Attractants for Pseudomonas bacteria and their uses

Abstract

This technology provides the ability to control the motility of microorganisms. [Solution] The attractant for Pseudomonas bacteria comprises at least one of polylysine and Bacillus bacteria, indicated by accession number NITE P-03755.

Description

Technical Field

[0001] The present disclosure relates to an attractant for Pseudomonas bacteria.

Background Art

[0002] Conventionally, it has been proposed to use microorganisms having an effect of promoting plant growth. For example, Patent Document 1 proposes a method of transplanting a plant, which is a terrestrial plant, characterized in that when transplanting the plant, a liquid containing a plant growth-promoting microorganism having a plant growth-promoting effect is added to the planting hole at the transplanting site.

[0003] In addition, various antibacterial agents have been proposed for pathogenic microorganisms. For example, Patent Document 2 discloses an antibacterial agent containing a terpene polymer.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] According to the technique described in Patent Document 1, there is room for improvement from the viewpoint of fixing plant growth-promoting microorganisms on the root surface of plants. For this reason, a technique capable of promoting the establishment of plant growth-promoting microorganisms, for example, a technique capable of controlling the motility of plant growth-promoting microorganisms is required. In addition, regarding the use of antibacterial agents as disclosed in Patent Document 2, the emergence of drug-resistant bacteria may be a problem. For this reason, other techniques capable of controlling the motility of pathogenic bacteria are required. Such problems are common problems not only for plant growth-promoting microorganisms and pathogenic bacteria but also for other microorganisms. [Means for solving the problem]

[0006] This disclosure can be implemented in the following forms:

[0007] (1) According to one embodiment of the present disclosure, an attractant for Pseudomonas bacteria is provided. The attractant comprises at least one of polylysine and Bacillus bacteria, as indicated by accession number NITE P-03755. This embodiment of the attractant can attract Pseudomonas bacteria, and thus the motility of Pseudomonas bacteria can be controlled.

[0008] (2) In the attractant described in (1) above, the polylysine may contain ε-polylysine. This form of attractant can attract Pseudomonas bacteria more effectively, and thus the motility of Pseudomonas bacteria can be controlled more effectively.

[0009] (3) In other forms of the present disclosure, a pesticide comprising the attractant described in (1) or (2) above is provided. This form of pesticide can attract Pseudomonas bacteria, thereby controlling the motility of plant growth-promoting microorganisms. As a result, the colonization of plant growth-promoting microorganisms on plants can be promoted.

[0010] (4) The pesticide described in (3) above may further contain the Pseudomonas bacteria. This form of pesticide, by further containing Pseudomonas bacteria, can further promote the establishment of plant growth-promoting microorganisms on plants.

[0011] (5) The pesticide described in (3) or (4) above may further contain at least one of Pseudomonas putida and Pseudomonas fluorescens. This form of pesticide further contains at least one of Pseudomonas putida and Pseudomonas fluorescens, which can further promote the establishment of Pseudomonas putida and Pseudomonas fluorescens on plants.

[0012] (6) In other forms of the present disclosure, a fertilizer comprising the attractant described in (1) or (2) above is provided. This form of fertilizer can attract Pseudomonas bacteria, thereby controlling the motility of plant growth-promoting microorganisms. As a result, the colonization of plant growth-promoting microorganisms on plants can be promoted.

[0013] (7) The fertilizer described in (6) above may further contain the Pseudomonas bacteria. This form of fertilizer, by further containing Pseudomonas bacteria, can further promote the establishment of plant growth-promoting microorganisms on plants.

[0014] (8) The fertilizer described in (6) or (7) above may further contain at least one of Pseudomonas putida and Pseudomonas fluorescens. This form of fertilizer, by further containing at least one of Pseudomonas putida and Pseudomonas fluorescens, can further promote the establishment of Pseudomonas putida and Pseudomonas fluorescens on plants.

[0015] (9) According to other forms of the present disclosure, plant seeds are provided. These plant seeds are coated on their surface with at least one of polylysine and a bacterium of the genus Bacillus, indicated by accession number NITE P-03755. According to these forms of plant seeds, it is possible to attract a bacterium of the genus Pseudomonas, thereby controlling the motility of plant growth-promoting microorganisms. As a result, the colonization of plant growth-promoting microorganisms on plants can be effectively promoted.

[0016] (10) According to another form of the present disclosure, a plant seedling is provided, in which at least one of polylysine and a bacterium of the genus Bacillus, indicated by accession number NITE P-03755, is applied to the surface of the plant roots. According to this form of plant seedling, the motility of plant growth promoting microorganisms can be controlled as Pseudomonas bacteria can be attracted. As a result, the colonization of plant growth promoting microorganisms on the plant roots can be effectively promoted.

[0017] (11) According to other forms of the present disclosure, a method for producing plants is provided. This method for producing plants includes the step of bringing at least one of polylysine and a Bacillus bacterium indicated by accession number NITE P-03755 into contact with the roots of a plant or the seeds of a plant. According to this form of the method for producing plants, Pseudomonas bacteria can be attracted, so the motility of plant growth promoting microorganisms can be controlled. As a result, the colonization of plant growth promoting microorganisms on plants can be effectively promoted, so the productivity of the plants can be increased.

[0018] (12) In the attractant described in (1) or (2) above, the Pseudomonas bacterium may be Pseudomonas aeruginosa. This form of attractant can control the motility of Pseudomonas aeruginosa. As a result, infections caused by Pseudomonas aeruginosa can be suppressed.

[0019] (13) According to other forms of the present disclosure, an article is provided having the attractant described in (1) or (2) above coated on its surface. According to this form of article, Pseudomonas bacteria can be attracted, and thus the motility of Pseudomonas bacteria can be controlled.

[0020] (14) According to another aspect of the present disclosure, a method for attracting Pseudomonas aeruginosa is provided. This method includes a step of applying polylysine to the surface of an article. According to the method of this aspect, Pseudomonas aeruginosa can be attracted.

[0021] (15) According to another aspect of the present disclosure, a method for producing ε-polylysine is provided. This method for producing ε-polylysine includes a step of secreting ε-polylysine by a Bacillus bacterium represented by accession number NITE P-03755, and a step of collecting the secreted ε-polylysine. According to the method for producing ε-polylysine of this aspect, ε-polylysine can be produced.

[0022] Note that the present disclosure can be implemented in various forms. For example, a method for producing an attractant for Pseudomonas bacteria, a method for producing a pesticide containing an attractant for Pseudomonas bacteria, a method for producing a fertilizer containing an attractant for Pseudomonas bacteria, a coating agent for plant seeds, a coating agent for plant seedlings, a method for producing plant seeds, a method for producing plant seedlings, a method for promoting plant growth, a method for controlling Pseudomonas aeruginosa, the use of ε-polylysine for producing an attractant for Pseudomonas bacteria, the use of a Bacillus bacterium represented by accession number NITE P-03755 for producing an attractant for Pseudomonas bacteria, etc.

Brief Description of Drawings

[0023] [Figure 1] It is an explanatory diagram showing the result of co-culturing the NB4 strain and Pseudomonas aeruginosa. [Figure 2] It is an explanatory diagram showing the result of culturing Pseudomonas aeruginosa together with the culture extract of the NB4 strain. [Figure 3] It is an explanatory diagram showing the attraction to the active fraction in the paper disk method [Figure 4] It is an explanatory diagram showing the 1H NMR spectrum of the active fraction. [Figure 5] It is an explanatory diagram showing the 13C NMR spectrum of the active fraction. [Figure 6]This is an explanatory diagram showing the COSY NMR spectrum of the active fraction. [Figure 7] This is an explanatory diagram showing the HMQC NMR spectrum of the active fraction. [Figure 8] This is a correlation diagram of the NMR analysis results. [Figure 9] This is an explanatory diagram showing the results of TLC analysis. [Figure 10] This is an explanatory diagram showing the results of culturing P. putida together with the active fraction. [Figure 11] This is an explanatory diagram showing the results of culturing P. fluorescens together with the active fraction. [Figure 12] This is an explanatory diagram showing the results of culturing Pseudomonas aeruginosa with ε-polylysine. [Modes for carrying out the invention]

[0024] The inventors of this application have revealed that Pseudomonas bacteria are attracted to a compound secreted by a Bacillus sp. bacterium, as shown in the examples described below, under accession number NITE P-03755 (Depositing organization: National Institute of Technology and Evaluation, Biotechnology Center, Patent Microorganism Depositary Center, Date of receipt: September 20, 2022). Furthermore, they have revealed that this compound is mainly ε-polylysine. Based on these findings, the inventors have completed the present invention.

[0025] According to one embodiment of the present disclosure, an attractant for Pseudomonas bacteria is provided. This attractant for Pseudomonas bacteria comprises at least one of polylysine and a Bacillus bacterium designated by accession number NITE P-03755. The attractant for Pseudomonas bacteria of the present disclosure can specifically attract Pseudomonas bacteria. For convenience, in the following description, the Bacillus bacterium designated by accession number NITE P-03755 will also be referred to as "NB4 strain".

[0026] Examples of Pseudomonas bacteria are not particularly limited and include bacteria that promote plant growth, bacteria that protect plants, pathogenic bacteria, bacteria that cause food poisoning, and bacteria that can break down chemical substances. However, it is preferable that the bacteria be at least one of either plant growth-promoting microorganisms or pathogenic bacteria.

[0027] Plant growth-promoting microorganisms are also called Plant Growth Promoting Microorganisms (PGPM) or Plant Growth Promoting Rhizobacteria (PGPR). Examples of plant growth-promoting microorganisms are not particularly limited and include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas chlororaphis, Pseudomonas aurantiaca, and Pseudomonas brushicacearum, but it is preferable that they be at least one of Pseudomonas putida and Pseudomonas fluorescens. Examples of fungi with plant-protective properties are not particularly limited, but include Pseudomonas protegens.

[0028] The attractant disclosed herein allows for the specific control of the motility of plant growth-promoting microorganisms, thereby promoting their establishment, for example, on the root surface of plants. Furthermore, because the attractant disclosed herein does not attract microorganisms other than Pseudomonas bacteria, it minimizes the impact on other microorganisms, suppresses the disruption of the microbial community, and reduces the environmental burden.

[0029] While not limited to specific species, examples of pathogenic bacteria include Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas acidovorans, Pseudomonas oryzihabitans, Pseudomonas plecoglossicida, Pseudomonas acidovorans, Pseudomonas syringae, Pseudomonas tolaasii, Pseudomonas anguilliseptica, Pseudomonas viridiflava, Pseudomonas plecoglossicida, Pseudomonas marginalis, Pseudomonas solanacearum, Pseudomonas agarici, Pseudomonas tomato, and Pseudomonas brushicacearum. The pathogen is preferably a bacterium that is pathogenic to humans, more preferably at least one selected from the group consisting of Pseudomonas aeruginosa, Pseudomonas paucimobilis, Pseudomonas putida, Pseudomonas fluorescens, and Pseudomonas acidovorans, and even more preferably Pseudomonas aeruginosa.

[0030] The attractant disclosed herein can specifically control the motility of pathogenic bacteria such as Pseudomonas aeruginosa, thereby suppressing infections caused by pathogens. More specifically, it can control the adhesion of pathogenic bacteria to medical devices and human body surfaces, thereby suppressing the formation of biofilms by pathogenic bacteria, and thus suppressing, for example, hospital-acquired infections. Furthermore, it can suppress the acquisition of drug resistance associated with biofilm formation, thus preventing the intractability and chronicity of infectious diseases. In addition, the attractant disclosed herein can be used as an antimicrobial material, antifouling material, and antibacterial material with a mechanism of action different from conventional antimicrobial agents. With the attractant disclosed herein, microorganisms other than Pseudomonas bacteria are not attracted, so the antimicrobial effect on other microorganisms such as commensal bacteria is not exerted, thereby suppressing the destruction of the microbiome. Furthermore, while the use of general antimicrobial agents and disinfectants has problems such as the emergence of resistant bacteria and ecosystem destruction due to release into the environment, the attractant disclosed herein can suppress the decrease in effectiveness caused by the emergence of resistant bacteria and reduce the burden on the environment.

[0031] While not limited to these, the bacteria that cause food poisoning include Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas fragi, Pseudomonas taetrolens, Pseudomonas mudicolens, and Pseudomonas lundensis.

[0032] Examples of bacteria capable of breaking down chemical substances include, but are not limited to, Pseudomonas putida, Pseudomonas alcaligenes, Pseudomonas mendocina, Pseudomonas pseudoalcaligenes, Pseudomonas resinovorans, Pseudomonas veronii, and Pseudomonas stutzeri.

[0033] The polylysine in the attractant of this disclosure includes at least one of ε-polylysine and α-polylysine, but from the viewpoint of enhancing the attractant effect, it is preferable to include ε-polylysine. Here, polylysine is a polyamino acid in which lysine is linked in a linear chain by peptide bonds. In ε-polylysine, the amino group at the ε position of lysine is peptide-bonded to a carboxyl group. In α-polylysine, the amino group at the α position of lysine is peptide-bonded to a carboxyl group.

[0034] The degree of polymerization of polylysine is not particularly limited, but from the viewpoint of enhancing the attractive effect, it is preferably 20 to 40 and more preferably 25 to 35. The molecular weight of polylysine is not particularly limited, but it is preferably 500 to 10000 and more preferably 1000 to 3000. Polylysine may be secreted from the NB4 strain, such as a culture extract of the NB4 strain, produced by the metabolism of other microorganisms, or chemically synthesized. When using polylysine derived from the NB4 strain and culture extracts of other microorganisms, it may be purified or not.

[0035] The Bacillus bacterium (NB4 strain) indicated by accession number NITE P-03755 can secrete ε-polylysine. The NB4 strain can be cultured in any medium suitable for the growth of Bacillus bacteria, such as YNB medium, LB medium, nutrient medium, tryptosoy medium, YM medium, YPD medium, PD medium, SD medium, etc.

[0036] The Pseudomonas bacterial attractant of this disclosure preferably contains at least polylysine, more preferably at least ε-polylysine, and may contain both ε-polylysine and strain NB4. In addition, the Pseudomonas bacterial attractant of this disclosure may contain one or more other optional components in addition to at least one of polylysine and strain NB4. These optional components are not particularly limited, but examples include lysine monomer, culture medium, carrier, buffer, solvent, stabilizer, thickener, preservative, antioxidant, emulsifier, surfactant, fragrance, colorant, etc. The form of the Pseudomonas bacterial attractant of this disclosure is not particularly limited, but may be, for example, liquid, gel, paste, powder, granules, etc.

[0037] The attractant of this disclosure may be used in combination with at least one of a pesticide and a fertilizer. In other forms of this disclosure, a pesticide containing the attractant is provided. This pesticide can attract Pseudomonas bacteria such as plant growth-promoting microorganisms, thereby promoting the colonization of these microorganisms on the plant surface and thus promoting plant growth. In other forms of this disclosure, a fertilizer containing the attractant is provided. This fertilizer can attract Pseudomonas bacteria such as plant growth-promoting microorganisms, thereby promoting the colonization of these microorganisms on the plant surface and thus promoting plant growth.

[0038] The pesticide or fertilizer of this disclosure preferably further contains plant growth-promoting microorganisms as Pseudomonas bacteria to be attracted, and more preferably further contains at least one of Pseudomonas putida and Pseudomonas fluorescens. According to this embodiment, plant growth-promoting microorganisms can be effectively attracted, so that the plant growth-promoting microorganisms can be more effectively fixed to surfaces such as the root surface of plants, and as a result, plant growth can be further promoted.

[0039] The application methods of the pesticides or fertilizers disclosed herein are not particularly limited and include, for example, mixing with soil or hydroponic water, applying to plant roots or seeds, or spraying onto plants. The form of the pesticides or fertilizers disclosed herein is not particularly limited and includes, for example, liquid, solid, gel, or paste. The timing of application of the pesticides or fertilizers disclosed herein is not particularly limited, but it is preferable to apply them to plants in the early stages of growth, such as seeds or seedlings. Furthermore, the pesticides or fertilizers disclosed herein can be applied to any part of the plant, such as roots, leaves, stems, branches, trunks, or seeds, but it is preferable to apply them to the roots (rhizosphere) or seeds.

[0040] The pesticides or fertilizers of this disclosure may contain one or more other optional components in addition to the attractant described above. These other optional components are not limited to, but include, for example, other pesticide components, other fertilizer components, soil conditioners, polymers, signaling molecules, and other microorganisms. Other pesticide components are not limited to, and may be either biological or chemical pesticides, such as fungicides, antimicrobial agents, insecticides, acaricides, insect repellents, herbicides, and plant growth regulators. Other fertilizer components are not limited to, but include, nitrogen (N), phosphorus (P), potassium (K), silicon (Si), magnesium (Mg), manganese (Mn), boron (B), calcium (Ca), sulfur (S), iron (Fe), copper (Cu), zinc (Zn), molybdenum (Mo), cobalt (Co), and chlorine (Cl). Soil improvement components are not particularly limited, but examples include mineral powders or clay components such as zeolite, vermiculite, bentonite, soft silica, perlite, peat moss, bark compost, clay, pumice, silica sand, calcium carbonate, diatomaceous earth, kaolin, and talc. High molecular weight substances are not particularly limited, but examples include polyethyleneimine, polyvinyl alcohol, and polyacrylic acid. Signal molecules are not particularly limited, but examples include chito-oligosaccharides, chitinous compounds, and flavonoids such as isoflavones and rutin.

[0041] The plants to which the pesticides or fertilizers disclosed herein are applied are not particularly limited and may include, for example, plants capable of producing agricultural products such as vegetables, fruits, and grains, as well as plants used for ornamental purposes or greening, such as flowers, ornamental trees, and other trees. More specifically, examples include Rosaceae plants such as strawberries, apples, pears, plums, and peaches; Brassicaceae plants such as cabbage, Chinese cabbage, broccoli, radish, bok choy, komatsuna, cauliflower, and Arabidopsis thaliana; Cucurbitaceae plants such as cucumbers, pumpkins, and melons; Solanaceae plants such as tomatoes, eggplants, bell peppers, chili peppers, tobacco, and potatoes; Poaceae plants such as rice and wheat; Fabaceae plants such as soybeans; Araceae plants such as taro; Actinidiaceae plants such as kiwifruit; and Amaryllidaceae plants such as onions and garlic.

[0042] In other forms of this disclosure, plant seeds are provided. These plant seeds are coated on their surface with at least one of polylysine and a Bacillus bacterium (NB4 strain) indicated by accession number NITE P-03755. The polylysine includes at least one of ε-polylysine and α-polylysine, but is preferably ε-polylysine from the viewpoint of enhancing the attractive effect. The plant is not particularly limited, and examples include the plants described above. The binder for coating is not particularly limited, but examples include polysaccharides such as CMC, HPC, and starch, PVA, MC, hydroxymethylcellulose (HPMC), gelatin, polyvinylpyrrolidone, gum arabic, polyacrylic acid, pullulan, polyethylene glycol, and alginic acid. The method for coating the surface of the plant seeds is not particularly limited, but may be carried out as follows, for example. For example, a solution or dispersion containing at least one of polylysine and the NB4 strain can be applied to the plant seeds as a coating solution, or the plant seeds can be impregnated with it and then dried. Alternatively, for example, a solution or dispersion containing at least one of polylysine and the NB4 strain may be used as a coating solution, applied to form a film, or molded into encapsulation and then used to coat the seeds. The polylysine used for coating may be secreted from the NB4 strain, such as a culture extract of the NB4 strain, produced by the metabolism of other microorganisms, or chemically synthesized. When using polylysine derived from the culture extracts of the NB4 strain and other microorganisms, it may be purified or not.

[0043] In other forms of this disclosure, plant seedlings are provided. These plant seedlings have at least one of ε-polylysine and a Bacillus bacterium (NB4 strain) indicated by accession number NITE P-03755 coated on the surface of the plant roots. The polylysine contains at least one of ε-polylysine and α-polylysine, but it is preferable to include ε-polylysine from the viewpoint of enhancing the attractive effect. The plant is not particularly limited, and examples include the plants described above. The method of coating the surface of the plant roots is not particularly limited, and for example, a method similar to the coating of seeds described above can be employed. The polylysine used for coating may be secreted from the NB4 strain, such as a culture extract of the NB4 strain, may be produced by the metabolism of other microorganisms, or may be chemically synthesized. When using polylysine derived from the NB4 strain and culture extracts of other microorganisms, it may be purified or not.

[0044] In other forms of this disclosure, a method for producing plants is provided. This method for producing plants includes the step of contacting at least one of polylysine and a Bacillus bacterium of the genus Bacillus (NB4 strain) indicated by accession number NITE P-03755 with the roots of a plant or the seeds of a plant. In the contact step, at least one of polylysine and the NB4 strain may be brought into direct contact with the roots of the plant or the seeds of the plant, or it may be brought into indirect contact via soil, water, etc., used for cultivating the plant. The polylysine includes at least one of ε-polylysine and α-polylysine, but from the viewpoint of enhancing the attractive effect, it is preferable to include ε-polylysine. This method for producing plants can attract Pseudomonas bacteria such as plant growth promoting microorganisms, thereby promoting the colonization of plant growth promoting microorganisms, etc., on the surface of the plant, and as a result, plant growth can be promoted. As a result, the productivity of the plant can be increased. The polylysine brought into contact with the roots of the plant or the seeds of the plant may be a culture extract of the NB4 strain or other substance secreted from the NB4 strain, may be produced by the metabolism of other microorganisms, or may be chemically synthesized. When using polylysine derived from NB4 strain and culture extracts of other microorganisms, it may be purified or not.

[0045] In other forms of this disclosure, articles are provided in which the attractant is coated on the surface. The attractant preferably contains polylysine, and more preferably contains ε-polylysine. The articles are not particularly limited, but include, for example, medical devices such as catheters and implants, pharmaceuticals, cooking utensils, household goods and their packaging materials. Articles of this disclosure can attract Pseudomonas bacteria such as pathogenic bacteria, and thus can control pathogenic bacteria. For example, by coating the surface of an article with the attractant, Pseudomonas bacteria such as pathogenic bacteria are attracted and become more mobile, which prevents them from remaining adhered to the surface of the article. As a result, infections that begin when pathogenic bacteria such as Pseudomonas aeruginosa adhere to medical devices, etc., can be suppressed.

[0046] Another embodiment of this disclosure provides a method for attracting Pseudomonas aeruginosa. This method includes the step of coating the surface of an article with polylysine. The polylysine includes at least one of ε-polylysine and α-polylysine, but it is preferable to include ε-polylysine from the viewpoint of enhancing the attracting effect. This method can attract Pseudomonas aeruginosa, and thus the motility of Pseudomonas aeruginosa can be controlled. The specific method in the coating step is not particularly limited, but may be carried out, for example, by spraying liquid polylysine onto the surface of the article or by spreading droplets of polylysine. The polylysine coated onto the surface of the article may be a culture extract of the NB4 strain, secreted from the NB4 strain, produced by the metabolism of other microorganisms, or chemically synthesized. When using polylysine derived from the NB4 strain and culture extracts of other microorganisms, it may be purified or not.

[0047] In other forms of this disclosure, a method for producing ε-polylysine is provided. This method includes the steps of causing a Bacillus bacterium (NB4 strain) indicated by accession number NITE P-03755 to secrete ε-polylysine, and collecting the secreted ε-polylysine. The specific method for causing ε-polylysine secretion is not particularly limited, but for example, ε-polylysine may be secreted by culturing the Bacillus bacterium in any medium suitable for its growth at, for example, 25°C to 37°C for 2 to 30 days. In the step of collecting the secreted ε-polylysine, for example, the culture may be centrifuged and precipitated with acetone to recover the precipitate. In addition, the components may be purified as needed. Purification may be carried out by, for example, subjecting the recovered precipitate to HP-20 column chromatography to recover the non-adsorbed fraction. Furthermore, the recovered non-adsorbed fraction may be subjected to CL-2B column chromatography to recover the active fraction. [Examples]

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0049] (1) Co-culture of NB4 strain and microorganisms We co-cultured Bacillus sp. NB4 strain (referred to as "NB4 strain" by accession number NITE P-03755) isolated from human epidermis with Pseudomonas aeruginosa and observed their attraction. Specifically, bacterial suspensions of NB4 strain and P. aeruginosa PAO1 strain, adjusted to a turbidity of 0.1, were dropped onto YNB agar medium supplemented with 1% by mass of casamino acid and 0.1% by mass of glucose. The cultures were then co-cultured at 30°C for 2 days, and P. aeruginosa colonies were visually observed. Similar co-cultures with NB4 strain were performed for Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus mutans, Candida albicans, and Cryptococcus neoformans under the same conditions, and the colonies were visually observed.

[0050] Figure 1 is an explanatory diagram showing the results of co-culturing Pseudomonas aeruginosa strain NB4. Figure 1 shows a photograph of the agar plate after co-culturing. As shown in Figure 1, morphological changes were observed in Pseudomonas aeruginosa colonies adjacent to NB4 colonies, indicating elongation toward the NB4 colonies. No morphological changes were observed in Pseudomonas aeruginosa colonies not adjacent to NB4 colonies. Furthermore, colony elongation similar to that of Pseudomonas aeruginosa was not observed in Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Streptococcus mutans, Candida albicans, and Cryptococcus neoformans.

[0051] (2) Attraction of Pseudomonas aeruginosa by culture extract of strain NB4 NB4 strain was inoculated onto YNB agar medium supplemented with 1% by mass of casamino acid and 0.1% by mass of glucose. The culture was then incubated at 30°C for 5 days. The supernatant was collected by centrifugation (10,000 × G, 10 minutes). The supernatant was sterilized using a 0.22 μm sterile filter (Millex-GV, Merck Millipore) and then immersed in an 8 mm diameter paper disc (Advantec). This paper disc was placed on YNB agar medium to which a bacterial suspension of P. aeruginosa strain PAO1 had been dropped, and incubated at 30°C for 2 days. Colonies were then visually observed.

[0052] Figure 2 is an explanatory diagram showing the results of culturing Pseudomonas aeruginosa with a culture extract of strain NB4. Figure 2 shows a photograph of the agar medium after culturing. As shown in Figure 2, morphological changes were observed in Pseudomonas aeruginosa colonies adjacent to the paper disc containing the culture extract of strain NB4, indicating elongation toward the paper disc. No morphological changes were observed in Pseudomonas aeruginosa colonies not adjacent to the paper disc.

[0053] (3) Purification and structural analysis of metabolites of strain NB4 NB4 strain was inoculated into YNB medium supplemented with 1% by mass casamino acid and 0.5% by mass glucose. The culture was then incubated at 30°C for 5 days. The culture was centrifuged (10,000 × G, 10 minutes) to collect the supernatant, and the precipitate was collected by acetone precipitation. This precipitate was subjected to HP-20 column chromatography to collect the non-adsorbed fraction. Further, it was subjected to CL-2B column chromatography, and the attractive activity to P. aeruginosa was evaluated by the paper disk method to obtain the active fraction. The obtained fraction was then subjected to structural analysis by NMR. 1 1H NMR analysis and 13For 13C NMR analysis, the sample was dissolved in deuterated methanol and measured at 600 MHz using an ECZ600 (JEOL Ltd.). COSY NMR and HMQC NMR spectra were also analyzed. Furthermore, TLC analysis was performed to determine the binding site. In the TLC analysis, the free amino group was dinitrophenylized (DNP) and then acid hydrolyzed. The TLC developing solvent was butanol:acetic acid:pyridine:water = 4:1:1:2. Ninhydrin reagent was used as the colorimetric reagent. Commercially available α-polylysine, ε-polylysine, and lysine monomers were used as standard reagents.

[0054] Figure 3 is an explanatory diagram illustrating the attraction to the active fraction in the paper disc method. Figure 3 shows a photograph of the agar plate after culture, showing the growth of Pseudomonas aeruginosa colonies. Figure 4 shows the active fraction 1 This is an explanatory diagram showing the 1H NMR spectrum. Figure 5 shows the active fraction. 13 Figure 4 is an explanatory diagram showing the 13C NMR spectrum. Figure 6 is an explanatory diagram showing the COSY NMR spectrum of the active fraction. Figure 7 is an explanatory diagram showing the HMQC NMR spectrum of the active fraction. Figure 8 is a correlation diagram of the NMR analysis results. As shown in Figures 4 to 8, the substance contained in the active fraction was found to be polylysine.

[0055] Figure 9 is an explanatory diagram showing the results of the TLC analysis. As shown in Figure 9, spots were obtained in the active fraction at the same Rf value as ε-polylysine. In other words, it was found that the polylysine contained in the active fraction is ε-polylysine, which has a structure in which the amino group at the α position of lysine is free and the amino group at the ε position is peptide-bonded to a carboxyl group. Therefore, it was revealed that the metabolite of the NB4 strain that attracts Pseudomonas bacteria is ε-polylysine.

[0056] (4) Attraction of other Pseudomonas bacteria by polylysine purified from metabolites of strain NB4 The active fraction, i.e., polylysine purified from the metabolites of strain NB4, was sterilized by filter using a 0.22 μm sterile filter (Millex-GV, Merck Millipore), and then immersed in an 8 mm diameter paper disc (Advantec). This paper disc was placed on YNB agar medium to which bacterial suspensions of P. putida and P. fluorescens were dropped, and the medium was incubated at 30°C for 2 days, after which the colonies were visually observed.

[0057] Figure 10 is an explanatory diagram showing the results of culturing P. putida with the active fraction. Figure 11 is an explanatory diagram showing the results of culturing P. fluorescens with the active fraction. Figures 10 and 11 show photographs of the agar plates after culturing. As shown in Figures 10 and 11, morphological changes were observed in colonies of P. putida and P. fluorescens adjacent to the paper disk containing the active fraction, indicating elongation toward the paper disk. No morphological changes were observed in colonies of P. putida and P. fluorescens not adjacent to the paper disk.

[0058] (5) Attraction of Pseudomonas aeruginosa by ε-polylysine and α-polylysine Paper discs (Advantec Corporation) with a diameter of 8 mm, soaked in ε-polylysine (JNC Corporation) or α-polylysine (Fujifilm Wako Pure Chemical Industries, Ltd.), were placed on YNB agar medium to which a bacterial suspension of P. aeruginosa strain PAO1 was dropped. The media were incubated at 30°C for 2 days, and the colonies were visually observed.

[0059] Figure 12 is an explanatory diagram showing the results of culturing Pseudomonas aeruginosa with ε-polylysine. Figure 12 shows a photograph of the agar medium after culturing. As shown in Figure 12, morphological changes were observed in Pseudomonas aeruginosa colonies adjacent to the paper disc containing ε-polylysine, indicating elongation toward the paper disc. No morphological changes were observed in Pseudomonas aeruginosa colonies not adjacent to the paper disc.

[0060] Figure 13 is an explanatory diagram showing the results of culturing Pseudomonas aeruginosa with α-polylysine. Figure 13 shows a photograph of the agar medium after culturing. As shown in Figure 13, morphological changes were observed in Pseudomonas aeruginosa colonies adjacent to the paper disc containing α-polylysine, indicating elongation toward the paper disc. No morphological changes were observed in Pseudomonas aeruginosa colonies not adjacent to the paper disc.

[0061] The results above demonstrate that the secretions of the Bacillus bacterium (NB4 strain) identified by accession number NITE P-03755 attract Pseudomonas bacteria. Furthermore, it was shown that the secretions exhibiting Pseudomonas attractant activity contain ε-polylysine. It was also shown that commercially available ε-polylysine or α-polylysine attract Pseudomonas bacteria.

[0062] The present invention is not limited to the embodiments described above, and can be realized in various configurations without departing from its spirit. For example, the technical features in the embodiments and examples corresponding to the technical features in each form described in the summary of the invention can be replaced or combined as appropriate in order to solve some or all of the above-described problems, or to achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate.

Claims

1. An attractant for Pseudomonas bacteria, comprising at least one of polylysine and a Bacillus bacterium indicated by accession number NITE P-03755.

2. The attractant according to claim 1, wherein the polylysine comprises ε-polylysine.

3. A pesticide comprising the attractant described in claim 1 or claim 2.

4. The aforementioned Pseudomonas bacteria further include The pesticide according to claim 3.

5. Further comprising at least one of Pseudomonas putida and Pseudomonas fluorescens, The pesticide according to claim 3.

6. A fertilizer comprising the attractant according to claim 1 or claim 2.

7. The aforementioned Pseudomonas bacteria further include The fertilizer according to claim 6.

8. Further comprising at least one of Pseudomonas putida and Pseudomonas fluorescens, The fertilizer according to claim 6.

9. Plant seeds coated on their surface with at least one of polylysine and a bacterium of the genus Bacillus, indicated by accession number NITE P-03755.

10. Plant seedlings in which at least one of polylysine and a bacterium of the genus Bacillus, indicated by accession number NITE P-03755, is applied to the surface of the plant roots.

11. The process includes bringing at least one of polylysine and a bacterium of the genus Bacillus, indicated by accession number NITE P-03755, into contact with the roots or seeds of a plant. Methods of plant production.

12. The attractant according to claim 1 or claim 2, wherein the Pseudomonas bacterium is Pseudomonas aeruginosa.

13. An article having the attractant described in claim 1 or claim 2 coated on its surface.

14. The process includes applying polylysine to the surface of an article. Methods for attracting Pseudomonas aeruginosa.

15. The process involves causing Bacillus bacteria, indicated by accession number NITE P-03755, to secrete ε-polylysine, The process of collecting secreted ε-polylysine, A method for producing ε-polylysine, including the above.

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