifically, the abiotic stress may be a drought stress.

The term “abiotic stress” is used herein in its ordinary sense as the negative effect of a non-biological factor on a living organism in a particular environment, and thus the negative effect of a non-biological factor on a plant in a specific environment with respect to the plant. Means impact.

Biological stress includes living obstructions such as fungi or harmful insects, while abiotic stressors can be naturally occurring or man-made and can be temperature, dry soil, osmotic stress, drought, salt or nutrient deficiencies. And, all of which can cause harm to plants in the affected area.

The microbial preparation of the present invention can reduce the drought stress of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledons and dicotyledons.

In addition, the microbial preparation of the present invention may have an effect of enhancing the growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledons and dicotyledons.

In an embodiment of the present invention, the plant may be a pepper as an example.

The growth enhancement of the plant means that it is related to the germination of the plant, the differentiation of each organ (leaf, stem, root, fruit, etc.), flowering or development, and an increase in size and weight, and an increase in yield.

The present invention uses a microbial preparation and the microbial preparation containing Bacillus butanolivorans KJ40 strain (KACC 92262P), spores of the strain, a culture solution of the strain or a mixture thereof as an active ingredient, In order to provide an effect of reducing drought stress or promoting plant growth, the present invention provides Bacillus butanolivorans KJ40 strain (KACC 92262P).

In the present invention, Bacillus butanolivorans KJ40 strain (KACC 92262P) was deposited with a microorganism at the National Academy of Agricultural Sciences on April 23, 2019, and was given KACC 92262P as the accession number.

1 shows the nucleotide sequence of a gene encoding 16S rRNA of Bacillus butanolivorans KJ40 strain (KACC 92262P).

The nucleotide sequence of the gene is represented by SEQ ID NO: 1.

Bacillus butanoliborans in the present invention may be a Bacillus butanolivorans KJ40 strain (KACC 92262P) comprising the gene sequence represented by SEQ ID NO: 1.

Figure 2 shows the results of phylogenetic analysis of Bacillus butanolivorans KJ40 strain (KACC 92262P).

The microbial preparation of the present invention can reduce the drought stress of plants in the field in the above-described Bacillus butanolivorans KJ40 strain (KACC 92262P), spores of the strain, culture medium of the strain, or a mixture thereof. Other optional ingredients used to enhance growth may further be included. For example, it may further include any one or more selected from the group consisting of a plant growth regulator, a plant growth stimulator, and a plant nutrient. However, it is not limited thereto.

The plant growth regulator, plant growth stimulator or plant nutrient may be a chemical or another strain of bacteria, and may be a herbicide, fungicide, pesticide, fertilizer, or any combination thereof.

The plant growth regulator may include other beneficial microorganisms, microbial activators, organic fertilizers, and chemical fertilizers.

Other beneficial microorganisms include Bacillus spp ., Azotobacter spp ., Trichoderma spp , and Saccharomyces spp , which are known to enhance plant growth. May be included. However, it is not limited to the microorganism species.

The microbial active material may include enzyme precursors, microbial metabolites, organic acids, carbohydrates, enzymes and/or trace elements. For example, microbial active substances include processed enzyme products such as yeast auto-solubilizers, humus (organic substances other than indicators and living organisms in the ground), seaweed extract, starch, amino acids, and zinc, iron, copper, manganese, Trace elements such as bromine and molybdenum may be included. However, it is not limited to the trace element.

Other beneficial microorganisms, microbial actives and organic fertilizers may be the same as described above. Chemical fertilizers can contain a variety of chemicals that can provide nitrogen, phosphorus, and potassium nutrients to support plant growth. For example, chemical fertilizers may include urea, calcium phosphate, potassium phosphate and mixed nitrogen-phosphate-potassium (N-P-K) fertilizers. Chemical fertilizers may also contain other materials known in the art.

The present invention relates to any one or more plants selected from the group consisting of microbial preparations containing Bacillus butanolivorans KJ40 strain (KACC 92262P), spores of the strain, culture medium of the strain, or a mixture thereof as an active ingredient. It provides a method for reducing drought stress of plants comprising the step of treating.

Specifically, the treating step may be performed by immersing, drenching, or spraying the strain, the culture medium in which the strain is cultivated, and the microorganism preparation using the strain in a seed or plant. In the case of the immersion method, the culture solution and the preparation may be drenched in the soil around the plant or the seeds may be immersed in the culture solution and the microbial preparation and inoculated. However, it is not limited to the above inoculation method.

With respect to the soil around the plant, the peripheral region to which the present invention is applied means a range in which the microbial preparation of the present invention can exert an effect, and includes both above and below ground. For example, it may be within about 2m, within about 1m, within about 70cm, within about 50cm, within about 25cm, within about 10cm, or within about 5cm around a plant, plant part or fruit. However, it is not limited to this area.

The drought stress reduction method of the plant is to reduce the drought stress of the plant to reduce the drought stress of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledons, and dicotyledons. That's the way. As an example, the plant can be applied to the pepper of the present invention.

The plant part to which the present invention is applied is not limited. Thus, not only the whole plant, but also a part of the plant, such as seeds, shoots, stems, roots, leaves, fruits, all of them, as well as all parts of them, are included.

The present invention relates to any one or more plants selected from the group consisting of microbial preparations containing Bacillus butanolivorans KJ40 strain (KACC 92262P), spores of the strain, culture medium of the strain, or a mixture thereof as an active ingredient. It provides a method to have a growth enhancing effect comprising the step of processing.

Specifically, the treating step may be performed by immersing, drenching, or spraying the strain, the culture medium in which the strain is cultivated, and the microorganism preparation using the strain in a seed or plant. In the case of the immersion method, the culture solution and the preparation may be drenched in the soil around the plant or the seeds may be immersed in the culture solution and the microbial preparation and inoculated. However, it is not limited to the above inoculation method.

The method for enhancing the growth of plants is a method for enhancing the growth of a plant that promotes the growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledons, and dicotyledons. . As an example, the plant can be applied to the pepper of the present invention.

The plant part to which the present invention is applied is not limited. Thus, not only the whole plant, but also a part of the plant, such as seeds, shoots, stems, roots, leaves, fruits, all of them, as well as all parts of them, are included.

The method of the present invention is to continuously apply the microbial agent at any point during the life cycle of the plant, during one or more stages of the life cycle of the plant, or at regular intervals in the life cycle of the plant, or throughout the life cycle of the plant. Includes that. Therefore, microbial preparations can be applied as needed. For example, the microbial preparation can be applied to the plant during growth, before and/or during flowering, and/or before and/or during seed development. In one example, the microbial agent may be applied before, during and/or immediately after the plant is transplanted from one location to another, such as from a greenhouse or a hotbed to a field. In another example, each microbial agent can be applied multiple times to the plant at a desired interval period.

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

Experimental Example 1: Effect of drought stress on pepper plants by irrigation treatment of Bacillus butanoliborance KJ40 strain (greenhouse assay)

In order to confirm the drought stress effect of Bacillus butanoliborance KJ40 strain, the plants were irrigated and tested in a greenhouse. Pepper was used as a plant. The control group was not treated with bacteria, and the experimental group was treated with Bacillus amyloliquefaciens GLC02 strain and Bacillus butanoliborance KJ40 strain.

Specifically, for testing in a greenhouse, peppers were planted and cultivated in soil in a 9cm pot, and then when the pepper plants became 5-6 leafy, the control group was not treated with specific bacteria, and the experimental group was Bacillus amyl. Loliquipes GLC02 strain and Bacillus butanoliborance KJ40 strain were irrigated (1ml/g of soil) on peppers, respectively. Thereafter, drought stress was applied for 5 days without water, and after 2 weeks, the shape of the above-ground parts (leaves and stems) of each pepper was checked, and the weight, porosity conductivity, and MDA values were evaluated.

3A is a photograph and a photograph of the shape of the pepper when drought stress is not applied, and FIG. 3B is a photograph of the shape after applying drought stress to each of the control group and the experimental group.

In FIG. 3A, when drought stress was not applied, the growth of pepper was active in both the control group and the experimental group, and no significant difference was found between the control group and the experimental group.

In FIG. 3B, when drought stress was applied, it was found that both the control and the experimental groups deteriorated the growth of peppers and dried the leaves or stems at the same time. However, in this case, in the experimental group treated with Bacillus butanoliborance KJ40 strain, the decrease in growth and the least drying of leaves or stems of pepper appeared in the experimental group treated with the control or Bacillus amyloliquefaciens GLC02 strain. Was able to confirm.

4A is a bar graph showing the weight of the above-ground parts (leaves and stems) of the peppers tested in a greenhouse.

When plants are subjected to drought stress, leaf moisture decreases due to transpiration, but when the Bacillus amyloliquefaciens GLC02 strain was treated, it decreased by 12.2% compared to the control group. On the other hand, when Bacillus butanolivorans KJ40 was treated, the weight of the above-ground part (leaves and stems) increased 43.7% compared to the control group, indicating that drought-induced stress was reduced.

Figure 4b is a bar graph showing the measurement of the pore conductivity of the peppers experimented in the greenhouse. Here, the pore conductivity refers to the velocity of gas passing through the pores.

When plants are subjected to drought stress, the pore conductivity gradually decreases and the CO2 absorption rate decreases, resulting in decreased leaf size, stem elongation, and root expansion, resulting in increased plant damage. Bacillus butanolivorans KJ40 In the case of treatment, the pore conductivity was maintained 55.7% higher than that of the control group.

4C is a bar graph showing the MDA values of the peppers tested in a greenhouse. Here, MDA is malondialdehyde, and refers to the degree of damage to the cell membrane lipids of plants. The higher the MDA level, the greater the cell destruction of the plant.

When plants are subjected to drought stress, free radicals such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide are generated, causing oxidative damage to cells through lipid peroxidation and membrane destruction. When treated with butanoliborans (Bacillus butanolivorans ) KJ40, malondialdehyde (MDA) decreased by 26.6% compared to the control group.

Experimental Example 2: Drought stress effect of pepper plants by irrigation treatment of Bacillus butanoliborance KJ40 strain (packaging assay)

Based on the results in the greenhouse, the effect test of Bacillus butanolivorans KJ40 strain (KACC 92262P) was expanded to perform an experiment in a green house packaging, which is shown in FIG. 5. In the field assay, the stress treatment method of pepper was performed in the same manner as the treatment method in the greenhouse assay described above. That is, in the case of the control group, specific bacteria were not treated, and in the case of the experimental group, Bacillus amyloliquefaciens GLC02 strain and Bacillus butanoliborance KJ40 strain were each irrigated (1 ml/g of soil) in peppers. Thereafter, drought stress was applied for 5 days without water, and after 2 weeks, the shape of the above-ground parts (leaves and stems) of each pepper was checked, and the porosity conductivity and moisture content were evaluated.

Figure 5 is a photograph showing the physiological activity of microorganisms in a green house packaging in which the actual crops are grown in a red pepper subjected to drought stress for 5 days.

In the case of the control group, no strain was treated, drought stress was given for 5 days, and then grown for 2 weeks. In the case of KJ40, Bacillus butanolivorans KJ40 strain (KACC 92262P) was irrigated (1ml/g of soil), drought stress was given for 5 days, and then raised for 2 weeks.

Referring to FIG. 5, in the case of the control group, the growth of the pepper is reduced, so that the leaf size is small and the leaf dryness occurs a lot. However, in the case of the pepper treated with Bacillus butanoliborance KJ40 strain, the leaf size is large and the mouth It can be seen that hardly any dryness occurs.

Figure 6a is a bar graph showing the pore conductivity of the red pepper irrigated (1ml/g of soil) with Bacillus butanolivorans KJ40 strain (KACC 92262P) in a green house.

As shown in Fig.6a, when Bacillus butanolivorans KJ40 strain (KACC 92262P) is irrigated in a field test (1ml/g of soil), similar to the results of a greenhouse test, the porosity was 36.4 compared to the control group. % Remained high.

6B is a bar graph showing the moisture content of the soil in which the peppers were planted in a green house by irrigation treatment (1 ml/g of soil) of Bacillus butanolivorans KJ40 strain (KACC 92262P).

As shown in Fig.6b, when the Bacillus butanolivorans KJ40 strain (KACC 92262P) is irrigated (1ml/g of soil) in a field test, similar to the results of a greenhouse test, the soil moisture content compared to the control group It was 23.9% higher.

Experimental Example 3: Effect of increasing the growth of pepper plants by irrigation treatment of Bacillus butanoliborance KJ40 strain

In order to confirm the effect of increasing the growth of Bacillus butanoliborance KJ40 strain, the plants were irrigated and tested in a greenhouse. Pepper was used as a plant. The control group was not treated with bacteria, and the experimental group was treated with Bacillus amyloliquefaciens GLC02 strain and Bacillus butanoliborance KJ40 strain.

In this experimental example, in the case of the control group, specific bacteria were not treated, and in the case of the experimental group, Bacillus amyloliquefaciens GLC02 strain and Bacillus butanoliborance KJ40 strain were drenched in peppers (1 ml/g of soil), respectively. . Thereafter, after 2 weeks, the weights of the roots and stems were measured to confirm the growth of the roots and stems of the pepper.

7A is a bar graph showing the weight of the roots of peppers grown in the above-described method.

As can be seen in FIG. 7a, when the pepper was treated with Bacillus butanolivorans KJ40 strain (KACC 92262P), the root growth was 81.4% higher than that of the control group. In contrast, when the Bacillus amyloliquefaciens GLC02 strain was treated, the root growth was rather lower than that of the control, and the root growth effect of the Bacillus butanoliborance strain was remarkable compared to other strains.

Figure 7b is a bar graph showing the weight of the stems of pepper cultivated by the above method.

As can be seen in FIG. 7B, when the pepper was treated with Bacillus butanolivorans KJ40 strain (KACC 92262P), stem growth was 29.2% higher than that of the control group. In contrast, when the Bacillus amyloliquefaciens GLC02 strain was treated, the stem growth was higher than that of the control group, but there was no significant difference. In contrast, it can be seen that there is a significant difference in the stem growth effect of the Bacillus butanoliborance strain compared to the control group.

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Claims (11)

For reducing drought stress of plants or for growing plants Bacillus butanolivorans KJ40 strain (KACC 92262P). The method of claim 1,
The plant is pepper, Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P). The method of claim 2,
The strain is Bacillus butanolivorans containing the 16s rRNA gene nucleotide sequence of SEQ ID NO: 1 KJ40 strain (KACC 92262P). A microbial preparation comprising at least one of the strain according to any one of claims 1 to 3, spores of the strain, culture medium of the strain, or a mixture thereof as an active ingredient. The method of claim 4,
The microbial preparation is a microbial preparation administered by irrigation treatment. The method of claim 5,
The microbial preparation further comprises any one or more selected from the group consisting of a plant growth regulator, a plant growth stimulator, and a plant nutrient. The method of claim 6,
The plant is a pepper, a microbial preparation. Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P), spores of the strain, a culture medium of the strain, or a microbial preparation containing a mixture thereof as an active ingredient. Drought stress reduction method comprising the steps. The method of claim 8,
The plant is pepper, drought stress reduction method. Bacillus butanolivorans (Bacillus butanolivorans) KJ40 strain (KACC 92262P), spores of the strain, a culture medium of the strain, or a microbial preparation containing a mixture thereof as an active ingredient. Growth enhancement method comprising the step. The method of claim 10,
The plant is a red pepper, growth enhancement method.

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