Insect control agent, insect pathogenic fungus, and use thereof

Abstract

To provide an insect control agent comprising an insect pathogenic fungus, which allows combination use with other agents.SOLUTION: An insect control agent comprising an insect pathogenic fungus has a DNA sequence of an ITS1 region consisting of (a) a base sequence represented by Sequence No. 1 or (b) a base sequence having 99.3% or more identity with the base sequence represented by Sequence No. 1, and a DNA sequence of an ITS2 region consisting of (c) a base sequence represented by Sequence No. 2 or (d) a base sequence having 99.3% or more identity with the base sequence represented by Sequence No. 2, and is viable in a condition containing 100 mM boric acid.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present disclosure relates to insect pathogens.

Background Art

[0002] Conventionally, chemically synthesized agents have been used to control pests. Some chemically synthesized agents are toxic to humans and livestock, and some remain in the environment and affect the ecosystem. Against this background, biological agrochemicals may be used as agents that are highly safe for humans and livestock and have a low environmental impact. For example, Non-Patent Document 1 discloses the Beauveria bassiana GHA strain of insect pathogens as a type of biological agrochemical.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] The insect pathogens disclosed in Non-Patent Document 1 have the problem of low immediate effectiveness. For this reason, combined use with other agents that are highly safe for humans and livestock and have a low environmental impact is desired. However, depending on the other agent, it is toxic to the insect pathogen, so combined use has been difficult. Therefore, there is a need for an insect control agent containing an insect pathogen that can be combined with other agents.

Means for Solving the Problems

[0005] The present invention can be realized in the following forms.

[0006] (1) According to one embodiment of the present invention, there is provided an insect control agent containing an insect pathogen. This insect control agent has a DNA sequence of the ITS1 region consisting of (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and a DNA sequence of the ITS2 region consisting of (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is viable in an environment containing 100 mM boric acid. According to the insect control agent containing the insect pathogen of this embodiment, since the insect pathogen can survive in an environment containing 100 mM boric acid, it can be used in combination with boric acid.

[0007] (2) In the insect control agent described in (1) above, the insect pathogen may have an accession number of NITE-AP-04029. According to the insect control agent containing the insect pathogen of this embodiment, since it has a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1 and a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, it has excellent boric acid resistance.

[0008] (3) In the insect control agent described in (1) or (2) above, the conidia of the insect pathogen may be contained at 1.0×10 3 cells / mL or more and 1.0×10 12 cells / mL or less. According to the insect control agent of this embodiment, a decrease in the control efficacy against insects can be suppressed.

[0009] (4) In the insect control agent according to any one of (1) to (3) above, it may further contain boric acid or a salt thereof. According to the insect control agent of this embodiment, since it contains boric acid or a salt thereof together with the insect pathogen, insects can be controlled by two modes of action. As a result, the control efficacy against insects can be improved.

[0010] (5) In the insect control agent according to (4) above, the boric acid or its salt may be contained in an amount of 100 mM or more and 800 mM or less. According to the insect control agent in this form, since the boric acid or its salt is contained in an amount of 100 mM or more and 800 mM or less, a decrease in the control efficacy against insects can be suppressed.

[0011] (6) According to another aspect of the present disclosure, a liquid containing an insect pathogen and boric acid or its salt is provided. This liquid contains the boric acid or its salt in an amount of 100 mM or more and 800 mM or less, and the insect pathogen has a DNA sequence of the ITS1 region consisting of (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and a DNA sequence of the ITS2 region consisting of (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2. According to the liquid in this form, since the growth of other microorganisms that do not have boric acid resistance can be suppressed, the preservability is excellent.

[0012] (7) According to another aspect of the present disclosure, a method for controlling insects is provided. This method for controlling insects includes a step of applying an insect pathogen and boric acid or its salt to a plant body, and the insect pathogen has a DNA sequence of the ITS1 region consisting of (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and a DNA sequence of the ITS2 region consisting of (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is capable of surviving in an environment containing 100 mM boric acid. According to the method for controlling insects in this form, since the insect pathogen and boric acid or its salt are applied to the plant body, insects can be controlled by two mechanisms of action. As a result, the control efficacy against insects can be improved.

[0013] (8) According to another aspect of the present disclosure, there is provided the use of an entomopathogenic bacterium, which has (a) a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and (c) a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is viable in an environment containing 100 mM boric acid, as an insecticide.

[0014] (9) According to another aspect of the present disclosure, there is provided an entomopathogenic bacterium. This entomopathogenic bacterium has (a) a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and (c) a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is viable in an environment containing 200 mM boric acid. According to this form of entomopathogenic bacterium, since it has boric acid tolerance, it can be used in combination with boric acid.

[0015] It should be noted that the present invention can be realized in various forms. For example, it can be realized in the form of an insect control composition containing an entomopathogenic bacterium, an insect control kit comprising an entomopathogenic bacterium and boric acid or a salt thereof, a method for producing an insecticide, a method for producing agricultural crops using the insecticide, a method for producing trees using the insecticide, and the like.

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] As shown in the following examples, the inventors of the present application have found that a filamentous fungus having an ITS1 region and an ITS2 region consisting of a predetermined base sequence has entomopathogenicity and boric acid resistance, and have completed the present invention.

[0018] According to an embodiment of the present disclosure, an entomopathogenic fungus is provided. This entomopathogenic fungus (a) a base sequence represented by SEQ ID NO: 1, or (b) a base sequence having an identity of 99.3% or more with the base sequence represented by SEQ ID NO: 1, and a DNA sequence of the ITS1 region consisting of (c) a base sequence represented by SEQ ID NO: 2, or (d) a base sequence having an identity of 99.3% or more with the base sequence represented by SEQ ID NO: 2, It has a DNA sequence of the ITS2 region consisting of, and is viable in an environment containing 100 mM boric acid. The ITS1 region is an intervening sequence between 18S rDNA and 5.8S rDNA, and the ITS2 region is an intervening sequence between 5.8S rDNA and 28S rDNA.

[0019] An entomopathogenic fungus having a DNA sequence of the ITS1 region consisting of the above (a) or the above (b) and a DNA sequence of the ITS2 region consisting of the above (c) or the above (d) is classified as Beauveria bassiana. The entomopathogenic fungus having a DNA sequence of the ITS1 region consisting of the above (a) or the above (b) and a DNA sequence of the ITS2 region consisting of the above (c) or the above (d) is not particularly limited, and for example, it may be a filamentous fungus represented by the accession number NITE-AP-04029. The identity of the nucleotide sequences in the above (b) and the above (d) means the maximum identity (%) of the resulting nucleotide sequences after aligning the two nucleotide sequences to be compared by introducing gaps as necessary. The identity of the nucleotide sequences can be calculated, for example, by blastn of NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ) implementing the BLAST (registered trademark) algorithm, or by Homolog search implemented in Genetyx Win.

[0020] The DNA sequence of the ITS1 region of the insect pathogen preferably has an identity of 99.4% or more, more preferably 99.5% or more, still more preferably 99.6% or more, even more preferably 99.7% or more, still even more preferably 99.8% or more, particularly preferably 99.9% or more, and particularly even more preferably 100% with respect to the nucleotide sequence represented by SEQ ID NO: 1 from the viewpoint of pathogenicity to insects. The DNA sequence of the ITS2 region of the insect pathogen preferably has an identity of 99.4% or more, more preferably 99.5% or more, still more preferably 99.6% or more, even more preferably 99.7% or more, still even more preferably 99.8% or more, particularly preferably 99.9% or more, and particularly even more preferably 100% with respect to the nucleotide sequence represented by SEQ ID NO: 2 from the viewpoint of boric acid resistance.

[0021] The insect pathogen of this embodiment attaches to the body surface of the host insect and exhibits an insecticidal effect by growing and propagating hyphae. Therefore, the insect pathogen of this embodiment can be used as an insect control agent.

[0022] The insect pathogen of this embodiment can survive in an environment containing 100 mM boric acid. Preferably, the insect pathogen can survive in an environment containing 150 mM boric acid, more preferably in an environment containing 200 mM boric acid, still more preferably in an environment containing 250 mM boric acid, even more preferably in an environment containing 300 mM boric acid, still even more preferably in an environment containing 350 mM boric acid, and particularly preferably in an environment containing 400 mM boric acid. In the present disclosure, "being able to survive in an environment containing boric acid at a predetermined concentration" means being able to survive for 4 days or more when left standing at 25°C in a medium, hydrate, aqueous solution, etc. containing boric acid at a predetermined concentration.

[0023] The entomopathogen of this embodiment has high resistance to boric acid and can be used in combination with boric acid. More specifically, for example, it can be used in combination with boric acid as a control agent. That is, in order to control the target insects, it can be used in combination with boric acid which has a different mechanism of action from the entomopathogen. In addition, since the entomopathogen of this embodiment has high resistance to boric acid, it can be stored in a liquid containing boric acid. As a result, in the liquid, the growth of other microorganisms that do not have boric acid resistance can be suppressed, so the storage stability is excellent.

[0024] According to the second aspect of the present disclosure, there is provided an insect control agent containing the above-mentioned entomopathogen. The insects to be controlled are not particularly limited, and any insect that can be parasitized by the above-mentioned entomopathogen can be a host. For example, aphids, thrips, aphids, ladybugs, mites, ants, diamondback moths, leaf beetles, etc. can be mentioned. According to the insect control agent of this embodiment, since the above-mentioned entomopathogen is pathogenic to the host insects, the insects can be controlled. In addition, according to the insect control agent of this embodiment, since the above-mentioned entomopathogen has transmissibility, it is possible to suppress a decrease in the persistence of the control effect. In addition, since the insect control agent of this embodiment contains an entomopathogen as an active ingredient, it is possible to suppress a decrease in safety to humans and livestock and reduce the environmental burden. Note that the "insect control agent" of the present disclosure includes insecticides.

[0025] The concentration of the conidia of the entomopathogen contained in the insect control agent of this embodiment is not particularly limited, and may be any concentration that can exert a control effect on the target insects. From the viewpoint of suppressing a decrease in controllability, the concentration of the conidia of the entomopathogen is preferably 1.0×10 3 or more per mL. Also, from the viewpoint of suppressing a decrease in economy, the concentration of the conidia of the entomopathogen is preferably 1.0×10 12 or less per mL. The insect control agent of this embodiment preferably contains the conidia of the entomopathogen at 1.0×10 3 or more and 1.0×10 12 or less per mL, and more preferably 1.0×10 3 or more and 1.0×10 10It is more preferable to contain not more than, and it is more preferably to contain not less than 1.0×10 4 per mL and not more than 1.0×10 10 per mL. It is still more preferable to contain not less than 1.0×10 4 per mL and not more than 1.0×10 8 per mL. It is even more preferable to contain not more than 1.0×10

[0026] The insect control agent of the present embodiment is preferably used in combination with boric acid or a salt thereof. Further, the insect control agent of the present embodiment preferably further contains boric acid or a salt thereof. By using the insect control agent in combination with boric acid or a salt thereof, or by containing boric acid or a salt thereof together with an insect pathogen, insects can be controlled by two mechanisms of action. As a result, it is expected to have both the immediate effect of boric acid and the transmissibility of the insect pathogen, so that the control performance can be improved.

[0027] The boric acid used in combination with the insect control agent, or the boric acid contained in the insect control agent, may be in the form of a salt. The salt is not particularly limited, and examples thereof include alkali metal salts such as sodium and potassium, alkaline earth metal salts such as calcium and magnesium, ammonium salts, and amine salts. From the viewpoint of suppressing a decrease in control performance, the concentration of boric acid or a salt thereof is preferably 50 mM or more. Further, from the viewpoint of suppressing a decrease in solubility, the concentration of boric acid or a salt thereof is preferably 800 mM or less. The insect control agent of the present embodiment preferably contains boric acid or a salt thereof at 50 mM or more and 800 mM or less, more preferably at 100 mM or more and 800 mM or less, and still more preferably at 100 mM or more and 600 mM or less.

[0028] Insect control agents may contain, in addition to entomopathogens, other components that contribute to insect control in order to further enhance the control effect against insects or to broaden the range of target insects. Also, insect control agents may be used in combination with other agents that contribute to insect control in order to further enhance the control effect against insects or to broaden the range of target insects. The other components or other agents are not particularly limited, but are preferably agents with high safety for humans and livestock and low environmental impact. Further, the insect control agent may further contain other optional components. The other optional components are not particularly limited, but examples include carriers, solvents, fixing agents, wetting agents, dispersants, antifungal agents, antibacterial agents, surfactants, stabilizers, pH adjusters, thickeners, defoaming agents, antifreeze agents, fillers, binders, disintegrants, lubricants, diluents, excipients, spreading agents, coloring agents, and the like.

[0029] The dosage form of the insect control agent can be variously selected according to the application form and the like, and is not particularly limited. Examples include liquids such as flowable agents and emulsions, and powders or granules such as wettable powders, water-soluble agents, powders, granules, and powder granules. The application form is not particularly limited, and examples include spraying and coating. From the viewpoint of enhancing the control effect, the application form to plants is preferably spraying or coating on the whole plant body, trunk, branches, leaves, etc., and more preferably spraying.

[0030] According to the third aspect of the present disclosure, a method for controlling insects is provided. This method for controlling insects includes a step of applying an entomopathogen and boric acid or a salt thereof to a plant body. The entomopathogen is the above-mentioned entomopathogen, (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, the DNA sequence of the ITS1 region consisting of (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, It has a DNA sequence of the ITS2 region consisting of, and is capable of surviving in an environment containing 100 mM boric acid. In the above step, the insect pathogen and boric acid or its salt may be applied together or separately, but it is preferable to apply them together. For this reason, the insect pathogen and boric acid or its salt may be applied at different locations or times, but it is preferable that the insect pathogen and boric acid or its salt are in a mixed state, or that the insect pathogen and boric acid or its salt are applied to the same location.

[0031] The application form to plants is not particularly limited, and examples include spraying, coating, etc. From the viewpoint of enhancing the control effect, as an application form to plants, it is preferably used by spraying or coating the whole plant body, the trunk, branches, leaves, etc., and more preferably used by spraying. The application time, number of applications, and frequency to the plant body are not particularly limited and may be appropriately set according to the target plant species, the environment of the cultivation area, the control guidelines, etc.

[0032] The plants to be applied are not particularly limited and may be plants for ornamental purposes such as flowers, flowering trees, and other trees, but are preferably plants capable of producing agricultural products such as vegetables, fruits, and grains. More specifically, for example, Solanaceae plants such as tomatoes, eggplants, peppers, chili peppers, tobacco, and potatoes, Brassicaceae plants such as cabbages, Chinese cabbages, broccoli, daikons, spinach, komatsuna, cauliflowers, and Arabidopsis thaliana, Cucurbitaceae plants such as cucumbers, pumpkins, and melons, Rosaceae plants such as strawberries, apples, pears, plums, and peaches, Poaceae plants such as rice and wheat, Fabaceae plants such as soybeans, Araceae plants such as taros, Actinidiaceae plants such as kiwifruits, Amaryllidaceae plants such as onions and garlic, etc.

[0033] According to the fourth aspect of the present disclosure, a liquid containing an insect pathogen and boric acid or its salt is provided. This liquid contains boric acid or its salt in an amount of 100 mM or more and 800 mM or less, and the insect pathogen is (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) A nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and the DNA sequence of the ITS1 region consisting of (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and the DNA sequence of the ITS2 region consisting of. According to the liquid of this form, since the growth of other microorganisms that do not have boric acid resistance can be suppressed, the preservability is excellent. Further, according to the liquid of this form, for example, the handleability is excellent as compared with a configuration in which an insect pathogen is suspended in an emulsified oily liquid or the like that does not contain boric acid. The liquid of the present disclosure may be an insect control composition or may be used in the production of an insect control agent. From the viewpoint of suppressing the growth of other microorganisms, the liquid of the present disclosure preferably contains boric acid or a salt thereof in an amount of 150 mM or more and 700 mM or less, and more preferably 200 mM or more and 600 mM or less. The liquid of the present disclosure may contain other optional components. The other optional components are not particularly limited, and examples thereof include carriers, solvents, fixing agents, wetting agents, dispersants, antifungal agents, antibacterial agents, surfactants, stabilizers, pH adjusters, thickeners, defoaming agents, antifreezing agents, fillers, binders, disintegrants, lubricants, diluents, excipients, spreading agents, coloring agents, and the like.

Examples

[0034] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples. In the following examples, “%” means “% by mass” unless otherwise specified.

[0035] 1. Isolation of bacteria 100 μl of TBE buffer (89 mM Tris-HCl, 89 mM boric acid, 2 mM EDTA, pH 8.0) in which bacterial growth was observed was applied to an LB solid medium (1% polypeptone, 0.5% yeast extract, 1% NaCl, 1.5% agar, adjusted to pH 7.5 with NaOH) supplemented with 100 mM boric acid, and statically cultured at 25° C. for 48 hours to separate colonies. In the following description, this separated bacterium is also referred to as “NIBB strain”.

[0036] 2. Confirmation Experiment of Boric Acid Tolerance For the soil suspension, Escherichia coli DH5α strain, and NIBB strain, a confirmation experiment of boric acid tolerance was conducted. LB solid media containing 0 mM, 25 mM, 50 mM, 100 mM, 200 mM, and 400 mM boric acid were prepared. 10 g of soil was collected from a field within the premises of the National Institute for Basic Biology in Okazaki City, Aichi Prefecture, and suspended in 100 ml of ultrapure water to obtain a soil suspension. 100 μl of each soil suspension was spread on the prepared LB solid media containing 0 - 400 mM boric acid, and statically cultured at 25°C for 48 hours. The Escherichia coli DH5α strain was cultured in 4 ml of LB liquid medium at 37°C, 150 rpm for 16 hours, and 100 μl of each was spread on the LB solid media containing 0 - 400 mM boric acid, and statically cultured at 37°C for 2 days. The isolated colonies (NIBB strain) were inoculated into 4 ml of LB liquid medium, cultured at 25°C, 150 rpm for 48 hours, and 100 μl of each was spread on the LB solid media containing 0 - 400 mM boric acid, and statically cultured at 37°C for 2 days.

[0037] Figure 1 is an explanatory diagram showing the results of the soil suspension in the confirmation experiment of boric acid tolerance. Figure 2 is an explanatory diagram showing the results of the Escherichia coli DH5α strain in the confirmation experiment of boric acid tolerance. Figure 3 is an explanatory diagram showing the results of the NIBB strain in the confirmation experiment of boric acid tolerance. The numerical values described in Figures 1 - 3 indicate the concentration of boric acid (mM). For the soil suspension and the Escherichia coli DH5α strain, no colony formation was observed in the medium containing 50 mM or more of boric acid. In contrast, for the NIBB strain, colony formation was observed after 2 days of culture in the medium containing 50 mM of boric acid and also in the medium containing 100 mM of boric acid. Additionally, through further long - term culture, colony formation was confirmed even in the medium containing 200 mM or more of boric acid (not shown). From the above results, it was shown that the NIBB strain has high boric acid tolerance.

[0038] 3. Molecular Phylogenetic Analysis PCR templates were prepared from the isolated colonies using Template Prepper for DNA (manufactured by Nippon Gene Co., Ltd.). The ITS1 and ITS2 regions were amplified according to the paper by Vancov et al. (2009) (Tony Vancov, Brad Keen, FEMS Microbiology Letters, Volume 296, Issue 1, July 2009, 91-96). The obtained PCR products were purified using NucleoSpin Gel and PCR Clean-up (manufactured by MACHEREY-NAGEL, "NucleoSpin" is a registered trademark), and the DNA sequences were determined by the Sanger sequencing method. Molecular phylogenetic analysis was performed on the obtained DNA sequences using BLAST (registered trademark) of NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi).

[0039] From the DNA sequences of the ITS1 and ITS2 regions used for the molecular phylogenetic analysis of fungi, the ITS1 and ITS2 regions of the NIBB strain had the highest homology with the Beauveria bassiana ARSEF 1564 strain registered in the database (Blast Internal transcribed spacer region (ITS) from Fungi type and reference materia), with a homology of 98% for both the ITS1 and ITS2 regions. According to this result, the NIBB strain was shown to be Beauveria bassiana.

[0040] 4. Confirmation experiment of growth in boric acid-added medium Growth confirmation experiments were conducted on the NIBB strain, the Beauveria bassiana GHA strain, and other Beauveria bassiana strains in a boric acid-supplemented medium. NBRC102367, NBRC102721, NBRC102737, NBRC102738, NBRC102739, NBRC102781, and NBRC102790 of Beauveria bassiana were obtained from NBRC. The Beauveria bassiana GHA strain was isolated from Botanigard ES (manufactured by Arysta LifeScience, "Botanigard" is a registered trademark). Each strain was spread on a malt solid medium (2% malt extract, 2% glucose, 0.1% polypeptone, 2.0% agar, adjusted to pH 6.0 with NaOH) and statically cultured at 25°C for 4 days. The cells were collected and a molecular suspension of 1×10 6 cells / ml was prepared according to the method of the paper by Nishi et al. (2021). 500 μl of the conidia suspension was inoculated into 50 ml of a malt liquid medium (2% malt extract, 2% glucose, 0.1% polypeptone, adjusted to pH 6.0 with NaOH) containing 0, 25, 50, 100, and 200 mM boric acid, and cultured with shaking at 25°C and 150 rpm for 4 days. The medium was filtered to collect the cells, and then the cell mass (g) was measured. The experiment was performed in triplicate.

[0041] Figure 4 is an explanatory diagram showing the results of the growth confirmation experiment in the boric acid-added medium. The error bars in Figure 4 indicate the standard deviation. Among Beauveria bassiana obtained from NBRC, many of NBRC102367, NBRC102737, NBRC102738, NBRC102739, NBRC102781, NBRC102790 and GHA strain could not grow in the medium containing 50 mM boric acid, and no growth was observed in the medium containing 100 mM or more boric acid. In contrast, for the NIBB strain, growth equivalent to that in the boric acid-free medium was confirmed in the medium containing 50 mM boric acid. Also, for the NIBB strain, about 50% growth was confirmed in the medium containing 100 mM boric acid compared to the boric acid-free medium, and about 25% growth was confirmed even in the medium containing 200 mM boric acid. Also, for NBRC102721, about 40% growth was confirmed in the medium containing 50 mM boric acid compared to the boric acid-free medium, and growth was also observed in the medium containing 100 mM boric acid. According to the above results, it was shown that boric acid tolerance is not a common feature of all Beauveria bassiana.

[0042] 5. Verification of the effect on termites Using the same method as the "Growth confirmation experiment in the boric acid-added medium", conidial suspensions of 1 × 10 6 cells / ml were prepared from the GHA strain and the NIBB strain. For the preparation of the conidial suspension of the NIBB strain, two types of colonies were used. Three pieces of filter paper cut into 1 × 1 cm were placed in each well of a 24-well plate, and 200 μl of the conidial suspension was dropped. A sample without conidia was used as a control. Individuals were collected from a single colony of Reticulitermes speratus captured in Okazaki City, Aichi Prefecture, and one individual was placed in each well and grown at 25°C in the dark. The number of surviving termites (individuals) up to the third day of growth is shown in Table 1. Note that the results using conidial suspensions prepared from two mutually different colonies of the NIBB strain are shown.

[0043]

Table 1

[0044] From the results shown in Table 1, the following was found. That is, the NIBB strain showed an insecticidal effect against termites, similar to the GHA strain isolated from the registered pesticide Botanigard (registered trademark). Therefore, it was shown that the NIBB strain retains entomopathogenicity.

[0045] Figure 5 is an image showing the pathogenicity of the GHA strain and the NIBB strain against termites. Termites grown in an environment containing a conidial suspension of the GHA strain showed hard white mycelia partially on the body surface. Also, termites grown in an environment containing a conidial suspension of the NIBB strain showed relatively soft mycelia over a wide area of the body surface.

[0046] 6. Confirmation test of insecticidal effect Figure 6 is an explanatory diagram showing a system using Formica japonica. Gypsum with a thickness of 2 cm was placed at the bottom of a plastic cup and left to stand for about 3 days to solidify. Jelly (2% sucrose, 0.5% yeast extract, 0.1% calcium chloride, 0.8% gellan gum) as bait was prepared and 1 g was placed. Separately from the jelly, a folded Kim towel with 1 ml of pure water dropped thereon was placed. Both the bait and the water were replaced with new ones every other day.

[0047] The insecticidal effect by adding boric acid, the insecticidal effect by adding entomopathogenic bacteria, and the insecticidal effect by the combined use of boric acid and the NIBB strain were examined respectively. Formica japonica collected in Okazaki City, Aichi Prefecture was used and acclimated for 3 days before the experiment. Jelly of the bait under each condition was placed in the cup and the ants were reared at 25 °C in the dark for 9 days. Every day at 12:00, immobile individuals were counted. In the confirmation of the insecticidal effect by adding boric acid, jelly containing 0 mM, 100 mM, and 200 mM boric acid was used. In the confirmation of the insecticidal effect by adding entomopathogenic bacteria, for the GHA strain or the NIBB strain, 1×10 6Jelly added with 100 μl of a conidia suspension at [number of conidia] per ml was used. In the confirmation of the insecticidal effect by the combined use of boric acid and the NIBB strain, jelly containing 200 mM boric acid and jelly added with 100 μl of a conidia suspension of the NIBB strain at [number of conidia] per ml were used. In the control group, jelly containing neither boric acid nor the conidia suspension was used. 6 Jelly added with 100 μl of a conidia suspension of the NIBB strain at [number of conidia] per ml was used. In the control group, jelly containing neither boric acid nor the conidia suspension was used.

[0048] Figure 7 is an explanatory diagram showing the results of the insecticidal effect by the addition of boric acid. Figure 8 is an explanatory diagram showing the results of the insecticidal effect by the addition of an entomopathogenic fungus. Figure 9 is an explanatory diagram showing the results of the insecticidal effect by the combined use of boric acid and the NIBB strain. In the Kaplan-Meier curves shown in Figures 7 to 9 and Figure 11 described later, the vertical axis indicates the survival rate (Probability of Survival, %), the horizontal axis indicates the elapsed time (Days elapsed, days), and the number of individuals (N) used in each test group is shown.

[0049] As shown in Figure 7, in the test group using jelly containing 100 mM boric acid, all individuals died on the 7th day, and in the test group using jelly containing 200 mM boric acid, all individuals died on the 3rd day. As shown in Figure 8, in the test groups using jelly containing the GHA strain or the NIBB strain, all individuals died on the 7th day. According to the results of Figures 7 and 8, it is shown that Beauveria bassiana exhibits an insecticidal effect later than boric acid. As shown in Figure 9, no difference in the insecticidal effect was observed between the test group using jelly with only boric acid added and the test group using jelly with boric acid and the NIBB strain added. Since the insecticidal effect of boric acid appears earlier than that of Beauveria bassiana, it is considered that the combined effect of boric acid and the NIBB strain could not be confirmed in this test.

[0050] Figure 10 is an image showing the pathogenicity of the NIBB strain against the black imported fire ant. In the black imported fire ants grown using jelly containing the NIBB strain as food, hyphae were observed over a wide area of the body surface.

[0051] 7. Examination of the transmissibility of the NIBB strain Since Beauveria bassiana is an insect pathogen, it is expected to spread from individual to individual, unlike boric acid. To confirm the transmission effect from individual to individual, black garden ants (Formica japonica) were grown for 1 day using jelly containing boric acid, the NIBB strain, or boric acid + the NIBB strain, and then grown in the same container with the same number of black garden ants that had been grown using normal food jelly, thereby verifying the transmissibility.

[0052] Black garden ants (Formica japonica) collected in Okazaki City, Aichi Prefecture were used and acclimated for 3 days before the experiment. As food jelly, those containing 200 mM boric acid, those with 100 μl of a conidial suspension of the NIBB strain at 1×10 6 cells / ml added to jelly without boric acid, those with 100 μl of a conidial suspension of the NIBB strain at 1×10 6 cells / ml added to jelly containing 200 mM boric acid, and those containing neither boric acid nor the NIBB strain were prepared, a total of 4 conditions of jelly. One condition of jelly was placed in each cup, and 24 black garden ants were placed in each and reared at 25°C in the dark for 1 day. At the same time, using normal food jelly containing neither boric acid nor the NIBB strain, each 24 black garden ants were reared at 25°C in the dark for 1 day. Thereafter, the groups of individuals reared using the above 4 conditions of jelly and the groups of individuals reared using normal jelly were combined and placed in one cup, and reared at 25°C in the dark for 9 days using normal jelly containing neither boric acid nor the NIBB strain. At 12:00 every day, the immobile individuals were counted.

[0053] Figure 11 is an explanatory diagram showing the results of examining the transmissibility of the NIBB strain. In the test group with only boric acid, half of the total individuals died by the 9th day. According to this result, it is considered that the individuals grown for 1 day on jelly containing boric acid died, indicating that the insecticidal effect of boric acid is not present in individuals that did not directly ingest the jelly containing boric acid. That is, it shows that boric acid does not have transmissibility. Also, in the test group with only the NIBB strain or the test group using a combination of boric acid and the NIBB strain, 90% of the total individuals died by the 9th day. According to this result, it shows that individuals that were not directly inoculated with the jelly containing the NIBB strain also died. Therefore, it is considered that the NIBB strain spread from individual to individual and exerted an insecticidal effect.

[0054] 8. Confirmation experiment on storage stability in boric acid solution When using entomopathogenic bacteria as pesticides, it is preferable that the bacterial cells can be stored in a liquid at room temperature or the like. Conventional pesticides such as Botanigard (registered trademark) are stored in a state mixed with oil in a form that can stably store the bacterial cells while preventing other bacteria from mixing and growing. Boric acid shows toxicity to bacteria and fungi. For this reason, since the boric acid solution is considered to be a storage form that can inhibit the growth of other bacteria, the storage stability in the boric acid solution was confirmed.

[0055] The GHA strain or NIBB strain was applied to a malt solid medium (2% malt extract, 2% glucose, 0.1% polypeptone, 2.0% agar, adjusted to pH 6.0 with NaOH) and statically cultured at 25°C for 4 days. The bacterial cells were collected, and according to the method in the paper by Nishi et al. (2021), a conidia suspension of 1×10 6 cells / ml was prepared. 0.4 ml of the conidia suspension was added to 3.6 ml of 100 mM Tris-HCl (pH 8.0) containing 0 mM, 400 mM, 600 mM, or 800 mM boric acid, and the mixture was shaken and cultured at 25°C and 150 rpm for 8 days. 100 μl was applied to the malt solid medium and statically cultured at 25°C for 4 days.

[0056] FIG. 12 is an explanatory diagram showing the results of the preservation confirmation experiment in a boric acid solution. When the GHA strain was incubated in a high-concentration boric acid solution of 400 mM to 800 mM for 8 days, only a few surviving conidia were observed. In contrast, even when the NIBB strain was incubated in a high-concentration boric acid solution of 400 mM to 800 mM for 8 days, many surviving conidia were confirmed. According to the above results, it was considered that the NIBB strain could be preserved in an aqueous solution containing boric acid and could be preserved while suppressing the growth of other bacteria.

[0057] 9. Analysis of DNA sequences of ITS1 and ITS2 regions As described in the above-mentioned "Molecular phylogenetic analysis", for the NIBB strain, the ITS1 and ITS2 regions were amplified by PCR, the obtained PCR products were purified, and then the DNA sequences were determined by the Sanger sequencing method. Similarly, for the Beauveria bassiana GHA strain, NBRC102367, NBRC102721, NBRC102737, NBRC102738, NBRC102739, NBRC102781, and NBRC102790 used in the above-mentioned "Growth confirmation experiment in boric acid-added medium", the DNA sequences of the ITS1 and ITS2 regions were analyzed. For ITS1 and ITS2, the identity between each strain was determined, and a phylogenetic tree of the genes was created. The identity of the nucleotide sequences was calculated using blastn of NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ) implementing the BLAST (registered trademark) algorithm.

[0058] SEQ ID NO: 1 is the DNA sequence of the ITS1 region of the NIBB strain. SEQ ID NO: 2 is the DNA sequence of the ITS2 region of the NIBB strain. SEQ ID NO: 3 is the DNA sequence of the ITS1 region of NBRC102367. SEQ ID NO: 4 is the DNA sequence of the ITS1 region of NBRC102721. SEQ ID NO: 5 is the DNA sequence of the ITS1 region of NBRC102737. SEQ ID NO: 6 is the DNA sequence of the ITS1 region of NBRC102738. SEQ ID NO: 7 is the DNA sequence of the ITS1 region of NBRC102739. SEQ ID NO: 8 is the DNA sequence of the ITS1 region of NBRC102781. SEQ ID NO: 9 is the DNA sequence of the ITS1 region of NBRC102790. SEQ ID NO: 10 is the DNA sequence of the ITS1 region of the GHA strain. SEQ ID NO: 11 is the DNA sequence of the ITS2 region of NBRC102367. SEQ ID NO: 12 is the DNA sequence of the ITS2 region of NBRC102721. SEQ ID NO: 13 is the DNA sequence of the ITS2 region of NBRC102737. SEQ ID NO: 14 is the DNA sequence of the ITS2 region of NBRC102738. SEQ ID NO: 15 is the DNA sequence of the ITS2 region of NBRC102739. SEQ ID NO: 16 is the DNA sequence of the ITS2 region of NBRC102781. SEQ ID NO: 17 is the DNA sequence of the ITS2 region of NBRC102790. SEQ ID NO: 18 is the DNA sequence of the ITS2 region of the GHA strain.

[0059] Figure 13 is an explanatory diagram showing the phylogenetic tree of ITS1. Figure 14 is an explanatory diagram showing the phylogenetic tree of ITS2. In both the ITS1 region and the ITS2 region, the identities of NBRC102721, the NIBB strain, and the GHA strain were relatively high. Also, among the Beauveria bassiana analyzed, the NIBB strain was found to be genetically closest to NBRC102721.

[0060] The present invention is not limited to the above-described embodiments, and can be realized in various configurations without departing from the gist thereof. For example, the embodiments and technical features in the examples corresponding to the technical features in each form described in the column of the summary of the invention can be appropriately replaced or combined in order to solve part or all of the above-described problems, or to achieve part or all of the above-described effects. Further, if the technical feature is not described as essential in this specification, it can be appropriately deleted.

Claims

1. An insect control agent containing an insect pathogenic bacterium, wherein the insect pathogenic bacterium (a) has a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1, or (b) has a DNA sequence of the ITS1 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and (c) has a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, or (d) has a DNA sequence of the ITS2 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is capable of surviving in an environment containing 100 mM boric acid. An insect control agent.

2. The insect control agent according to Claim 1, wherein the insect pathogenic bacterium has an accession number of NITE-AP-04029. An insect control agent.

3. The insect control agent according to Claim 1 or Claim 2. An insect control agent. The conidia of the entomopathogenic fungus are contained at 1.0×10 ３ or more and 1.0×10 １２ or less per mL,

4. The insect control agent according to Claim 1 or Claim 2, further comprising boric acid or a salt thereof. An insect control agent.

5. The insect control agent according to Claim 4, wherein the boric acid or a salt thereof is contained in an amount of 100 mM or more and 800 mM or less. An insect control agent.

6. A liquid containing an insect pathogenic bacterium and boric acid or a salt thereof, wherein the boric acid or a salt thereof is contained in an amount of 100 mM or more and 800 mM or less, and the insect pathogenic bacterium (a) has a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1, or (b) has a DNA sequence of the ITS1 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and (c) has a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, or (d) has a DNA sequence of the ITS2 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO:

2. A liquid.

7. A method for controlling insects, comprising the step of applying an insect pathogenic bacterium and boric acid or a salt thereof to a plant body, wherein the insect pathogenic bacterium (a) has a DNA sequence of the ITS1 region consisting of the nucleotide sequence represented by SEQ ID NO: 1, or (b) has a DNA sequence of the ITS1 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, and (c) has a DNA sequence of the ITS2 region consisting of the nucleotide sequence represented by SEQ ID NO: 2, or (d) has a DNA sequence of the ITS2 region consisting of a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and is capable of surviving in an environment containing 100 mM boric acid. A method for controlling insects.

8. (a) The nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 ​ ​ ​ The DNA sequence of the ITS1 region consisting of, (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, the DNA sequence of the ITS2 region consisting of, having, and the use of an entomopathogenic bacterium capable of surviving in an environment containing 100 mM boric acid as an insecticide.

9. An entomopathogenic bacterium, (a) the nucleotide sequence represented by SEQ ID NO: 1, or (b) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, the DNA sequence of the ITS1 region consisting of, (c) the nucleotide sequence represented by SEQ ID NO: 2, or (d) a nucleotide sequence having 99.3% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, the DNA sequence of the ITS2 region consisting of, having, and being capable of surviving in an environment containing 200 mM boric acid, an entomopathogenic bacterium.

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