KR102411304B1 - Bacillus zanthoxyli strain promoting tolerance of plants and use thereof - Google Patents

Abstract

The present invention relates to a novel Bacillus mesonae strain for enhancing the resistance of plants and their use, and the Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), which has an excellent effect of enhancing the resistance of plants to various stresses, It relates to a composition for enhancing plant stress resistance, including the strain or a culture medium thereof, a method for promoting stress resistance of a plant using the strain, and a method for promoting plant growth using the strain.
The strain of the present invention shows an excellent effect of increasing the stress resistance of plants, and by using it, plants can grow well even under conditions of high temperature and salt accumulation or under adverse conditions for plant growth in which pathogens exist, thereby increasing plant production. .

Description

Bacillus zanthoxyli strain promoting tolerance of plants and use thereof

The present invention relates to a Bacillus xanthoxyli HS1 strain for promoting plant resistance, disease prevention and growth, and uses thereof, and resistance to salt stress and plant resistance among various abiotic stresses, and plant resistance enhancement, growth promotion Bacillus zanthoxyli with excellent effect HS1 (Accession No.: KCTC18833P) and a composition for promoting stress resistance of plants using the same, a method for promoting stress resistance of plants, and a method for promoting plant growth.

According to the data released by the National Statistical Office in 2018, the cultivated area of facility houses in Korea was about 51,997ha, followed by Gyeongnam region with about 9,566ha, Gyeongbuk region with about 9,004ha, Chungnam region with about 7,939ha, and Gyeonggi region about It is said to have been counted as 6,507 ha. The Rural Development Administration of the Republic of Korea estimates that about 55% of all cultivated fields are suffering damage from salt accumulation.

In agricultural land, such salt accumulation can be seen as the main cause of indiscriminate use of chemical fertilizers and pesticides. In particular, when chemical fertilizers are used, Ca ²⁺ , Mg ²⁺ , K ⁺ , Na ⁺ , etc. are not leached out and salt accumulation occurs in the facility cultivation site where rainfall is less than the amount of evapotranspiration. As a result, soil hardening occurs and plant root growth is inhibited, making it impossible to absorb nutrients in a balanced way. As an indirect damage caused by salt accumulation, the soil microbiome changes, which changes the plant growth environment, which can negatively affect plant growth and crop yield.

In order to solve the above problems, plants (crops) resistant to salt stress are being developed through traditional breeding and genetic modification. However, each requires a large investment in terms of time and cost. In particular, it is difficult to obtain a variety license for genetically modified organisms, and it is difficult to provide an appropriate solution to the problem due to negative perceptions of genetically modified organisms.

Recently, research on enhancing salt resistance of plants using plant growth promoting rhizobacteria ( PGPR ) is increasing, and representative microorganisms are Azospirillum, Burkholderia , and Rhizobium. ( Rhizobium ), Pseudomonas ( Pseudomonas ), Acetobacter ( Acetobacter ) and Bacillus ( Bacillus ) Research results are being published that microorganisms belonging to the genus enhance the resistance to salt stress of plants.

Many types of microorganisms inhabiting the soil play important roles in terms of plant growth promotion, material circulation, and environmental purification. Among them, research has been conducted on root-knot bacteria that fix nitrogen in the air by making symbiotic plants with plants, mycorrhizal fungi and fungi that promote phosphate absorption in plants, mycorrhizal bacteria that promote plant growth, and microorganisms related to pest control. Attempts to use it as a microbial agent are being made in various fields.

에서 6개월 정도만 보관이 가능하며 유통기간이 경과함에 따라 미생물의 생존율이 감소하고 접종된 미생물이 사용한 토양에서 우점적으로 정착하여 안정적으로 작용하는 경우에만 그 효과를 나타낸다는 한계점들이 있다. Microbial preparations are composed of live microorganisms and in some cases, carriers that have adsorbed the inoculum, organic nutrients for inoculum activation, and other adjuvants. It depends on the function of microorganisms. Therefore, unlike general products, which are finished products, microbial preparations are progressive products that have to maintain their vitality until the moment they are consumed and exhibit a living function.

It can be stored for only about 6 months, and as the shelf life elapses, the survival rate of microorganisms decreases, and there are limitations in that the effect is only shown when the inoculated microorganisms dominately settle in the used soil and act stably.

In addition, microbial preparations that do not use the intrinsic function of microorganisms are just general organic fertilizers, and nutrients included for the survival of inoculated microorganisms may also accumulate in the soil, causing secondary soil problems. Finally, problems caused by changes in the microbial community in the soil when these microbial agents are used have not yet been accurately identified.

The technical problem to be solved by the present invention is to provide a novel strain effective for increasing the resistance of plants, a composition for promoting resistance and growth of plants using the strain, and a method for promoting resistance and growth of plants using the same .

Another technical problem to be solved by the present invention is to increase the resistance of plants even when the activity of the strain is inhibited, suppressed or stopped, and to provide a composition for promoting growth and a method for promoting resistance and growth of plants using the same.

Objects of the present invention are not limited to those mentioned above, and other objects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description.

According to an aspect of the present invention in order to solve the above technical problem, Bacillus zanthoxyli ) HS1 strain (Accession No.: KCTC18833P) is provided.

Here, the strain according to the present invention is characterized in that it has 16S rRNA of the nucleotide sequence represented by SEQ ID NO: 1.

Here, the present invention provides a composition for enhancing stress resistance of a plant comprising Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), a culture medium of the strain, or both as an active ingredient.

Here, the composition for promoting stress resistance of plants according to the present invention is characterized in that it enhances resistance, disease occurrence, and growth of plants to any one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrition and pathogens do it with

Here, the composition for enhancing the stress resistance of plants according to the present invention is selected from the group consisting of Ralstonia solanacearum and Phytophthora capsici . It is characterized in that it inhibits development and promotes growth.

Here, the composition for enhancing plant stress resistance according to the present invention is any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledonous plants and dicotyledonous plants resistance, disease occurrence It is characterized by inhibiting and promoting growth.

Here, the composition for enhancing plant stress resistance according to the present invention is characterized in that it further comprises any one or more selected from the group consisting of a plant growth regulator, a plant growth stimulator, and a plant nutrient.

Here, the present invention is Bacillus zanthoxyli ( Bacillus zanthoxyli ) HS1 strain (Accession No.: KCTC18833P), the culture solution of the strain, a composition comprising the strain and a composition comprising the culture solution of the strain Any one or more selected from the group consisting of It provides a method for enhancing stress resistance of a plant, comprising the step of treating the plant, the area surrounding the plant, or both.

Here, the method for promoting stress resistance of plants according to the present invention is characterized in that the resistance of plants to any one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrition and pathogens, inhibition of disease occurrence and growth do.

Here, the method for enhancing the stress resistance of plants according to the present invention is a pathogen that causes one or more plant diseases selected from the group consisting of Ralstonia solanacearum and Phytophthora capsici . Resistance of plants to stress, disease occurrence It is characterized by inhibiting and promoting growth.

Here, the method for enhancing the stress resistance of the plant according to the present invention is one or more selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledonous plants, and dicotyledonous plants Resistance, disease occurrence It is characterized by inhibiting and promoting growth.

Here, the present invention is Bacillus zanthoxyli ( Bacillus zanthoxyli ) HS1 strain (Accession No.: KCTC18833P), the culture solution of the strain, a composition comprising the strain and a composition comprising the culture solution of the strain Any one or more selected from the group consisting of A method for promoting plant growth is provided, comprising the step of treating the plant, the area surrounding the plant, or both.

Here, the method for accelerating plant growth according to the present invention promotes the growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocot plants and dicotyledonous plants. do.

According to an embodiment of the present invention, a novel strain that increases the resistance (resistance) of plants to various stresses, Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), stress of plants comprising the strain or its culture medium It is possible to provide a composition for enhancing resistance, a method for promoting stress resistance of plants using the same, and a method for promoting plant growth. Accordingly, by using the specific strain according to the present invention, it is possible to increase the stress resistance of the plant, thereby promoting plant growth even in an environment unfavorable for plant growth.

According to another embodiment of the present invention, a composition that increases the resistance of plants and promotes growth even when the activity of a novel strain, Bacillus zanthoxyli HS1 strain is inhibited, suppressed or stopped, may be provided.

Effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those of ordinary skill in the art to which the present invention belongs from the following description.

1 is a schematic diagram illustrating molecular phylogenetic classification using 16S rRNA nucleotide sequence information of Bacillus zanthoxyli HS1 strain.
Figure 2 is Bacillus zantoxyli ( B. zanthoxyli ) The test results of disease resistance of tomato and paprika plants induced by HS1. Figure 2a is an experimental result for tomato blight caused by Ralstonia solanacearum ( R. solanacearum ), Figure 2b is a phytophthora capsaici ( P. capsici ) For paprika blight caused by It is the result of an experiment on the disease resistance inducing efficacy of the strain of the present invention. BTH (Benzo(1,2,3) thiadiazole-7-carbothioic acid(S)-methyl ester) and the present invention, a representative substance known to impart disease resistance to untreated plants and plants 1 week before inoculation of pathogens on tomatoes and paprika The disease incidence rate was confirmed for each of the strains treated. As a result, it was confirmed that the strain treatment of the present invention has a higher effect than BTH for late blight caused by Phytophthora capsici ( P. capsici ), and R. solanacearum ( R. solanacearum ) It was confirmed that the strain-treated group of the present invention showed high disease resistance compared to the untreated group. This shows that the strain of the present invention imparts disease resistance to plants.
Figure 3 is a B. zanthoxyli ( B. zanthoxyli ) It is a photograph and the test result of the enhanced resistance to salt stress of cabbage, cucumber, tomato plants induced by HS1. 3a, 3b and 3c are views confirming the growth degree of seedlings of vegetable crops under salinity stress conditions. Figure 3a is Chinese cabbage ( Brassica rapa subsp. pekinensis cv. lyeekwang ), Figure 3b is cucumber ( Cucumis sativus cv. It is the result of comparing the weight of total seedling, fresh weight of root, and fresh weight of shoot. 10 mM MgSO4 (white bars) and B. zanthoxyli HS1 strain (OD ₆₀₀ = 0.25/g soil) (grey bars) were used as a standard solution commonly used in salt stress studies, The Balanced Salinity Solution (TBSS) (125 mM KNO ₃ , 82 mM Ca(NO ₃ ) ₂ , 41mM MgSO ₄ , 22mM KH ₂ PO ₄ ) was dispensed into plant cultivation soil 7 days after treatment with the invention strain. Since the germination rate, plant growth, etc. are suppressed in plants under salt stress conditions, the above results show that plants infused with the strain of the present invention had higher measured values of germination rate, shoot growth, and root length even under salt stress compared to the control group. can check that These results mean that the strain of the present invention relieves growth inhibition caused by salt stress of plants.
4 is a result of measuring the content of photosynthetic pigments in Chinese cabbage and cucumber plants grown under salt stress conditions treated with Bacillus zanthoxyli ( B. zanthoxyli ). 4a, 4b, 4c and 4d are results of measuring the chlorophyll and carotenoid content of Chinese cabbage and cucumber grown under salt stress conditions. Salt stress can reduce chlorophyll content by enhancing chlorophyllase activity as a chlorophyll-degrading enzyme and destabilizing the pigment-protein complex. Chlorophyll and carotenoids in Chinese cabbage and cucumber plants after application of 50 mM NaCl or TBSS (125 mM KNO ₃ , 82 mM Ca(NO ₃ ) ₂ , 41 mM MgSO ₄ , 22 mM KH ₂ PO ₄ ) to examine the effects of enhancing salt stress tolerance in plants. content was confirmed. Chlorophyll content in Chinese cabbage plants after salt stress treatment was higher in plants treated with the present strain than in simulated plants (FIG. 4a). The carotenoid level of the Chinese cabbage plants treated with the invention strain was relatively higher than that of the mock-treated Chinese cabbage plants after 50 mM NaCl treatment, but was similar to the untreated group in the TBSS treatment ( FIG. 4b ). However, pretreatment of the inventive strain did not protect photosynthetic pigment degradation in cucumber plants from salinity stress ( FIGS. 4c and 4d ). These results suggest that the strain of the present invention has a different effect on the pigment stability of Chinese cabbage and cucumber plants, and may be more useful for Chinese cabbage plants than cucumber and tomato plants to control salinity stress.
Figure 5 is an experimental result that Bacillus xanthoxyli ( B. zanthoxyli ) HS1 dead cell treatment can confer resistance to seed germination and seedling growth of Arabidopsis plants under salt stress. Figures 5a and 5b show the salt stress response of Arabidopsis wild-type and Arabidopsis mutant sos2-2 ( salt overly sensitive 2-2 ) plants measured according to the dead cell concentration of the strain of the present invention by measuring the chlorophyll content. 5c and 5d are results of verifying the effect of imparting resistance to salt stress of the dead strain of the present invention.
In order to confirm whether the strain of the present invention imparts salt resistance to plants even in the dead cell state, an experiment was conducted using Arabidopsis thaliana , a model plant, and Arabidopsis thaliana mutant sos2-2 ( salt overly sensitive 2 ) -2 ) was used to re-verify its efficacy. The wild-type and mutant seeds were immersed in a liquid plant culture medium (Murashige & Skoog (MS) media) treated with NaCl, a typical salt, at concentrations of 0mM, 50mM, 75mM, and the dead cells of the present strain were OD ₆₀₀ = 0, 0.01, 0.05 and 0.1, respectively. Referring to FIG. 5c , when the present strain is not treated, it can be seen that both wild-type Columbia-0 (Col-0) and mutant sos2-2 are delayed in growth or suppressed in germination from the 50 mM NaCl treatment group. In contrast, as can be seen from FIG. 5d, when dead cells were treated at a concentration of OD ₆₀₀ = 0.01, both wild-type and mutant germination and growth at a NaCl concentration of 50 mM, the same as that of a plant having a NaCl concentration of 0 mM. have. Results as shown in the figure suggest that the present strain imparts salt stress resistance to plants and promotes growth in the dead cell state.

Objects and effects of the present invention, and technical configurations for achieving them will become clear with reference to the embodiments described below in detail in conjunction with the accompanying drawings. In describing the present invention, if it is determined that a detailed description of a well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. And the terms described below are terms defined in consideration of donation in the present invention, which may vary according to the intention or custom of the user or operator.

However, the present invention is not limited to the embodiments disclosed below and may be implemented in various different forms. Only the present embodiments are provided so that the disclosure of the present invention is complete, and to completely inform those of ordinary skill in the art to which the present invention belongs, the scope of the invention, the present invention is defined by the scope of the claims will only be Therefore, the definition should be made based on the content throughout this specification.

Hereinafter, embodiments of the present invention will be described in detail.

The present invention for achieving the above-mentioned object is Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), a composition for enhancing stress resistance of a plant comprising the strain or its culture medium, the strain or the composition, etc. It is characterized in that it is a method for promoting stress resistance of plants using a method and a method for promoting plant growth of plants using the strain or the composition.

The present invention provides a Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P). The strain may have 16S rRNA of the nucleotide sequence shown in SEQ ID NO: 1.

The present invention provides a composition for enhancing stress resistance of a plant comprising Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), a culture medium of the strain, or both as an active ingredient.

As used herein, "plant" refers to any living organism belonging to the plant kingdom (i.e., 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 the composition can be applied include brassica, bulb vegetables, grains, citrus, cotton, gourds, fruit vegetables, leaf vegetables, legumes, oil seed crops, peanuts, pome fruits, root vegetables, tuber vegetables, caliber vegetables , nuclear fruits, tobacco, strawberries and other berries and various ornamental plants. Plants herein may be pepper, tomato or paprika.

As used herein, the term "culture medium" refers to a result obtained by inoculating a strain on a medium and culturing for a certain period of time, the culture medium itself culturing the strain according to the present invention in a suitable liquid medium, the filtrate obtained by filtering or centrifuging the culture medium to remove the strain (filtration solution or centrifuged supernatant), a cell lysate obtained by sonicating the culture solution or treating the culture solution with a lysozyme, and a concentrate obtained by concentrating the culture solution, filtrate, lysate, etc., but is limited thereto it is not In addition, the probiotic or antibacterial composition may contain a culture such as a solid medium culture or a dried product or an extract of the culture medium, but is not limited thereto, and may further include a suitable excipient or carrier.

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

The composition for promoting stress resistance of plants of the present invention can enhance resistance, disease prevention and growth of plants to any one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrition and pathogens.

As used herein, “stress” may be biotic or abiotic or biotic stress on a plant, and specifically, the abiotic stress may be dryness, high temperature, low temperature, high salinity, or nutrient deficiency. The term “abiotic stress” is used herein in its ordinary sense as the negative effect of a non-biotic factor on a living organism in a specific environment, and thus in relation to plants, the negative effect of a non-biotic factor on a plant in a specific environment means influence. Biological stressors include living obstructions such as fungi or harmful insects, whereas abiotic stressors can be naturally occurring or man-made, and include temperature, dry soil, osmotic stress, drought, salt or nutrient deficiencies. , all of which can cause harm to plants in the affected area. For example, the salt stress can include increased salt concentrations or drought. A nutrient deficiency may be a lack of soil nutrients, such as potassium, phosphate or iron, in the soil region around the plant. An increase in stress resistance to (soil) nutrients, such as phosphate deficiency, can be provided by improved solubilization of nutrients lacking in the soil.

The term “resistance (or tolerance) to salt” is used herein in its ordinary meaning to refer to the resistance (tolerance) of a plant to a salt concentration. Thus, "increasing the salt tolerance or resistance of a plant" typically refers to reducing the salt concentration (in its environment, such as in soil or water) when the plant is exposed to a salt concentration higher than a physiologically acceptable salt concentration. It means to increase or improve the ability of a plant to tolerate or tolerate.

The term "water resistance (or tolerance)" or "dry resistance (or tolerance)" refers to its general capacity for a plant's ability to avoid stress and its consequences by maintaining a desirable water balance and swelling, even when exposed to normally dry conditions. is also used herein in the sense of Thus, "increasing the moisture (or drying) tolerance" of a plant maintains a more desirable water balance and swelling than if it were exposed to drought conditions where the plant does not receive the amount of water it needs regularly to maintain water balance and swelling. It means to increase or improve the ability of a plant to

The term "plant pathogen" means a pathogen that invades a plant and causes loss and damage to the leaves, stems, roots or fruits of plants, such as fungi, bacteria, viruses and phytoplasma ) is included. Information on plant pathogens is well known in the art and is disclosed on the Korean Plant Disease Name List (Korean Society for Plant Pathology; Book Publishing Academy) and the RDA website (www.rda.go.kr).

The term "plant pathogen resistance" or "pathogen resistance" refers to the ability to improve the growth of a plant, usually free from infection and growth of pathogens, and enhancing resistance to such pathogens means control, sterilization, Contains the meaning of insect repellent.

The composition for enhancing the stress resistance of plants according to the present invention is a pathogen that induces any one or more plant diseases selected from the group consisting of Ralstonia solanacearum and Phytophthora capsici . Resistance of plants to stress, disease inhibition and growth.

The composition for enhancing the stress resistance of plants of the present invention is selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledonous plants and dicotyledonous plants. Resistance of any one or more plants, disease inhibition and growth can promote

The present invention exerts the enhancement or promotion of plant growth through the enhancement of stress resistance of plants. As used herein, the term 'plant growth' refers to the germination of plants, differentiation of each organ (root, stem, leaf, fruit, etc.), flowering or growth, and size and weight improvement, and related to quantity improvement.

The composition for enhancing the stress resistance of plants of the present invention is added to the above-described strain of the present invention, the strain culture medium or a mixture thereof, other optional components used to enhance the stress resistance of plants in the art or to promote growth can be included as As an example, it may further include any one or more selected from the group consisting of plant growth regulators, plant growth stimulants and plant nutrients.

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

In the present invention, the composition for promoting plant growth may include other beneficial microorganisms, microbial active substances, organic fertilizers, and chemical fertilizers.

The other beneficial microorganisms include Bacillus spp., Azotobacter spp., Trichoderm a spp, and Saccharomyces spp, which are known to promote plant growth. ) may be included.

The microbial active material may include enzyme precursors, microbial metabolites, organic acids, carbohydrates, enzymes and/or trace elements, and the like. For example, microbial active substances may include processed enzyme products such as yeast autolysate, humus materials, seaweed extracts, starches, amino acids, and trace elements such as zinc, iron, copper, manganese, bromine, and molybdenum. .

Other beneficial microorganisms, microbial active substances and organic fertilizers may be the same as those 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 include other materials known in the art.

The present invention is Bacillus zanthoxyli ( Bacillus zanthoxyli ) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain, a composition comprising the strain, and a composition comprising a culture solution of the strain Any one or more plants selected from the group consisting of, Provided is a method for enhancing stress resistance of a plant, comprising the step of treating an area around the plant or both.

The part of the plant applied to the embodiment of the present invention is not limited. Accordingly, it includes the whole plant as well as parts of the plant such as seeds, shoots, stems, roots, leaves, fruits, all of them as well as parts of them.

An embodiment of the present invention can be carried out by immersing or irrigating seeds or plants, that is, spraying the strain, the culture solution culturing the strain, and the composition using the strain. In the case of the immersion method, the culture solution and the preparation can be drenched in the soil around the plant or the seeds are immersed in the culture solution and composition and shaken inoculated.

The peripheral area applied by the embodiment of the present invention means a range in which the composition of the present invention can exert the effect, and includes both above ground and underground. For example, it may be an area within about 2 m, within about 1 m, within about 70 cm, within about 50 cm, within about 25 cm, within about 10 cm, or within about 5 cm of a plant, plant part, or fruit.

A method according to an embodiment of the present invention comprises administering a composition comprising the Bacillus zanthoxyli HS1 strain at any time during the life cycle of a plant, during one or more stages of the life cycle of the plant or at regular intervals in the life cycle of the plant. This includes continuous application at intervals or throughout the life of the plant. Accordingly, the composition can be applied as needed. The composition may be applied to the plant, for example, during growth, before and/or during flowering and/or before and/or during seed development. In one example, the composition may be applied before, during and/or immediately after the plant is transplanted from one location to another, such as in a greenhouse or hotbed, into the field. As another example, each composition may be applied to the plant several times at a desired interval period.

The method for enhancing stress resistance of a plant of the present invention can enhance the resistance of plants to any one or more stresses selected from the group consisting of moisture, temperature, salt, pH, nutrition and pathogens, suppression of disease occurrence and growth.

The method for promoting stress resistance of plants according to the present invention can enhance the resistance of plants to the stress of pathogens that cause any one or more plant diseases selected from the group consisting of blight and late blight, suppression of disease occurrence and growth. Specific details of the pathogen are the same as described above.

The method for enhancing stress resistance of the plant of the present invention is a tree, herb, shrub, grass, vine, fern, moss, green algae, monocotyledonous plant and dicotyledonous plant resistance of any one or more plants selected from the group consisting of resistance, disease prevention and growth can promote

The present invention is Bacillus zanthoxyli ( Bacillus zanthoxyli ) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain, a composition comprising the strain, and a composition comprising a culture solution of the strain Any one or more plants selected from the group consisting of, A method for promoting plant growth is provided, comprising the step of treating an area around the plant or both.

The method for promoting plant growth of the present invention can promote the growth of any one or more plants selected from the group consisting of trees, herbs, shrubs, grasses, vines, ferns, moss, green algae, monocotyledonous plants and dicotyledonous plants.

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 thereto.

[Example 1] Selection and identification of microorganisms

The strain of the present invention was isolated from cucumber and Chinese cabbage rhizome soil in Gyeonggi-do, and total DNA was isolated using Qiagen genomic tip kip for classification identification. was amplified.

에서 4분, 58

에서 1분, 72

에서 2분으로 35회 반복하였으며 이 후 72

에서 8분 동안 유지하였다. 증폭된 PCR 부분의 염기서열을 분석한 후 National Center for Biotechnology Information (NCBI)에서 BLAST 네트워크 서비스를 이용하여 염기서열이 유사한 종을 확인하였다.PCR conditions are 95

at 4 min, 58

at 1 min, 72

was repeated 35 times in 2 minutes, after which 72

was maintained for 8 minutes. After analyzing the nucleotide sequence of the amplified PCR part, a species having a similar nucleotide sequence was identified using the BLAST network service at the National Center for Biotechnology Information (NCBI).

The 16S rRNA information of the strain of the present invention obtained as a result of the above experiment was described in SEQ ID NO: 1, and it was identified as Bacillus xanthoxyli through phylogenetic analysis (see FIG. 1). Accordingly, the obtained strain was named Bacillus xanthoxili HS1, and was deposited and given an accession number KCTC18833P.

1 is a phylogenetic diagram showing the taxonomic classification using 16S rRNA of Bacillus zanthoxyli (B. zanthoxyli ) HS1 strain. Table 1 shows the taxonomic classification using carbon sources of B. zanthoxyli HS1 . In the case of bacterial species belonging to the genus Bacillus , taxonomic classification using only the 16S rRNA nucleotide sequence is difficult. 1 and Table 1, it can be seen that the strain of the present invention belongs to Bacillus zanthoxyli .

Characteristics ^a Bacillus sp. HS1 B. zanthoxyli 1433 ^T* B. aryabhattai B8W22 ^T* pH growth range 6.0-10.0 6.0-10.0 6.0-9. NaCl tolerance (%) 0-7 0-7 0-11 Acid production from Erythritol + - + Ribose + + + Galactose - - - D-sorbitol + + - D-lyxose - - - D-fucose - - - L-fucose - - - D-arabitol + - - Utilization of D-maltose - + + D-melibiose - - + D-glucose - + + D-mannose - - + D-fructose - - + D-sorbitol - - + D-mannitol - - +

[Example 2] Bacillus zanthoxyli Plant disease resistance test induced by HS1

After sowing tomato and paprika seeds in a 72-hole seedling tray, the plants were grown until cotyledons appeared. The strain of the present invention was cultured in tryptic soy agar medium at 28 ° C. for 24 hours, then single colonies were cultured in 100 ml tryptic soy broth at 28 ° C., 150 rpm for 48 hours with shaking, the culture medium was centrifuged (5,000 rpm, 5 min), the supernatant was removed, and only the precipitated bacteria were harvested with a 10mM magnesium sulfate (MgSO ₄ ) solution, re-diluted with OD600=0.25/gram soil, and irrigated at a radius of 2 cm around the seedlings.

After 7 days of irrigation with the strain suspension of the present invention, the green blight pathogen ( Ralstonia solanacearum ) (OD600=0.2, 1 ml/gram soil) and the late blight ( Phytophthora capsici ) ) (2,000 spores/gram soil) was inoculated using the soil drench method and the disease incidence rate (%) was evaluated.

Figure 2a is an experimental result for tomato blight caused by Ralstonia solanacearum ( R. solanacearum ), Figure 2b is a phytophthora capsaici ( P. capsici ) For paprika blight caused by It is the result of an experiment on the disease resistance inducing efficacy of the strain of the present invention.

BTH (Benzo(1,2,3) thiadiazole-7-carbothioic acid(S)-methyl ester) and the present invention, a representative material known to impart disease resistance to untreated plants and plants one week before inoculation of pathogens on tomatoes and paprika The disease incidence rate was confirmed for each of the strains treated.

As a result, it was confirmed that the strain treatment of the present invention has a higher effect than BTH for late blight caused by Phytophthora capsici ( P. capsici ), and R. solanacearum ( R. solanacearum ) It was confirmed that the strain-treated group of the present invention showed high disease resistance compared to the untreated group. This shows that the strain of the present invention imparts disease resistance to plants.

[Example 3] Bacillus zanthoxyli Evaluation of tolerance to salt stress induced by HS1

Cabbage, cucumber, and tomato seedlings were grown for 7 days at 24 degrees (12 hours light, 12 hours dark) in a 12 hole seedling tray with BioPlug provided by Nongwoo Bio. The strain suspension of the present invention prepared as described in Example 2 was irrigated with a radius of 2 cm around the seedlings of Chinese cabbage, cucumber, and tomato.

The balanced salinity solution (TBSS) (125mM KNO ₃ , 82mM Ca(NO ₃ ) ₂ , 41mM MgSO ₄ , 22mM KH ₂ PO ₄ ) is a standard solution commonly used in salt stress studies after 7 days of treatment with the strain of the present invention was treated three times at an interval of 2 days at 1 ml/soil, and the results in FIG. 3 are the results measured after 10 days have elapsed after the last treatment.

Figure 3a is Chinese cabbage ( Brassica rapa subsp. pekinensis cv. hyukwang ), Figure 3b is cucumber ( Cucumis sativus cv. It is the result of comparing the weight of total seedling, fresh weight of root, and fresh weight of shoot. 10 mM MgSO4 (white bars) and B. zanthoxyli HS1 strain (OD ₆₀₀ = 0.25/g soil) (grey bars) were used as a standard solution commonly used in salt stress studies, The Balanced Salinity Solution (TBSS) (125 mM KNO ₃ , 82 mM Ca(NO ₃ ) ₂ , 41mM MgSO ₄ , 22mM KH ₂ PO ₄ ) was dispensed into plant cultivation soil 7 days after treatment with the invention strain. Since the germination rate, plant growth, etc. are suppressed in plants under salt stress conditions, the above results show that plants infused with the strain of the present invention had higher measured values of germination rate, shoot growth, and root length even under salt stress compared to the control group. can check that These results mean that the strain of the present invention relieves growth inhibition caused by salt stress of plants.

[Example 4] Bacillus zanthoxyli Determination of chlorophyll and carotenoid content in Chinese cabbage and cucumber grown in saline stress conditions through HS1 treatment

Plant growth and salt stress treatment conditions of the experiment for Example 4 were the same as in Example 3, and 50 mM NaCl was additionally used in addition to TBSS used in Example 3 for salt stress treatment. 1mL of 50mM NaCl treatment was also applied to 1 gram of soil, and the chlorophyll and carothionide contents were also carried out on the day 10 days after the last treatment.

4a, 4b, 4c and 4d are results of measuring the chlorophyll and carotenoid content of Chinese cabbage and cucumber grown under salt stress conditions. Salt stress can reduce chlorophyll content by enhancing chlorophyllase activity as a chlorophyll-degrading enzyme and destabilizing the pigment-protein complex. Chlorophyll and carotenoids in Chinese cabbage and cucumber plants after application of 50 mM NaCl or TBSS (125 mM KNO ₃ , 82 mM Ca(NO ₃ ) ₂ , 41 mM MgSO ₄ , 22 mM KH ₂ PO ₄ ) to examine the effects of enhancing salt stress tolerance in plants. content was confirmed. Chlorophyll content in Chinese cabbage plants after salt stress treatment was higher in plants treated with the present strain than in simulated plants (FIG. 4a). The carotenoid level of the Chinese cabbage plants treated with the invention strain was relatively higher than that of the mock-treated Chinese cabbage plants after 50 mM NaCl treatment, but was similar to the untreated group in the TBSS treatment ( FIG. 4b ). However, pretreatment of the inventive strain did not protect photosynthetic pigment degradation in cucumber plants from salinity stress ( FIGS. 4c and 4d ). These results suggest that the strain of the present invention has a different effect on the pigment stability of Chinese cabbage and cucumber plants, and may be more useful for Chinese cabbage plants than cucumber and tomato plants to control salinity stress.

[Example 5] Bacillus zanthoxyli Evaluation of salt stress tolerance induced by HS1R treatment with dead cells

After disinfection of seeds of Arabidopsis thaliana wild-type (Columbia-0) and sos2-2 ( salt overly sensitive 2-2 ), a mutant plant with very strong salt sensitivity, 0mM, 50mM, 75mM NaCl and bone Shaking culture (75 rpm) in liquid plant culture medium (Murashige & Skoog (MS) media) containing dead cell suspension (OD ₆₀₀ = 0, 0.01, 0.05, 0.1) of the strain of the invention was performed (24 degrees, 16 hours) -light, 8 hours - dark). About 10 days after incubation, chlorophyll content and germination of seeds were compared.

Figures 5a and 5b show the salt stress response of Arabidopsis wild-type and Arabidopsis mutant sos2-2 ( salt overly sensitive 2-2 ) plants measured according to the dead cell concentration of the strain of the present invention by measuring the chlorophyll content. 5C and 5D are the results of verifying the effect of imparting resistance to salt stress of the dead strain of the present invention.

In order to confirm whether the strain of the present invention imparts salt resistance to plants even in the dead cell state, an experiment was conducted using Arabidopsis thaliana, a model plant, and Arabidopsis thaliana mutant sos2-2 ( salt overly sensitive 2-2 ), which is sensitive to salt stress was used to re-verify its efficacy.

The wild-type and mutant seeds were immersed in a liquid plant culture medium (Murashige & Skoog (MS) media) treated with NaCl, a typical salt, at concentrations of 0mM, 50mM, and 75mM, and the dead cells of the present strain were OD ₆₀₀ = 0, 0.01, 0.05 and 0.1, respectively.

Referring to Figure 5C, when the present strain is not treated, both wild-type Columbia-0 (Col-0) and mutant sos2-2 can be confirmed that growth is delayed or germination is inhibited from the 50 mM NaCl treatment group. In contrast, as can be seen from FIG. 5D, when dead cells were treated at a concentration of OD ₆₀₀ = 0.01, both wild-type and mutant germination and growth were observed at a NaCl concentration of 50 mM, the same as that of a plant having a NaCl concentration of 0 mM. have. Results as shown in the figure suggest that the present strain imparts salt stress resistance to plants and promotes growth in the dead cell state.

In the present specification and drawings, preferred embodiments of the present invention have been disclosed, and although specific terms are used, these are only used in a general sense to easily explain the technical content of the present invention and help the understanding of the present invention. It is not intended to limit the scope. It will be apparent to those of ordinary skill in the art to which the present invention pertains that other modifications based on the technical spirit of the present invention can be implemented in addition to the embodiments disclosed herein.

Korea Institute of Biotechnology and Biotechnology KCTC18833P 20200723

<110> Dong-A University Research Foundation for Industry-Academy Cooperation <120> Bacillus zanthoxyli strain promoting tolerance of plants and use it <130> GP200069 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1473 <212> DNA <213> Unknown <220> <223> 16S rRNA of Bacillus zanthoxyli HS1 <400> 1 atgaacgctg gcggcgtgcc taatacatgc aagtcgagcg aactgattag aagcttgctt 60 ctatgacgtt agcggcggac gggtgagtaa cacgtgggca acctgcctgt aagactggga 120 taacttcggg aaaccgaagc taataccgga taggatcttc tccttcatgg gagatgattg 180 aaagatggtt tcggctatca cttacagatg ggcccgcggt gcattagcta gttggtgagg 240 taacggctca ccaaggcaac gatgcatagc cgacctgaga gggtgatcgg ccacactggg 300 actgagacac ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg 360 aaagtctgac ggagcaacgc cgcgtgagtg atgaaggctt tcgggtcgta aaactctgtt 420 gttagggaag aacaagtacg agagtaactg ctcgtacctt gacggtacct aaccagaaag 480 ccacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggaa 540 ttattgggcg taaagcgcgc gcaggcggtt tcttaagtct gatgtgaaag cccacggctc 600 aaccgtggag ggtcattgga aactggggaa cttgagtgca gaagagaaaa gcggaattcc 660 acgtgtagcg gtgaaatgcg tagagatgtg gaggaacacc agtggcgaag gcggcttttt 720 ggtctgtaac tgacgctgag gcgcgaaagc gtggggagca aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctgc 840 agctaacgca ttaagcactc cgcctgggga gtacggtcgc aagactgaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 960 cttaccaggt cttgacatcc tctgacaact ctagagatag agcgttcccc ttcgggggac 1020 agagtgacag gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaacccttg atcttagttg ccagcattta gttgggcact ctaaggtgac 1140 tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa tcatcatgcc ccttatgacc 1200 tgggctacac acgtgctaca atggatggta caaagggctg caagaccgcg aggtcaagcc 1260 aatcccataa aaccattctc agttcggatt gtaggctgca actcgcctac atgaagctgg 1320 aatcgctagt aatcgcggat cagcatgccg cggtgaatac gttcccgggc cttgtacaca 1380 ccgcccgtca caccacgaga gtttgtaaca cccgaagtcg gtggagtaac cgtaaggagc 1440 tagccgccta aggtgggaca gatgattggg gtg 1473

Claims (12)

Bacillus zanthoxyli (Bacillus zanthoxyli) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain or a composition for enhancing stress resistance of plants comprising both as an active ingredient,
The composition for enhancing the stress resistance of the plant enhances the resistance of tomatoes to the tomato blight caused by the green blight fungus (Ralstonia solanacearum) or paprika resistance to the paprika blight caused by the late blight (Phytophthora capsici) A composition for promoting stress resistance of plants, characterized in that it promotes.
According to claim 1,
The strain is a composition for promoting stress resistance of plants, characterized in that it has 16S rRNA of the nucleotide sequence represented by SEQ ID NO: 1.
Bacillus zanthoxyli (Bacillus zanthoxyli) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain or a composition for enhancing stress resistance of plants comprising both as an active ingredient,
The composition for enhancing the stress resistance of the plant is a composition for promoting stress resistance of a plant, characterized in that it relieves the germination rate, shoot growth or root growth inhibition of cabbage, cucumber or tomato against salt stress.
Bacillus zanthoxyli (Bacillus zanthoxyli) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain or a composition for enhancing stress resistance of plants comprising both as an active ingredient,
The composition for enhancing stress resistance of plants is a composition for enhancing stress resistance of plants, characterized in that it alleviates the decrease in chlorophyll or carotenoid content of Chinese cabbage plants for salinity stress.
delete delete Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain, a composition comprising the strain, and a composition comprising the culture solution of the strain Any one or more selected from the group consisting of a plant, the area surrounding the plant Or in the method for enhancing stress resistance of plants comprising the step of treating them both,
The method for enhancing the stress resistance of the plant is a plant, characterized in that it enhances the resistance of the plant to the pathogen causing any one or more plant diseases selected from the group consisting of Ralstonia solanacearum and Phytophthora capsici. How to improve stress resistance.
delete delete delete Bacillus zanthoxyli (Bacillus zanthoxyli) HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain, a composition comprising the strain, and a composition comprising the culture solution of the strain Any one or more selected from the group consisting of Chinese cabbage under saline stress conditions A method for promoting plant growth comprising the step of treating an area around a plant, cucumber plant or tomato plant,
The method for promoting plant growth is a method for promoting plant growth, characterized in that the germination rate, shoot growth, or root growth of the cabbage plant, the cucumber plant or the tomato plant is promoted.
Bacillus zanthoxyli HS1 strain (Accession No.: KCTC18833P), a culture solution of the strain, a composition comprising the strain, and a composition comprising the culture solution of the strain Any one or more selected from the group consisting of Chinese cabbage under salt stress conditions In the plant growth promotion method comprising the step of treating a plant or an area around the cabbage plant,
The method for promoting plant growth is a method for promoting plant growth, characterized in that increasing the chlorophyll or carotenoid content of the Chinese cabbage plant.

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