Azospirillum sp. strain having plant growth promoting ability and use thereof

Abstract

The present application relates to a novel Azospirillum sp. strain and use thereof, and the novel Azospirillum sp. strain can be specifically used for promoting plant growth.

Description

Azospirillum strain having plant growth-promoting ability and its uses

[0001] This application claims the benefit of priority based on Korean Patent Application No. 10-2024-0191841 filed on December 19, 2024, and all contents disclosed in the documents of said Korean patent application are incorporated as part of this application.

[0002] Throughout this application, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this disclosure in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0003] The present application relates to a strain of the genus Azospirillum having plant growth-promoting ability and its uses.

[0004] Strains of the genus Azospirillum belong to the Alpha-Proteobacteria class of bacteria; they are Gram-negative bacteria that do not form spores. Although Azospirillum microorganisms are aerobic, they can convert inert atmospheric nitrogen into a biologically usable form under low-oxygen conditions. Azospirillum strains inhabit freshwater and soil, and are known to reside particularly in the plant rhizosphere, playing a close role in the growth of root hairs and plant growth, and promoting plant growth through various mechanisms. Furthermore, they promote growth by fixing nitrogen and supplying it to the rhizosphere, thereby enabling plants to utilize more of it. In addition, they play a role in protecting plant roots from stress caused by drought and floods (Fukami, Josiane, Paula Cerezini, and Mariangela Hungria. "Azospirillum: benefits that go far beyond biological nitrogen fixation." Amb Express 8.1 (2018): 73.).

[0005] Azospirillum brasilense, belonging to the genus Azospirillum, is a non-photosynthetic microorganism that inhabits the rhizosphere of a wide range of plants in the Rhodospirillaceae family. Azospirillum brasilense is well known as a plant growth-promoting microbe (PGPM) that promotes plant growth by fixing atmospheric nitrogen and producing siderophores and phytohormones. PGPMs promote (1) increased mineral nutrient solubility and nitrogen fixation; (2) the production of plant hormones such as auxin (indole-3-acetic acid; IAA), and the production of ACC deaminase, an enzyme that hydrolyzes 1-aminocyclopropane-1-carboxylate (ACC), a direct precursor of ethylene, thereby lowering plant ethylene concentration and promoting root length growth; (3) Plant growth is promoted through mechanisms such as suppression of soil-borne pathogens (e.g., hydrogen cyanide production, siderophores, nutrient competition), and improvement of plant stress tolerance to drought, floods, salinity, and metal toxicity. The use of such PGPMs to improve crop production is currently considered a promising agricultural approach, and as environmental problems such as groundwater contamination due to excessive fertilizer use increase, biological alternatives have become promising and necessary. In response to this need, Azospirillum brasilense is being used as a microbial fertilizer (inoculant) to promote growth and increase yields of various plants such as corn, soybeans, and millet. However, since microbial fertilizer products are mainly produced in liquid form, there is a continuous need for the development of Azospirillum brasilense strains that have enhanced plant growth-promoting activity while maintaining high storage stability in liquid form at room temperature.

[0006] The present application provides a novel strain of Azospirillum brasilense having plant growth-promoting activity and uses thereof.

[0007] One example provides a strain of Azospirillum brasilense deposited under accession number KCCM13420P. The strain may have carotenoid production ability, plant growth-promoting activity, storage stability, or a combination of these characteristics.

[0008] Another example provides a composition comprising the strain, a culture of the strain, or a combination thereof. The composition may be used for carotenoid production, plant growth promotion, and / or storage stabilization.

[0009] Another example is,

[0010] The above strain,

[0011] A culture of the above strain, and

[0012] A fertilizer composition is provided comprising one or more selected from the group consisting of the above strain, a culture of the above strain, or a combination thereof.

[0013] Another example provides a method for cultivating plants or a method for promoting plant growth, comprising the step of treating a plant and / or soil with one or more selected from the group consisting of the strain, a culture of the strain, the composition, and the fertilizer composition.

[0014] Another example provides a plant growth-promoting use of a composition comprising the above strain, a culture of the above strain, or a combination thereof, or a use for manufacturing a plant growth-promoting composition (e.g., fertilizer).

[0015] Another example provides a composition for producing carotenoids comprising the strain, a culture of the strain, or a combination thereof.

[0016] Another example provides a use for producing carotenoids of a composition comprising the strain, a culture of the strain, or a combination thereof, or a use for preparing a composition for producing carotenoids.

[0017] Each description and embodiment disclosed in this application may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not to be limited by the specific descriptions provided below. Additionally, a person skilled in the art may recognize or identify numerous equivalents to the specific embodiments of this application described herein using only ordinary experimentation. Moreover, such equivalents are intended to be included in this application.

[0018]

[0019] One example of the present application provides an Azospirillum brasilense strain deposited under accession number KCCM13420P.

[0020] The Azospirillum brasilense strain deposited under the accession number KCCM13420P may be a mutant strain obtained by inducing a mutation in the Azospirillum brasilense strain (parent strain) through external stimulation. Specifically, the strain may be a mutant strain obtained by irradiating with radiation (e.g., gamma rays).

[0021] The Azospirillum brasilense strain deposited under accession number KCCM13420P may be a mutant strain obtained by mutating the parent strain Azospirillum brasilense, and may be a strain with significantly improved carotenoid productivity, plant growth-promoting activity, and / or storage stability compared to the parent strain.

[0022] As used in this specification, the term “parent strain” refers to a strain prior to mutation induction and may be used interchangeably with “wild-type” strain. The parent strain may be a strain that has not undergone mutation to obtain the Azospirillum brasilense strain deposited under accession number KCCM13420P of this application, and in one embodiment, it may be the Azospirillum brasilense strain deposited under accession number KCCM13327P.

[0023] The above mutagenic method may be carried out by various means known in the art, and may use either a physical or chemical mutagenic method. For example, a physical mutagenic method suitable for the present invention may use a method of irradiating with gamma rays or ultraviolet rays, but is not limited thereto. Additionally, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), diepoxybutane, ethylmethanesulfonate, mustard compounds, hydrazine, and nitrous acid may be used as chemical mutagenic methods, but are not limited thereto.

[0024] In one embodiment, the mutation for producing the mutant strain may be by a method of irradiating with radiation. The radiation for inducing the mutation may be gamma rays or ultraviolet rays, for example, gamma rays. The radiation (e.g., gamma rays) may be a dose of 2 kGy to 5 kGy per hour, a dose of 2 kGy to 4 kGy, a dose of 3 kGy to 5 kGy, or a dose of 3 kGy to 4 kGy (e.g., 3 kGy or 4 kGy dose), but is not limited thereto. The irradiation time of the radiation (e.g., gamma rays) may be obtained by irradiating for 0.5 to 2 hours or 0.5 to 1.5 hours, for example, about 1 hour, but is not limited thereto.

[0025] In one embodiment of the present application, a mutation was induced in an Azospirillum brasilense strain deposited under accession number KCCM13327P by irradiating it with gamma rays of 3 kGy / hr or 4 kGy / hr for 1 hour, and among the mutant strains, a strain having or with enhanced carotenoid production ability was selected and named Azospirillum brasilense CP03-0008, and deposited with the Korean Culture Collection of Microorganisms, an international depositary institution under the Treaty of Budapest, on November 16, 2023, and assigned accession number KCCM13420P.

[0026] In one embodiment, the Azospirillum brasilense strain deposited under accession number KCCM13420P of the present application may have genomic DNA containing one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 9 to 19.

[0027] In one embodiment, the coding DNA sequence of a gene present in the genome of the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P according to one example of the present application may include one or more nucleotide sequences selected from the group consisting of SEQ ID NOs 9 to 19, but is not limited thereto.

[0028] The nucleotide sequence of SEQ ID NO. 9 above may be a DNA sequence of a 16S rRNA gene, and

[0029] The nucleotide sequence of SEQ ID NO. 10 above may be the sequence of the atpD gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding the ATP synthase F1 complex beta subunit, and

[0030] The nucleotide sequence of SEQ ID NO. 11 above may be the sequence of the dnaK gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding the chaperone protein DnaK, and

[0031] The nucleotide sequence of SEQ ID NO. 12 above may be the sequence of the fumC gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding fumarate hydratase, and

[0032] The nucleotide sequence of SEQ ID NO. 13 above may be the sequence of the gyrA gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding DNA gyrase subunit A, and

[0033] The nucleotide sequence of SEQ ID NO. 14 above may be the sequence of the gyrB gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding DNA gyrase subunit B.

[0034] The nucleotide sequence of SEQ ID NO. 15 above may be the sequence of the recA gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding recombinase A, and

[0035] The nucleotide sequence of SEQ ID NO. 16 above may be the sequence of the rpoA gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding the RNA polymerase alpha subunit, and

[0036] The nucleotide sequence of SEQ ID NO. 17 above may be the sequence of the rpoB gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding the RNA polymerase beta subunit, and

[0037] In addition, the nucleotide sequence of SEQ ID NO. 18 may be the sequence of the rpoD gene derived from Azospirillum brasilense, i.e., a nucleotide sequence encoding the RNA polymerase sigma-70 factor, and

[0038] The nucleotide sequence of SEQ ID NO. 19 above may be the sequence of the trpB gene derived from Azospirillum brasilense, that is, the nucleotide sequence encoding the tryptophan synthase beta chain.

[0039] As demonstrated in the examples to be described later, when the Azospirillum brasilense strain deposited under accession number KCCM13420P was compared with the parent strain prior to irradiation (e.g., the Azospirillum brasilense strain deposited under accession number KCCM13327P described above), it was confirmed that the nucleotide sequences of SEQ ID NOs 9 to 19 contained in the genomic DNA matched each other. Accordingly, it was confirmed that the strain deposited under accession number KCCM13420P is the Azospirillum brasilense strain, which is the same species as the parent strain.

[0040]

[0041] The Azospirillum brasilense strain deposited under accession number KCCM13420P according to one example of the present application may have carotenoid production activity or increased carotenoid production activity compared to the unmodified or wild-type Azospirillum brasilense strain (see Examples 2 and 3). Additionally, the strain may have plant growth-promoting activity or increased plant growth-promoting activity compared to the unmodified or wild-type Azospirillum brasilense strain (see Example 4). Furthermore, the Azospirillum brasilense strain deposited under accession number KCCM13420P may have excellent storage stability or increased storage stability compared to the unmodified or wild-type Azospirillum brasilense strain (see Example 5).

[0042] In this application, the term “pre-modification strain” does not exclude strains containing mutations that may naturally occur in microorganisms, and refers to the natural (wild) strain itself or a strain prior to a change in traits caused by genetic variation due to natural or artificial factors. It may be, for example, an existing known strain or a parent strain prior to irradiation (e.g., the strain *Azospirillum brasilense* deposited under accession number KCCM13327P mentioned above), but is not limited thereto. The term “pre-modification strain” may be used interchangeably with “pre-modification microorganism,” “non-modified strain,” “non-mutated strain,” “non-modified microorganism,” “non-mutated strain,” “CW01-0108,” or “parent strain.”

[0043]

[0044] In one embodiment, the Azospirillum brasilense strain deposited under accession number KCCM13420P according to one example of the present application may include, but is not limited to, one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8 in its genomic DNA.

[0045]

[0046] In one embodiment, the coding DNA sequence of a gene present in the genome of the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P according to one example of the present application may include one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8, but is not limited thereto.

[0047] In one embodiment, the nucleotide sequence of SEQ ID NO. 2 is a nucleotide sequence encoding glutamate 5-kinase, and may include, but is not limited to, a variant in which the 651st base (Cytosine, C) is substituted with T (Thymine) and CC is inserted between the 796th base (Cytosine, C) and the 797th base (Thymine, T) of the nucleotide sequence encoding glutamate 5-kinase of the parent strain prior to irradiation (e.g., the Azospirillum brasilense strain deposited under accession number KCCM13327P described above) (SEQ ID NO. 1) encoding glutamate 5-kinase, but is not limited to. The sequence of SEQ ID NO. 1 may be a sequence corresponding to a sequence encoding glutamate 5-kinase derived from Azospirillum brasilense registered in the NCBI database (e.g., NCBI Gene ID: 56452319).

[0048] In one embodiment, the nucleotide sequence of SEQ ID NO. 4 is a nucleotide sequence encoding lysylphosphatidylglycerol synthase, and may include, but is not limited to, a variant in which the 1605th base (Cytosine, C) is deleted and C is inserted between the 1607th base (Guanine, G) and the 1608th base (Cytosine, C) in the nucleotide sequence encoding lysylphosphatidylglycerol synthase of the parent strain prior to irradiation (e.g., the Azospirillum brasilense strain deposited under accession number KCCM13327P described above) is inserted between the 1607th base (Guanine, G) and the 1608th base (Cytosine, C) in the nucleotide sequence encoding lysylphosphatidylglycerol synthase of the parent strain prior to irradiation (e.g., SEQ ID NO. 3). The sequence of SEQ ID NO. 3 above may be a sequence corresponding to a sequence encoding Azospirillum brasilense-derived lysylphosphatidylglycerol synthase registered in the NCBI database (e.g., NCBI Gene ID: 56451010), but is not limited thereto.

[0049] In one embodiment, the nucleotide sequence of SEQ ID NO. 6 is a nucleotide sequence encoding glycerol kinase, and may include, but is not limited to, a variant in which the base at the 1138th position (Guanine, G) in the nucleotide sequence encoding glycerol kinase of the parent strain prior to irradiation (e.g., the Azospirillum brasilense strain deposited under accession number KCCM13327P described above) is substituted with A (Adenine). The sequence of SEQ ID NO. 5 may be a sequence corresponding to a sequence encoding glycerol kinase derived from Azospirillum brasilense registered in the NCBI database (e.g., NCBI Gene ID: 56450763), but is not limited thereto.

[0050] In one embodiment, the nucleotide sequence of SEQ ID NO. 8 is a nucleotide sequence encoding PTS fructose-like transporter subunit IIB, and may include, but is not limited to, a variant in which the base at the 1840th position (Thymine, T) in the nucleotide sequence encoding PTS fructose-like transporter subunit IIB of the parent strain prior to irradiation (e.g., the Azospirillum brasilense strain deposited under accession number KCCM13327P described above) is substituted with C. The sequence of SEQ ID NO. 7 may be a sequence corresponding to the sequence encoding PTS fructose-like transporter subunit IIB derived from Azospirillum brasilense registered in the NCBI database (e.g., NCBI Gene ID: 56453471), but is not limited thereto.

[0051] According to one example of the present application, the Azospirillum brasilense strain deposited under accession number KCCM13420P may have increased carotenoid production activity compared to an Azospirillum brasilense strain that does not contain one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8 (see Examples 2 and 3). Additionally, the Azospirillum brasilense strain deposited under accession number KCCM13420P may have increased plant growth-promoting activity compared to an Azospirillum brasilense strain that does not contain one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8 (see Example 4). Additionally, the Azospirillum brasilense strain deposited under accession number KCCM13420P may have increased storage stability compared to an Azospirillum brasilense strain that does not contain one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8 (see Example 5).

[0052]

[0053] The strain *Azospirillum brasilense* deposited under accession number KCCM13420P is characterized by carotenoid production activity, plant growth-promoting activity, and / or storage stability.

[0054] The Azospirillum brasilense strain deposited under accession number KCCM13420P may possess carotenoid production activity or may have increased carotenoid production capacity. According to an embodiment of the present invention, it was confirmed that the strain has superior carotenoid production capacity compared to existing known strains or other Azospirillum brasilense strains, and that crop cultivation experiments can significantly promote plant growth.

[0055] Carotenoids are natural pigments belonging to the isoprenoid (also known as "tetraterpenoid") family, which are most widely synthesized by all photosynthetic organisms, including macroalgae, microalgae, plants, some non-photosynthetic bacteria, insects, and fungi. Carotenoids can be utilized as natural pigments because they absorb wavelengths in the range of approximately 400 to 520 nm and exhibit yellow or orange colors. Carotenoids possess a unique polyene chain (-C=C-)n structure with 8 to 13 conjugated double bonds, acting as a stabilizing antioxidant that effectively inhibits various types of cellular oxidants present within cells. Carotenoids, possessing these antioxidant properties, are well known to enhance resistance to oxidative stress in the cells of microorganisms and higher organisms through activities such as photosynthesis, photoprotection, cell membrane stabilization, and the inhibition of reactive oxygen species (ROS). In addition, carotenoids are known to play an important role in microbial survival / growth by acting as precursors for the biosynthesis of substances involved in root-mycorrhizal interactions (e.g., strigolactone), reproduction (e.g., trisporic acid in Mucor), and microbial adaptation under biological and abiotic stress (e.g., abscisic acid).

[0056] As used herein, the term “plant” refers to an organism that possesses a cell wall and chlorophyll and performs photosynthesis. It may be interpreted broadly to encompass not only multicellular eukaryotic organisms that perform photosynthesis but also unicellular organisms that perform photosynthesis, such as algae, or narrowly to refer specifically to multicellular eukaryotic organisms that perform photosynthesis. The crops to which the strain of the present application, the composition containing the same, and / or the method using the same can be applied are not particularly limited as long as they are plants that grow using carotenoids. For example, as a specific example, the plants include food crops such as rice, wheat, barley, corn, soybeans, potatoes, red beans, oats, and sorghum; vegetable crops such as napa cabbage, radish, chili pepper, strawberry, tomato, watermelon, cucumber, cabbage, Korean melon, pumpkin, green onion, onion, and carrot; and specialty crops such as ginseng, tobacco, cotton, fodder, pasture grass, sesame, sugarcane, sugar beet, perilla, peanut, rapeseed, grass, and castor oil plant. The plants may be one or more selected from the group consisting of fruit trees including apple trees, pear trees, jujube trees, peaches, kiwifruit, grapes, citrus fruits, persimmons, plums, apricots, and bananas; woody plants including pine trees, palm oil plants, and eucalyptus; flowering plants including roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, and tulips; and fodder crops including ryegrass, red clover, orchardgrass, alfalfa, tall fescue, and perennial ryegrass, but are not limited thereto. As another example, the plants may be dicotyledonous plants such as Arabidopsis thaliana, potatoes, eggplants, tobacco, chili peppers, tomatoes, burdock, crown daisies, lettuce, balloon flower roots, spinach, Swiss chard, sweet potatoes, celery, carrots, water celery, parsley, Chinese cabbage, cabbage, mustard greens, watermelons, Korean melons, cucumbers, pumpkins, gourds, strawberries, soybeans, mung beans, kidney beans, or peas; One or more selected from the group consisting of monocotyledonous plants such as rice, wheat, barley, corn, and sorghum may be used, but are not limited thereto. In one embodiment of the present invention, corn was used, but is not limited thereto.

[0057] As used in this specification, the term “plant growth” refers to the process in which a plant seed germinates to produce roots, stems, and leaves, or subsequently gradually increases in size and weight. Another term in this specification, “plant growth promotion,” includes all actions that promote (increase, enhance) the length growth, volume growth, and weight gain of a plant body, and can be interpreted to encompass all actions that increase the biomass (indicated by the bioweight or bio-viscosity) or content of the plant body as a result thereof. Specifically, the strain of this application has the advantage of excellent carotenoid production capacity, thereby enhancing the plant growth promotion effect.

[0058] Meanwhile, the Azospirillum brasilense strain deposited under accession number KCCM13420P may have increased storage stability compared to the pre-modification or wild-type Azospirillum brasilense strain (or an Azospirillum brasilense strain not containing one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8).

[0059] According to an embodiment of the present invention, the strain was confirmed to have excellent storage stability, as the number of viable cells over the storage period in the form of a liquid culture medium at room temperature was maintained at a higher level than that of existing known strains or other Azospirillum brasilense strains.

[0060] As used herein, the term “storage stability” refers to the ability of a strain to maintain viability and / or activity during storage under conditions such as room temperature or a low-oxygen environment. Here, viability means that the strain can grow and proliferate again when transferred to conditions suitable for growth. Analytical methods for evaluating the storage stability of a strain include, but are not limited to, plating and counting of colony forming units (CFU) or most probable number (MPN) analysis.

[0061] The storage stability of the strain can be tested under various conditions, with or without specific packaging, regardless of the presence or absence of a formulation. Important determinants of conditions include, but are not limited to, temperature, relative humidity, chemical composition (e.g., oxygen content of the gaseous atmosphere), and environmental radiation levels. In one embodiment of the present invention, the number of viable cells of the strain of the present invention was measured according to storage time after culture in a liquid culture medium at room temperature, but is not limited thereto.

[0062] In one embodiment, the storage stability of the Azospirillum brasilense strain deposited under accession number KCCM13420P may be 10°C to 40°C, more specifically 10°C to 40°C, 10°C to 30°C, 10°C to 27.5°C, 10°C to 25°C, 20°C to 40°C, 20°C to 30°C, 20°C to 27.5°C, 20°C to 25°C, 22.5°C to 40°C, 22.5°C to 30°C, or 22.5°C to 27.5°C, 22.5°C to 25°C, for example, at room temperature (25°C), but is not limited thereto.

[0063] In one embodiment, the storage stability of the strain is 1 to 12 months, 1 to 9 months, 1 to 24 weeks, 1 to 20 weeks, 1 to 16 weeks, 1 to 12 weeks, 1 to 8 weeks, 1 to 7 weeks, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 2 to 12 months, 2 to 9 months, 2 to 24 weeks, 2 to 20 weeks, 2 to 16 weeks, 2 to 12 weeks, 2 to 8 weeks, 2 to 7 weeks, 2 to 6 weeks, 2 to 5 weeks, 2 to 4 weeks, 2 to 3 weeks, 3 to 12 months, 3 to 9 months, 3 to 24 weeks, 3 to 20 weeks, 3 to 16 weeks, 3 to 12 weeks, 3 to 8 weeks, 3 to 7 weeks, 3 to 6 weeks, 3 to 5 weeks, 3 to 4 weeks, 3 weeks, 5 to 12 months, 5 to 9 months, 5 to 24 weeks, 5 to 20 weeks, 5 to 16 weeks, 5 to 12 weeks, 5 to 8 weeks, 5 to 7 weeks, 7 to 12 months, 7 to 9 months, 7 to 24 weeks, 7 to 20 weeks, 7 to 16 weeks, 7 to 12 weeks, 7 to 2 months, 7 to 11 weeks, 7 to 10 weeks, 7 to 8 weeks, 8 to 12 months, 8 to 9 months, 8 to 24 weeks, 8 to 20 weeks, 8 to 16 weeks, 8 to 12 weeks, 8 to 11 weeks, 8 to 10 weeks, 9 weeks to 12 months, 9 weeks to 9 months, 9 to 24 weeks, 9 to 20 weeks, 9 to 16 weeks, 9 weeks to 3 months, 9 to 12 weeks, 9 to 11 weeks, 9 to 10 weeks, or after 10 weeks, the viable cell count of the strain is 0.01% or more, 0.05% or more, 0.1% or more, 0.4% or more, 0.5% or more, 0.6% or more, 0.65% or more, 0.66% or more, 0.67% or more, 0.68% or more, 0.It may mean maintaining 69% or more, 1% or more, 5% or more, 10% or more, 20% or more, 25% or more, 30% or more, 35% or more, 37.5% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, or 46.5% or more, but is not limited thereto. There are no specific restrictions on the upper limit value, and it may be, for example, 100% or less, 90% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 48% or less, 47% or less, 40% or less, 35% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.75% or less, or 0.7% or less, but is not limited thereto. In this case, the storage start time may be 24 hours from the culture of the strain.

[0064] At this time, the strain may be cultured in a solid medium, cultured in peat, cultured in a slurry, in a cell suspension, in a dry state, in the form of a film or gel, or in a liquid culture medium; more specifically, it may be in a liquid culture medium, but is not limited thereto.

[0065]

[0066] The term “carotenoid productivity or production activity” as used in this specification refers to the function and / or ability to produce carotenoids, and may be used interchangeably with “carotenoid production capacity,” and can be confirmed by measuring the content of carotenoids contained in the culture medium or strain-derived biomass after culturing a carotenoid-producing strain.

[0067] For example, the Azospirillum brasilense strain deposited under accession number KCCM13420P of the present application may have a carotenoid production (or production capacity) that is increased by about 1.5 times or more, about 2 times or more, about 3 times or more, about 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, about 11 times or more, about 12 times or more, about 13 times or more, about 14 times or more, about 15 times or more, or about 16 times or more compared to the carotenoid production (or production capacity) of the parent strain before mutation (or Azospirillum brasilense strain not containing one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6 and SEQ ID NO. 8), but is not limited thereto. There are no specific restrictions on the upper limit, and it may be, for example, about 100 times or less, about 75 times or less, about 50 times or less, about 40 times or less, about 30 times or less, about 25 times or less, or about 20 times or less, but is not limited thereto.

[0068] The term “about” above may be interpreted to include all values ​​within a range equivalent to or similar to the value that follows, for example, including a range of ±20%, ±15%, ±10%, ±5%, ±2%, or ±1% of the value that follows, but is not limited thereto.

[0069]

[0070] Another example of the present application provides a composition comprising the strain of Azospirillum brasilense deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof.

[0071] In one embodiment, the composition may be used for carotenoid production and / or plant growth promotion.

[0072] In one embodiment, the composition may be a composition for producing carotenoids comprising the strain of Azospirillum brasilense deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof.

[0073] In one embodiment, the composition may be a plant growth-promoting composition comprising the Azospirillum brasilense strain deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof.

[0074] Another example is,

[0075] Azospirillum brasilense strain deposited under accession number KCCM13420P according to one example of the present application described above,

[0076] A culture of the above strain, and

[0077] A fertilizer composition comprising one or more selected from the group consisting of the above strain, a culture of the above strain, or a combination thereof.

[0078] In the above composition and fertilizer composition, since the storage stability of the strain and / or the culture of the strain is increased compared to the strain before modification or the wild-type Azospirillum brasilense strain, the desired effect / activity can be maintained for a longer period.

[0079] The Azospirillum brasilense strain and plant deposited under the above accession number KCCM13420P are as described above.

[0080] The uses of the above composition for carotenoid production and plant growth promotion are as previously described.

[0081] The above strain may be interpreted to include one or more selected from the group consisting of the cell itself as well as derivatives derived therefrom, such as biomass derived from the cell and / or dried product of the cell and / or biomass, and crushed product of the cell, biomass, or dried product.

[0082] The “culture of the strain” above refers to a product obtained by culturing the strain in a medium (liquid, solid, semi-solid) using a conventional culture method, and may or may not contain the strain, and there are no restrictions on its form. In one example, the “culture of the strain” above may include both the medium and the strain (culture containing the strain); culture in which the strain has been removed and only the medium has been removed (culture with strain removed; for example, if the medium is liquid, it may be expressed as “culture supernatant,” which means the culture supernatant from which the strain has been removed from the culture (culture medium)); and may include one or more selected from the group consisting of the culture containing the strain and / or culture with strain removed, or the dried product of the dried product.

[0083] The above composition may contain preservatives, stabilizers, wetting agents or emulsifiers, cryoprotectants, or excipients. The preservatives, stabilizers, or excipients may be included in the composition in an effective amount sufficient to reduce the deterioration of the composition. As used herein, the term “deterioration” may include a decrease in the number, decrease in activity, or a combination thereof of the strain of Azospirillum brasilense deposited under accession number KCCM13420P included in the composition. For example, by including an effective amount of preservatives, stabilizers, or excipients sufficient to reduce the deterioration of the composition, the strain of Azospirillum brasilense deposited under accession number KCCM13420P within the composition may survive for a longer period or maintain its activity, unlike the strain of Azospirillum brasilense deposited under accession number KCCM13420P existing in its natural state.

[0084] The above preservatives, stabilizers, or excipients may be used without limitation if they are commonly used in the art, provided that they can reduce the deterioration of the quality of the above composition. For example, the above stabilizer may be one or more selected from the group consisting of alginate, sodium alginate, casein, sodium casein, cellulose, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, pectin, gelatin, gum arabic, xanthan gum, PVP (polyvinylpyrrolidone), dextran, cyclodextrin, and starch.

[0085] The cryoprotectant may be included in the composition in an effective amount sufficient to reduce quality degradation of the composition when the composition is in a freeze-dried state. For example, unlike the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P existing in its natural state, the composition may contain an effective amount of cryoprotectant sufficient to reduce quality degradation of the composition when freeze-dried, thereby allowing the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P to survive or maintain its activity for a longer period within the freeze-dried composition.

[0086] The above cryoprotective agent may be used without limitation if it is intended to reduce the quality degradation of the composition in a freeze-dried state, and is commonly used in the art. For example, the above cryoprotective agent may be one or more selected from the group consisting of glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), glucose, trehalose, maltodextrin, dextrin, skim milk, and starch.

[0087] In the above composition, the strain and / or culture of the strain are included as an active ingredient that exerts a desired effect / activity (plant growth promotion or carotenoid production), and may be included at an effective concentration to exert the said effect / activity.

[0088] The above composition can be prepared according to methods generally applied in all fertilizer and pesticide chemistry, and preferably, the composition of the present application can be formulated in a conventional manner to be used with a plant growth-promoting agent, i.e., a conventional fertilizer, and the scope of application of the composition to a plant or the soil on which the plant is grown may vary depending on the type of plant, age, climatic conditions, etc.

[0089] The above composition may be applied to plants by conventional methods such as immersion or drenching (spraying), and may be applied to plants alone or mixed with fertilizer, but is not limited thereto.

[0090] In the above fertilizer composition, the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P or a culture thereof may be included as a main component of the fertilizer (microbial fertilizer) itself, or added to and / or mixed into the fertilizer in the form of a fertilizer additive.

[0091] The term “fertilizer” above refers collectively to nutrient substances applied to plants and / or soil to promote plant growth. Fertilizers may be manufactured by manufacturing methods commonly used in the art. For example, the fertilizer may further include excipients and / or carriers commonly used in the art and may be manufactured in a conventional fertilizer formulation known in the art. The fertilizer composition is not limited in its physical properties and may be in liquid or solid form, but is not limited thereto.

[0092] The above fertilizer may be an eco-friendly fertilizer that can replace chemical fertilizers (e.g., microbial fertilizer).

[0093] The strain of the present invention or its culture solution is 10 per weight of fertilizer 5 It is preferable to contain at least CFU / ml, but is not limited thereto.

[0094] The above fertilizer may be intended to promote plant growth.

[0095]

[0096] Another example provides a microbial preparation for promoting plant growth comprising the strain of Azospirillum brasilense deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof.

[0097] The Azospirillum brasilense strain, culture, and plant deposited under accession number KCCM13420P are as described above.

[0098] In this specification, the term “microbial preparation” refers to a type of biological preparation that, when used in soil or the like, possesses the effects described in the intended use due to the activity of the contained beneficial microorganisms and ultimately exhibits an effect that aids in plant growth. Accordingly, the microbial preparation of this application relates to a microbial preparation that promotes plant growth by providing carotenoids. The microbial preparation comprising the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P or a culture thereof can subsequently be manufactured in the form of a liquid fertilizer. In other words, the microbial preparation of this application can be formulated as a biofertilizer to overcome the limitations of environmentally friendly organic farming where the supply of chemical fertilizers is restricted.

[0099] The above-mentioned microbial preparation may be manufactured in various forms known in the art; for example, when manufactured in the form of a dry powder or a liquid fertilizer, the strain of *Azospirillum brasilense* deposited under accession number KCCM13420P (e.g., live bacteria) 10 per g of dry powder or per 1 ml of liquid 7 It may contain more than CFU / ml, for example 10 7 to 10 12 CFU / ml or 10 8 to 10 10 It may contain CFU / ml, but is not limited thereto.

[0100]

[0101] Another example provides a method for promoting plant growth, comprising the step of treating a plant and / or soil with one or more selected from the group consisting of an Azospirillum brasilense strain deposited under accession number KCCM13420P, a culture of said strain, the composition described above, and the fertilizer composition described above.

[0102] The Azospirillum brasilense strain, culture, composition, plant, and fertilizer deposited under the above accession number KCCM13420P are as described above.

[0103] The strain, culture, and composition of *Azospirillum brasilense* deposited under accession number KCCM13420P may be prepared using suitable methods known in the art. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC may be used, and may further include a purification process, and may include methods known in the art such as concentration and drying of the strain or culture thereof.

[0104] The step of treating the above-mentioned plants and / or soil may be performed by a method of treating the strain, culture, composition, and / or fertilizer to the soil and / or plants (e.g., whole plant, roots, stems, leaves, seeds, etc.). For example, the step may be performed without limitation in any conventional manner, such as spraying (spraying) the strain, culture, and / or composition onto the soil and / or plants, or drenching or applying it to the plants.

[0105]

[0106] Another example provides a plant growth-promoting use of a composition comprising the strain of Azospirillum brasilense deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof, or a use for the preparation of a plant growth-promoting composition (e.g., a fertilizer).

[0107] Another example provides a use for producing carotenoids of a composition comprising the strain of Azospirillum brasilense deposited under accession number KCCM13420P, a culture of said strain, or a combination thereof, or a use for preparing a composition for producing carotenoids.

[0108] The Azospirillum brasilense strain, culture, composition, plant, and carotenoid deposited under accession number KCCM13420P are as described above.

[0109]

[0110] Other examples provide a composition, method, product, process, or use characterized by one or more elements disclosed in the present application.

[0111] This application relates to a novel strain of the genus Azospirillum with enhanced plant growth-promoting ability and its uses. According to one aspect of this application, the novel strain of Azospirillum brasilense exhibits excellent carotenoid production ability, plant growth-promoting ability, and storage stability. Therefore, the novel strain of the present invention can be usefully employed for plant growth promotion as a substitute for chemical fertilizers.

[0112] Figure 1 shows the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) of the present application cultured by streaking on a TYG plate medium, compared with the parent strain CW01-0108 (accession number KCCM13327P).

[0113] Figure 2 shows the appearance of a culture medium in which the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) of the present application is cultured in TYG medium, compared with the parent strain CW01-0108 (accession number KCCM13327P).

[0114] FIG. 3a is a graph showing the carotenoid production capacity of the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) of the present application compared with the parent strain CW01-0108 (accession number KCCM13327P).

[0115] FIG. 3b shows a photograph of the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) of the present application after carotenoid extraction.

[0116] FIG. 4a is a graph showing the results of measuring the above-ground length of corn after treating corn with a diluted culture solution of the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) and the parent strain CW01-0108 (accession number KCCM13327P), respectively.

[0117] FIG. 4b is a graph showing the results of measuring the above-ground fresh weight of corn after treating corn with a diluted culture solution of the novel Azospirillum brasilense strain CP03-0008 (accession number KCCM13420P) and the parent strain CW01-0108 (accession number KCCM13327P), respectively.

[0118] The present application will be described in more detail below through examples. These examples are intended solely to illustrate the present application more specifically, and it will be obvious to those skilled in the art that the scope of the present application is not limited by these examples according to the gist of the application.

[0119]

[0120] Examples

[0121]

[0122] (Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight / weight) % for solid / solid, (weight / volume) % for solid / liquid, and (volume / volume) % for liquid / liquid, unless otherwise noted.)

[0123]

[0124] Example 1. Preparation of Azospirillum mutant strains using artificial mutagenesis

[0125] To obtain a variety of random microbial mutant strains, mutants were screened by constructing a mutation library using gamma irradiation, one of the physical mutation induction methods, on the Azospirillum brasilense strain.

[0126] More specifically, the wild-type Azospirillum brasilense strain CW01-0108, deposited under accession number KCCM13327P, was inoculated into a 500 mL flask containing 50 mL of TYG (Tryptone-Yeast Extract-Glucose) medium and cultured in a shaking incubator at 30°C for 24 hours to obtain a culture medium with an absorbance value of 10.08 at 562 nm. Then, using TYG medium, a culture medium with an absorbance value of 6.0 at 562 nm and a cell count of 2 x 10 9 A culture diluent was prepared by diluting to cells / ml.

[0127] Gamma irradiation was performed on the culture dilution obtained above. The gamma irradiation experiment was conducted at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute. Using a high-level gamma irradiation device of Nordion, which uses Co-60 as a source and is located at the Advanced Radiation Research Institute of the Korea Atomic Energy Research Institute, 30 mL of the prepared culture dilution was irradiated with gamma rays at 3 kGy / hr and 4 kGy / hr for 1 hour each. Subsequently, 100 μL of the gamma-irradiated stock solution was divided and plated onto TYG plate media in 30 and 20 90x15 mm Petri dishes, respectively. The Petri dishes were incubated in a static incubator at 30°C for 96 hours, and a total of approximately 24,300,400 individual colonies were obtained under gamma irradiation conditions of 3 kGy / hr or 4 kGy / hr. To ensure variant stability, all individual colonies generated in the entire petri dish were collected, suspended in a 5% DMSO solution, and then aliquoted into 1 mL portions in 1.5 mL microtubes and stored in an ultra-low temperature freezer at -80°C.

[0128] The composition of the TYG medium and TYG plate medium used in this embodiment is as follows:

[0129] <TYG 배지>

[0130] Tryptone (BD 211705) 3.0 g / L, yeast extract 3.0 g / L, glucose 3.0 g / L, K2HPO4 1.0 g / L

[0131] <TYG plate 배지>

[0132] Tryptone (BD 211705) 3.0 g / L, Yeast extract 3.0 g / L, Glucose 3.0 g / L, K2HPO4 1.0 g / L, Agar 20 g / L

[0133]

[0134] Example 2. Screening of Azospirillum mutant strains with excellent carotenoid production capacity

[0135] Example 2-1. Primary screening of Azospirillum mutant strains with excellent carotenoid production capacity

[0136] For screening carotenoid high-production variants, the sample obtained in Example 1 was removed from the ultra-low temperature freezer, and the 5% DMSO suspension thawed at room temperature was 10 using a 1X PBS solution -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 After serial dilution, 100 μL of the dilution corresponding to each factor was plated onto TYG plates and reactivated into individual colonies by incubating in a 30°C static incubator for 24 hours. Among the reactivated individual colonies, reddish colonies were selected and streaked onto TYG plates. Through this process, the CP03-0008 strain was selected (see Fig. 1).

[0137]

[0138] Example 2-2. Comparative analysis of gene sequences of selected mutant strains (Strain CP03-0008).

[0139] To identify the above-mentioned selected strain CP03-0008 Izospirillum brasilense (accession number KCCM13420P), the DNA nucleotide sequence of the 16s rRNA gene was analyzed. As a result of Multi-Locus Sequence Analysis (MLSA) comparing the nucleotide sequences of housekeeping genes—which are essential for basic cellular functions and widely distributed in bacteria—with those of atpD: ATP synthase F1 complex beta subunit (SEQN 10), dnaK: Chaperone protein DnaK (SEQN 11), fumC: Fumarate hydratase (SEQN 12), gyrA: DNA gyrase subunit A (SEQN 13), gyrB: DNA gyrase subunit B (SEQN 14), and recA: Recombinase A (SEQN 15), rpoA: RNA polymerase alpha subunit (sequence number 16), rpoB: RNA polymerase beta subunit (sequence number 17), rpoD: RNA polymerase sigma-70 factor (sequence number 18), trpB: Tryptophan synthase beta chain sequence (sequence number 19) matched that of the parent strain (CW01-0108; accession number KCCM13327P), and accordingly, it was confirmed that the strain is the same Azospirillum brasilense strain as the parent strain.

[0140] In addition, to analyze the DNA sequences of genes essential for microbial growth, whole genome sequencing (WGS) was performed on strain CP03-0008 and the parent strain (CW01-0108; accession number KCCM13327P).

[0141] Some of the variants identified in WGS were selected and analyzed, and it was confirmed that the CP03-0008 strain contains specific nucleotide variants in four genes (glutamate 5-kinase, lysylphosphatidylglycerol synthase, glycerol kinase, PTS fructose-like transporter subunit IIB) compared to the wild type. These variants are sequence changes that are not present in the wild type and can be used as markers to define the genetic distinctiveness of the CP03-0008 variant.

[0142] The specific details of the variants identified in the CP03-0008 strain are as shown in Table 1 below. The following sequence numbers 1 to 8 are based on the coding DNA sequence (cDNA) of each gene.

[0143] No. Protein Name NCBIGene ID Wild type (CW01-0108) Nucleotide sequence variant (CP03-0008) Nucleotide variation details in nucleotide sequence variant 1 glutamate 5-kinase 56452319 Sequence No. 1 Sequence No. 2 Substitution of the 651st base (Cytosine, C) of Sequence No. 1 with T (Thymine); and CC insertion between the 796th base (Cytosine, C) and the 797th base (Thymine, T) of Sequence No. 1 2 lysylphosphatidylglycerol synthase 56451010 Sequence No. 3 Sequence No. 4 Deletion of the 1605th base (Cytosine, C) of Sequence No. 3; and insertion of C between the 1607th base (Guanine, G) and the 1608th base (Cytosine, C) of SEQ ID NO. 3 3 glycerol kinase 56450763 SEQ ID NO. 5 SEQ ID NO. 6 Substitution of the 1138th base (Guanine, G) of SEQ ID NO. 5 with A (Adenine) 4 PTS fructose-like transporter subunit IIB 56453471 SEQ ID NO. 7 SEQ ID NO. 8 Substitution of the 1840th base (Thymine, T) of SEQ ID NO. 7 with C

[0144] CP03-0008, selected as described above, was deposited with the Korean Culture Collection of Microorganisms, an international depositary institution under the Treaty of Budapest, on November 16, 2023, and was assigned accession number KCCM13420P.

[0145]

[0146] Example 2-3. Reconfirmation of carotenoid production capacity of a selected mutant strain (Strain CP03-0008)

[0147] A single colony of the CP03-0008 strain, confirmed as a reddish colony, was inoculated into 10 ml of TYG and cultured at 30°C for 24 hours. 2 ml of the culture medium was inoculated into 50 ml of TYG medium in a 300 ml flask and cultured at 30°C for 24 hours. In the case of the culture medium, it was confirmed that the culture medium of the CP03-0008 strain was redder than that of the parent strain, CW01-0108 (Fig. 2).

[0148] The composition of the 1X PBS buffer used in this embodiment is as follows:

[0149] <1X PBS buffer>

[0150] Potassium Phosphate, Monobasic, Anhydrous (KH2PO4) 144 mg / L, Sodium Chloride (NaCl) 9000 mg / L, Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4) 421.62 mg / L

[0151]

[0152] Example 3. Test of carotenoid production ability of strain CP03-0008

[0153] 50 ml of the culture medium of the CP03-0008 strain isolated in Example 2 above, cultured in TYG medium, was centrifuged at 13,000 rpm for 3 minutes, and the supernatant was removed. 50 ml of distilled water was added to the cell pellet from which the supernatant had been removed and sufficient resuspension was achieved. The cell pellet was then washed by centrifuging again at 13,000 rpm for 3 minutes and removing the supernatant. Another 50 ml of distilled water was added to resuspension, and 1 ml of the resuspension was placed in a 1.5 ml tube and centrifuged at 13,000 rpm for 3 minutes, after which the supernatant was removed. Beads (0.3 mm size) with a volume of approximately 300 μL were added to the obtained cell pellet, and 500 μL of DMSO was added to sufficiently resuspense the pellet.

[0154] A 1.5 ml tube containing the above-mentioned resuspended solution was placed in an Eppendorf ThermoMixer® C and incubated at 55°C at 2000 rpm for 10 minutes to lyse the cells in the tube. After cell lysis, 500 μL of acetone containing 0.04% (w / v) BHT (Butylated hydroxytoluene) was added and vortexed sufficiently. Subsequently, the 1.5 ml tube was placed back into the Eppendorf ThermoMixer® C and mixed well at 45°C at 2000 rpm for 15 minutes to extract carotenoids. Since carotenoids can be easily degraded by light, the Thermomixer lid was tightly closed to block light during the extraction process. After extraction, the resuspended solution was centrifuged at 13,000 rpm for 3 minutes, and 500 μL of the supernatant was placed in a brown vial (photodegradation resistant). Afterwards, carotenoids were analyzed by measuring wavelength values ​​from 400 nm to 700 nm using a UV / VIS (Ultraviolet visible) spectrophotometer (Evolution 201, Thermo scientific).

[0155] The results obtained above are shown in Fig. 3a. When comparing the values ​​at 500 nm, which is the wavelength range showing the maximum wavelength value shown in Fig. 3a, it was confirmed that the variant strain CP03-0008 of the present application was 0.41, which is about 16 times higher than the parent strain (control strain) CW01-0108, which was 0.24.

[0156] In addition, a photograph of the re-suspended solution after extraction is shown in Fig. 3b. As shown in Fig. 3b, the culture medium of the variant strain CP03-0008 of the present application was orange compared to the culture medium of the parent strain (accession number KCCM13327P), indicating that the carotenoid production was significantly higher.

[0157] As such, it was confirmed that the variant strain CP03-0008 (accession number KCCM13420P) of the present application possesses significantly increased carotenoid production activity compared to the parent strain CW01-0108 (accession number KCCM13327P).

[0158]

[0159] Example 4. Test of plant growth-promoting effect of CP03-0008 strain

[0160] In order to confirm the plant growth-promoting effect of the CP03-0008 strain, which was confirmed to have improved carotenoid production capacity compared to the parent strain (CW01-0108) in Example 3 above, the parent strain and the CP03-0008 strain were applied to corn by drenching and cultivated, and the degree of corn growth was measured.

[0161] Specifically, the corn seeds used in this embodiment were selected as the Happy Chal No. 3 variety (seller: Danong). The corn seeds were sterilized with 70% (v / v) ethanol and then washed several times with sterile distilled water. Horticultural potting soil and vermiculite were mixed in a 1:2 ratio and placed in 2L horticultural pots measuring 17cm × 16.5cm × 11cm. The prepared corn seeds were then sown one per pot and cultivated. After culturing the mother strain and the CP03-0008 strain in TYG medium for 24 hours, the viable cell count of the culture solution was 5 x 10⁶ using sterile distilled water. 9 The solution was diluted to a concentration of cfu / ml. The diluted culture solution was further diluted 500-fold in sterile distilled water, and 2 ml was applied as a drench to each pot where corn had germinated. At least 10 corn plants treated with the strain were randomly placed in each treatment group and cultivated. The cultivation period was 40 days, during which no pesticides or fertilizers other than the strain treatment were applied. Soil moisture was supplied via groundwater whenever necessary. Results were analyzed by harvesting all plants from each treatment group in the 5th week of cultivation and measuring the length and weight of the above-ground parts.

[0162] The results obtained above are shown in Figures 4a (average above-ground length; cm / number of plants) and 4b (average above-ground fresh weight; g / number of plants). As shown in Figures 4a and 4b, the above-ground length of the corn treated with the CP03-0008 strain increased by approximately 2.5% compared to the parent strain (CW01-0108) treatment group, and the above-ground fresh weight of the corn treated with the CP03-0008 strain increased by approximately 6.0% compared to the parent strain (CW01-0108) treatment group. The above results demonstrate that the CP03-0008 strain has a superior plant growth-promoting effect compared to the parent strain.

[0163]

[0164] Example 5. Storage stability test of liquid culture medium of strain CP03-0008

[0165] Since microbial fertilizer products are primarily produced in liquid form, higher storage stability of the strain in liquid form at room temperature is advantageous for product development. Therefore, the storage stability of the CP03-0008 strain in a liquid culture state for 6 months (24 weeks) was analyzed as follows.

[0166] After inoculating a single colony of the CP03-0008 strain isolated in Example 2 above into 10 ml of TYG, the culture was incubated in 50 ml of TYG medium in a 300 ml flask for 24 hours. The liquid culture was stored at 25°C for 24 weeks, during which time the liquid culture was retrieved every 4 weeks to measure the number of viable bacteria (survival count) by plating it onto a TYG plate. The preserved liquid culture was [prepared] using 1X PBS buffer solution 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 10 after serial dilution -4 , 10 -5 , 10 -6 Three plates of 100 µl each of the dilution solution corresponding to the dilution factor were plated onto TYG plates and incubated in a 30°C static incubator for 24 hours. Plates with between 20 and 200 colonies were selected and counted, and the viable cell count was determined by calculating the cfu value per mL. The results of the viable cell count measurement are shown in Table 1.

[0167] The composition of the 1X PBS buffer and TYG plate medium used in this example is as follows:

[0168] <1X PBS buffer>

[0169] Potassium Phosphate, Monobasic, Anhydrous (KH2PO4) 144 mg / L, Sodium Chloride (NaCl) 9000 mg / L, Sodium Phosphate, Dibasic, Anhydrous (Na2HPO4) 421.62 mg / L

[0170] <TYG plate 배지>

[0171] Tryptone (BD 211705) 3.0 g / L, Yeast extract 3.0 g / L, Glucose 3.0 g / L, K2HPO4 1.0 g / L, Agar 20 g / L

[0172] Culture Conditions Start 4 weeks 8 weeks 12 weeks 16 weeks 24 weeks CW01-0108 (Parent strain) 7.85 x 10⁻⁶ 9 1.66x10 9 8.50x10 8 3.80x10 8 4.00x10 8 3.50x10 8 CP03-00088.40x10 9 3.18x10 9 1.57x10 9 6.75x10 8 6.70x10 8 4.60x10 8 CP03-00087.55x10 9 3.30x10 9 2.08x10 9 1.00E+099.15x10 8 6.55x10 8

[0173] As shown in Table 2, for the parent strain CW01-0108, the viable cell count of the liquid culture after 24 hours was 7.85 x 10⁶ 9 After storage at 25°C for 24 weeks at cfu / ml, the viable cell count was 3.50 x 10⁶ 8 It was found to be cfu / ml. On the other hand, the viable cell count of the liquid culture of strain CP03-0008 after 24 hours was 8.44 x 10⁶ 9 cfu / ml, 7.55x10 9It was cfu / ml, and after storage at 25°C for 24 weeks, the viable cell count was 4.6 x 10⁶ 8 cfu / ml, 6.55x10 8 It was confirmed that the storage stability of the CP03-0008 strain in cfu / ml was significantly superior compared to the parent strain CW01-0108.

[0174] From the foregoing description, those skilled in the art to which this application pertains will understand that this application may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of this application should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

[0175]

[0176] [Consignment Number]

[0177] Depository Name: Korean Culture Collection Center (KCCM)

[0178] Trustee Number: KCCM13420P

[0179] Date of Trust: 20231116

[0180]

Claims

1. Azospirillum brasilense strain deposited under accession number KCCM13420P.

2. In paragraph 1, the strain is, Carotenoid production capacity, Plant growth-promoting activity, Storage stability, or Their combination A strain that possesses 3. A strain according to paragraph 2, wherein the carotenoid production capacity, plant growth-promoting activity, storage stability, or a combination thereof is increased compared to the strain before modification or the wild-type Azospirillum brasilense strain.

4. The strain according to claim 1, wherein the strain comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, and SEQ ID NO. 8 in its genomic DNA.

5. A composition comprising the strain of claim 1, a culture of said strain, or a combination thereof.

6. In paragraph 5, the composition is used for carotenoid production, plant growth promotion, or a combination thereof.

7. The strain of paragraph 1, A culture of the above strain, and A fertilizer composition comprising one or more selected from the group consisting of the strain, a culture of the strain, or a combination thereof.

8. The strain of paragraph 1, Culture of the above strain, A composition comprising the above strain, a culture of the above strain, or a combination thereof, and A fertilizer composition comprising one or more selected from the group consisting of the strain, a culture of the strain, and the composition. A method for cultivating plants, comprising the step of treating a plant, soil, or both of one or more selected from a group consisting of 9. The strain of paragraph 1, Culture of the above strain, A composition comprising the above strain, a culture of the above strain, or a combination thereof, and A fertilizer composition comprising one or more selected from the group consisting of the strain, a culture of the strain, and the composition. A method for promoting plant growth comprising the step of treating a plant, soil, or both of one or more selected from the group consisting of

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