Novel burkholderia strain and use thereof

Abstract

The present invention relates to a novel a Burkholderia cepacia JK6 strain (KCTC 16020BP) identified from the roots of Robinia pseudoacacia L. The Burkholderia cepacia JK6 strain according to the present invention exhibits antibacterial activity against a Fusarium oxysporum strain. In addition, the Burkholderia cepacia JK6 strain exhibits phosphate solubilization ability and siderophore production ability. Therefore, the Burkholderia cepacia JK6 strain according to the present invention can be effectively used, in the future, as an antagonistic microorganism against Fusarium oxysporum strains causing various plant fungal diseases including banana panama disease, and as a microorganism for promoting plant growth.

Description

Novel Bulkholderia strain and its uses

[0001] The present invention relates to a novel Bulkholderia strain and a composition for controlling plant pathogenic fungi and a composition for promoting plant growth containing the same as an active ingredient.

[0002] Plant pathogenic fungi cause damage to many crops, significantly reducing their productivity. To protect crops from these fungi, chemical pesticides have been consistently used worldwide. However, the misuse and overuse of chemical pesticides have raised various issues, including soil, water, and agricultural product contamination, toxicity, and ecosystem disruption. Consequently, research aimed at developing biopesticides to replace these chemical pesticides is actively underway globally. In line with this, numerous microorganisms that engage in beneficial interactions with plants are being discovered, and efforts to utilize them as biofertilizers or biocontrol agents are continuing.

[0003] As described above, microorganisms that exert beneficial effects on plant growth in the rhizosphere are called plant growth-promoting rhizobacteria (PGPR). Numerous studies have reported that these microorganisms enhance plant growth and vitality, and increase yields. These microorganisms are known to aid plant growth by decomposing organic matter in the soil or by converting nutrients that are difficult for plants to utilize into forms that are easily absorbed and utilized. Furthermore, they protect plants by exhibiting antagonistic effects against harmful bacteria that cause plant diseases (Olanrewaju et al. (2017) Mechanisms of action of plant growth promoting bacteria. World J Microbiol Biotechnol 33:197).

[0004] Among these, there are products developed to utilize bacteria that exhibit antifungal activity and promote plant growth in agriculture, but most are limited to Bacillus, Pseudomonas, and Streptomyces.

[0005] In Korea, although Enterobacter, which promotes plant growth, was disclosed in Korean Registered Patent No. 10-1611537, the development of effective, low-toxicity antifungal agents for clinical use and domestic microbial pesticides remains insufficient. Therefore, there is a need for more active research on effective microorganisms that can be used as biopesticides to control plant pathogenic fungi.

[0006] Accordingly, the inventors conducted research to secure a novel microorganism that promotes plant growth while exhibiting antifungal activity against plant pathogenic fungi, and completed the present invention by confirming that the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP), isolated from the roots of acacia trees, inhibits the growth of the Fusarium oxysporum strain.

[0007] To achieve the above objective, one aspect of the present invention provides a Burkholderia cepacia JK6 strain (accession number KCTC 16020BP) and a culture thereof.

[0008] Another aspect of the present invention provides a composition for controlling plant pathogens or a composition for promoting plant growth, comprising the above-mentioned Burkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient.

[0009] Another aspect of the present invention provides a kit for controlling plant pathogens or a kit for promoting plant growth comprising the above-mentioned Burkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof.

[0010] The Bulkholderia cepacia JK6 strain (KCTC 16020BP) according to the present invention exhibited antibacterial activity against Fusarium oxysporum strains. In addition, it was confirmed that the strain possesses phosphate solubilizing ability and siderophore production ability. Therefore, the Bulkholderia cepacia JK6 strain according to the present invention can be usefully utilized in the future as an antagonistic microorganism against Fusarium oxysporum strains, which cause various plant fungal diseases including banana Panama disease, and as a microorganism for promoting plant growth.

[0011] Figure 1 is a diagram showing the results of culturing (2 days) microorganisms isolated from the roots of an acacia tree.

[0012] Figure 2 is a diagram showing the results of confirming the antimicrobial activity of a microorganism isolated from the roots of a black locust tree against Fusarium oxysporum f.sp. cubense tropical race 4 (FocTR4).

[0013] Figures 3a and 3b show the results of blast analysis of the 16s rRNA gene sequence of Gj-5 among the 13 microorganisms isolated from the roots of acacia trees.

[0014] Figures 4a and 4b show the results of blast analysis of the 16s rRNA gene sequence of Gj-8 among the 13 types of microorganisms isolated from the roots of acacia trees.

[0015] Figures 5a and 5b show the results of blast analysis of the 16s rRNA gene sequences of 11 types of microorganisms (13 types) isolated from the roots of acacia trees.

[0016] Figures 6a to 6d show the results of multiple sequence alignment analysis of the 16S rRNA gene sequences of 11 microorganisms predicted to be Burkholderia cepacia species among microorganisms isolated from the roots of black locust trees.

[0017] Figure 7 is a diagram showing phylogenetic groups classified based on 16s rRNA gene sequences for 11 species of microorganisms predicted to be Bulkholderia cepacia species among microorganisms isolated from the roots of black locust trees.

[0018] Figure 8 is a diagram showing the results of comparing the colony colors of Bulkholderia cepacia JK6 strain (KCTC 16020BP) and Bulkholderia cepacia JK11 strain (KCTC 14199BP), which are strains of the same genus.

[0019] Figure 9 is a figure showing the results of comparing the motility and antibacterial activity against Escherichia coli of the Bulkholderia cepacia JK6 strain (KCTC 16020BP) and the genus Bulkholderia cepacia JK11 strain (KCTC 14199BP).

[0020] Figure 10 is a diagram showing the phylogenetic tree of the Bulkholderia cepacia JK6 strain and the Bulkholderia cepacia JK11 strain.

[0021] Figure 11 is a figure showing the results of confirming the antibacterial activity of the Bulkholderia cepacia JK6 strain according to the present invention against the F. oxysporum f.sp. lactucae strain.

[0022] Figure 12 is a figure showing the results of confirming the antibacterial activity of the Bulkholderia cepacia JK6 strain according to the present invention against the F. oxysporum f.sp. lagenariae strain.

[0023] Figure 13 is a figure showing the results of confirming the antibacterial activity of the Bulkholderia cepacia JK6 strain according to the present invention against the F. oxysporum f.sp. cucumerinum strain.

[0024] Figure 14 is a figure showing the results of confirming the antibacterial activity of the Bulkholderia cepacia JK6 strain according to the present invention against the F. oxysporum f.sp. cubense strain.

[0025] Figure 15 is a figure showing the results of confirming the antibacterial activity of the Bulkholderia cepacia JK6 strain according to the present invention against seven types of Fusarium oxysporum strains (F. oxysporum).

[0026] Figure 16 is a diagram showing the results of confirming the phosphate solubilization ability of the Bulkholderia cepacia JK6 strain according to the present invention.

[0027] Figure 17 is a diagram showing the results of confirming the siderophore production ability of the Bulkholderia cepacia JK6 strain according to the present invention.

[0028] Figure 18 is a diagram showing the results of confirming the nitrogen fixation ability of the Bulkholderia cepacia JK6 strain according to the present invention.

[0029] Bulkholderia cepacia JK6 strain

[0030] One aspect of the present invention provides a Burkholderia cepacia JK6 strain (accession number KCTC 16020BP).

[0031] The above-mentioned Bulkholderia cepacia JK6 strain is a novel strain deposited at the National Institute of Biotechnology and Bioengineering’s Center for Biological Resources on August 27, 2024, under accession number KCTC 16020BP. The above-mentioned JK6 strain is a fungus isolated from the roots of a black locust tree collected from Mt. Geonji (coordinates: 35.86N 127.13E), Deokjin-gu, Jeonju-si, Jeonbuk, South Korea.

[0032] As used herein, the term "Burkholderia cepacia" refers to a Gram-negative bacterium that is widely distributed in nature and commonly found. Although it exists alongside humans in daily life, it is an opportunistic pathogen that infects humans under specific circumstances. In particular, it has been reported to cause cystic fibrosis in conjunction with Pseudomonas aeruginosa. Additionally, it acts as an antagonist against plant pests, is known as a plant growth-promoting rhizosphere microorganism (PGPR), and can decompose harmful substances, thereby playing an important role in increasing agricultural productivity.

[0033] In the present invention, the Bulkholderia cepacia JK6 strain may include the 16s rRNA gene sequence of SEQ ID NO. 1. Additionally, the strain may include three chromosomes.

[0034] The above Bulkholderia cepacia JK6 strain can be obtained by isolating and identifying it through culture on selective agar media, and the strain can exhibit antifungal activity.

[0035] Specifically, the above-mentioned Bulkholderia cepacia JK6 strain can inhibit the growth of Fusarium oxysporum strains, but is not limited thereto. More specifically, the above-mentioned Bulkholderia cepacia JK6 strain can inhibit the growth of any one or more plant pathogens selected from the group consisting of F. oxysporum f.sp. cubense, F. oxysporum f.sp. radicis-lycopersici, F. oxysporum f.sp. gladioli, F. oxysporum f.sp. lactucae, F. oxysporum f.sp. cucumerinum, and F. oxysporum f.sp. melonis, but is not limited thereto. Accordingly, the above-mentioned Bulkholderia cepacia JK6 strain can be used as an antifungal agent against Fusarium oxysporum strains.

[0036] As used herein, the term "Fusarium oxysporum" refers to a root-infecting fungal pathogen comprising over 120 specific fungi that infect more than 100 different plant species. The pathogen initiates its infection cycle during the biotrophic stage, infects the plant through the roots, moves to the plant's xylem, and accumulates fungal mycelia and defense-related compounds within the plant body in the xylem, thereby causing wilting of the xylem. As the infection progresses, F. oxysporum is known to transform into a necrotic pathogen, causing leaf necrosis and lesions that eventually lead to the death of the plant. In the case of cucurbit crops, the diseased xylem ruptures, exhibiting a phenomenon where the stem splits vertically (vine splitting).

[0037] The above "F. oxysporum f.sp. cubense (Foc)" strain is a pathogenic fungus that causes banana Panama disease, also known as wilt disease (or fusarium wilt). It is currently classified into four races, three of which (races 1, 2, and 4) are known to cause wilt disease. In particular, race 4 (FocTR4) is known to cause wilt disease regardless of banana variety, but no fungicide for it is known yet.

[0038] The above strain "F. oxysporum f.sp. radicis-lycopersici(FUSARL)" is a pathogenic fungus that causes root rot in tomatoes. Upon infection, dark brown lesions appear at the soil-stem junction and browning occurs in the xylem; it causes leaves to wilt and die, or wilts and dark brown discoloration at the base of the stems of mature plants. Thirty-seven plant species, including 10 species from the Solanaceae, Leguminosae, Cucurbitaceae, and Chenopodiaceae families, are susceptible to the above strain.

[0039] The above strain "F. oxysporum f.sp. gladioli (FUSAGL)" is a pathogenic fungus that causes bulb rot and yellow disease. Infected plants turn yellow, stop growing, discolor flowers, and destroy bulbs.

[0040] The aforementioned "F. oxysporum f.sp. lactucae (FUSALC)" strain is a soil-borne pathogenic fungus that causes lettuce wilt disease. It is currently classified into four races, most of which belong to race 4. It causes host-specific infection; upon infection, the leaves turn yellow and wilt, and brown or black streaks appear in the vascular tissue of the stem. It has been reported that the above strain also infects melons, tomatoes, watermelons, cotton, broccoli, cauliflower, and spinach roots.

[0041] The above "F. oxysporum f.sp. cucumerinum (FUSACC)" strain is a soil-borne pathogenic fungus that causes cucumber wilt. The strain is currently classified into four races.

[0042] The above "F. oxysporum f.sp. melonis (FUSAM)" strain is a soil-borne pathogenic fungus that causes melon wilt disease. It is currently classified into four races.

[0043] In addition, the Bulkholderia cepacia JK6 strain of the present invention may have i) the ability to decompose insoluble phosphate (phosphate solubilization ability) and ii) the ability to produce siderophores that capture iron. Therefore, the Bulkholderia cepacia JK6 strain can be used as a plant growth-promoting rhizosphere microorganism (PGPR) to promote plant growth.

[0044] As used in this specification, the term "plant growth-promoting rhizosphere microorganisms (PGPR)" refers to microorganisms that inhabit the area around plant roots and promote plant growth. These microorganisms are known to aid in growth by producing indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC), as well as by supplying minerals through nitrogen fixation, siderophore production, and solubilization of phosphate, calcium, and zinc. Known PGPRs to date include Azotobacter, Azospirillus, Bacillus, Bulkholeria, Enterobacter, Klebsiella, Pantoea, Pseudomonas, and Rhizobium.

[0045] As used herein, the term "siderophore" refers to a compound capable of solubilizing insoluble minerals in the soil. In particular, siderophores synthesized and secreted by microorganisms in the soil bind to iron (Fe), a mineral found in the soil, allowing it to be absorbed by plant roots and utilized for plant growth. Since iron plays a role in promoting enzymatic reactions, oxygen metabolism, the electron transport chain, and DNA and RNA synthesis within the plant body, the absorption of iron into the plant body by siderophores promotes plant growth. Furthermore, it inhibits the growth of plant pathogens by competitively inhibiting their absorption of iron ions.

[0046] Therefore, the above-mentioned Bulkholderia cepacia JK6 strain can promote plant growth as a plant growth-promoting rhizosphere microorganism (PGPR).

[0047] Bulkholderia cepacia JK6 strain culture

[0048] Another aspect of the present invention provides a culture of the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP).

[0049] As used in this specification, the term "culture" refers to a product obtained by inoculating a strain into a culture medium and culturing it for a certain period of time; it may be used interchangeably with "culture solution" and may refer to a substance containing all materials contained within the culture medium in which the strain was cultured. For example, it may refer to a substance containing metabolites or secretions that are products of strain culture, or a lysate thereof, and the strain itself may also be contained within the culture solution. Furthermore, it may include a filtrate (filtrate or supernatant after centrifugation) obtained by filtering or centrifuging the culture solution to remove the strain, and a concentrate obtained by concentrating the culture solution, filtrate, cell lysate, etc.

[0050] In addition, the above culture medium can be cultured according to methods commonly used in the industry, preferably using nutrient broth (NB), but is not limited thereto.

[0051] Composition for controlling plant pathogenic fungi

[0052] Another aspect of the present invention provides a composition for controlling plant pathogenic fungi comprising a Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient. In this case, the Bulkholderia cepacia JK6 strain and the culture thereof are the same as those described above.

[0053] The term "control" above refers to preventing plants, such as crops, from being damaged by pests and diseases, eliminating pathogens that have occurred, or preventing the spread of pathogens.

[0054] The Bulkholderia cepacia JK6 strain of the present invention or a culture thereof can inhibit the growth of fungi that can cause plant diseases.

[0055] The term "plant disease" above refers to a disease occurring in a plant, signifying an abnormality in the plant's natural form and physiological functions. The cause of the disease may be pathogenic factors, nutrient deficiencies in the soil, weather conditions, toxic gases, etc. The pathogen may be a fungal or bacterial pathogen, but is not limited thereto.

[0056] Specifically, the above-mentioned Bulkholderia cepacia JK6 strain or a culture thereof may be included as an active ingredient in a composition for controlling plant pathogenic fungi and act as a fungicide against plant pathogenic fungi. More specifically, the fungal pathogen may be a Fusarium oxysporum strain, but is not limited thereto. The Fusarium oxysporum strain is the same as described above.

[0057] A composition for controlling plant pathogenic fungi containing the above-mentioned Bulkholderia cepacia JK6 strain or a culture thereof as an active ingredient can be applied to plant seeds, leaves, stems, roots, or surrounding soil to control plant pathogenic fungi.

[0058] The above-mentioned control composition may include, without limitation, any preparations necessary for controlling plant diseases, such as pesticide preparations, microbial preparations, fertilizers, and control agents.

[0059] With respect to the total weight of the above composition, the active ingredient, the Bulkholderia cepacia JK6 strain or a culture thereof, may be included at an effective concentration, for example, about 10 3 cfu (colony forming unit) to about 10 8 It may contain a content of CFU / g or a culture having an equivalent number of viable bacteria.

[0060] In the present invention, the composition may be prepared in a powder or liquid formulation. In the case of a powder formulation, it may be the cell itself of the strain of the present invention, or a powder formed by adding and mixing additives such as an extender or a cryoprotectant and freeze-drying it; in the case of a liquid formulation, it may be the culture of the strain of the present invention itself, a diluted solution obtained by diluting the culture with a solvent, or a suspension obtained by separating the cell from the culture and suspending it in another solvent.

[0061] All methods commonly used in the field can be applied to formulate the above composition as an agricultural composition. Specifically, for the purpose of stable formulation of microorganisms, it may be formulated into wettable powders, granules, powders, emulsions, sprays, fogging agents, capsules, and gels, but is not limited thereto. In addition, when the strain or culture solution thereof, which is the active ingredient, is supplied separately for long-term preservation, the strain or culture solution thereof may be used by preserving it in a glycerol storage solution at -70°C or lower, or by freeze-drying it at -20°C to -80°C.

[0062] In addition to the active ingredient, the above composition for controlling plant pathogenic fungi may further include substances commonly used for controlling plant diseases. It may further include pharmaceutically acceptable solid carriers, liquid carriers, liquid diluents, liquefied gaseous diluents, solid diluents, or other adjuvants such as surfactants, disintegrants, extenders, and nutrients as excipients. At this time, as the surfactant, one or more selected from the group consisting of polycarboxylate, sodium lignosulfonate, calcium lignosulfonate, sodium dialkyl sulfosuccinate, sodium alkyl aryl sulfonate, polyoxyethylene alkyl phenyl ether, sodium tripolyphosphate, polyoxyethylene alkyl aryl phosphoric ester, polyoxyethylene alkyl aryl ether, polyoxyethylene alkyl aryl polymer, polyoxyalkylone alkyl phenyl ether, polyoxyethylene nonyl phenyl ether, sodium sulfonate naphthalene formaldehyde, Triton 100, and Tween 80 may be used.

[0063] As the above-mentioned filler and nutrient, one or more selected from the group consisting of soybean flour, rice, wheat, red clay, diatomaceous earth, dextrin, glucose, and starch may be used, and as the disintegrant, one or more selected from the group consisting of bentonite, talc, dialite, kaolin, and calcium carbonate may be used.

[0064] In addition, the above composition may be prepared by further adding one or more selected from the group consisting of a surfactant, an inert carrier, a preservative, a wetting agent, a feed promoter, an attractant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protective agent, a buffer, and a flow agent to the microorganism.

[0065] Control methods for plant pathogenic fungi

[0066] Another aspect of the present invention provides a method for controlling plant pathogenic fungi comprising the step of treating a plant or soil adjacent to a plant with a composition for controlling plant pathogenic fungi comprising the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient. In this case, the Bulkholderia cepacia JK6 strain, the culture thereof, and the composition for controlling plant pathogenic fungi are the same as those described above.

[0067] The above treatment method can be carried out by methods generally used, namely spraying (e.g., spraying, misting, atomizing, powder spraying, granule spraying, surface application, constant application, etc.), soil application (e.g., incorporation, drenching, etc.), surface application (e.g., coating, smearing, covering, etc.), immersion, poisoning, fumigation, etc.

[0068] As one embodiment of the above control method, the composition may be directly sprayed or immersed on plant seeds, leaves, stems, roots, or soil adjacent thereto. More specifically, the method for controlling plant pathogenic fungi may involve directly soaking or spraying the control composition, adsorbing the strain or its culture solution onto vermiculite, diatomite, or perlite and spreading it, or spraying the strain culture solution or the adsorbed Bulkholderia cepacia JK6 strain before tilling the cultivated land and then tilling and mixing it. In this case, the amount of the composition used may be appropriately determined according to the formulation, damage conditions, application method, application location, etc.

[0069] For example, the effective amount of microorganisms contained in the composition treated according to the above method is approximately 1 × 10 per cultivated land area (㎡). 3 to about 1×10 8It may include a culture having a number of microorganisms in cfu (colony forming unit) or an equivalent number of viable bacteria. In addition, the effective amount of microorganisms contained in the composition treated by spraying in the above method is approximately 1 × 10⁶ per ml. 3 to about 1×10 8 It may contain a culture having a microbial count of cfu or an equivalent number of viable bacteria, and the effective amount of microorganisms contained in the composition treated by immersion is approximately 1 × 10⁶ per ml. 3 to about 1×10 8 It may include a culture containing a number of microorganisms of cfu or an equivalent number of viable bacteria.

[0070] composition for promoting plant growth

[0071] Another aspect of the present invention provides a composition for promoting plant growth comprising the above-mentioned Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient. In this case, the composition for promoting plant growth may be used as a microbial preparation.

[0072] As used herein, the term “plant growth” may mean, but is not limited to, increasing leaf fresh weight, root fresh weight, number of leaves, number of lateral roots, root length, or chlorophyll content.

[0073] As used herein, the term "microbial preparation" refers to a biological preparation used to effectively promote plant growth by directly utilizing the microorganism itself or by utilizing a preparation containing a microorganism.

[0074] In one embodiment of the present invention, the Bulkholderia cepacia JK6 strain exhibited excellent phosphate solubilization ability and siderophore production ability for degrading insoluble phosphate (Figs. 16 and 17). The phosphate solubilization ability and siderophore production ability are characteristics of a plant growth-promoting rhizosphere microorganism (PGPR), and the Bulkholderia cepacia JK6 strain can promote plant growth as a plant growth-promoting rhizosphere microorganism (PGPR). Therefore, the Bulkholderia cepacia JK6 strain or a culture thereof can be usefully used as a plant growth promoter.

[0075] Based on the total weight of the above composition, the active ingredient, the Bulkholderia cepacia JK6 strain or a culture thereof, is included at an effective concentration, approximately 10 3 cfu (colony forming unit) to about 10 8 It may contain a content of CFU / g or a culture having an equivalent number of viable bacteria.

[0076] In addition, the target plants for growth promotion in the present invention may be fruit crops such as grapes, apples, and pears, grass crops such as rice, wheat, and barley, vegetable crops such as chili peppers and tomatoes, and flower crops such as lilies, chrysanthemums, and roses, but are not limited thereto.

[0077] The plant growth-promoting composition according to the present invention may additionally include, in addition to the Bulkholderia cepacia JK6 strain or its culture, substances necessary for formulation, such as substances for maintaining the activity of said strain (e.g., buffers, etc.), substances for preventing contamination by other microorganisms (e.g., antibiotics, etc.), and preservatives for storage (e.g., glycerol for freezing storage), as needed, and may additionally include other substances necessary for use in promoting plant growth. The composition may be easily selected by a person skilled in the art.

[0078] In addition, the above plant growth-promoting composition may further include commonly used excipients and other auxiliary agents (e.g., surfactants such as dispersants or foaming agents, disintegrants, fillers, and nutrients).

[0079] In the present invention, the composition may be prepared in a powder or liquid formulation. In the case of a powder formulation, it may be the cell itself of the strain of the present invention, or a powder formed by adding and mixing additives such as an extender or a cryoprotectant and freeze-drying it; in the case of a liquid formulation, it may be the culture of the strain of the present invention itself, a diluted solution obtained by diluting the culture with a solvent, or a suspension obtained by separating the cell from the culture and suspending it in another solvent.

[0080] Methods to promote plant growth

[0081] Another aspect of the present invention provides a method for promoting plant growth comprising the step of treating a plant or soil adjacent to a plant with a composition for promoting plant growth that includes the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient. Specifically, plant growth can be promoted by treating the plant seeds, leaves, stems, roots, or soil adjacent thereto with the plant growth-promoting composition according to the present invention. The Bulkholderia cepacia JK6 strain, the culture thereof, and the composition for promoting plant growth are the same as those described above.

[0082] At this time, the treatment method may be carried out by generally practiced methods, namely by spraying (e.g., spraying, misting, atomizing, powder spraying, granule spraying, surface application, continuous application, etc.), soil application (e.g., incorporation, drenching, etc.), surface application (e.g., coating, smearing, covering, etc.), immersion, poisoning, fumigation, etc. The amount of the above composition used can be appropriately determined according to the formulation, damage condition, application method, application location, etc.

[0083] Kit

[0084] Another aspect of the present invention provides a kit for controlling plant pathogenic fungi comprising the above-mentioned Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof.

[0085] Another aspect of the present invention provides a kit for promoting plant growth comprising the above-mentioned Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof.

[0086] The above-mentioned Bulkholderia cepacia JK6 strain and its culture are identical to those described above.

[0087] In the present invention, the kit may be provided such that the Bulkholderia cepacia JK6 strain or its culture and each other constituent composition are stored in separate containers and can be mixed and used at the time of use, but is not limited thereto.

[0088] Additionally, the above kit may include one or more other component compositions, solutions, or devices for controlling plant pathogenic fungi or promoting plant growth, depending on the respective application, but is not limited thereto. Examples of such other components may include, but are not limited to, suitable carriers, solvents, buffers, stabilizers, etc. The carrier may include soluble carriers or insoluble carriers, and may be, for example, a physiologically acceptable buffer known in the art (e.g., PBS), but is not limited thereto.

[0089] In addition, each of the above kits may additionally include a user guide describing the optimal reaction execution conditions. The guide is a printed document explaining how to use the kit, for example, reaction conditions. The guide includes instructions in the form of a pamphlet or leaflet, a label attached to the kit, and descriptions on the surface of a package containing the kit. Furthermore, the guide includes information disclosed or provided through electronic media, such as the Internet.

[0090] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples.

[0091] Example 1. Isolation and culture of microorganisms

[0092] Example 1.1. Preparation of rhizosphere samples and isolation of endophytes

[0093] To secure novel microorganisms that possess antifungal activity against plant pathogenic fungi while simultaneously promoting plant growth, endophytes present within healthy plant root tissues were isolated. As the plant samples, root samples from 1 to 2-year-old herbaceous and woody acacia trees were collected from Mt. Geonji (coordinates: 35.86N 127.13E), Deokjin-gu, Jeonju-si. The collected roots were placed in zip-lock bags, labeled, and brought to the laboratory for microbial isolation.

[0094] After removing excess soil around the roots of the collected herbaceous and woody plants, surface soil particles were removed by washing several times with sterile water. The roots were cut into pieces approximately 2 cm or smaller and transferred to an Erlenmeyer flask; commercial bleach (1 / 4 dilution + 0.1% Triton X-100) was added, and the surface was sterilized in a shaker for 15 minutes. Subsequently, the bleach solution was removed, the roots were washed three times with sterile distilled water, and finally, the roots were transferred to a new ziplock bag and an appropriate amount of physiological saline (0.85% NaCl) was added (10 ml / g saline (root)). The root tissue was crushed by pounding with a plastic hammer, and the extract was transferred to a new tube. After leaving the root sections at room temperature for 5 minutes to allow impurities to settle, the supernatant was transferred to a new tube and used for microbial culture.

[0095] Example 1.2. Microbial Culture

[0096] The supernatant (microorganism extract supernatant) obtained in Example 1.1 above was serially diluted tenfold using saline solution. 100 µl of the diluted microorganism extract was inoculated onto LB agar medium, spread, and cultured at 25°C for 2 to 5 days (Fig. 1). The composition of the LB agar medium used is as shown in Table 1 below.

[0097] Ingredients (g / l) Tryptone 10 Yeast extract 5 Sodium chloride (NaCl) 10 Agar (add after adjusting to pH 7.0) 15

[0098] Example 2. Identification of Bulkholderia cepacia JK6 strain

[0099] A single colony formed by culturing the microorganism isolated in Example 1 was selected, inoculated into a new medium, and cultured in a pure culture. At this time, the isolated bacterial colonies were classified and cultured according to their morphological characteristics (shape, color, size, etc.). After inoculating each colony into LB broth and culturing at 25°C for 1 day, the strain culture solution (5 µl) was inoculated into a PDA (potato dextrose agar) medium in which the hyphae of Fusarium oxysporum f.sp. cubense tropical race 4 (FocTR4) grow, and the colonies were cultured together at 25°C for 5 days. After 5 days of culture, the strains exhibiting antimicrobial activity were sent to Cosmogentech for 16S rRNA gene sequencing analysis (sequencing analysis was performed using a 27F primer) (Fig. 2).

[0100] As a result, as shown in FIGS. 3a to 5b, among the total 13 strains (Gj-1 to Gj-13) exhibiting antibacterial activity, 11 strains (Gj-1 to Gj-4, Gj-6, Gj-7, Gj-9 to Gj-13) exhibiting strong antibacterial activity were identified as Burkholderia sp. (Figs. 5a and 5b), and among the remaining two strains, Gj-5 was predicted to be Enterobacteriaceae or Rahnella sp., and Gj-8 was predicted to be Pseudomonas sp. (Figs. 3a to 4b).

[0101] In addition, as a result of multiple sequence alignment of the 16S rRNA base sequences of the 11 strains mentioned above, the strains could be classified into at least three groups (Figs. 6a to 7). Among the strains mentioned above, Gj-2, Gj-6, and Gj-10, in particular, showed strong antibacterial activity.

[0102] The 16s rRNA gene sequence of Gj-6 among the strains above (sequence number 1) was identical to that of the previously isolated Burkholderia cepacia JK11 strain (KCTC 14199BP), but showed distinct differences in colony color and motility (Figs. 8 and 9).

[0103] Accordingly, the above Gj-6 was named "Bulkholderia cepacia JK6 strain (KCTC 16020BP)," and analysis for further identification and evaluation of antimicrobial activity were performed on the said strain. In this specification, "JK6" and "Bulkholderia cepacia JK6 strain (KCTC 16020BP)" may be used interchangeably. The above Bulkholderia cepacia JK6 strain may include the 16s rRNA gene sequence of SEQ ID NO. 1.

[0104] Example 3. Genome analysis of Bulkholderia cepacia JK6

[0105] To accurately identify the species of Bulkholderia cepacia JK6 (KCTC 16020BP), genomic sequencing analysis was performed by commissioning PacBio NGS (next generation sequencing) analysis to Macrogen. The results are shown in Tables 2 and 3 below.

[0106] Sample IDTotal bases (bp)Total readsGC (%)AT (%)Q20 (%)Q30 (%)JK-62,391,663,93415,838,83466.633.496.290.1JK-6_filtered1,648,016,74610,923,62465.834.299.497.8

[0107] Genome featuresValueTotal genome size (bp)8,104,269Chromosome 13,600,495Chromosome 23,307,321Chromosome 31,196,453G + C content (%)66.9Number of protein-coding genes7261Number of tRNA genes77Number of rRNA genes18Number of tmRNA1

[0108] The genome of Bulkholderia cepacia JK6 (KCTC 16020BP) consists of three chromosomes and was analyzed to be 8,104,269 bp in total.

[0109] The above genomic information was registered in the following official database (Table 4).

[0110] Database TypeNumberGenbankCP163499-CP163501BioprojectPRJNA1141197BiosampleSAMN42883091SRASRR30058561

[0111] In addition, genome blast analysis results showed the highest similarity to the Bulkholderia cepacia CMCC9B23005 strain (Table 5).

[0112] ContigContig LengthSubject DescriptionSubject LengthE-valuecontig13,600,495CP090608.1 Burkholderia cepacia strain CMCC(B)23005 chromosome 13,739,9380contig23,307,321CP034554.1 Burkholderia cepacia ATCC 25416 chromosome 23,396,9580contig31,196,453CP034555.1 Burkholderia cepacia ATCC 25416 chromosome 31,187,9770

[0113] As a result of ANI (average nucleotide identity) analysis, the Bulkholderia cepacia JK6 strain showed a value of 99% or higher (generally, 95% or higher is considered the same species) with Bulkholderia cepacia BC00085-00086 strain and BC92_19-21 strain (Table 6).

[0114] RankingSimilar GenomeANI (%)Aln. Cov. (%)1GCA_034306125.1_B.cepacia_BC0008599.1996.032GCA_034309305.1_B.cepacia_BC0008699.1995.793GCF_036861785.1_B.c epacia_BC92_2099.1896.114GCF_036861765.1_B.cepacia_BC92_2199.1796.395GCF_036861805.1_B.cepacia_BC92_1999.1796.35

[0115] A molecular phylogenetic positional investigation was conducted between the above JK6 strain and a closely related strain of the same genus, Bulkholderia cepacia JK11 strain (KCTC 14199BP) (or mixed with the JK11 strain) and is shown in Fig. 10.

[0116] Example 4. Verification of the antimicrobial activity of Bulkholderia cepacia JK6 strain against Fusarium oxysporum strain

[0117] Example 4.1. Preparation of Fusarium oxysporum strain

[0118] The FocTR4 strain was obtained from ATCC (F. oxysporum f.sp.cubense 23486, item number 96289) (ATCC 23486), and six strains of Fusarium oxysporum (F. oxysporum) were obtained from the Rural Development Administration. The six strains obtained from the Rural Development Administration are shown in Table 7 below.

[0119] Number KACC no. Scientific name Culture temperature (℃) 140031Fusarium oxysporumf.sp.radicis-lycopersici25240051Fusarium oxysporumf.sp.gladioli25342795Fusarium oxysporumf.sp.lactucae30447084Fusarium oxysporumf.sp.lagenariae25547667Fusarium oxysporumf.sp.cucumerinum25647669Fusarium oxysporumf.sp.melonis25

[0120] To isolate fungal spores from the Fusarium oxysporum strain distributed by the Rural Development Administration, the strains were inoculated onto PDA (potato dextrose agar) medium and cultured at 25–28°C for one day. Afterward, spores grown on the medium were collected using sterile distilled water or saline solution. At this time, the spores were suspended in distilled water or an aqueous solution by gently scraping the culture surface with a sterile brush or plastic loop.

[0121] Large mycelia were removed from the spore suspension collected as described above using a sterile filter or gauze. Then, the spore concentration in the suspension was measured using a hemocytometer. The spore suspension prepared as described above contained 10 spores per 1 ml to suit the experimental purpose. 6 ~10 8 It was prepared and used at the spore concentration. In addition, glycerol (concentration) was added to the spore suspension and stored frozen at -70℃ (long-term storage).

[0122] Example 4.2. Confirmation of the antimicrobial activity of Bulkholderia cepacia JK6 strain against Fusarium oxysporum strain spores

[0123] After inoculating the middle of a PDA medium with each of the Fusarium oxysporum strain spores (7 types) prepared in the same manner as in Example 4.1 above, Escherichia coli (E. coli), Bulkholderia cepacia JK6 strain (KCTC 16020BP), and Bulkholderia cepacia JK11 strain (KCTC 14199BP) (7 ul, 10 6 cfu / ml) was inoculated as described in FIGS. 11 to 15. Afterward, each medium was cultured at 25–28°C for 6 days. At this time, the Escherichia coli (DH5a, Enzynomics, Inc.) and the genus strain Bulkholderia cepacia JK11 (KCTC 14199BP) were used as a control.

[0124] As a result, it was confirmed that the Bulkholderia cepacia JK6 strain inhibited the growth of spores of all inoculated Fusarium oxysporum strains. The spore growth inhibitory activity of the Bulkholderia cepacia JK6 strain was superior compared to the Bulkholderia cepacia JK11 strain (KCTC 14199BP) used as a control strain (Figs. 11 to 15).

[0125] Example 5. Confirmation of the activity-promoting effect of PGPR on Bulkholderia cepacia JK6 strain

[0126] To confirm the activity of the Bulkholderia cepacia JK6 strain (KCTC 16020BP) as a plant growth-promoting rhizosphere microorganism (PGPR), the phosphate solubilization, siderophore synthesis, and nitrogen fixation abilities of the Bulkholderia cepacia JK6 strain were examined.

[0127] Strains of Bulkholderia cepacia JK6 (KCTC 16020BP) and Bulkholderia cepacia JK11 (KCTC 14199BP) used in the experiment were inoculated into LB broth, respectively, and cultured overnight at 25–28°C to prepare strain suspensions, which were then used in each experiment. At this time, the Bulkholderia cepacia JK11 strain was used as a control.

[0128] Example 5.1. Measurement of Phosphate Solubilization Activity

[0129] 7 µl of each cultured strain suspension from Example 5 above was inoculated into a selective medium containing phosphate (Pikovskaya's agar medium), cultured at 25–28°C for 6 days, and the phosphate solubilization ability was confirmed. The composition (pH 7.0) of the Pikovskaya's agar medium is as shown in Table 8 below.

[0130] Ingredient Name Content (g / l) Glucose 10 Tricalcium Phosphate (Ca3(PO4)2)5 Ammonium Sulfate ((NH4)2SO4) 0.5 Sodium Chloride (NaCl) 0.2 Magnesium Sulfate Hydrate (MgSO4·7H2O) 0.1 Potassium Chloride (KCl) 0.2 Yeast Extract 0.5 Manganese Sulfate Hydrate (MnSO4·H2O) 0.002 Iron Sulfate Hydrate (FeSO4·7H2O) 0.002

[0131] As a result, as shown in Fig. 16, it was confirmed that the Bulkholderia cepacia JK6 strain solubilizes insoluble phosphate at the inoculation site. The phosphate solubilization activity of the Bulkholderia cepacia JK6 strain was at a level almost identical to that of the Bulkholderia cepacia JK11 strain (KCTC 14199BP) used as a comparative strain.

[0132] Example 5.2. Confirmation of Sidelophore Synthesis Ability

[0133] 7 µl of each strain suspension prepared in Example 5 above was inoculated into CAS medium and cultured at 25–28°C for 6 days to confirm the siderophore synthesis ability. The CAS medium used was prepared by the following method. First, solutions 1, 2, and 3 of Table 9 below were prepared separately and mixed. The mixture was diluted 1 / 20 times with distilled water, agar (final concentration 1.2%) was added, and then autoclaved. The sterilized medium was prepared by adding 10 ml of CAS agar to a Petri dish to solidify it, and then overlaying 10–15 ml of nutrient agar medium.

[0134] Composition solution: 150 ml distilled water, 60.5 mg CAS (chrome azurol S) solution; 210 ml HCl (10 mM), 1 mM FeCl3·6H2O solution; 340 ml distilled water, 72.9 mg CTAB (cetrimonium bromide)

[0135] As a result, as shown in Fig. 17, the siderophore production capacity of the Bulkholderia cepacia JK6 strain was confirmed by CAS decolorization of the inoculation site. The siderophore production rate of the Bulkholderia cepacia JK6 strain was almost the same as that of the Bulkholderia cepacia JK11 strain (KCTC 14199BP) used as a control strain.

[0136] Example 5.3. Confirmation of Nitrogen Fixation Ability

[0137] 7 µl of each strain suspension prepared in Example 5 above was inoculated into N-free basal medium, cultured at 25–28°C for 6 days, and nitrogen fixation ability was confirmed. The composition of the N-free basal medium used is as shown in Table 10 below.

[0138] Ingredient Name Content (g / l) Sucrose 10 Potassium diphosphate (K2HPO4) 0.5 Magnesium sulfate hydrate (MgSO4·7H2O) 0.2 Sodium chloride (NaCl) 0.2 Calcium carbonate (CaCO3) 0.1 Iron sulfate hydrate (FeSO4·7H2O) 0.01

[0139] As a result, as shown in Fig. 18, no nitrogen fixation ability was observed in the Bulkholderia cepacia JK6 strain. Through the above results, the Bulkholderia cepacia JK6 strain (KCTC 16020BP) of the present invention was identified as a Bulkholderia cepacia species with distinct strain characteristics and antimicrobial activity against Fusarium oxysporum strains compared to the Bulkholderia cepacia JK11 strain (KCTC 14199BP), a strain of the same genus. The above results suggest that the Bulkholderia cepacia JK6 strain (KCTC 16020BP) of the present invention can be usefully employed as an antagonistic microorganism against banana Panama disease and various plant fungal diseases in the future.

[0140]

Claims

1. Burkholderia cepacia JK6 strain (accession number KCTC 16020BP).

2. In Paragraph 1, The strain above is a strain comprising the 16s rRNA gene sequence of SEQ ID NO.

1.

3. In Paragraph 1, The strain above is a strain having antifungal activity or plant growth-promoting activity.

4. Culture of Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP).

5. A composition for controlling plant pathogenic fungi comprising the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient.

6. In Paragraph 5, A composition for controlling plant pathogenic fungi, wherein the above-mentioned plant pathogenic fungus is a strain of Fusarium oxysporum.

7. In Paragraph 6, A composition for controlling plant pathogenic fungi, wherein the above-mentioned Fusarium oxysporum strain is one or more selected from the group consisting of F. oxysporum f.sp. cubense, F. oxysporum f.sp. radicis-lycopersici, F. oxysporum f.sp. gladioli, F. oxysporum f.sp. lactucae, F. oxysporum f.sp. cucumerinum, and F. oxysporum f.sp. melonis.

8. A method for controlling plant pathogenic fungi comprising the step of applying the composition for controlling plant pathogenic fungi of claim 5 to a plant or soil adjacent to the plant.

9. A composition for promoting plant growth comprising the Bulkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof as an active ingredient.

10. A method for promoting plant growth comprising the step of treating a plant or soil adjacent to a plant with the plant growth-promoting composition of claim 9.

11. A kit for controlling plant pathogens comprising the Burkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof.

12. A kit for promoting plant growth comprising the Burkholderia cepacia JK6 strain (accession number KCTC 16020BP) or a culture thereof.

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