KR20190044484A - Bacillus siamensis strain promoting resistance of plants against biotic and abiotic stress and use thereof - Google Patents

Abstract

The present invention relates to a novel strain of Bacillus siamensis H30-3 which promotes the resistance of plants against biotic or abiotic stress and uses thereof. A composition for enhancing the resistance of plants against stress and a method for enhancing the resistance of the present invention significantly increases the resistance of plants against biotic or abiotic stress using Bacillus siamensis H30-3 strain, thereby promoting the growth of plants even in environments where plant growth is unfavorable.

Description

The present invention relates to a Bacillus cyamensis strain and a use thereof for promoting biotic or abiotic stress resistance of a plant,

The present invention relates to novel Bacillus Xiamensis strains and their use for enhancing plant resistance to biological or abiotic stress.

Environmental stresses such as low temperature, high temperature, salt, or drying have a great influence on the survival and growth of plants, as plants have a significantly lower ability to avoid environmental stresses in the course of survival than animals. These environmental stresses are important factors that greatly reduce the crop production of the farmers, and biological stresses such as the onset of the plant diseases may also be seriously damaged by the cultivation of the crops.

Cabbage is a low temperature vegetable that likes a cool climate. When it reaches high temperature in the growing season, it is damaged by various plant diseases or insects. Among them, Chinese cabbage blight occurs during the growth of Chinese cabbage, and penetrates directly through the surface of the plant from the surface of the plant near the root, spreading to the whole underground or cabbage, causing rotting of the cabbage leaves, causing serious damage to the entire cabbage field. The cabbage, celery, radish, tobacco, onion, tomato, carrots, potatoes, lettuce or wasabi, as well as cabbage, are the most important limiting factors in the production of summer cabbage. It is also a serious disease that causes damage to various major vegetables. In addition, the Chinese cabbage blight is a bacterial and soil-borne plant disease, and the bacterial disease has a very low control effect of the medicine, so that it is difficult to prevent the control effect unless a large amount of medicine is sprayed proactively.

On the other hand, as the social interest in the side effects of pesticides increases, researches on biological control methods using microorganisms for various diseases, which are difficult to control, have been attempted. Microorganisms that have antimicrobial activity against pathogens may be used for biological control, or they may induce resistant responses in crops by inoculating microorganisms in advance to the crops for the purpose of inducing disease resistance to the crops. These biological control methods do not cause problems such as environmental pollution, weakness and residual toxicity, and they are evaluated as a preferable control method because the control effect lasts for a long time.

At present, many domestic farmers are suffering from severe damage due to abiotic environmental stress such as high temperature drying, or biological stress such as cabbage blight, for example, which inhibits crop production. It is necessary to develop a technique for promoting the production of crops by promoting the resistance of plants to such biological or abiotic stresses.

It is a technical object of the present invention to provide a novel strain which increases the resistance of a plant to biological or abiotic stress.

It is another object of the present invention to provide a method for improving the resistance to biological or abiotic stress of a plant by using the novel strain to facilitate cultivation of the crop in an unstable environment.

The present invention provides a novel strain, Bacillus siamensis H30-3 strain (Accession No .: KACC 92187P).

The inventors of the present invention have intensively studied to find a novel strain that promotes resistance to biological or abiotic stress of a plant, and as a result, found a new strain of Bacillus cyamensis H30-3 of the present invention, The present inventors have found that the strain remarkably improves the resistance of the plant to a plant disease or a high temperature drying environment, thereby completing the present invention.

The Bacillus cyamensis H30-3 strain of the present invention was deposited with the Institute of Agricultural Biotechnology on Aug. 21, 2017 and received the accession number KACC 92187P. The Bacillus cyamensis H30-3 strain of the present invention comprises the 16S rRNA of the nucleotide sequence shown in SEQ ID NO: 1.

The present invention provides a composition for promoting stress resistance of a plant comprising a strain of Bacillus cyamensis H30-3 as an active ingredient. In addition, the present invention provides a plant disease inhibiting composition comprising a strain of Bacillus cyamensis H30-3 as an active ingredient.

The composition may contain, as an active ingredient, a Bacillus cyamensis H30-3 strain, a cell, a spore, a culture solution of the strain, a concentrated culture solution, a dried product of the culture solution, an extract of the culture solution, an extracellular polysaccharide or a volatile substance. The active ingredient can be suitably selected by a person skilled in the art in consideration of the hair growth environment, the kind of the target plant, or the economy. When the composition is treated in a plant, the composition can enhance the resistance of the plant to biological or abiotic stress and promote the growth of the plant.

In the present invention, the term " cell " means a cell wall fraction of a strain, a dead cell, or a dried cell. The culture medium in the culture broth can be removed and centrifugation or filtration can be performed to recover only the concentrated cells, and this step can be carried out according to need by a general practitioner. The concentrated microbial cells can be preserved by freezing or freeze-drying according to a conventional method so as not to lose their activity.

In the present invention, the term 'culture extract' means a culture obtained by extracting various cultures using various organic solvents so as to include various physiologically active substances, and includes C _1-4 alcohol (for example, methanol, ethanol, butanol, etc.) Acetone, ethyl acetate, and the like may be used alone or two or more kinds of organic solvents may be successively used for extraction.

The Bacillus cyamensis H30-3 strain of the present invention can be cultured in a large amount by a conventional culture method of microorganisms of the genus Bacillus. The medium for the culture may be a solid medium such as NA (Nutrient Agar) or TSA (Tryptic Soy Agar), or a medium such as Nutrient Broth (NB), King's medium B Broth (KB), Peptone Sucrose Broth (PSB) ) Or the like may be used.

In the present invention, the composition for enhancing the stress resistance of the plant may enhance the resistance of the plant to biological or abiotic stress. The biological stress includes the occurrence of plant diseases due to plant pathogens and the like, and the abiotic stress may include any one or more environmental stresses in the group consisting of drying, high temperature, low temperature, salt, pH, .

In the present invention, the plant to which the composition can be treated may be any one or more selected from the group consisting of tree, herb, shrub, grass, vine, fern, moss, green algae, monocotyledonous plants and dicotyledonous plants. It is not. Preferably, when the composition is processed into Chinese cabbage, the resistance of plants to cabbage blight or high temperature drying stress can be significantly improved.

The plant pathogens to which the Bacillus cyamensis strain H30-3 of the present invention imparts resistance to the plant are selected from the group consisting of Alternaria alternata , Phytophthora capsici , Fusarium oxysporum , Colletotrichum acutatum , and Pectobacterium carotovorum pv. Carotovara . However, the present invention is not limited thereto. Preferably, the plant disease to which the Bacillus cyamensis H30-3 strain of the present invention imparts resistance to the plant may be Chinese cabbage blight disease, and when the H30-3 strain is treated with the plant, the plant resistance to cabbage blight disease is remarkable .

In addition, the composition according to the present invention can be used as a wetting agent, a granulating agent, a wetting agent, a wetting agent or the like using a surfactant or an extender, which can be used as a biochemical pesticide for the purpose of stable formulation suitable for actual packaging, For example, in the form of a liquid wettable agent, a liquid agent, a water-soluble agent, a water-soluble granule, or an encapsulating agent.

The present invention provides a method for enhancing resistance of a plant to stress, preferably biological or abiotic stress, comprising treating the plant with a strain of Bacillus cyamensis H30-3. In addition, the present invention provides a plant disease suppressing method comprising treating a plant with a strain of Bacillus cyamensis H30-3. The method can treat Bacillus cyamensis H30-3 strain in any one or more selected from the group consisting of plant seeds, roots, stem, leaves and whole plants.

In addition, the method may include a method of treating the tissue of a plant in a plant breeding period, or a method of treating the plant by planting a H30-3 strain in a medium for controlling a disease before and after planting or drinking water, and may be carried out by various methods known in the art It can be applied to the above-mentioned plant or soil around the plant. In addition, the treatment of the plant or the soil around the plant can be carried out by inoculation, spraying, coating, crossing, infiltration, spraying, coating, dusting or dipping.

In addition, the present invention provides a method for producing a composition for promoting stress resistance of a plant comprising culturing a strain of Bacillus cyamensis H30-3. Any method known in the art can be used for culturing the strain, and the method is not particularly limited.

The Aspergillus terreus strain H30-3 (deposited) of the present invention significantly enhances plant resistance to plant biological or abiotic stress.

In addition, the composition of the present invention for enhancing resistance to stress and resistance-promoting method of the present invention significantly increases the resistance of a plant to biological or abiotic stress using the Bacillus cyamensis strain H30-3, It is possible to promote the growth of the plant even in this unfavorable environment.

Brief Description of the Drawings Fig. 1 is a system analysis chart comparing 16S rRNA base sequences of Bacillus cyamensis H30-3 strain of the present invention with other Bacillus strains.
Fig. 2 shows the result of evaluating the antimicrobial activity of volatile substances produced by Bacillus cyamensis strain H30-3.
Fig. 3 shows the result of measuring the inhibitory effect on the Chinese cabbage blight of Bacillus cyamensis H30-3.
FIG. 4 shows the results of evaluating the survival rate of plants in high temperature drying stress when treating Bacillus cyamensis H30-3 strain.
FIG. 5 shows the results of measurement of soil moisture and chlorophyll content around a plant treated with Bacillus cyamensis H30-3 strain under high temperature drying conditions.
Fig. 6 shows the results of evaluation of plant survival rate against high temperature drying stress in the treatment of extracellular polysaccharide of Bacillus cyamensis strain H30-3.
FIG. 7 shows the results of evaluation of plant survival rate against high temperature drying stress caused by volatile substances of Bacillus cyamensis strain H30-3.
FIG. 8 shows the results of evaluation of the plant survival rate against high temperature drying stress by the methanol extract of Bacillus cyamensis H30-3 culture medium.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  One: Bacillus Cyamensis  Screening and Identification of H30-3 Strain

The H30-3 strain was sampled in 2004 from the Oi cultivation area in Gongju city, Chungcheongnam-do, and the soil was sampled and diluted by 1/10 TSA. The strains used for the assay were cultured in TSB at 25 ° C for 24 hours, and the strains were harvested by centrifugation. Thereafter, the suspension was made with 10 mM MgSO ₄ , and the absorbance was adjusted to A600 = 0.2, and used uniformly as the inoculum.

(5'-GAG TTT GAT CCT GGC TCA-3 ') and rP2 (5'-ACG GCT AAC TTG TTA CGA) were used to isolate the total RNAs using Qiagen genomnic tip kip CT-3 ') primers were used to amplify the 16S rRNA region by PCR. The PCR conditions were repeated 35 times at 95 ° C for 4 minutes, 58 ° C for 1 minute, and 72 ° C for 2 minutes, and then kept at 72 ° C for 8 minutes. After analyzing the nucleotide sequence of the amplified PCR fragment, we identified the species with similar nucleotide sequence using EZ BioCloud network service.

As a result of the phylogenetic analysis, H30-3 was identified as Bacillus cyamensis because it has 99.93% similarity with Bacillus cyamensis KCTC13613T (type strain) in the 16S rRNA sequence (1381 bp) (Fig. 1). The 16S rRNA information of H30-3 is shown in Sequence Listing 1.

Example  2: Induction effect of plant resistance on biological stress of H30-3 strain

To evaluate the antimicrobial activity of H30-3, TSA was added to one side of the I-plate, PDA was added to the other side, and H30-3 strain (200 μl) was applied to TSA. PDA were cultured with Alternaria alternata, Phytophthora capsici, Fusarium oxysporum or Colletotrichum acutatum , respectively , and mycelial growth was inhibited compared to the control. As a control, 10 mM MgSO ₄ buffer and Bacillus vallismortis strain BS07M known to have antibacterial activity were used. Among the four fungi tested, the mycelial growth of Alternaria alternata and Colletotrichum acutatum was inhibited by 13.7% and 20.3% of H30-3 volatiles, respectively, compared to the control (Fig. 2).

Chinese cabbage rot was tested with Chinese cabbage varieties. Pectobacterium carotovorum pv. 21 strains of carotovara were used. The cabbage was cultivated for 2 weeks in a 9 cm diameter vial containing the supernatant and treated with 20 ml of H30-3 bacterial suspension. The bacterial suspension was first cultured in TSA at 28 DEG C for 24 hours, and the single colonies were shake-cultured in 100 mL of TSB at 28 DEG C and 150 rpm for 48 hours. Then, the culture was centrifuged (5,000 rpm, 5 minutes), and the supernatant was removed. Only the precipitated bacteria were dissolved in 10 mM MgSO ₄ Solution, and used as a bacterial suspension at 10 < ⁷ > cells / ml. After 7 days of bacterial rooting treatment, 10-20 cells / ml of the Chinese cabbage root rot (10 ⁷ cells / ㎖) was mixed with mineral oil and inoculated 20ml each time. The disease severity (%) was evaluated 7 days after inoculation. When two lobes appeared on the main leaf, 10 ⁷ cells / ㎖ of bacterial suspension was treated with 20 ml of the control, followed by cultivation for 7 days, followed by high temperature drying stress treatment. As a control, 10 mM MgSO4 and BTH (0.1 mM), which is known to induce plant resistance, were used. In the Chinese cabbage treated with strain H30-3, the invention of bacterial blight was reduced by 44.4% (FIG. 3).

Example  3: Inducible effect of plant resistance to high temperature drying stress of strain H30-3

The H30-3 strain and the control (10 mM MgSO ₄ ) were treated in the greenhouse condition, and the Chinese cabbage was subjected to high temperature drying stress and cabbage growth was compared.

For this purpose, Chinese cabbage seeds (cultivar: Deam, Gyeonggwang) were planted in a 9cm plastic pot containing soil and cultivated in a greenhouse for 2 weeks. The bacterial suspension was first cultured in TSA at 28 DEG C for 24 hours, then single colonies were shake-cultured in 100 mL TSB at 28 DEG C and 150 rpm for 48 hours. Then, the culture was centrifuged (5,000 rpm, 5 minutes), and the supernatant was removed. Only the precipitated bacteria were suspended in ₁₀ mM MgSO ₄ Solution, and used as a bacterial suspension at 10 < ⁷ > cells / ml. When two lobes were emerged from the main leaves, 10 ⁷ cells / ㎖ of bacterial suspension was treated with 20 ml of each strain, cultured for 7 days, and subjected to high temperature drying stress treatment.

High temperature drying stress was maintained at 35 ℃, which could affect cabbage growth, for 5 ~ 7 days. The plant survival index was evaluated as 0 (complete death) to 5 (surviving), and after the high temperature drying stress condition, water was again given to 40 ml / 0 to 5).

As a result, the survival rate of plants in control (control) was 1.9, and the value of H30-3 was 2.7, which was significantly higher than that of control. After rehydration, the H30-3 biomass increased by 9.6% compared to the control (Fig. 4). The plant survival rate after the high temperature drying was about 1.65 in the control group, whereas the H30-3 was remarkably high at 2.5 and the bioavailability was increased by 28.0% compared to the control (Fig. 4).

In the greenhouse condition, the soil moisture was higher at 5.2% than the control at 4.5% compared with H30-3. The SPAD value for measuring chlorophyll content was not significantly different from that of control (48.4) and H30-3 (47.8). In the case of the temperate cultivars, H30-3 was 2.9% higher when the soil moisture was 2.1%, and H30-3 was 49.0 when the control was 34.4 (Fig. 5).

Example  4: of the strain H30-3 To extracellular polysaccharide (EPS)  Induced resistance to high temperature drying stress

In the same greenhouse conditions as the above-mentioned high temperature drying stress, the seedlings and the seedlings were seeded and cultivated, and the exopolysaccharide of the strain H30-3 was treated.

The high temperature drying stress was given under the same conditions as in Example 3, and the viability of the Chinese cabbage was evaluated as 0-5. After the evaluation, water was given at 40 ml / pot and allowed to recover for 1 day, and the degree of survival and bioavailability were evaluated.

H30-3 strain was cultured as in Example 3 to obtain extracellular polysaccharide of strain H30-3. The culture was centrifuged, the supernatant was collected, and 2.5% of cold 100% ethanol was added to precipitate overnight. Then, the supernatant was discarded by centrifugation (6,000 g, 30 minutes), and the precipitated extracellular polysaccharide was lyophilized. After lyophilization, the extracellular polysaccharide was weighed to 10 ppm. 20 ml of the extracellular polysaccharide was treated and treated for 7 days, and the degree of survival and fresh weight of the plant were evaluated under the high temperature drying stress as in Example 3.

Experimental results showed that plant survival was not different between cultivars, however, the fresh weight after rehydration increased by 43.5% when treated with the extracellular polysaccharide of H30-3. The survival rate of the plant was significantly higher than that of control (1.25, 2.25) in the extracellular polysaccharide (10ppm) of H30-3, and the fresh weight was significantly increased (251%).

Example  5: Induction effect of plant resistance to high temperature drying stress by volatile substances of H30-3 strain

In the same greenhouse conditions as the above-mentioned high temperature drying stress, the cultivars of seedlings and seeds were cultivated and the high temperature drying stress due to the volatile substances of the strain H30-3 was evaluated. For this purpose, a sprout vegetable container with octagonal upper and lower ends was used, and the sterilized soil was filled in the upper and lower sides so as not to touch each other. In the lower part, 40 ml of bacterial suspension of H30-3 strain was treated, and the resistance induction effect of H30-3 by volatile substances was evaluated by physically separating the Chinese cabbage seeds at the upper part.

The high temperature drying stress was given under the same conditions as in Example 3, and the viability of the Chinese cabbage was evaluated as 0-5. After the evaluation, water was given at 40 ml / pot and allowed to recover for 1 day, and the degree of survival and biomass were evaluated.

As a result, the survival rate of the plants was significantly increased from 1.3 to 3.4 when the volatile substances of H30-3 strain were treated, and increased from 1.9 to 3.4 after rehydration. The survival rate of the plant was increased to 2.1 when the control was 1.0, and to 1.9 when the control was 1.1 after reutilization. The biomass increased in the control cultivar at the control level of 0.6 at 0.8 and increased slightly to 0.5 at the control cultivar at the control cultivar, but there was no statistical significance (Fig. 7).

Example  6: Induction effect of plant resistance to high temperature drying stress by culture concentrate of H30-3 strain

In the same greenhouse conditions as the above-described high temperature drying stress, the seedlings and the seedlings were seeded and cultured, and the high temperature drying stress by the culture concentrate of the strain H30-3 was evaluated. To this end, the bacterial suspension was cultured in the same manner as described above, and the culture supernatant was collected by centrifugation and fractionated with n-butanol (1: 1, v / v) and concentrated at 50 ° C. The final concentrate was prepared at 1, 10, and 100 ppm with methanol as a solvent. Seedlings were cultivated in H30-3 culture concentrate (1, 10, 100ppm) for 3 hours and seeded. After 3 weeks of sowing, high temperature drying stress was treated as in Example 3, and plant viability and fresh weight of the plant parts were evaluated.

As a result, the survival rate of plant was significantly increased at 1, 10, and 100ppm concentration of H30-3 strain after high temperature drying stress and rehydration. The fresh weight of the plant part was increased by 145.3% when treated with 10ppm seed concentrate (FIG. 8). The survival rate of the plants grown in the temperate zone was significantly increased by 1, 10, and 100ppm treatment of H30-3 strain after high temperature drying stress and reutilization. The fresh weight of the plant was increased by 34.5% after 100ppm treatment (Fig. 8).

Agricultural Biotechnology Research Institute KACC92187P 20170821

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Bacillus siamensis strain promoting resistance of plants against          biotic and abiotic stress and use thereof <130> P18-075, 2017-0339-10-A <150> KR 10-2017-0136728 <151> 2017-10-20 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1381 <212> DNA <213> Artificial Sequence <220> <223> Bacillus siamensis H30-3 16S rRNA <400> 1 gggagcttgc tccctgatgt tagcggcgga cgggtgagta acacgtgggt aacctgcctg 60 taagactggg ataactccgg gaaaccgggg ctaataccgg atggttgttt gaaccgcatg 120 gttcagacat aaaaggtggc ttcggctacc acttacagat ggacccgcgg cgcattagct 180 agttggtgag gtaacggctc accaaggcga cgatgcgtag ccgacctgag agggtgatcg 240 gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc 300 cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt 360 aaagctctgt tgttagggaa gaacaagtgc cgttcaaata gggcggcacc ttgacggtac 420 ctaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt aggtggcaag 480 cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg tttcttaagt ctgatgtgaa 540 agcccccggc tcaaccgggg agggtcattg gaaactgggg aacttgagtg cagaagagga 600 gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg tggaggaaca ccagtggcga 660 aggcgactct ctggtctgta actgacgctg aggagcgaaa gcgtggggga gcgaacagga 720 ttagataccc tggtagtcca cgccgtaaac gatgagtgct aagtgttagg gggtttccgc 780 cccttagtgc tgcagctaac gcattaagca ctccgcctgg ggagtacggt cgcaagactg 840 aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgaag 900 caacgcgaag aaccttacca ggtcttgaca tcctctgaca atcctagaga taggacgtcc 960 ccttcggggg cagagtgaca ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt 1020 gggttaagtc ccgcaacgag cgcaaccctt gatcttagtt gccagcattc agttgggcac 1080 tctaaggtga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa atcatcatgc 1140 cccttatgac ctgggctaca cacgtgctac aatggacaga acaaagggca gcgaaaccgc 1200 gaggttaagc caatcccaca aatctgttct cagttcggat cgcagtctgc aactcgactg 1260 cgtgaagctg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1320 ccttgtacac accgcccgtc acaccacgag agtttgtaac acccgaagtc ggtgaggtaa 1380 c 1381

Claims (12)

Bacillus siamensis strain H30-3 (KACC 92187P). A composition for promoting stress resistance of a plant comprising Bacillus cyamensis H30-3 strain (KACC 92187P) as an active ingredient. The composition according to claim 2, wherein the stress of the plant is a biological or an abiotic stress. 4. The composition according to claim 3, wherein the abiotic stress is at least one selected from the group consisting of dryness, high temperature, low temperature, salt, pH and malnutrition. 5. The composition of claim 4, wherein the abiotic stress is high temperature drying. The composition according to claim 2, wherein the plant is a cabbage. [3] The composition according to claim 2, wherein the composition comprises at least one selected from the group consisting of a concentrated culture solution of Bacillus cyamensis strain H30-3, an extracellular polysaccharide, and a volatile substance as an active ingredient. A composition for suppressing plant diseases, which comprises Bacillus ciliosensis H30-3 strain (KACC 92187P) as an active ingredient. 9. The plant of claim 8, wherein the plant disease is selected from the group consisting of Alternaria alternata , Phytophthora capsici , Fusarium oxysporum , Colletotrichum acutatum and Pectobacterium carotovorum pv. carotovara ) which is a plant disease caused by a plant pathogenic bacterium. The composition according to claim 9, wherein the plant disease is Chinese cabbage blight caused by Pectobacterium carotovorum pv. Carotovara . A method for enhancing the stress resistance of a plant comprising the step of treating the plant with a strain of Bacillus cyamensis H30-3 (KACC 92187P). A method for inhibiting a plant disease, comprising treating the plant with a strain of Bacillus cyamensis H30-3 (KACC 92187P).

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